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Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control

Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control YEASTBOOK CELL SIGNALING & DEVELOPMENT Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control ,1 †,1 Robbie Loewith* and Michael N. Hall *Department of Molecular Biology and National Centers of Competence in Research and Frontiers in Genetics and Chemical Biology, University of Geneva, Geneva, CH-1211, Switzerland, and Biozentrum, University of Basel, Basel CH-4056, Switzerland ABSTRACT TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae. TABLE OF CONTENTS Abstract 1177 Introduction 1178 The Early Days 1178 TOR Complex 1 1181 Composition of TOR complex 1 1181 Localization of TORC1 1183 Upstream of TORC1 1183 Physiological regulators (carbon, nitrogen, phosphate, stress, caffeine): 1183 The EGO complex: 1184 Feedback loop/ribosome biogenesis homeostasis: 1184 Downstream of TORC1 1185 Proximal TORC1 effectors: 1185 Characterization of Sch9 as a TORC1 substrate: 1185 Characterization of Tap42‐PP2A as a TORC1 effector: 1185 Other TORC1 substrates: 1186 Distal readouts downstream of TORC1: 1186 TORC1 promotes cell growth: 1186 Protein synthesis: 1186 Ribosome biogenesis: 1186 Regulation of cell cycle/cell size: 1188 Continued Copyright © 2011 by the Genetics Society of America doi: 10.1534/genetics.111.133363 Manuscript received July 29, 2011; accepted for publication September 12, 2011 Available freely online through the author-supported open access option. Corresponding authors: Biozentrum, University of Basel, Klingelbergstrasse 70, Basel CH-4056, Switzerland. E-mail: m.hall@unibas.ch; and Department of Molecular Biology, NCCRs Chemical Biology and Frontiers in Genetics, University of Geneva, 30 quai Ernest Ansermet Geneva CH-1211, Switzerland. E-mail: Robbie.Loewith@unige.ch Genetics, Vol. 189, 1177–1201 December 2011 1177 CONTENTS, continued TORC1 inhibits stress responses: 1189 Environmental stress response: 1189 Nutrient uptake and intermediary metabolism: 1189 Autophagy: 1190 Cell-wall integrity pathway: 1191 TORC1 accelerates aging: 1191 Less-characterized effectors identified in phosphoproteomic studies: 1192 TOR Complex 2 1192 Composition and localization of TOR complex 2 1192 Upstream of TORC2 1193 TORC2 substrates 1193 Distal readouts downstream of TORC2 1193 Future Directions 1194 What is upstream of the two complexes? 1194 What is downstream of the TORCs? 1194 HE contributors to this GENETICS set of reviews were nutrients to the tumor cells (Guba et al. 2002). Finally, Tasked to focus on the developments in their field since rapamycin-eluting stents prevent restenosis after angio- plasty. Thus, rapamycin has clinical applications in three 1991, the year the last yeast monographs were published. major therapeutic areas: organ transplantation, cancer, and Coincidentally, Target Of Rapamycin (TOR) was discovered coronary artery disease. What do fungi and the seemingly in 1991. We thus have the whole TOR story to tell, from the very different conditions of transplant rejection, cancer, and beginning, in a review that marks the 20th anniversary of restenosis have in common in their underlying biology such TOR. As we review TOR signaling in Saccharomyces cerevi- that all can be treated with the same drug? All three con- siae, the reader is referred to other reviews for descriptions ditions (and the spread of pathogenic fungi) are due to of TOR in other organisms (Wullschleger et al. 2006; Polak ectopic or otherwise undesirable cell growth, suggesting and Hall 2009; Soulard et al. 2009; Caron et al. 2010; Kim that the molecular target of rapamycin is a central controller and Guan 2011; Zoncu et al. 2011). of cell growth. TOR is indeed dedicated to controlling cell The story of the TOR-signaling network begins with a re- growth, but what is this target and how does it control cell markable drug, rapamycin (Abraham and Wiederrecht growth? 1996; Benjamin et al. 2011). Rapamycin is a lipophilic mac- rolide and a natural secondary metabolite produced by Streptomyces hygroscopicus, a bacterium isolated from a soil The Early Days sample collected in Rapa-Nui (Easter Island) in 1965— hence the name rapamycin. Rapamycin was originally puri- Studies to identify the cellular target of rapamycin and to fied in the early 1970s as an antifungal agent. Although it elucidate the drug’s mode of action were initiated in the late effectively inhibits fungi, it was discarded as an antifungal 1980s by several groups working with yeast (Heitman et al. agent because of its then undesirable immunosuppressive 1991a; Cafferkey et al. 1993; Kunz et al. 1993) and mam- side effects. Years later, it was “rediscovered” as a T-cell malian cells (Brown et al. 1994; Chiu et al. 1994; Sabatini inhibitor and as an immunosuppressant for the treatment et al. 1994; Sabers et al. 1995). At that time, rapamycin was of allograft rejection. Preclinical studies subsequently known to inhibit the vertebrate immune system by blocking showed that rapamycin and its derivatives, CCI-779 a signaling pathway in helper T cells that mediates cell cycle (Wyeth-Ayerst) and RAD001 (Novartis), also strongly in- (G1) progression in response to the lymphokine IL-2. How- hibit the proliferation of tumor cells. Rapamycin received ever, the molecular mode of action of the drug was not clinical approval in 1999 for use in the prevention of organ known other than it possibly involved binding and inhibiting rejection in kidney transplant patients, and additional appli- the cytosolic peptidyl-prolyl cis-trans isomerase FKBP12 cations as an immunosuppressive agent have since been de- (FK506-binding protein 12), also known as an immunophi- veloped. CCI-779 (Torisel) and RAD001 (Afinitor) were lin (Schreiber 1991). Furthermore, the observation that approved in 2007 and 2009, respectively, for treatment of rapamycin inhibited cell cycle progression in yeast as in advanced kidney cancer. Rapamycin is effective against mammalian cells suggested that the molecular target was tumors because it blocks the growth of tumor cells directly conserved from yeast to vertebrates and that yeast cells and because of the indirect effect of preventing the growth could thus be exploited to identify the target of rapamycin of new blood vessels (angiogenesis) that supply oxygen and (Heitman et al. 1991a). It should be noted that the early 1178 R. Loewith and M. N. Hall researchers were interested not only in understanding rapa- mutants defective in FKBP were recovered, but also obtained mycin’s mechanism of action, but also in using rapamycin as were mutants altered in either one of two novel genes a probe to identify novel proliferation-controlling signaling termed TOR1 and TOR2. The fpr1 mutations were common pathways (Kunz and Hall 1993). In the late 1980s, signifi- and recessive. Interestingly, the TOR1 and TOR2 mutations cantly less was known about signaling pathways than today; were rare and dominant. The TOR1 and TOR2 genes were indeed, few and only incomplete pathways were known. cloned, on the basis of the dominant rapamycin-resistance The early studies in yeast first focused on identifying an phenotype of the mutant alleles, and sequenced (Cafferkey FKBP (FK506-binding protein) (Heitman et al. 1991b; Koltin et al. 1993; Kunz et al. 1993; Helliwell et al. 1994). Both et al. 1991; Tanida et al. 1991; Wiederrecht et al. 1991). TOR1 and TOR2 proteins are 282 kDa in size (2470 and FKBP12 had previously been identified in mammalian cell 2474 amino acids, respectively) and 67% identical. TOR1 extracts as a rapamycin (and FK506)-binding protein. Yeast and TOR2 are also the founding members of the PI kinase- FKBP was purified to homogeneity using an FK506 column related protein kinase (PIKK) family of atypical Ser/Thr- and partially sequenced. The protein sequence information specific kinases (other members include TEL1, ATM, DNA- was used to design degenerate oligonucleotides that were PK, and MEC1) (Keith and Schreiber 1995). Although the then used to isolate the FKBP-encoding gene FPR1 (Heitman catalytic domain of all members of this protein kinase family et al. 1991b). The predicted amino acid sequence of yeast resembles the catalytic domain of lipid kinases (PI3K and Fpr1 was 54% identical to that of the concurrently charac- PI4K), no PIKK family member has lipid kinase activity, and terized human FKBP12, providing further support that the the significance of the resemblance to lipid kinases is un- mode of action of rapamycin was conserved from yeast to known. Two reports in 1995—before TOR was shown to be humans. Curiously, disruption of the FKBP gene in yeast a protein kinase—claimed that yeast and mammalian TOR (FPR1) revealed that FKBP is not essential for growth had lipid kinase (PI4K) activity, but these findings were (Heitman et al. 1991b; Koltin et al. 1991; Tanida et al. never confirmed and are now thought to have been due to 1991; Wiederrecht et al. 1991). Additional FKBPs and cyclo- a contaminating PI4K. Disruption of TOR1 and TOR2 in philins (also an immunophilin and proline isomerase) were combination caused a growth arrest similar to that caused subsequently discovered and cloned, and again single and by rapamycin treatment, suggesting that TOR1 and TOR2 multiple disruptions were constructed without consequen- are indeed the targets of FKBP–rapamycin and that the tial loss of viability (Heitman et al. 1991b, 1992; Davis et al. FKBP–rapamycin complex inhibits TOR activity (Kunz 1992; Kunz and Hall 1993; Dolinski et al. 1997). The finding et al. 1993). It was subsequently demonstrated that the that FPR1 disruption did not affect viability was paradoxical FKBP–rapamycin complex binds directly to TOR1 and because FKBP was believed to be the in vivo binding protein/ TOR2 (Stan et al. 1994; Lorenz and Heitman 1995; Zheng target for the toxic effect of rapamycin. Why did inhibition et al. 1995) and that TOR is widely conserved both struc- of FKBP by rapamycin block growth whereas inhibition of turally and as the target of FKBP–rapamycin (Schmelzle and FKBP by disruption of the FPR1 gene have no effect on Hall 2000). However, S. cerevisiae is unusual in having two growth? The subsequent finding that an FPR1 disruption TOR genes whereas almost all other eukaryotes, including confers rapamycin resistance (Heitman et al. 1991a,b), com- plants, worms, flies, and mammals, have a single TOR gene. bined with the observation that some drug analogs are not As described below, this additional complexity in S. cerevi- immunosuppressive despite being able to bind and inhibit siae helped the analysis of TOR signaling because it allowed FKBP12 proline isomerase (Schreiber 1991), provided the differentiating two functionally different signaling branches answer to the above question and led to the well-established on the basis of different requirements for the two TORs. model of immunosuppressive drug action: an immunophilin- It should be noted that there is no evidence to indicate drug complex (e.g., FKBP-rapamycin) gains a new toxic ac- that FKBP has a role in normal TOR signaling, i.e., in the tivity that acts on another target. In other words, FKBP is absence of rapamycin. Rapamycin hijacks or corrupts FKBP only a cofactor or receptor required by the drug to act on to interact with TOR. In addition, some have speculated that something else; FKBP itself is not the target required for rapamycin mimics an endogenous metabolite that normally viability. This mode of drug action also applies to the well- regulates TOR with or without FKBP. Although this would known immunosuppressants cyclosporin A and FK506 (from provide an explanation for the evolution of the mechanism cyclophilin–cyclosporin A and FKBP–FK506 complexes) and of action of rapamycin, no evidence has been reported for an is conserved from yeast to humans (Schreiber 1991). These endogenous rapamycin-like compound or for such a mode of early studies in yeast were the first of many that have since TOR regulation. contributed to an understanding of rapamycin action and All TORs have a similar domain structure (Figure 1A). TOR signaling even in mammalian cells (Crespo and Hall The domains found in TOR—in order from the N to the C 2002), illustrating that a model organism such as yeast is terminus of TOR—compose the so-called HEAT repeats, the valuable in both basic and biomedical research. FAT domain, the FRB domain, the kinase domain, and the To identify the target of the FKBP–rapamycin complex, FATC domain (Schmelzle et al. 2002). The HEAT repeats rapamycin-resistant yeast mutants were selected (Heitman (originally found in huntingtin, elongation factor 3, the A et al. 1991a; Cafferkey et al. 1993). As expected, fpr1 subunit of PP2A, and TOR1) consist of 20 HEAT motifs, TOR Function 1179 each of which is 40 residues that form a pair of interacting antiparallel a-helices (Andrade and Bork 1995; Perry and Kleckner 2003). The HEAT repeats occupy the N-terminal half of TOR and are the binding region for subunits of the TOR complexes (Wullschleger et al. 2005) (see below). The central FAT domain (500 residues) and the extreme C-terminal FATC domain (35 residues), flanking the FRB and kinase domains, are always paired and found in all PIKK family members (Alarcon et al. 1999; Bosotti et al. 2000; Dames et al. 2005). The FRB domain (100 residues) is the FKBP–rapamycin-binding region. All rapamycin resis- tance-conferring TOR mutations fall within the FRB domain, thereby directly preventing the binding of FKBP–rapamycin without otherwise affecting TOR activity (Heitman et al. 1991a; Cafferkey et al. 1993; Helliwell et al. 1994; Stan et al. 1994; Chen et al. 1995; Lorenz and Heitman 1995; Choi et al. 1996). Interestingly, all the original rapamycin- resistance conferring mutations in TOR1 and TOR2 are mis- sense mutations confined to a single, equivalent codon encoding a critical serine residue (Ser1972Arg or Ser1972- Asn in TOR1 and Ser1975Ile in TOR2) (Cafferkey et al. 1993; Helliwell et al. 1994), which explains why the rapamycin-resistance TOR mutations were rare. Recreating an equivalent mutation (Ser2035Ile) in mammalian TOR (mTOR) was instrumental in demonstrating that mTOR is the target of FKBP–rapamycin in mammalian cells (Brown Figure 1 (A) Conserved domain structure of TOR. The N-terminal half of et al. 1995). Thus, the early rapamycin-resistant yeast TOR is composed of two blocks of 20 HEAT repeats, 40 aa that form mutants turned out to be very informative. They not only pairs of interacting antiparallel a-helices. The 500-aa FAT (FRAP-ATM- identified TOR, but also identified the FKBP–rapamycin- TRRAP) domain contains modified HEAT repeats. Missense mutations in binding site in TOR and contributed to elucidating the the 100-aa FRB (FKBP12-rapamycin-binding) domain confer complete resistance to rapamycin. The kinase domain phosphorylates Ser/Thr resi- mechanism of action of rapamycin. The kinase domain is dues in protein substrates, but at the sequence level resembles the cat- the catalytic domain and resembles the kinase domain of alytic domain of phosphatidylinositol kinases. The 35-aa FATC domain is PI3K and PI4K lipid kinases. Despite high interest in a struc- always found C-terminal to the FAT domain and is essential for kinase ture of the kinase domain, no such structure exists for any activity. (B) Composition of TOR complex 1. TORC1 is 2 MDa in size and TOR, which is likely due to technical difficulties in express- contains Kog1, Tco89, Lst8, and either TOR1 or TOR2. The HEAT repeats found in Kog1 and the seven-bladed propellers of the WD-40 repeats ing this domain for structural studies. In the absence of found in Kog1 and Lst8 are depicted. The binding of Kog1 to TOR is a true model, a homology model based on the crystal struc- complex, involving multiple domains on each protein. Lst8 binds to the ture of related PI3K has been elaborated (Sturgill and Hall kinase domain of TOR. Each component is likely present in two copies. (C) 2009). A number of groups have identified activating, mis- Composition of TOR complex 2. TORC2 is 2 MDa in size and contains sense mutations in S. cerevisiae and Schizosaccharomyces Avo1, Avo2, Avo3, Bit61, and/or its paralog Bit2, Lst8, and TOR2 but not TOR1. The RasGEFN domain of Avo3 and the PH domain of Avo1 are pombe TORs (Reinke et al. 2006; Urano et al. 2007; Ohne indicated. Each component is likely present in two copies. et al. 2008). These mutations fall within the FAT, FRB, and kinase domains, and, interestingly, one of the hotspots in the kinase domain corresponds to a region for oncogenic muta- within a few generations as small-budded cells in the G2/M tions in PI3K (Sturgill and Hall 2009; Hardt et al. 2011). phase of the cell cycle and with a randomized actin cyto- In the mid-1990s, research in the TOR field focused on skeleton (Kunz et al. 1993; Helliwell et al. 1994, 1998a; elucidating the cellular roles of TOR1 and TOR2. It was Schmidt et al. 1996). These and other findings led to the found that TOR1 and TOR2 play a central role in controlling model that TOR2 has two essential functions: one function cell growth as part of two separate signaling branches. Al- is redundant with TOR1 (TOR shared) and the other func- though structurally similar, TOR1 and TOR2 are not func- tion is unique to TOR2 (TOR2 unique) (Hall 1996; Helliwell tionally identical (Kunz et al. 1993; Helliwell et al. 1994). et al. 1998a). As described below, these two TOR2 functions Combined disruption of TOR1 and TOR2, or rapamycin turned out to be two separate signaling branches (each cor- treatment, mimics nutrient deprivation including causing responding to a structurally and functionally distinct TOR a G0 growth arrest within one generation (Barbet et al. complex) that control cell growth in different ways (Barbet 1996). Disruption of TOR1 alone has little-to-no effect, et al. 1996; Schmidt et al. 1997, 1998; Bickle et al. 1998; and disruption of TOR2 alone causes cells to arrest growth Helliwell et al. 1998a; Loewith et al. 2002; Loewith and Hall 1180 R. Loewith and M. N. Hall 2004; De Virgilio and Loewith 2006; Breitkreutz et al. 2010; this way of thinking about the two branches has subsided in Kaizu et al. 2010). recent years as the TOR2-unique pathway was shown to The early characterization of TOR disruptions and rapa- control sphingolipid synthesis and endocytosis in addition mycin treatment led to two more important conclusions. to the actin cytoskeleton (Powers et al. 2010). First, as described in more detail below, the finding that Another major breakthrough in the TOR field occurred in TOR inhibition mimics starvation led to the notion that 2002: the identification of the two multiprotein complexes TOR controls cell growth in response to nutrients (Barbet termed TOR complex 1 (TORC1) and TORC2 (Loewith et al. 1996; Rohde et al. 2001). Subsequent studies con- et al. 2002; Wedaman et al. 2003; Reinke et al. 2004; firmed this notion and demonstrated that TOR in higher Wullschleger et al. 2006). The two structurally and function- eukaryotes also controls cell growth in response to ally distinct TOR complexes were biochemically purified nutrients; i.e., TOR is conserved in structure and function from yeast cells and subsequently shown to correspond (Thomas and Hall 1997; Hara et al. 1998; Schmelzle and to the two genetically defined TOR-signaling branches. Hall 2000). Second, the observation that inhibition specifi- TORC1, which contains either TOR1 or TOR2 and is rapa- cally of the TOR-shared signaling branch (disruption of both mycin sensitive, mediates the TOR-shared pathway. TORC2, TORs but not of TOR2 alone) or rapamycin treatment which specifically contains TOR2 and is rapamycin insensi- mimics starvation suggested that only the TOR-shared path- tive, mediates the TOR2-unique pathway. The TORCs were way is nutrient responsive and rapamycin sensitive (Zheng a major breakthrough because they provided a molecular et al. 1995; Barbet et al. 1996; Schmidt et al. 1996; Rohde basis for the functional complexity and selective rapamycin et al. 2001). The molecular basis of these findings would sensitivity of TOR signaling. The biochemical identification remain a mystery until the discovery of the two structurally of the TORCs and the genetic definition of the two signaling and functionally distinct TOR complexes (see below). branches also, gratifyingly, cross-validated each other such The realization that TOR controls growth (increase in cell that there is a high level of confidence in the current “two size or mass) was a particularly important development branch-two complex” model of TOR signaling. The subse- (Barbet et al. 1996; Thomas and Hall 1997; Schmelzle et al. quent identification of TORCs in other eukaryotes, including 2002). Rapamycin or loss of TOR function causes a cell cycle plants, worms, flies, and mammals (Table 1), showed that arrest, and TOR was thus originally thought to be a control- the two complexes, like TOR itself, are conserved and gave ler of cell division (increase in cell number). Furthermore, at further support to the above model (Hara et al. 2002; that time, growth was largely thought to be controlled pas- Kim et al. 2002; Loewith et al. 2002; Jacinto et al. 2004; sively: i.e., the simple availability of nutrients (building Sarbassov et al. 2004). Below we focus on the structure, blocks) led to cell growth. As described below, the realiza- function, and regulation of the two TOR complexes. We tion that TOR controls many cellular processes that collec- discuss some downstream readouts of the TORCs that were tively determine mass accumulation, combined with the fact originally described before the discovery of the TORCs but that there was no direct role for TOR in the cell cycle ma- are now retroactively attributed to TORC1 or TORC2 on the chinery then being characterized, led to the notions that basis of their TOR requirement or rapamycin sensitivity. TOR controls growth and that growth is thus actively con- trolled. The originally confusing defect in cell cycle progres- sion observed upon TOR inhibition is in fact an indirect TOR Complex 1 effect of growth inhibition: a growth defect is dominant over Composition of TOR complex 1 cell cycle progression. Since the late 1990s, many groups have been character- TORC1 consists of Kog1, Lst8, Tco89,and either TOR1 or izing the two separate TOR2-signaling branches. It was TOR2 (Figure 1B) (Loewith et al. 2002; Wedaman et al. found that the TOR-shared signaling branch is composed 2003; Reinke et al. 2004). Gel filtration chromatography of various effector pathways that control a wide variety of (R.Loewith,W.Oppliger, and M. Hall, unpublished readouts that collectively determine the mass of the cell. results) indicated that TORC1 has a size of 2MDa,sug- The readouts controlled by this branch include protein syn- gesting that the entire complex is likely dimeric. This thesis and degradation, mRNA synthesis and degradation, wouldbeconsistentwiththe dimericstructuresproposed ribosome biogenesis, nutrient transport, and autophagy for TORC2 (Wullschleger et al. 2005) and mTORC1 (Yip (Schmelzle and Hall 2000). This branch is viewed as medi- et al. 2010). The names of mammalian and invertebrate ating temporal control of cell growth. The TOR2-unique orthologs of TORC1 subunits and the salient features of S. cerevisiae TORC1 subunits are summarized in Table 1 branch controls the polarized organization of the actin cy- and Table 2, respectively. Although all subunits are thought toskeleton, endocytosis, and sphingolipid synthesis. This to act positively with TOR1/2 in vivo, by and large their second branch is viewed as mediating spatial control of cell growth, on the basis largely of early work showing that it functions await characterization. In the presence of rapamy- controls the actin cytoskeleton. Thus, the logic of the two cin, all components of TORC1 can be coprecipitated with branches appears to be to integrate temporal and spatial FKBP12 (Loewith et al. 2002), demonstrating that, unlike control of cell growth (Loewith and Hall 2004). However, mammalian TORC1 (Yip et al. 2010), the structural integrity TOR Function 1181 1182 R. Loewith and M. N. Hall Table 1 TORC1, TORC2, and EGO complex orthologs in various genera S. cerevisiae S. pombe C. albicans D. discoideum A. thaliana C. elegans D. melanogaster Mammals TORC1 TOR1 or TOR2 Tor1 or Tor1 Tor TOR TOR/let-363 TOR mTOR Tor2 Kog1/Las24 Mip1 Kog1 Raptor RAPTOR1A and daf-15 Raptor Raptor RAPTOR1B Lst8 Wat1/Pop3 Orf19.3862 lst8? AT2G22040 lst-8? CG3004 mLST8 AT3G18140 Tco89 Tco89 Tco89 pcr25kl1p3887 —— — — — Toc1 —— — — — — —— — — — — — PRAS40 —— — — — — — DEPTOR TORC2 TOR2 Tor1 or Tor2 Tor1 tor TOR TOR/let-363 TOR mTOR Avo1 Sin1 orf19.5221 piaA sinh-1 Sin1 mSIN1 Avo2 — Avo2 —— — — Avo3/Tsc11 Ste20 Tsc11 rip3 rict-1 Rictor Rictor Lst8 Wat1/Pop3 Orf19.3862 lst8 AT2G22040 lst-8 CG3004 mLST8 AT3G18140 Bit61 Bit61 —— — — — PRR5/Protor —— — — — — — DEPTOR EGO complex Gtr1 Gtr1 Gtr1 ragA — raga-1 RagA RagA,B Gtr2 Gtr2 Gtr2 ragC — ragc-1 RagC RagC,D Ego1/Meh1/Gse2 —— — — — CG14184 LAMTOR1/p18 Ego3/Slm4/Nir1/Gse1 —— — — lamtor-2, ? CG5189, CG5110 LAMTOR2/p14, LAMTOR3/ MP1 Orthologs listed are from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Dictyostelium discoideum, Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium melanogaster, and mammals. P-POD: Princeton Protein Orthology Database/BLAST. We note that TORC2 appears to be absent in plants, e.g., A. thaliana. —, no demonstrated/obvious ortholog. Table 2 Salient features of TORC1 components Protein Size Motifs/domains Potential function TOR1 2470 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain TOR2 2474 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain Kog1 1557 aa An N-terminal conserved region 4, HEAT Present substrate to TOR repeats, 7 C-terminal WD-40 repeats Tco89 799 aa None obvious Receive signals from EGO complex Lst8 303 aa 7 WD-40 repeats Stabilize kinase domain of yeast TORC1 is not compromised by this macrolide. De- of yeast cells to rapamycin results in a dramatic drop in spite recent molecular reconstructions from low resolution protein synthesis, induction of autophagy, and exit from (25 Å) electron microscopy of a TOR1–Kog1 subcomplex the cell cycle and entrance into a quiescent G0 state. This (Adami et al. 2007), the molecular mechanism by which was the first indication that TOR, actually TORC1, might binding of FKBP-rapamycin inhibits TORC1 activity is enig- regulate growth downstream of nutrient cues. This hypoth- matic and remains a fascinating question. esis was strengthened when TORC1, in response to nitrogen and carbon cues, was found to promote the sequestration of Localization of TORC1 several nutrient-responsive transcription factors in the cyto- Tagging of Kog1, Tco89, Lst8, and TOR1 with GFP demon- plasm (Beck and Hall 1999). Consistently, transcriptome strates that TORC1 is concentrated on the limiting mem- profiling demonstrated a highly similar transcriptional re- brane of the vacuole (Urban et al. 2007; Sturgill et al. sponse of yeast cells exposed to rapamycin, nutrient starva- 2008; Berchtold and Walther 2009; Binda et al. 2009). tion, or noxious stressors (Cardenas et al. 1999; Hardwick These observations are consistent with previous studies that et al. 1999; Komeili et al. 2000; Shamji et al. 2000; Gasch localized TORC1 via immunogold electron microscopy and and Werner-Washburne 2002). Although suggestive, these cellular fractionation (Chen and Kaiser 2003; Reinke et al. observations provided only correlative evidence that TORC1 2004). Artificial tethering of a TORC1 peptide substrate to activity is regulated in response to environmental cues. the vacuole demonstrates that vacuole-localized TORC1 is Characterization of a bona fide substrate of TORC1 allowed catalytically competent (Urban et al. 2007). This localization this model to be tested directly. appears to be constitutive (Binda et al. 2009), suggesting As detailed below, Sch9 presently remains the best- that changes in “geography” play no obvious role in regulat- characterized substrate of TORC1, and monitoring its phos- ing yeast TORC1-signaling output. The yeast vacuole is a ma- phorylation by Western blotting serves as a convenient proxy jor nutrient reservoir and TORC1 signaling is responsive to for TORC1 activity. In addition to exposure to rapamycin, nutrient cues (see below). Thus, vacuolar localization of Sch9 is rapidly dephosphorylated in cells experiencing acute TORC1 seems logical. Although convincing, these studies starvation of carbon, nitrogen, phosphate, or amino acids do not exclude the possibility that a fraction of TORC1 (Urban et al. 2007; Binda et al. 2009). These and other obser- may also be active elsewhere in the cell. Li et al. (2006), vations confirm that TORC1 is responsive to both the abun- for example, have reported that TOR1 dynamically associ- dance and the quality of nutrients in the environment; but, ates with the rDNA locus to regulate 35S rRNA with few exceptions (see The EGO complex), how nutrient transcription. cues are sensed and how this information is transduced to TORC1 remain unknown. Upstream of TORC1 TORC1 activity is also regulated in response to noxious Physiological regulators (carbon, nitrogen, phosphate, stressors. When cells are subjected to various stress con- stress, caffeine): A major breakthrough in the TOR field ditions, including high salt, redox stress, a shift to a higher came with the observation that rapamycin treatment alters temperature, or caffeine, Sch9 phosphorylation is reduced yeast physiology in much the same way as nutrient dramatically (Kuranda et al. 2006; Urban et al. 2007). With starvation (Barbet et al. 1996). Like starvation, exposure the exception of caffeine, which directly inhibits TORC1 Table 3 Salient features of EGO Complex components Protein Size Motifs/domains Potential function Gtr1 310 aa Ras-family GTPase GTP-bound form activates TORC1 Gtr2 341 aa Ras-family GTPase GDP-bound form activates TORC1 Ego1/Meh1/ Gse2 184 aa N-terminal palmitoylation/myristolation Vacuolar recruitment Ego3/Slm4/Nir1/Gse1 162 aa Transmembrane domain, PtdIns(3,5)P2 binding Vacuolar recruitment Described in Dubouloz et al. (2005), Hou et al. (2005), and references therein. TOR Function 1183 and the Rags mediate amino acid sufficiency signals to mTORC1 (reviewed in Kim and Guan 2011). Like its mammalian counterpart, the EGO complex resides on the vacuolar/lysosomal membrane and is thought to couple amino acid signals to TORC1 (Binda et al. 2009). Curiously, GTP GDP the Gtr1 Gtr2 combination activates TORC1 with the nucleotide-binding status of Gtr1 seemingly dominant over the nucleotide-binding status of Gtr2. TORC1 activity in both metazoans and yeast appears to be particularly responsive to glutamine (Crespo et al. 2002) and the branched-chain amino acid leucine (Binda et al. 2009; Cohen and Hall 2009). In yeast, leucine starvation destabilizes Gtr1-TORC1 association and causes a reduction Q65L in Sch9 phosphorylation whereas GTP-locked Gtr1 remains bound to TORC1 and Sch9 dephosphorylation is Figure 2 The EGO complex is a major regulator of TORC1. The EGO delayed in cells expressing this mutant (Binda et al. 2009). complex (EGOC) is composed of four proteins: the palmitoylated and Loss of Gtr1 results in reduced Sch9 phosphorylation and myristolated protein Ego1, the transmembrane protein Ego3, and two S20L slow growth whereas GDP-locked Gtr1 is dominant neg- Ras-family GTPases, Gtr1 and Gtr2. Like TORC1, the EGO complex is S20L ative. When Gtr1 is expressed as the sole version of Gtr1, localized to the vacuolar membrane where it appears to sense/respond cells are extremely sick. This near inviability is suppressed to intracellular leucine levels and potentially to intravacuolar amino acid levels. Vam6 has been identified as a guanine nucleotide exchange factor by deletion of the TCO89 gene encoding the TORC1 subunit for Gtr1 but no other GEFs or GAPS for this GTPase system have been Tco89. Collectively, these observations suggest that the EGO GTP GDP reported. In the Gtr1 and Gtr2 configuration, the EGO complex complex can both positively and negatively regulate TORC1 somehow activates TORC1; the reverse conformation inactivates TORC1. activity via Tco89. The fact that the EGO complex can neg- Activated TORC1, via its two main effector branches, the AGC kinase atively regulate TORC1 activity seems to be at odds with the Sch9 and the Tap42-PP2a and PP2a-like protein phosphatases, stimulates growth by favoring anabolic processes and by antagonizing catabolic current metazoan model according to which the EGO com- processes and stress-response programs. plex counterpart serves only to localize TORC1 to the vacu- ole. Indeed, in contrast to the results obtained in metazoans, kinase activity (Kuranda et al. 2006; Reinke et al. 2006; in yeast, TORC1 appears to stably localize to the vacuolar membrane regardless of nutrient conditions. Thus, how the Wanke et al. 2008), how environmental stress signals are EGO complex influences TORC1 activity remains a mystery transduced to TORC1 is also unclear. although the crystal structure of the Gtr1–Gtr2 complex, The EGO complex: When environmental conditions are in- reported very recently, provides some mechanistic insights appropriate for growth, cells stop dividing, slow their me- (Gong et al. 2011). tabolism, induce the expression of stress-responsive proteins, Also mysterious are the mechanisms by which amino acid and accumulate energy stores. This nondividing but meta- sufficiency modulates Gtr1/2 guanine nucleotide loading. bolically active state is known as quiescence (G0). How cells Given its localization, it is tempting to postulate that the enter into quiescence is relatively well characterized. In EGO complex responds to levels of intravacuolar amino contrast—and despite its medical relevance (inappropriate acids, possibly via the recently described Gtr1 guanine– exit from quiescence can lead to cancer or reactivation of nucleotide exchange factor (GEF) Vam6/Vps39 (Binda a latent infection)—how quiescent cells reinitiate growth is et al. 2009). It is equally plausible, however, that this signal poorly understood. To shed light on this process, a clever is mediated by an as-yet-unidentified GTPase-activating pro- screen was performed to identify mutants that are unable to tein (GAP) activity. Consistent with the conserved function escape from rapamycin-induced growth arrest (EGO) of the EGO/Ragulator complex, and like its yeast ortholog, mutants (Dubouloz et al. 2005). This and a follow-up study hVPS39 has been found to function positively upstream of (Binda et al. 2009) identified the EGO complex as an impor- mTORC1 (Flinn et al. 2010). tant regulator of TORC1. The EGO complex is composed of four proteins: Ego1, Feedback loop/ribosome biogenesis homeostasis: Although Ego3, Gtr1, and Gtr2 (Table 3 and Figure 2). Gtr1 and most recognized as a target of signals emanating from Gtr2 are Ras-family GTPases and orthologs of the metazoan extracellular nutrients and noxious stresses, it is becoming Rag GTPases (Kim et al. 2008; Sancak et al. 2008) (Table 1). increasingly apparent that TORC1 also responds to intracel- Although they lack obvious sequence homologies, Ego1 and lular cues. In addition to the sensing of intracellular amino Ego3 are likely the functional homologs of vertebrate p18 acids as described above, outputs from distal effectors also (LAMTOR1) and p14 + MP1 (LAMTOR2 + LAMTOR3), regulate TORC1 in apparent feedback loops. For example, respectively, which function together as the “Ragulator” in both yeast and mammalian cells, it is well documented complex (Kogan et al. 2010; Sancak et al. 2010). Ragulator that TORC1 activity stimulates translation initiation 1184 R. Loewith and M. N. Hall (Wullschleger et al. 2006). Interestingly, inhibition of trans- kinase activity, i.e., activity even in the absence of TORC1 lation with cycloheximide causes a pronounced increase in (Urban et al. 2007). Presumably, phosphorylation of the turn (m)TORC1 activity presumably by triggering an increase in motif helps to stabilize Sch9 while phosphorylation of the the concentration of free amino acids in the cytoplasm hydrophobic motif stabilizes Sch9 in an active conformation. (Beugnet et al. 2003; Urban et al. 2007; Binda et al. Curiously, although their in vivo functions are unknown, 2009). Ribosome biogenesis (described in more detail be- in vitro TORC1 preferentially phosphorylates Ser758 and low) is a second example. TORC1 regulates ribosome bio- Ser765 within the hydrophobic-like motifs (R. Loewith, un- genesis in part via two substrates, Sch9 and the transcription published results). That TORC1 can phosphorylate amino factor Sfp1. Reduced ribosome biogenesis resulting from de- acids found within such diverse sequence contexts, which letion of SCH9 or SFP1 results in a dramatic increase in is rather atypical for protein kinases, is also curious. TORC1 activity (Lempiainen et al. 2009). It is possible that Characterization of Tap42‐PP2A as a TORC1 effector: In blocking ribosome biogenesis, like translation inhibition, addition to Sch9, TORC1 also regulates type 2A (Pph21, causes an increase in free amino acids that subsequently Pph22, and Pph3—generically PP2Ac) and 2A-related phos- activates TORC1. Alternatively, other mechanisms could be phatases (Sit4, Ppg1). These partially redundant yet pleio- at play. Regardless of mechanism, such feedback loops pro- tropic enzymes are notoriously difficult to study. Analysis of vide an elegant means by which growth homeostasis can be Sit4 function, and therefore of TORC1 function, is further maintained by TORC1. complicated by strain-dependent polymorphisms at the SSD1 (Suppressor of SIT4 Deletion) locus (Reinke et al. Downstream of TORC1 2004). In general terms, when growth conditions permit, TORC1 A role for these phosphatases downstream of TORC1 was is active and its signals promote the accumulation of cel- first described by the Arndt lab (Di Como and Arndt 1996). lular mass. However, as both proximal and distal TORC1 In this work, a subpopulation of these enzymes was found to effectors continue to be described, the extent of this tem- interact in a TORC1-dependent manner with a regulatory poral regulation of growth control is only starting to be protein known as Tap42. Rrd1 and Rrd2, two peptidyl- appreciated. prolyl cis/trans isomerases, were subsequently also found to be present in these Tap42 complexes (Zheng and Jiang Proximal TORC1 effectors: Characterization of Sch9 as 2005; Jordens et al. 2006). Work, done in large part by the a TORC1 substrate: Arguably, the best-characterized sub- Jiang group, posits that when TORC1 is active, Tap42 is strates of both yeast and metazoan TOR complexes are the phosphorylated and bound tightly to the phosphatase–Rrd AGC family kinases. This rather well-studied family of complex (Di Como and Arndt 1996; Jiang and Broach 1999; kinases is so named on the basis of its mammalian members Zheng and Jiang 2005). Inactivation of TORC1 results in PKA, PKG, and PKC (Pearce et al. 2010). Typically, activation Tap42 dephosphorylation and a weakened association with of AGC family kinases requires phosphorylation of two con- phosphatases that presumably results in their activation served regulatory motifs, the “T,” or “activation,” loop lo- and/or change in substrate preference (Duvel et al. 2003; cated in the catalytic domain and the “hydrophobic” motif Yan et al. 2006). How TORC1 maintains Tap42 phosphory- found toward the C terminus. Phosphorylation of these lation is mechanistically unclear. It may phosphorylate motifs helps stabilize the kinase domain in an active confor- Tap42 directly (Jiang and Broach 1999), or it may act via mation. Several AGC family kinases additionally contain the Tap42 interacting phosphoprotein Tip41 (Jacinto et al. a “turn” motif located between the kinase domain and the 2001). Interestingly, Tip41 has been proposed to both an- hydrophobic motif, phosphorylation of which is thought to tagonize and cooperate with Tap42 in controlling TORC1 promote protein stability. While the T loop is phosphorylated signaling (Jacinto et al. 2001; Kuepfer et al. 2007). by PDK1 or its ortholog Pkh in mammalian or yeast cells, Although the mechanisms coupling TORC1 to Tap42– respectively, phosphorylation of the hydrophobic and possi- PPase complexes remain to be elucidated, genetic argu- bly the turn motifs is often mediated by TORC1 or TORC2. ments clearly position Tap42 as a prominent effector of Analogous to S6K for mTORC1, the AGC kinase Sch9 was TORC1. Specifically, several alleles of TAP42 (e.g., TAP42- recently found to be a direct substrate for yeast TORC1 11) that confer strong resistance to rapamycin by blocking (Powers 2007). Six target sites in the C terminus of Sch9 a subset of rapamycin-induced readouts have been identi- are phosphorylated by TORC1: Thr737 found in a classical fied (Di Como and Arndt 1996; Duvel et al. 2003). hydrophobic motif; Thr723 and Ser726, Ser/Thr-Pro sites Curiously, TAP42-11 does not provide rapamycin resis- found in what appears to be a turn motif; Ser758 and tance in all strain backgrounds. However, co-expression of Ser765 found in sequences that resemble the hydrophobic genetically activated Sch9 (described above) in rapamycin- motif; and Ser711 in a region that partially resembles a hy- sensitive TAP42-11 backgrounds results in a very strong syn- drophobic motif. TORC1-mediated phosphorylation is nec- thetic resistance to rapamycin (Urban et al. 2007). From this essary for Sch9 activity. Replacing the target amino acids observation, it appears that Sch9 and Tap42-PPase com- with alanine yields a nonfunctional Sch9, whereas replacing plexes are major effector branches downstream of TORC1 them with a phosphomimetic residue confers constitutive with each branch, at least in some backgrounds, performing TOR Function 1185 one or more essential function. The readouts mediated by lation of Ser577 and, consequently, an increase in Gcn2 these two TORC1 branches are discussed below. activity and a reduction in 59CAP-dependent translation Other TORC1 substrates: In addition to the regulation of (Cherkasova and Hinnebusch 2003). It is possible that one these two major effector branches, TORC1 has been or more Tap42-associated phosphatases directly dephos- reported to directly phosphorylate other substrates includ- phorylates Ser577, but this has not been formally demon- ing Sfp1(Lempiainen et al. 2009), Gln3 (Bertram et al. strated. The nature of the kinase that phosphorylates Gcn2 2000), and Atg13 (Kamada et al. 2010). The roles that these Ser577 is unknown other than it is not Sch9 (M. Stahl and proteins play downstream of TORC1 are discussed below. R. Loewith, unpublished results). Sch9 inhibition, however, Tyers and colleagues have recently defined a global also leads to eIF2a phosphorylation via an undefined path- protein kinase and phosphatase interaction network in yeast way (Urban et al. 2007). (Breitkreutz et al. 2010). This study, consisting of affinity Studies with rapamycin suggest that, in addition to purification followed by mass spectrometry, included TOR1 eIF2a, TORC1 may target additional translation factors and TOR2. They found and confirmed that TORC1 physi- such as the 59CAP-binding protein (eIF4E) interacting pro- cally interacts with the following proteins: Mks1, a protein teins Eap1 and/or the eIF4G scaffold (Berset et al. 1998; involved in retrograde (RTG) mitochondria-to-nucleus sig- Cosentino et al. 2000). Finally, recent phosphoproteomics naling (see below); curiously, FMP48, an uncharacterized studies (Huber et al. 2009; Loewith 2010; Soulard et al. protein presumed to localize to the mitochondria (Reinders 2010) have identified several translation-related proteins et al. 2006); Npr1, a protein kinase involved in the intracel- whose phosphorylation is altered by rapamycin treatment, lular sorting of nutrient permeases (see below); Ksp1, suggesting that these factors could also couple TORC1 to a protein kinase involved in nutrient-regulated haploid fila- protein synthesis. mentous growth (Bharucha et al. 2008); Nap1, a chromatin Ribosome biogenesis: In optimal conditions, yeast cells assembly factor and a mitotic factor involved in regulation of grow and divide approximately every 100 min. Such rapid bud formation (Calvert et al. 2008); Nnk1, the nitrogen growth requires robust protein synthesis, which of course network kinase presumably involved in intermediate nitro- requires ribosomes. Indeed, rapidly growing yeast cells gen metabolism (Breitkreutz et al. 2010); Sky1, an Ser/Arg contain 200,000 ribosomes, implying that each cell must domain kinase involved in pre-mRNA splicing (Shen and produce and assemble 2000 ribosomes per minute Green 2006); and Bck1 and Kdx1, which are involved in (Warner 1999). This is not a trivial feat as each ribosome MAPK signaling (Breitkreutz et al. 2010). Given their phys- contains 78 unique proteins (encoded by 137 RP genes) in ical interaction with TORC1, all of these proteins, in addi- addition to four rRNA molecules, three derived from the tion to multiple other, as-yet-unconfirmed interactors, are RNA Pol I-transcribed 35S pre-rRNA and one transcribed potential substrates (or regulators) of TORC1. These results by RNA Pol III. Fifty percent of RNA Pol II transcription is underscore the central role that TORC1 plays in cell growth. devoted to ribosomal proteins. In addition, numerous pro- tein and small RNA trans-acting factors, known as ribosome Distal readouts downstream of TORC1: TORC1 promotes biogenesis (RiBi) factors, are required for the correct pro- cell growth: When environmental conditions are favorable, cessing, folding, assembly, nuclear export of pre-ribosomal TORC1 coordinates the production and accumulation particles to the cytoplasm, and final maturation events into of cellular mass, i.e., growth, via regulation of several 40S and 60S particles. The production of all these abundant processes. molecules represents a huge energetic investment. Not sur- Protein synthesis: The first realization that TORC1 serves prisingly, yeast cells have developed elaborate measures to to couple environmental cues to the cell growth machinery coordinate the expression of rRNA, tRNA, RPs, and RiBi came with the observation that rapamycin treatment elicits factors in response to environmental conditions. Much of a marked drop in protein synthesis by blocking translation this regulation is mediated by TORC1 at the level of tran- initiation (Barbet et al. 1996). A major target for this regu- scription. As ribosome biogenesis has clear links to diseases lation appears to be the translation initiation factor eIF2. such as cancer, anemia, and aging, dissection of its regula- Upon amino acid starvation or rapamycin treatment, the tion will undoubtedly have clinical ramifications (Lempiainen a-subunit of eIF2 is phosphorylated and this dominantly and Shore 2009). interferes with 59CAP-dependent mRNA translation In S. cerevisiae, the rDNA locus consists of 150 tandemly (reviewed in Hinnebusch 2005). TORC1 signals to eIF2a repeated transcription units on chromosome XII, and yet via both the Sch9 and Tap42-PPase branches. The sole rRNA production is still limiting for cell growth (Warner eIF2a kinase is the conserved Gcn2 protein. Gcn2 binds 1999). Each of these rDNA units comprises the RNA poly- and is activated by uncharged tRNAs that accumulate when merase III transcribed 5S rRNA gene, the intergenic spacer cells are starved for an amino acid (detailed below). region, and the RNA Pol I-transcribed 35S rRNA gene, Gcn2 activity is also regulated by phosphorylation. encoding the 35S precursor of the mature 18S, 5.8S, and Gcn2 phosphorylation on Ser577 reduces tRNA binding 25S rRNAs. RNA Pol III also transcribes tRNA genes as well and, consequently, kinase activity. Treating cells with rapa- as several additional genes encoding small noncoding RNAs. mycin elicits a rapid, Tap42-PPase-dependent dephosphory- In the late 1990s, it was reported that rapamycin results in 1186 R. Loewith and M. N. Hall Figure 3 Control of RiBi and RP gene transcription by TORC1. RiBi factors are required for the proper expression, processing, assembly, export, and maturation of rRNA and RPs into ribosomes. This energetically costly procedure is under tight regulation, particularly at the transcription level. TORC1 regulates RiBi and RP gene transcription via multiple pathways: (1) TORC1 directly phosphorylates the Split Zn-finger transcription factor Sfp1, which presumably regulates its nuclear localization and/or binding to RP and possibly RiBi gene promoters to stimulate their expression. (2) Fhl1 and Rap1 bind constitutively to RP promoters. When TORC1 is active, phosphorylated Ifh1 binds to Fhl1 to stimulate transcription, possibly by recruiting the NuA4 histone acetyltransferase. When TORC1 is inactive, Yak1 phosphorylates Crf1, which subsequently outcom- petes Ifh1 for binding to Fhl1. (3) Sch9 phosphorylates and thus inhibits Stb3 and the paralogs Dot6 and Tod6. Inhibition of TORC1/Sch9 results in the dephosphorylation of these three transcription repressors, which subsequently bind to RRPE and PAC elements found in RiBi promoters. Stb3 additionally binds RP promoters. Bound to pro- moters, these repressors recruit the RPD3L histone deace- tylase complex to repress transcription. a rapid and pronounced drop in 5S, 35S, and tRNA produc- represses RNA Pol III in a Maf1-dependent fashion (Huber tion (Zaragoza et al. 1998; Powers and Walter 1999). Re- et al. 2009; Michels 2011). Maf1 is conserved and also func- cently, the relevant signaling pathways in this regulation tions downstream of mTORC1 to regulate RNA Pol III activ- have become clearer. ity. However, in mammalian cells, and perhaps in yeast cells TORC1 regulates the accumulation of RNA Pol I tran- too, Maf1 is directly phosphorylated by mTORC1 rather scripts at multiple levels. Processing of the 35S pre-rRNA than by the Sch9 ortholog S6K1 (Wei et al. 2009; Wei and occurs cotranscriptionally and is dependent on the presence Zheng 2010; Michels 2011). of ribosomal proteins (Tschochner and Hurt 2003). The fast A total of 137 genes encode the 78 proteins that make up drop in RNA Pol I-dependent transcripts observed upon a yeast ribosome (most RPs are encoded by two genes rapamycin treatment is apparently due to decreased trans- yielding nearly identical proteins). TORC1 coordinately lation (described above) of ribosomal proteins (Reiter et al. regulates the expression of these genes through several 2011). The majority of mRNAs being translated in a rapidly mechanisms (Figure 3) (Lempiainen and Shore 2009). A growing cell encode ribosomal proteins (Warner 1999), and central component of this regulation is the Fhl1 protein thus a drop in translation will rapidly reduce the levels of (Lee et al. 2002; Martin et al. 2004; Schawalder et al. free ribosomal proteins that are themselves needed stoichio- 2004; Wade et al. 2004; Rudra et al. 2005). Fhl1 has a fork- metrically for processing of rRNA into pre-ribosome par- head DNA-binding domain, and its constitutive association ticles. rRNA that is not efficiently processed is immediately to ribosomal protein gene (RP) promoters is facilitated by degraded, presumably to prevent imbalances in structural the DNA-binding protein Rap1 and the high mobility group components of the ribosome. At later time points following protein Hmo1 (Hall et al. 2006; Berger et al. 2007). TORC1 rapamycin treatment, RNA Pol I no longer associates with regulates RP transcription by determining the association the rDNA and transcription stops. This late effect could be between Fhl1 and either one of two FHB-containing pro- the result of rapamycin-induced degradation of the essential teins, Ifh1 and Crf1. Both Ifh1 and Crf1 are phosphopro- RNA Pol I transcription factor Rrn3 (Claypool et al. 2004; teins. When cells are growing and TORC1 is active, Ifh1 is Laferte et al. 2006; Reiter et al. 2011). phosphorylated and binds to Fhl1 to stimulate RP transcrip- TORC1 regulates RNA Pol III apparently exclusively via tion. Conversely, inhibition of TORC1 results in the phos- Sch9 and a repressor protein named Maf1 (Upadhya et al. phorylation of Crf1, which displaces Ifh1 to repress RP 2002; Oficjalska-Pham et al. 2006; Reina et al. 2006; Huber transcription. The signaling events upstream of Ifh1 are et al. 2009; Lee et al. 2009). Sch9 directly phosphorylates not known, whereas TORC1 seems to signal to Crf1 via seven sites in Maf1 that prevent it from interacting with and the Ras/PKA pathway target Yak1 (Martin et al. 2004). thus inhibiting RNA Pol III (Vannini et al. 2010). Phospho- However, it should be noted that the crosstalk between mimetic variants of Maf1 clearly fail to associate with RNA TORC1 signals and Ras/PKA signals has been debated. Pol III, but, curiously, Sch9 inhibition still causes a reduction While it is clear that hyperactivation of Ras/PKA can sup- in RNA Pol III activity in these strains but not in maf1D press many rapamycin-induced phenotypes (Schmelzle et al. strains. This and other observations suggest that an addi- 2004), suggesting that PKA is downstream of TORC1, it has tional Sch9 target exists that, when dephosphorylated, also been proposed that TORC1 and PKA signal in parallel TOR Function 1187 pathways that impinge on common targets (Zurita-Martinez active role in mRNA stability and, via its potential substrate and Cardenas 2005; Ramachandran and Herman 2011). Re- Sky1, in pre-mRNA splicing. This observation is significant cently, Soulard et al. (2010) have provided some clarifica- when one considers that 90% of all mRNA splicing events tion of this dilemma by proposing that TORC1 functions occur on RP transcripts (Warner 1999). Thus, TORC1 is well upstream of PKA but only for a subset of PKA targets. Thus, positioned to coordinate multiple aspects of ribosome biogen- TORC1 may be both upstream and parallel to PKA. esis in response to growth stimuli. As introduced above, TORC1-dependent regulation of RP gene transcription TORC1 activity is dramatically increased in sfp1 and sch9 cells still occurs in the absence of the Fhl1/Ifh1/Crf1 system, (Lempiainen et al. 2009), suggesting that some aspect of ribo- suggesting the existence of additional regulatory mecha- some biogenesis must also signal in a feedback loop to TORC1. nisms. One of these is the split zinc (Zn)-finger protein It will be interesting to see what steps of ribosome biogenesis Sfp1 (Fingerman et al. 2003; Jorgensen et al. 2004; Marion contribute to TORC1 regulation. et al. 2004; Lempiainen et al. 2009; Singh and Tyers 2009). Regulation of cell cycle/cell size: Although distinct pro- TORC1 binds and directly phosphorylates Sfp1 to promote cesses, cell growth and cell division are often intimately its binding to a subset of RP gene promoters. Curiously, un- linked. Yeast cells, for example, commit to a new round of like Sch9, TORC1-mediated Sfp1 phosphorylation appears cell division only after attaining a critical size. This cell-size to be insensitive to osmotic or nutritional stress, suggesting threshold is dictated in large part by environmental growth that TORC1 regulates these two substrates via very different conditions (Cook and Tyers 2007). How cells couple envi- mechanisms (Lempiainen et al. 2009). Sfp1 also interacts ronmental cues to the cell cycle machinery is fascinating but with the conserved Rab escort protein Mrs6, an essential poorly understood. Interestingly, sfp1 and sch9 were the top protein functioning in membrane sorting (Lempiainen two hits in a systematic search for mutations conferring et al. 2009; Singh and Tyers 2009). Sfp1-Mrs6 association small cell size (Jorgensen et al. 2002, 2004). This and fol- is important for the nuclear localization of Sfp1, but its low-up observations demonstrated that ribosome biogenesis functional implications are otherwise unclear. Intriguingly, plays a major role in cell-size determination. These results this association may underlie the presently unexplained further predict that environmental cues regulate the cell-size genetic and biochemical interactions between TORC1 and threshold via TORC1, i.e., that poor growth conditions re- vesicular transport machineries (Aronova et al. 2007; Zurita- duce the activity of TORC1 and subsequently the activities of Martinez et al. 2007). Although physical interaction with Sfp1 and Sch9. Consequently, this would decrease ribosome RiBi promoters has not been reported, overexpression of biogenesis, which, in mysterious ways, would lower the cell- Sfp1 causes a rapid upregulation of most RiBi genes, sug- size threshold required for cell division. In contrast, acute gesting that Sfp1 also regulates this important regulon inhibition of TORC1 with high concentrations of rapamycin (Jorgensen et al. 2004). Better understood is the regulation leads to an arrest in G1 due to reduced translation of the of RiBi gene expression downstream of Sch9. RiBi promoters cyclin Cln3 (Barbet et al. 1996) and a paradoxical increase typically possess polymerase A and C (PAC) and/or ribo- in cell size. This increase in cell size is actually due to swell- somal RNA processing element (RRPE) elements. PAC ele- ing of the vacuole as a consequence of increased autophagy ments are bound by the myb-family transcription factors (see below; sfp1 or sch9 deletions presumably do not induce Dot6 and Tod6 (Freckleton et al. 2009; Zhu et al. 2009) autophagy). whereas RRPE elements are bound by Stb3 (Liko et al. Although best appreciated for its role in G1 regulation, 2007). Stb3 seems to bind to T-rich elements in RP pro- TORC1 additionally regulates the transition through other moters as well (Huber et al. 2011). All three transcription phases of the cell cycle. TORC1 promotes S phase by factors are phosphorylated by Sch9 and thus are under maintaining deoxynucleoside triphosphate pools. Deoxy- TORC1 control (Lippman and Broach 2009; Liko et al. nucleoside triphosphates are the obligate building blocks 2010; Huber et al. 2011). When TORC1 is inactivated, for DNA synthesis, and a role for TORC1 in their synthesis Dot6, Tod6, and Stb3 are dephosphorylated, allowing becomes apparent under conditions of DNA replication them to bind to their cognate promoter elements and recruit stress or DNA damage when elevated deoxynucleoside the RPD3L histone acetyltransferase complex to repress triphosphate pools are necessary for error-prone trans- transcription. lesion DNA polymerases (Shen et al. 2007). Via the Tap42- In summary, TORC1 plays a central role in regulating PPase branch, TORC1 also influences the G2/M transition ribosome biogenesis, particularly at the transcriptional level. (Nakashima et al. 2008). Specifically, TORC1 regulates the However, it is now clear that TORC1 also influences ribosome subcellular localization of the polo-like kinase Cdc5. Cdc5 biogenesis post-transcriptionally. Phosphoproteomics as well as activity destabilizes Swe1, a kinase that phosphorylates and more directed studies suggest that TORC1 regulates various thus inactivates the mitotic cyclin-dependent kinase Cdc28. catalytic steps of ribosome assembly per se (Honma et al. 2006; Inhibition of TORC1 mislocalizes Cdc5, causing an inappro- Huber et al. 2009; Loewith 2010). Phosphoproteomics and priate stabilization of Swe1 and, consequently, inactivation biochemical studies (Albig and Decker 2001; Grigull et al. of Cdc28 and prolonged G2/M. Although TORC1 signals 2004; Huber et al. 2009; Breitkreutz et al. 2010; Loewith likely impinge upon additional nodes in the cell division 2010; Soulard et al. 2010) also suggest that TORC1 plays an cycle (Huber et al. 2009; Soulard et al. 2010), the above 1188 R. Loewith and M. N. Hall observations already exemplify the intricate connections be- ers, enabling them to selectively import only the desired tween cell growth signals and the cell division cycle. Recip- nutrients (Van Belle and Andre 2001). In general terms, rocal, but less well described, cues and/or outputs from the in good growth conditions, many high-affinity, substrate- cell division cycle regulate cell growth, likely in part via selective permeases are expressed and sorted to the plasma TORC1 (Goranov and Amon 2010). membrane to actively pump in nutrients that are used TORC1 inhibits stress responses: In addition to stimulating directly in ATP production and/or anabolism of nitrogenous anabolic processes, TORC1 also promotes growth by sup- compounds. Shift to poor growth conditions results in the pressing a variety of stress-response programs. Although replacement of high-affinity permeases, which are targeted essential for surviving environmental insults, activation of to the vacuole for degradation with few low-affinity, broad- stress-responsive pathways is incompatible with rapid specificity permeases that facilitate uptake of a wide range growth, and constitutive activation of these pathways of carbon and nitrogenous compounds that can be catabo- generally results in cell death. As described below, the lized by the cell. For example, in response to nitrogen star- best-characterized stress-response programs under the in- vation, the high-affinity tryptophan-specific permease, Tat2, fluence of TORC1 are transcriptional in nature. However, it localized to the plasma membrane, is ubiquitinated, endo- is clear that TORC1 also regulates post-transcriptional cytosed, and ultimately degraded. In contrast, the general aspects of stress responses such as mRNA stability, protein amino acid permease Gap1 is re-routed to the plasma mem- trafficking, and the activities of metabolic enzymes. brane instead of to the vacuole/endosomes. Although Environmental stress response: Exposure of yeast cells to details are still emerging, TORC1 appears to regulate such noxious stressors, including nutrient limitation and entry permease-sorting events primarily via Tap42-PPase and into stationary phase, elicits a stereotypic transcriptional its (potentially direct) effector Npr1 (Schmidt et al. 1998; response known as the environmental stress response Beck et al. 1999; De Craene et al. 2001; Jacinto et al. 2001; (ESR) (Gasch and Werner-Washburne 2002). This includes Soetens et al. 2001; Breitkreutz et al. 2010). Npr1 is a heavily 300 upregulated genes that encode activities such as pro- phosphorylated, seemingly fungal-specific, Ser/Thr kinase tein chaperones and oxygen radical scavengers that help that upon TORC1 inactivation is rapidly dephosphorylated cells endure stressful environments. The central compo- and activated (Gander et al. 2008). Although genetic studies nents of this pathway are the Zn-finger transcription fac- clearly imply a role for Npr1 in protein-sorting events, the tors Msn2/4 and Gis1, the LATS family kinase Rim15, and mechanisms of this regulation have remained elusive. It is the a-endosulfine family paralogs Igo1 and Igo2 (De Virgilio possible that the permeases themselves are Npr1 substrates. 2011). TORC1 via Sch9, and possibly also Tap42-PPase, Indeed, several nutrient and cation permeases have been promotes cytoplasmic anchoring of Rim15 to 14-3-3 pro- identified as rapamycin-sensitive phosphoproteins (Huber teins by maintaining Rim15 phosphorylated on Ser1061 et al. 2009; Soulard et al. 2010). Also identified in these and Thr1075 (Wanke et al. 2005, 2008). Inhibition of phosphoproteomics studies were several a-arrestin-related TORC1 results in nuclear localization of Rim15, which sub- proteins. These phosphoproteins function as clathrin adap- sequently triggers the activation, in a poorly understood tor molecules and have been implicated in mediating the fashion, of the expression of Msn2/4- and Gis1-dependent sorting fates of a number of different permeases; and, one, ESR genes. However, TORC1 inhibition results in a marked Aly2, has recently been reported to be an Npr1 substrate turnover of mRNAs (Albig and Decker 2001), and, as noted (Lin et al. 2008; Nikko et al. 2008; Nikko and Pelham above, in a dramatic drop in translation. Thus it would 2009; O’Donnell et al. 2010). Whether this observation is appear that increasing transcription of protein-coding genes indicative of a more general trend in Npr1-meditated per- in TORC1-inhibited cells would be futile as these mRNA mease trafficking remains to be seen. would likely be degraded before ever being translated. This TORC1 regulates permease activity by regulating not only appears not to be the case as Rim15 phosphorylates Igo1 permease localization but also expression. This was shown in and its paralog Igo2, allowing them to associate with newly early transcriptomics experiments, which clearly demon- transcribed Msn2/4- and Gis1-regulated mRNAs to protect strated that TORC1 regulates the expression of a large these transcripts from degradation via the 59-39 mRNA number of permeases and other factors required for the decay pathway (Talarek et al. 2010; Luo et al. 2011). assimilation of alternative nitrogenous sources (Cardenas Nutrient uptake and intermediary metabolism: To best et al. 1999; Hardwick et al. 1999; Komeili et al. 2000; Shamji compete with other microbes in their environment, yeast et al. 2000). TORC1 regulates the expression of nitrogen have optimized the use of available nutrients to accommo- catabolite repression (NCR)-sensitive genes via the Tap42- date fast growth (De Virgilio and Loewith 2006). Although PPase branch. The proteins encoded by these genes (e.g., a wide variety of compounds can be utilized as carbon or Gap1) enable cells to import and metabolize poor nitrogen nitrogen sources, yeast cells will exclusively assimilate pre- sources such as proline and allantoin. In the presence of ferred nutrient sources before using nonpreferred, subopti- preferred nitrogen sources such as glutamine, glutamate, or am- mal ones. To attain this dietary specificity, and to respond to monia, active TORC1 promotes the association of the GATA- nutritional stress, yeast cells carefully regulate the expres- family transcription factor Gln3 with its cytoplasmic anchor sion and sorting of their many (.270) membrane transport- Ure2. Mechanistically, this involves both TORC1-dependent TOR Function 1189 and TORC1-independent regulation of Gln3, and possibly of well as genetic studies have implicated TORC1 as a negative Ure2, phosphorylation (Beck and Hall 1999; Cardenas et al. regulator of Rtg1/3-dependent transcription (Komeili et al. 1999; Hardwick et al. 1999; Carvalho and Zheng 2003; 2000; Shamji et al. 2000; Chen and Kaiser 2003). However, Georis et al. 2009a; Tate et al. 2009, 2010). Two other it is presently unclear how TORC1 influences this pathway; less-characterized GATA factors, Gat1 and Dal81, also have TORC1 inhibition could indirectly influence retrograde re- roles in the regulation of NCR-sensitive genes (Georis et al. sponse signaling via alterations in metabolite levels. Alter- 2009b). natively, the direct association between TORC1 and Mks1 In addition to the NCR pathway, TORC1 also regulates observed by the Tyers group and described above and the the expression of amino acid permeases by modulating the fact that Mks1 is a rapamycin-sensitive phosphoprotein in- activity of the SPS-sensing pathway. This pathway consists of stead suggest that TORC1 could play a much more direct a plasma-membrane-localized sensor of external amino role in regulating this pathway (Liu et al. 2003; Breitkreutz acids, Ssy1, and two downstream factors, Ptr3 and Ssy5 et al. 2010). Finally, phosphoproteomics studies suggest that (Ljungdahl 2009). Upon activation of the pathway, Ssy5 TORC1 regulates intermediate metabolism by directly alter- catalyzes an endoproteolytic processing event that cleaves ing the activities of metabolic enzymes, particularly those and releases an N-terminal regulatory domain from two involved in the early steps of glycolysis (Loewith 2011). transcription factors, Stp1 and Stp2, the shortened forms Autophagy: As described above, starved cells express of which translocate to the nucleus and activate the tran- a suite of stress-responsive proteins to help them negotiate scription of a number of amino acid permease-encoding hostile environmental conditions. This new synthesis genes. TORC1 via Tap42-PPase modulates this pathway by requires both energy and amino acids that yeast cells obtain promoting the stability of Stp1 and thus the ability of cells to by inducing autophagy. Autophagy refers to a variety of utilize external amino acids (Shin et al. 2009). mechanisms by which cytosplasmic material, including In contrast to the SPS-sensing pathway that is activated proteins and lipids, is translocated to the vacuole and by amino acids, the Gcn4 transcription factor is activated catabolized. Amino acids and fatty acids thus acquired are, upon amino acid starvation (Hinnebusch 2005). As men- respectively, used to synthesize new proteins and oxidized tioned above, rapamycin treatment or amino acid starvation by mitochondria to produce ATP. Mechanistically, there are results in a rapid decline in translation initiation by trigger- two different modes of autophagy in yeast. One is micro- ing phosphorylation of the a-subunit of eIF2. Although autophagy, which involves the direct transfer of cytoplasm eIF2a phosphorylation results in the repression of bulk into the vacuole via invaginations of the vacuolar mem- translation, due to the presence of four short upstream open brane. The other is macroautophagy, which involves the de reading frames in its leader sequence, the mRNA encoding novo formation of double-membrane vesicles called auto- Gcn4 is, in contrast, preferentially translated. Subsequent phagosomes. Autophagosomes encapsulate cytoplasm and accumulation of Gcn4 protein leads to the transcriptional then fuse with the vacuole. Both forms of autophagy are induction of nearly all genes encoding amino acid biosyn- regulated by TORC1 (De Virgilio and Loewith 2006) thetic enzymes. although, mechanistically, macroautophagy is better under- TORC1 also regulates amino acid biosynthesis, in partic- stood (reviewed in Cebollero and Reggiori 2009; Nakatogawa ular glutamine and glutamate homeostasis, via the retro- et al. 2009; Inoue and Klionsky 2010). grade response pathway (Komeili et al. 2000; Crespo and Autophagy is conserved across eukarya, and there is Hall 2002; Crespo et al. 2002; Liu and Butow 2006). This much interest in understanding how macroautophagy is signaling pathway serves to communicate mitochondrial regulated as it has been linked to several pathologies dysfunction to the nucleus to induce an appropriate tran- including cancer, neurological disorders, and longevity scriptional response. In addition to hosting the aerobic en- (Yang and Klionsky 2010). In yeast, many autophagy- ergy production machinery, mitochondria are also the sites related (ATG) genes encode proteins that participate in the of amino acid precursor, nucleotide, and lipid production. induction of autophagy, the nucleation of the autophagosome, Signals, possibly changes in glutamate or glutamine levels, elongation and completion of the autophagosome, and, emanating from dysfunctional mitochondria impinge upon finally, in fusion of the autophagosome with the vacuole to a cytosolic regulatory protein, Rtg2. Thus activated, Rtg2 release the autolysosome into the vacuolar lumen (Chen and antagonizes the ability of Mks1 to sequester the heterodi- Klionsky 2011; Reiter et al. 2011). TORC1 regulates macro- meric bZip/HLH transcription factor complex composed of autophagy by signaling to the Atg1 kinase complex that is Rtg1 and Rtg3 in the cytoplasm. Allowed to enter the nu- required for the induction of macroautophagy. Specifically, cleus, Rtg1/3 activates genes encoding enzymes required for when TORC1 is active, Atg13 is hyperphosphorylated, pre- anaplerotic reactions that resupply tri-carboxylic acid cycle sumably directly by TORC1 (although Tap42-PPase has also intermediates that have been extracted for biosynthetic been implicated in this regulation), and this prevents the reactions. Key among these intermediates is a-ketoglutarate, association of Atg13 with Atg1, Atg17, Atg31,and Atg29 the precursor of glutamate and glutamine from which (Yorimitsu et al. 2009; Kamada et al. 2010). Inhibition of all nitrogen-containing metabolites evolve (Magasanik and TORC1 results in dephosphorylation of Atg13, assembly Kaiser 2002). Both transcriptome-profiling experiments as of the Atg1 protein kinase complex, phosphorylation and 1190 R. Loewith and M. N. Hall activation of Atg1 (Kijanska et al. 2010; Yeh et al. 2010), and, 2009; Bjedov et al. 2010). These observations have created subsequently, macroautophagy mediated by as-yet-unidenti- much excitement in that aging is now thought of as a dis- fied Atg1 substrates. Although metazoan homologs exist for ease, which, like other diseases, can be ameliorated through many of the Atg1 kinase complex components, a unifying pharmaceutical intervention. These observations have also model of how TORC1 regulates this complex in different spe- raised the important question, what are the downstream cies has yet to emerge (Chen and Klionsky 2011; Reiter et al. function(s) of TORC1 that modulate life span? The answer 2011). to this question is presently unclear, and it is very likely that Cell-wall integrity pathway: The cell wall is essential for multiple TORC1 effector pathways contribute (Blagosklonny yeast cells to survive hostile environments and, more and Hall 2009). Studies in many model systems are pres- importantly, to prevent internal turgor pressure from rup- ently underway to address this issue. Below are some of the turing the plasma membrane. Although a thickening of the highlights from studies in yeast. cell wall helps protect stressed or stationary-phase cells, this Yeast life span is assayed in one of two ways. Replicative rigid structure must also be remodelled to accommodate cell life span (RLS) is a measure of the number of progeny that growth. Homeostasis of this structure is maintained by the a single mother cell can produce before senescence. Chro- cell-wall integrity (CWI) pathway (Levin 2005). Cell-wall nological life span (CLS) is a measure of the length of time integrity is monitored by WSC (cell-wall integrity and stress a population of yeast cells can remain in stationary phase response component) family proteins. WSCs, which are in- before they lose the ability to restart growth following re- tegral plasma membrane proteins, function upstream of the inoculation into fresh media. RLS is thought to be a para- Rho1 GTPase by modulating the activity of the GEFs Rom1 digm for aging of mitotic cells while CLS is thought to be GTP and Rom2. Rho1 has several effectors including the yeast a paradigm for aging of quiescent cells. Consistent with protein kinase C homolog, Pkc1. The best-characterized bigger eukaryotes, where newborns are obviously born Pkc1 effector is a mitogen-activated protein kinase (MAPK) young, gametogenesis (i.e., cells derived from meiotic cell cascade composed of Bck1 (a MAPKKK), Mkk1 and -2 divisions) resets RLS in yeast (Unal et al. 2011). (redundant MAPKKs), and Slt2/Mpk1 (a MAPK). Activation Kaeberlein et al. (2005) have recently attempted labor- of this pathway leads to the expression of many cell-wall intensive approaches to identify genes involved in both rep- biosynthetic enzymes, which helps to remodel the cell wall licative and chronological life span. A random screen of 564 both during normal growth and in response to stress. yeast strains, each lacking a single nonessential gene, impli- Both TORC1 and TORC2 (discussed below) appear to cated both TOR1 and SCH9 in RLS downstream of caloric impinge upon the CWI pathway. Entry into stationary phase, restriction. Also identified in this screen were a number of carbon starvation, nitrogen starvation, and rapamycin treat- genes encoding ribosomal proteins. Further analyses of RP ment all elicit activation of the CWI pathway, demonstrating genes subsequently demonstrated that specific depletion of that TORC1 negatively regulates the CWI pathway (Ai et al. 60S ribosomal protein subunits extends RLS (Steffen et al. 2002; Krause and Gray 2002; Torres et al. 2002; Reinke 2008). Curiously, RLS extension observed upon TORC1 inhi- et al. 2004; Araki et al. 2005; Soulard et al. 2010). Further- bition and 60S subunit depletion seems to be mediated by more, pkc1, bck1, and mpk1 mutants rapidly lose viability Gcn4, the TORC1-dependent transcription factor that regu- upon carbon or nitrogen starvation, demonstrating that the lates the expression of amino acid biosynthetic genes as de- CWI pathway is required for viability in G0. Mechanistically, scribed above. The relevant Gcn4 target genes/processes how TORC1 signals impinge on the CWI pathway is not involved in RLS are not yet known, but an interesting candi- clear. Soulard et al. (2010) have implicated the Sch9 effector date could be macroautophagy. Induction of macroautophagy, branch while Torres et al. (2002) have postulated that sig- like TORC1 and Sch9 inhibition, increases both RLS and CLS nals through the Tap42-PPase branch causes membrane (Madeo et al. 2010a,b; Morselli et al. 2011; and see below), stress that, via WSC family members, activates downstream and Gcn4 is required for amino acid-starvation-induced mac- components of the CWI pathway. roautophagy (Ecker et al. 2010). Furthermore, spermidine, TORC1 accelerates aging: Arguably one of the most a potent inducer of macroautophagy, potentially via Gcn4 interesting functions of TORC1 is its involvement in the (Teixeira et al. 2010), appears to promote longevity not only regulation of life span. It is well established that, in virtually in yeast but also in several other model organisms (Eisenberg every biological system, aging, i.e., the progressive deterio- et al. 2009). Since TORC1, Sch9,and Gcn4 homologs are ration of cell, tissue, and organ function, can be delayed found in most eukaryotes, this appears to represent a con- through calorie or dietary restriction. Epistasis studies have served aging pathway (Kaeberlein and Kennedy 2011). led many to believe that this is due to reduced TORC1 Sch9 was one of the first genes to be implicated in CLS signaling (reviewed in Weindruch and Walford 1988; (Fabrizio et al. 2001). A subsequent high-throughput assay Kapahi et al. 2010; Zoncu et al. 2010, 2011; Kaeberlein involving 4800 viable single-gene yeast mutants further impli- and Kennedy 2011). Indeed, genetic or chemical targeting cated TORC1 in CLS (Powers et al. 2006). These and other of TORC1 has been demonstrated to increase life span in studies (Wanke et al. 2008; Wei et al. 2008) provided evidence yeast, worms, flies, and mice (Vellai et al. 2003; Jia et al. that reduced TORC1-Sch9-signaling activity promotes life span 2004; Kapahi et al. 2004; Wanke et al. 2008; Harrison et al. by increasing the Rim15-dependent expression of environmental TOR Function 1191 stress-response genes (described above). Later, Burtner et al. et al. 2003; Reinke et al. 2004; Zinzalla et al. 2010) (Figure (2009) demonstrated that acetic acid-induced mortality is the 1C). The names of mammalian and invertebrate orthologs of primary mechanism of chronological aging in yeast under stan- TORC2 subunits and the salient features of S. cerevisiae dard conditions and that this toxicity is better tolerated when TORC2 subunits are summarized in Table 1 and Table 4, environmental stress-response genes are artificially induced, respectively. The highly conserved, essential core subunits for example, upon inhibition of TORC1 or Sch9 activities. How- are TOR2, Avo1, Avo3, and Lst8. Avo1 and Avo3 bind co- ever, this model is not universally accepted. Pan et al. (2011) operatively to the N-terminal HEAT repeat region in TOR2 have proposed that TORC1 inhibition leads to increased mito- and are required for TORC2 integrity (Wullschleger et al. chondrial function and a consequent increase in reactive oxy- 2005). TORC2 autophosphorylates sites in Avo1 and Avo3, gen species that elicit a Rim15-independent pro-survival signal. but the purpose of this phosphorylation is unknown. Avo1 Furthermore, acetic acid accumulation appears not to be a con- has a C-terminal PH-like domain that mediates binding tributing factor in CLS in this study. Given its apparent conser- to the plasma membrane (Berchtold and Walther 2009). vation across eukarya (Blagosklonny and Hall 2009), Avo3 has a RasGEFN domain, a subdomain often found in elucidation of the mechanisms by which TORC1 regulates life the N-terminal part of a larger GDP/GTP exchange domain span is eagerly awaited. of exchange factors for Ras-like GTPases, but the function of Less-characterized effectors identified in phosphoproteomic the RasGEFN domain is unknown. Lst8 binds to the kinase studies: As alluded to above, large-scale mass spectrometry- domain in TOR2 and is required for TOR2 kinase activity based phosphoproteomic studies have recently been per- (Wullschleger et al. 2005). Lst8 is a Gb-like propeller pro- formed to identify the rapamycin-sensitive phosphoproteome tein consisting of seven WD40 motifs. TORC2 is rapamycin (Huber et al. 2009; Soulard et al. 2010). The major limitation insensitive whereas TORC1 is rapamycin sensitive because of these studies was their poor coverage as evidenced by their FKBP-rapamycin binds only TORC1 (Loewith et al. 2002). rather modest overlap, although this could be partly explained This selective FKBP-rapamycin binding is presumably due to by the different growth conditions and technical approaches Avo1 masking the FRB domain in TOR2 in TORC2. Finally, employed. Rapamycin exposure times were chosen such that co-immunoprecipitation and gel filtration experiments sug- layers of signaling events (e.g., kinase/phosphatase cascades) gest that TORC2 is a multimer, likely a TORC2-TORC2 di- would be observed. These events should have been triggered mer (Wullschleger et al. 2005). as a direct consequence of TORC1 inhibition and not as a sec- The cellular localization of TORC2 has been studied by ondary consequence of cell cycle delays or changes in tran- subcellular fractionation, indirect immunofluorescence, scription. Hundreds of rapamycin-sensitive phosphorylation immunogold electron microscopy, and visualization of GFP- sites were mapped, the majority of which are in proteins tagged TORC2 components (Kunz et al. 2000; Wedaman not previously implicated in TORC1 signaling. However, as et al. 2003; Aronova et al. 2007; Sturgill et al. 2008; Berchtold sufficient time elapsed to activate entire signaling cascades, and Walther 2009). In considering these studies, it is impor- a potential TORC1 consensus target motif was not evident tant to realize that the vast majority of TOR2 (90%) is in from the data analyses. Still, the data from these studies TORC2 (vs. TORC1), and thus TOR2 localization studies pre- will be instrumental in both elucidating how TORC1 signals sumably detect mainly, if not exclusively, TORC2. All studies to its known distal readouts and discovering new TORC1 indicate that TORC2 is at or near the plasma membrane. functions. Berchtold and Walther (2009) suggest that TORC2 is dynam- ically localized to a previously unrecognized plasma mem- TOR Complex 2 brane domain termed the MCT (membrane compartment containing TORC2). Furthermore, they conclude that TORC2 Composition and localization of TOR complex 2 plasma membrane localization is essential for viability and is TOR complex 2 (TORC2) is rapamycin insensitive and mediated by the C-terminal PH domain in Avo1.Mostof the consists of TOR2, Avo1, Avo2, Avo3, Bit61 (and/or its localization studies have found that TORC2 is also at another, paralog Bit2), and Lst8 (Loewith et al. 2002; Wedaman ill-defined cellular location(s). For example, Kunz et al. Table 4 Salient features of TORC2 components Protein Size Motifs/domains Potential function Tor2 2470 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain Avo1 1176 aa PH Recruit TORC2 to plasma membrane Avo2 426 aa None obvious Unknown Avo3/Tsc11 1430 aa RasGEFN Scaffold Bit61 543 aa None obvious Paralogs with unknown function Bit2 545 aa None obvious Paralogs with unknown function Lst8 303 aa 7 WD-40 repeats Stabilize kinase domain Data for this table were obtained from Cybulski and Hall (2009). 1192 R. Loewith and M. N. Hall some. As ribosomes determine the growth capacity of a cell, this mechanism ensures that TORC2 is active only in growing cells. There are also indications that environmental stress inhibits TORC2 signaling, possibly to prevent growth in unfavorable conditions. The mechanism of this regulation and the level at which it intersects with the TORC2 pathway are poorly defined, but it may involve the Slm proteins (see below) and the stress-activated phosphatase calcineurin (Bultynck et al. 2006; Mulet et al. 2006). TORC2 substrates Figure 4 Signaling by TORC2. TORC2 directly phosphorylates the AGC kinase family member Ypk (Ypk1 and 2) and the PH domain containing The best-characterized and possibly the major TORC2 sub- protein Slm (Slm1 and -2). Downstream effectors include the phospha- strate is the protein kinase Ypk. Ypk1 and Ypk2 are an es- tase calcineurin, the transcription factor Crz1, and Pkc1. TORC2 controls organization of the actin cytoskeleton, endocytosis, sphingolipid biosyn- sential pair of homologous kinases and members of the AGC thesis, and stress-related transcription. The effector pathways by which kinase family (Roelants et al. 2004) (Figure 4). Kamada TORC2 controls these processes are incompletely understood (see Distal et al. (2005) linked Ypk to TORC2 signaling upon isolating readouts downstream of TORC2 for further details). YPK2 as a multicopy suppressor of a TORC2 defect. They then showed that immunopurified TOR2 directly phosphor- ylates Ypk2 at Ser641 in the turn motif and Thr659 in the (2000) report that part of TOR2 is also in an unknown sub- hydrophobic motif. TORC2 phosphorylates and activates cellular membrane fraction distinct from Golgi, vacuoles, Gad8 and SGK1, the S. pombe and mammalian orthologs mitochondria, and the nucleus. Wedaman et al. (2003) of Ypk, respectively, in a similar manner (Matsuo et al. showed that TOR2 can be in the cell interior often in asso- 2003; Garcia-Martinez and Alessi 2008). It is well estab- ciation with membrane tracks. Sturgill et al. (2008) detected lished that TORC1 or TORC2 phosphorylates the turn and a cytoplasmic fluorescent signal in cells expressing GFP- hydrophobic motifs in several kinases as a conserved mech- tagged TOR2. In conclusion, TORC2 appears to be at mul- anism of activation of AGC kinase family members (see tiple cellular locations, the plasma membrane, and one or above) (Jacinto and Lorberg 2008). Ypk/Gad8/SGK1 possibly more other sites. A plasma membrane location is appears to be a major TORC2 substrate as an ypk, gad8,or consistent with the role of TORC2 in controlling the actin sgk1 mutation phenocopies a TORC2 defect, and overex- cytoskeleton and endocytosis (see below). pression of Ypk2, Gad8, or SGK1 is sufficient to suppress Upstream of TORC2 a TORC2 defect in S. cerevisiae, S. pombe,or Caenorhabditis elegans, respectively (Matsuo et al. 2003; Kamada et al. The upstream regulation of TORC2 is poorly characterized 2005; Jones et al. 2009; Soukas et al. 2009). The two ho- (Cybulski and Hall 2009). Several lines of evidence in many mologous, TORC2- and phosphoinositide (PI4,5P )-binding different organisms indicate that nutrients regulate TORC1 proteins Slm1 and Slm2 have also been reported to be phos- (see above). On the other hand, there is no reported evidence phorylated in a TORC2-dependent manner both in vivo and supporting the notion that TORC2 is controlled by nutrients. in vitro (Audhya et al. 2004; Fadri et al. 2005). However, the Knockout of TORC2 does not confer a starvation-like pheno- physiological relevance of Slm phosphorylation is unknown type, and the nutrient-sensitive EGO complex appears not to other than that it appears to be required for localization of be upstream of TORC2. Zinzalla et al. (2011) recently devised Slm to the plasma membrane (Audhya et al. 2004; Fadri a “reverse” suppressor screen to identify upstream regulators et al. 2005). of TORC2. This screen was based on the observation that constitutively active Ypk2 (Ypk2*) suppresses the loss of via- Distal readouts downstream of TORC2 bility due to a TORC2 defect. Ypk2 is a protein kinase nor- mally phosphorylated and activated by TORC2 (see below). The first described and best-characterized TORC2 readout is Zinzalla et al. (2011) screened for mutants that require Ypk2* the actin cytoskeleton (Figure 4). TORC2 controls the cell for viability. As predicted, this screen isolated several mutants cycle-dependent polarization of the actin cytoskeleton. As defective in genes encoding essential TORC2 components, the polarized actin cytoskeleton directs the secretory path- but also in the gene NIP7. Subsequent experiments confirmed way and thereby newly made protein and lipid to the grow- that Nip7, a ribosome maturation factor, is required for ing daughter bud, this is a mechanism by which TORC2 TORC2 kinase activity. The role of Nip7 in the activation of mediates spatial control of cell growth. The first indication yeast TORC2 has so far not been pursued further, but experi- that TOR2 is linked to the actin cytoskeleton came from the ments in mammalian cells suggest that mNip7 is required for isolation of TCP20, which encodes an actin-specific chaper- mTORC2 activation indirectly via its role in ribosome matu- one, as a dosage suppressor of a dominant-negative TOR2 ration. In mammalian cells, and presumably also in yeast “kinase-dead” mutation (Schmidt et al. 1996). This, in turn, cells, TORC2 is activated by direct association with the ribo- led to the discovery that tor2 mutants display an actin TOR Function 1193 organization defect (Schmidt et al. 1996). The subsequent mutations in genes encoding components of the sphingolipid isolation of sac7, which encodes a Rho-GAP (GTPase- biosynthetic pathway, suppress a csg2 mutation. Sur1/Csg1 activating protein), as a second-site suppressor of a tor2- and Csg2 are subunits, probably the catalytic and regulatory temperature-sensitive (ts) mutation suggested that TOR2 subunits, respectively, of mannosylinositol phosphorylcera- is linked to the actin cytoskeleton via a signaling pathway mide synthase that mediates a late step in sphingolipid bio- containing a Rho GTPase. It was later demonstrated that synthesis. The Slm proteins were subsequently also linked to Sac7 is indeed a GAP for Rho1 and that TOR2 activates sphingolipid metabolism (Tabuchi et al. 2006; Daquinag et al. the Rho1 GTPase switch via the Rho1-GEF Rom2 (Schmidt 2007). Most recently, Aronova et al. (2008) profiled sphingo- et al. 1997; Bickle et al. 1998). Rom2 GEF activity is reduced lipids in a conditional avo3 mutant and thereby confirmed in extracts from a tor2-ts mutant (Schmidt et al. 1997; Bickle that TORC2 plays a positive role in sphingolipid biosynthesis. et al. 1998). The finding that overexpression of Rom2 sup- Aronova et al. (2008) also investigated the molecular mech- presses a tor2-ts mutation, whereas overexpression of cata- anism by which TORC2 controls sphingolipids. They found lytically active Rom2 lacking its lipid-binding PH domain that TORC2 regulates sphingolipid production via Ypk2 and does not suppress, suggested that TOR2 signals to Rom2 suggest a model wherein TORC2 signaling is coupled to via the PH domain. It was subsequently shown that TOR2 sphingoid long-chain bases (early intermediates in sphingoli- signals to the actin cytoskeleton mainly, if not exclusively, pid synthesis) to control Ypk2 and late steps in sphingolipid via the Rho1 effector Pkc1 (protein kinase C) and the Pkc1- synthesis. Furthermore, the biosynthetic step controlled controlled cell-wall integrity MAP kinase cascade (Helliwell by TORC2 and Ypk2 is antagonized by the phosphatase cal- et al. 1998b). cineurin that is functionally linked to the Slm proteins How might TORC2 signal to Rom2 to activate the Rho1 (Bultynck et al. 2006; Mulet et al. 2006; Aronova et al. GTPase switch? The PH domain in Rom2 suggests that it 2008). Another potential target for the regulation of sphingo- may involve a lipid intermediate. This possibility is sup- lipid biosynthesis by TOR are the Orm1 and Orm2 proteins. ported by the observation that overexpression of the PI-4- The conserved Orm proteins, identified as a potential risk P 5-kinase Mss4 suppresses a tor2-ts mutation (Desrivieres factor for childhood asthma, form a complex that negatively et al. 1998; Helliwell et al. 1998a) and that PI4,5P at the regulates the first and rate-limiting step in sphingolipid bio- plasma membrane is required to recruit/activate Rom2 synthesis (Breslow et al. 2010; Han et al. 2010). Both Orm1 (Audhya and Emr 2002). The mechanism by which TORC2 and Orm2 are phosphoproteins and at least Orm1 phosphor- mayactivatePI4,5P signaling or possibly a parallel path- ylation changes upon rapamycin treatment (Huber et al. wayconverging on thecell-wall integritypathway is un- 2009; Soulard et al. 2010). Furthermore, loss of Orm2 sup- known, but likely involves the well-established TORC2 presses a Ypk deficiency (Roelants et al. 2002; Schmelzle et al. substrate Ypk (Roelants et al. 2002; Schmelzle et al. 2002; Kamada et al. 2005; Mulet et al. 2006). These findings 2002; Kamada et al. 2005; Mulet et al. 2006). The phos- suggest that both TORC1 and TORC2 may control sphingoli- phoinositide-binding Slm proteins and sphingolipids may pid synthesis via Orm proteins. also be functionally related to TORC2-mediated control of the actin cytoskeleton (Sun et al. 2000; Friant et al. Future Directions 2001; Roelants et al. 2002; Audhya et al. 2004; Fadri et al. 2005; Liu et al. 2005; Tabuchi et al. 2006; Daquinag What is upstream of the two complexes? et al. 2007). How TORC activities are altered in response to environ- A second downstream process controlled by TORC2 is mental cues remains a major void in our understanding of endocytosis. Efficient internalization of cell-surface compo- the TOR-signaling network. The TOR complexes are regu- nents is an important aspect of cell growth control. deHart lated by nutrients, stress, or ribosomes, but the mechanisms et al. (2003) identified a tor2 mutation in a screen for by which these inputs are sensed and how this information mutants defective in ligand-stimulated internalization of is transduced, with the notable exceptions discussed above, a cell-surface receptor. TORC2 appears to control endocyto- to ultimately regulate kinase activity remain largely un- sis via Rho1, Ypk1, and possibly the Slm proteins, but how known. Genetic screens, such as the reverse suppressor Rho1, Ypk1, and the Slm proteins are functionally related in screen described above, should help to further elucidate mediating TORC2-controlled endocytosis is unknown these signaling pathways. Unlike growth factor-signaling (deHart et al. 2002, 2003; Bultynck et al. 2006). pathways, which are present only in metazoans, nutrient A third TORC2-regulated process is sphingolipid biosyn- and stress-responsive pathways are found in all eukaryotic thesis (Powers et al. 2010). Sphingolipids serve as essential cells, and thus their characterization in model organisms structural components in lipid bilayers and as signaling mol- would have far-reaching implications. ecules. The first indication that TORC2 controls sphingolipid synthesis was the finding that overexpression of SUR1 sup- What is downstream of the TORCs? presses a temperature-sensitive tor2 mutation (Helliwell et al. 1998a). In a parallel study, Beeler et al. (1998) reported that The TORCs play a central role in the regulation of cell a mutation in TOR2 or AVO3 (also known as TSC11), or growth by signaling to a staggering number of distal 1194 R. Loewith and M. N. Hall Audhya, A., and S. D. Emr, 2002 Stt4 PI 4-kinase localizes to the downstream processes. Recent phosphoproteomics studies plasma membrane and functions in the Pkc1-mediated MAP have begun to illuminate the relevant phosphorylation kinase cascade. Dev. Cell 2: 593–605. cascades and, in addition, have suggested the existence of Audhya, A., R. Loewith, A. B. Parsons, L. Gao, M. Tabuchi et al., novel growth-related effectors downstream of TORC1. Sim- 2004 Genome-wide lethality screen identifies new PI4,5P2 ilar studies describing the TORC2-dependent phosphopro- effectors that regulate the actin cytoskeleton. EMBO J. 23: 3747–3757. teome are eagerly anticipated. Elucidating these downstream Barbet, N. C., U. Schneider, S. B. Helliwell, I. Stansfield, M. F. Tuite signaling events is both academically interesting and medi- et al., 1996 TOR controls translation initiation and early G1 cally important; cell growth, like cell birth (division) and cell progression in yeast. Mol. Biol. 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Thorner TOR Function 1201 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genetics Pubmed Central

Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control

Genetics , Volume 189 (4) – Dec 1, 2011

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Copyright © 2011 by the Genetics Society of America
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1943-2631
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10.1534/genetics.111.133363
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Abstract

YEASTBOOK CELL SIGNALING & DEVELOPMENT Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control ,1 †,1 Robbie Loewith* and Michael N. Hall *Department of Molecular Biology and National Centers of Competence in Research and Frontiers in Genetics and Chemical Biology, University of Geneva, Geneva, CH-1211, Switzerland, and Biozentrum, University of Basel, Basel CH-4056, Switzerland ABSTRACT TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae. TABLE OF CONTENTS Abstract 1177 Introduction 1178 The Early Days 1178 TOR Complex 1 1181 Composition of TOR complex 1 1181 Localization of TORC1 1183 Upstream of TORC1 1183 Physiological regulators (carbon, nitrogen, phosphate, stress, caffeine): 1183 The EGO complex: 1184 Feedback loop/ribosome biogenesis homeostasis: 1184 Downstream of TORC1 1185 Proximal TORC1 effectors: 1185 Characterization of Sch9 as a TORC1 substrate: 1185 Characterization of Tap42‐PP2A as a TORC1 effector: 1185 Other TORC1 substrates: 1186 Distal readouts downstream of TORC1: 1186 TORC1 promotes cell growth: 1186 Protein synthesis: 1186 Ribosome biogenesis: 1186 Regulation of cell cycle/cell size: 1188 Continued Copyright © 2011 by the Genetics Society of America doi: 10.1534/genetics.111.133363 Manuscript received July 29, 2011; accepted for publication September 12, 2011 Available freely online through the author-supported open access option. Corresponding authors: Biozentrum, University of Basel, Klingelbergstrasse 70, Basel CH-4056, Switzerland. E-mail: m.hall@unibas.ch; and Department of Molecular Biology, NCCRs Chemical Biology and Frontiers in Genetics, University of Geneva, 30 quai Ernest Ansermet Geneva CH-1211, Switzerland. E-mail: Robbie.Loewith@unige.ch Genetics, Vol. 189, 1177–1201 December 2011 1177 CONTENTS, continued TORC1 inhibits stress responses: 1189 Environmental stress response: 1189 Nutrient uptake and intermediary metabolism: 1189 Autophagy: 1190 Cell-wall integrity pathway: 1191 TORC1 accelerates aging: 1191 Less-characterized effectors identified in phosphoproteomic studies: 1192 TOR Complex 2 1192 Composition and localization of TOR complex 2 1192 Upstream of TORC2 1193 TORC2 substrates 1193 Distal readouts downstream of TORC2 1193 Future Directions 1194 What is upstream of the two complexes? 1194 What is downstream of the TORCs? 1194 HE contributors to this GENETICS set of reviews were nutrients to the tumor cells (Guba et al. 2002). Finally, Tasked to focus on the developments in their field since rapamycin-eluting stents prevent restenosis after angio- plasty. Thus, rapamycin has clinical applications in three 1991, the year the last yeast monographs were published. major therapeutic areas: organ transplantation, cancer, and Coincidentally, Target Of Rapamycin (TOR) was discovered coronary artery disease. What do fungi and the seemingly in 1991. We thus have the whole TOR story to tell, from the very different conditions of transplant rejection, cancer, and beginning, in a review that marks the 20th anniversary of restenosis have in common in their underlying biology such TOR. As we review TOR signaling in Saccharomyces cerevi- that all can be treated with the same drug? All three con- siae, the reader is referred to other reviews for descriptions ditions (and the spread of pathogenic fungi) are due to of TOR in other organisms (Wullschleger et al. 2006; Polak ectopic or otherwise undesirable cell growth, suggesting and Hall 2009; Soulard et al. 2009; Caron et al. 2010; Kim that the molecular target of rapamycin is a central controller and Guan 2011; Zoncu et al. 2011). of cell growth. TOR is indeed dedicated to controlling cell The story of the TOR-signaling network begins with a re- growth, but what is this target and how does it control cell markable drug, rapamycin (Abraham and Wiederrecht growth? 1996; Benjamin et al. 2011). Rapamycin is a lipophilic mac- rolide and a natural secondary metabolite produced by Streptomyces hygroscopicus, a bacterium isolated from a soil The Early Days sample collected in Rapa-Nui (Easter Island) in 1965— hence the name rapamycin. Rapamycin was originally puri- Studies to identify the cellular target of rapamycin and to fied in the early 1970s as an antifungal agent. Although it elucidate the drug’s mode of action were initiated in the late effectively inhibits fungi, it was discarded as an antifungal 1980s by several groups working with yeast (Heitman et al. agent because of its then undesirable immunosuppressive 1991a; Cafferkey et al. 1993; Kunz et al. 1993) and mam- side effects. Years later, it was “rediscovered” as a T-cell malian cells (Brown et al. 1994; Chiu et al. 1994; Sabatini inhibitor and as an immunosuppressant for the treatment et al. 1994; Sabers et al. 1995). At that time, rapamycin was of allograft rejection. Preclinical studies subsequently known to inhibit the vertebrate immune system by blocking showed that rapamycin and its derivatives, CCI-779 a signaling pathway in helper T cells that mediates cell cycle (Wyeth-Ayerst) and RAD001 (Novartis), also strongly in- (G1) progression in response to the lymphokine IL-2. How- hibit the proliferation of tumor cells. Rapamycin received ever, the molecular mode of action of the drug was not clinical approval in 1999 for use in the prevention of organ known other than it possibly involved binding and inhibiting rejection in kidney transplant patients, and additional appli- the cytosolic peptidyl-prolyl cis-trans isomerase FKBP12 cations as an immunosuppressive agent have since been de- (FK506-binding protein 12), also known as an immunophi- veloped. CCI-779 (Torisel) and RAD001 (Afinitor) were lin (Schreiber 1991). Furthermore, the observation that approved in 2007 and 2009, respectively, for treatment of rapamycin inhibited cell cycle progression in yeast as in advanced kidney cancer. Rapamycin is effective against mammalian cells suggested that the molecular target was tumors because it blocks the growth of tumor cells directly conserved from yeast to vertebrates and that yeast cells and because of the indirect effect of preventing the growth could thus be exploited to identify the target of rapamycin of new blood vessels (angiogenesis) that supply oxygen and (Heitman et al. 1991a). It should be noted that the early 1178 R. Loewith and M. N. Hall researchers were interested not only in understanding rapa- mutants defective in FKBP were recovered, but also obtained mycin’s mechanism of action, but also in using rapamycin as were mutants altered in either one of two novel genes a probe to identify novel proliferation-controlling signaling termed TOR1 and TOR2. The fpr1 mutations were common pathways (Kunz and Hall 1993). In the late 1980s, signifi- and recessive. Interestingly, the TOR1 and TOR2 mutations cantly less was known about signaling pathways than today; were rare and dominant. The TOR1 and TOR2 genes were indeed, few and only incomplete pathways were known. cloned, on the basis of the dominant rapamycin-resistance The early studies in yeast first focused on identifying an phenotype of the mutant alleles, and sequenced (Cafferkey FKBP (FK506-binding protein) (Heitman et al. 1991b; Koltin et al. 1993; Kunz et al. 1993; Helliwell et al. 1994). Both et al. 1991; Tanida et al. 1991; Wiederrecht et al. 1991). TOR1 and TOR2 proteins are 282 kDa in size (2470 and FKBP12 had previously been identified in mammalian cell 2474 amino acids, respectively) and 67% identical. TOR1 extracts as a rapamycin (and FK506)-binding protein. Yeast and TOR2 are also the founding members of the PI kinase- FKBP was purified to homogeneity using an FK506 column related protein kinase (PIKK) family of atypical Ser/Thr- and partially sequenced. The protein sequence information specific kinases (other members include TEL1, ATM, DNA- was used to design degenerate oligonucleotides that were PK, and MEC1) (Keith and Schreiber 1995). Although the then used to isolate the FKBP-encoding gene FPR1 (Heitman catalytic domain of all members of this protein kinase family et al. 1991b). The predicted amino acid sequence of yeast resembles the catalytic domain of lipid kinases (PI3K and Fpr1 was 54% identical to that of the concurrently charac- PI4K), no PIKK family member has lipid kinase activity, and terized human FKBP12, providing further support that the the significance of the resemblance to lipid kinases is un- mode of action of rapamycin was conserved from yeast to known. Two reports in 1995—before TOR was shown to be humans. Curiously, disruption of the FKBP gene in yeast a protein kinase—claimed that yeast and mammalian TOR (FPR1) revealed that FKBP is not essential for growth had lipid kinase (PI4K) activity, but these findings were (Heitman et al. 1991b; Koltin et al. 1991; Tanida et al. never confirmed and are now thought to have been due to 1991; Wiederrecht et al. 1991). Additional FKBPs and cyclo- a contaminating PI4K. Disruption of TOR1 and TOR2 in philins (also an immunophilin and proline isomerase) were combination caused a growth arrest similar to that caused subsequently discovered and cloned, and again single and by rapamycin treatment, suggesting that TOR1 and TOR2 multiple disruptions were constructed without consequen- are indeed the targets of FKBP–rapamycin and that the tial loss of viability (Heitman et al. 1991b, 1992; Davis et al. FKBP–rapamycin complex inhibits TOR activity (Kunz 1992; Kunz and Hall 1993; Dolinski et al. 1997). The finding et al. 1993). It was subsequently demonstrated that the that FPR1 disruption did not affect viability was paradoxical FKBP–rapamycin complex binds directly to TOR1 and because FKBP was believed to be the in vivo binding protein/ TOR2 (Stan et al. 1994; Lorenz and Heitman 1995; Zheng target for the toxic effect of rapamycin. Why did inhibition et al. 1995) and that TOR is widely conserved both struc- of FKBP by rapamycin block growth whereas inhibition of turally and as the target of FKBP–rapamycin (Schmelzle and FKBP by disruption of the FPR1 gene have no effect on Hall 2000). However, S. cerevisiae is unusual in having two growth? The subsequent finding that an FPR1 disruption TOR genes whereas almost all other eukaryotes, including confers rapamycin resistance (Heitman et al. 1991a,b), com- plants, worms, flies, and mammals, have a single TOR gene. bined with the observation that some drug analogs are not As described below, this additional complexity in S. cerevi- immunosuppressive despite being able to bind and inhibit siae helped the analysis of TOR signaling because it allowed FKBP12 proline isomerase (Schreiber 1991), provided the differentiating two functionally different signaling branches answer to the above question and led to the well-established on the basis of different requirements for the two TORs. model of immunosuppressive drug action: an immunophilin- It should be noted that there is no evidence to indicate drug complex (e.g., FKBP-rapamycin) gains a new toxic ac- that FKBP has a role in normal TOR signaling, i.e., in the tivity that acts on another target. In other words, FKBP is absence of rapamycin. Rapamycin hijacks or corrupts FKBP only a cofactor or receptor required by the drug to act on to interact with TOR. In addition, some have speculated that something else; FKBP itself is not the target required for rapamycin mimics an endogenous metabolite that normally viability. This mode of drug action also applies to the well- regulates TOR with or without FKBP. Although this would known immunosuppressants cyclosporin A and FK506 (from provide an explanation for the evolution of the mechanism cyclophilin–cyclosporin A and FKBP–FK506 complexes) and of action of rapamycin, no evidence has been reported for an is conserved from yeast to humans (Schreiber 1991). These endogenous rapamycin-like compound or for such a mode of early studies in yeast were the first of many that have since TOR regulation. contributed to an understanding of rapamycin action and All TORs have a similar domain structure (Figure 1A). TOR signaling even in mammalian cells (Crespo and Hall The domains found in TOR—in order from the N to the C 2002), illustrating that a model organism such as yeast is terminus of TOR—compose the so-called HEAT repeats, the valuable in both basic and biomedical research. FAT domain, the FRB domain, the kinase domain, and the To identify the target of the FKBP–rapamycin complex, FATC domain (Schmelzle et al. 2002). The HEAT repeats rapamycin-resistant yeast mutants were selected (Heitman (originally found in huntingtin, elongation factor 3, the A et al. 1991a; Cafferkey et al. 1993). As expected, fpr1 subunit of PP2A, and TOR1) consist of 20 HEAT motifs, TOR Function 1179 each of which is 40 residues that form a pair of interacting antiparallel a-helices (Andrade and Bork 1995; Perry and Kleckner 2003). The HEAT repeats occupy the N-terminal half of TOR and are the binding region for subunits of the TOR complexes (Wullschleger et al. 2005) (see below). The central FAT domain (500 residues) and the extreme C-terminal FATC domain (35 residues), flanking the FRB and kinase domains, are always paired and found in all PIKK family members (Alarcon et al. 1999; Bosotti et al. 2000; Dames et al. 2005). The FRB domain (100 residues) is the FKBP–rapamycin-binding region. All rapamycin resis- tance-conferring TOR mutations fall within the FRB domain, thereby directly preventing the binding of FKBP–rapamycin without otherwise affecting TOR activity (Heitman et al. 1991a; Cafferkey et al. 1993; Helliwell et al. 1994; Stan et al. 1994; Chen et al. 1995; Lorenz and Heitman 1995; Choi et al. 1996). Interestingly, all the original rapamycin- resistance conferring mutations in TOR1 and TOR2 are mis- sense mutations confined to a single, equivalent codon encoding a critical serine residue (Ser1972Arg or Ser1972- Asn in TOR1 and Ser1975Ile in TOR2) (Cafferkey et al. 1993; Helliwell et al. 1994), which explains why the rapamycin-resistance TOR mutations were rare. Recreating an equivalent mutation (Ser2035Ile) in mammalian TOR (mTOR) was instrumental in demonstrating that mTOR is the target of FKBP–rapamycin in mammalian cells (Brown Figure 1 (A) Conserved domain structure of TOR. The N-terminal half of et al. 1995). Thus, the early rapamycin-resistant yeast TOR is composed of two blocks of 20 HEAT repeats, 40 aa that form mutants turned out to be very informative. They not only pairs of interacting antiparallel a-helices. The 500-aa FAT (FRAP-ATM- identified TOR, but also identified the FKBP–rapamycin- TRRAP) domain contains modified HEAT repeats. Missense mutations in binding site in TOR and contributed to elucidating the the 100-aa FRB (FKBP12-rapamycin-binding) domain confer complete resistance to rapamycin. The kinase domain phosphorylates Ser/Thr resi- mechanism of action of rapamycin. The kinase domain is dues in protein substrates, but at the sequence level resembles the cat- the catalytic domain and resembles the kinase domain of alytic domain of phosphatidylinositol kinases. The 35-aa FATC domain is PI3K and PI4K lipid kinases. Despite high interest in a struc- always found C-terminal to the FAT domain and is essential for kinase ture of the kinase domain, no such structure exists for any activity. (B) Composition of TOR complex 1. TORC1 is 2 MDa in size and TOR, which is likely due to technical difficulties in express- contains Kog1, Tco89, Lst8, and either TOR1 or TOR2. The HEAT repeats found in Kog1 and the seven-bladed propellers of the WD-40 repeats ing this domain for structural studies. In the absence of found in Kog1 and Lst8 are depicted. The binding of Kog1 to TOR is a true model, a homology model based on the crystal struc- complex, involving multiple domains on each protein. Lst8 binds to the ture of related PI3K has been elaborated (Sturgill and Hall kinase domain of TOR. Each component is likely present in two copies. (C) 2009). A number of groups have identified activating, mis- Composition of TOR complex 2. TORC2 is 2 MDa in size and contains sense mutations in S. cerevisiae and Schizosaccharomyces Avo1, Avo2, Avo3, Bit61, and/or its paralog Bit2, Lst8, and TOR2 but not TOR1. The RasGEFN domain of Avo3 and the PH domain of Avo1 are pombe TORs (Reinke et al. 2006; Urano et al. 2007; Ohne indicated. Each component is likely present in two copies. et al. 2008). These mutations fall within the FAT, FRB, and kinase domains, and, interestingly, one of the hotspots in the kinase domain corresponds to a region for oncogenic muta- within a few generations as small-budded cells in the G2/M tions in PI3K (Sturgill and Hall 2009; Hardt et al. 2011). phase of the cell cycle and with a randomized actin cyto- In the mid-1990s, research in the TOR field focused on skeleton (Kunz et al. 1993; Helliwell et al. 1994, 1998a; elucidating the cellular roles of TOR1 and TOR2. It was Schmidt et al. 1996). These and other findings led to the found that TOR1 and TOR2 play a central role in controlling model that TOR2 has two essential functions: one function cell growth as part of two separate signaling branches. Al- is redundant with TOR1 (TOR shared) and the other func- though structurally similar, TOR1 and TOR2 are not func- tion is unique to TOR2 (TOR2 unique) (Hall 1996; Helliwell tionally identical (Kunz et al. 1993; Helliwell et al. 1994). et al. 1998a). As described below, these two TOR2 functions Combined disruption of TOR1 and TOR2, or rapamycin turned out to be two separate signaling branches (each cor- treatment, mimics nutrient deprivation including causing responding to a structurally and functionally distinct TOR a G0 growth arrest within one generation (Barbet et al. complex) that control cell growth in different ways (Barbet 1996). Disruption of TOR1 alone has little-to-no effect, et al. 1996; Schmidt et al. 1997, 1998; Bickle et al. 1998; and disruption of TOR2 alone causes cells to arrest growth Helliwell et al. 1998a; Loewith et al. 2002; Loewith and Hall 1180 R. Loewith and M. N. Hall 2004; De Virgilio and Loewith 2006; Breitkreutz et al. 2010; this way of thinking about the two branches has subsided in Kaizu et al. 2010). recent years as the TOR2-unique pathway was shown to The early characterization of TOR disruptions and rapa- control sphingolipid synthesis and endocytosis in addition mycin treatment led to two more important conclusions. to the actin cytoskeleton (Powers et al. 2010). First, as described in more detail below, the finding that Another major breakthrough in the TOR field occurred in TOR inhibition mimics starvation led to the notion that 2002: the identification of the two multiprotein complexes TOR controls cell growth in response to nutrients (Barbet termed TOR complex 1 (TORC1) and TORC2 (Loewith et al. 1996; Rohde et al. 2001). Subsequent studies con- et al. 2002; Wedaman et al. 2003; Reinke et al. 2004; firmed this notion and demonstrated that TOR in higher Wullschleger et al. 2006). The two structurally and function- eukaryotes also controls cell growth in response to ally distinct TOR complexes were biochemically purified nutrients; i.e., TOR is conserved in structure and function from yeast cells and subsequently shown to correspond (Thomas and Hall 1997; Hara et al. 1998; Schmelzle and to the two genetically defined TOR-signaling branches. Hall 2000). Second, the observation that inhibition specifi- TORC1, which contains either TOR1 or TOR2 and is rapa- cally of the TOR-shared signaling branch (disruption of both mycin sensitive, mediates the TOR-shared pathway. TORC2, TORs but not of TOR2 alone) or rapamycin treatment which specifically contains TOR2 and is rapamycin insensi- mimics starvation suggested that only the TOR-shared path- tive, mediates the TOR2-unique pathway. The TORCs were way is nutrient responsive and rapamycin sensitive (Zheng a major breakthrough because they provided a molecular et al. 1995; Barbet et al. 1996; Schmidt et al. 1996; Rohde basis for the functional complexity and selective rapamycin et al. 2001). The molecular basis of these findings would sensitivity of TOR signaling. The biochemical identification remain a mystery until the discovery of the two structurally of the TORCs and the genetic definition of the two signaling and functionally distinct TOR complexes (see below). branches also, gratifyingly, cross-validated each other such The realization that TOR controls growth (increase in cell that there is a high level of confidence in the current “two size or mass) was a particularly important development branch-two complex” model of TOR signaling. The subse- (Barbet et al. 1996; Thomas and Hall 1997; Schmelzle et al. quent identification of TORCs in other eukaryotes, including 2002). Rapamycin or loss of TOR function causes a cell cycle plants, worms, flies, and mammals (Table 1), showed that arrest, and TOR was thus originally thought to be a control- the two complexes, like TOR itself, are conserved and gave ler of cell division (increase in cell number). Furthermore, at further support to the above model (Hara et al. 2002; that time, growth was largely thought to be controlled pas- Kim et al. 2002; Loewith et al. 2002; Jacinto et al. 2004; sively: i.e., the simple availability of nutrients (building Sarbassov et al. 2004). Below we focus on the structure, blocks) led to cell growth. As described below, the realiza- function, and regulation of the two TOR complexes. We tion that TOR controls many cellular processes that collec- discuss some downstream readouts of the TORCs that were tively determine mass accumulation, combined with the fact originally described before the discovery of the TORCs but that there was no direct role for TOR in the cell cycle ma- are now retroactively attributed to TORC1 or TORC2 on the chinery then being characterized, led to the notions that basis of their TOR requirement or rapamycin sensitivity. TOR controls growth and that growth is thus actively con- trolled. The originally confusing defect in cell cycle progres- sion observed upon TOR inhibition is in fact an indirect TOR Complex 1 effect of growth inhibition: a growth defect is dominant over Composition of TOR complex 1 cell cycle progression. Since the late 1990s, many groups have been character- TORC1 consists of Kog1, Lst8, Tco89,and either TOR1 or izing the two separate TOR2-signaling branches. It was TOR2 (Figure 1B) (Loewith et al. 2002; Wedaman et al. found that the TOR-shared signaling branch is composed 2003; Reinke et al. 2004). Gel filtration chromatography of various effector pathways that control a wide variety of (R.Loewith,W.Oppliger, and M. Hall, unpublished readouts that collectively determine the mass of the cell. results) indicated that TORC1 has a size of 2MDa,sug- The readouts controlled by this branch include protein syn- gesting that the entire complex is likely dimeric. This thesis and degradation, mRNA synthesis and degradation, wouldbeconsistentwiththe dimericstructuresproposed ribosome biogenesis, nutrient transport, and autophagy for TORC2 (Wullschleger et al. 2005) and mTORC1 (Yip (Schmelzle and Hall 2000). This branch is viewed as medi- et al. 2010). The names of mammalian and invertebrate ating temporal control of cell growth. The TOR2-unique orthologs of TORC1 subunits and the salient features of S. cerevisiae TORC1 subunits are summarized in Table 1 branch controls the polarized organization of the actin cy- and Table 2, respectively. Although all subunits are thought toskeleton, endocytosis, and sphingolipid synthesis. This to act positively with TOR1/2 in vivo, by and large their second branch is viewed as mediating spatial control of cell growth, on the basis largely of early work showing that it functions await characterization. In the presence of rapamy- controls the actin cytoskeleton. Thus, the logic of the two cin, all components of TORC1 can be coprecipitated with branches appears to be to integrate temporal and spatial FKBP12 (Loewith et al. 2002), demonstrating that, unlike control of cell growth (Loewith and Hall 2004). However, mammalian TORC1 (Yip et al. 2010), the structural integrity TOR Function 1181 1182 R. Loewith and M. N. Hall Table 1 TORC1, TORC2, and EGO complex orthologs in various genera S. cerevisiae S. pombe C. albicans D. discoideum A. thaliana C. elegans D. melanogaster Mammals TORC1 TOR1 or TOR2 Tor1 or Tor1 Tor TOR TOR/let-363 TOR mTOR Tor2 Kog1/Las24 Mip1 Kog1 Raptor RAPTOR1A and daf-15 Raptor Raptor RAPTOR1B Lst8 Wat1/Pop3 Orf19.3862 lst8? AT2G22040 lst-8? CG3004 mLST8 AT3G18140 Tco89 Tco89 Tco89 pcr25kl1p3887 —— — — — Toc1 —— — — — — —— — — — — — PRAS40 —— — — — — — DEPTOR TORC2 TOR2 Tor1 or Tor2 Tor1 tor TOR TOR/let-363 TOR mTOR Avo1 Sin1 orf19.5221 piaA sinh-1 Sin1 mSIN1 Avo2 — Avo2 —— — — Avo3/Tsc11 Ste20 Tsc11 rip3 rict-1 Rictor Rictor Lst8 Wat1/Pop3 Orf19.3862 lst8 AT2G22040 lst-8 CG3004 mLST8 AT3G18140 Bit61 Bit61 —— — — — PRR5/Protor —— — — — — — DEPTOR EGO complex Gtr1 Gtr1 Gtr1 ragA — raga-1 RagA RagA,B Gtr2 Gtr2 Gtr2 ragC — ragc-1 RagC RagC,D Ego1/Meh1/Gse2 —— — — — CG14184 LAMTOR1/p18 Ego3/Slm4/Nir1/Gse1 —— — — lamtor-2, ? CG5189, CG5110 LAMTOR2/p14, LAMTOR3/ MP1 Orthologs listed are from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Dictyostelium discoideum, Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium melanogaster, and mammals. P-POD: Princeton Protein Orthology Database/BLAST. We note that TORC2 appears to be absent in plants, e.g., A. thaliana. —, no demonstrated/obvious ortholog. Table 2 Salient features of TORC1 components Protein Size Motifs/domains Potential function TOR1 2470 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain TOR2 2474 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain Kog1 1557 aa An N-terminal conserved region 4, HEAT Present substrate to TOR repeats, 7 C-terminal WD-40 repeats Tco89 799 aa None obvious Receive signals from EGO complex Lst8 303 aa 7 WD-40 repeats Stabilize kinase domain of yeast TORC1 is not compromised by this macrolide. De- of yeast cells to rapamycin results in a dramatic drop in spite recent molecular reconstructions from low resolution protein synthesis, induction of autophagy, and exit from (25 Å) electron microscopy of a TOR1–Kog1 subcomplex the cell cycle and entrance into a quiescent G0 state. This (Adami et al. 2007), the molecular mechanism by which was the first indication that TOR, actually TORC1, might binding of FKBP-rapamycin inhibits TORC1 activity is enig- regulate growth downstream of nutrient cues. This hypoth- matic and remains a fascinating question. esis was strengthened when TORC1, in response to nitrogen and carbon cues, was found to promote the sequestration of Localization of TORC1 several nutrient-responsive transcription factors in the cyto- Tagging of Kog1, Tco89, Lst8, and TOR1 with GFP demon- plasm (Beck and Hall 1999). Consistently, transcriptome strates that TORC1 is concentrated on the limiting mem- profiling demonstrated a highly similar transcriptional re- brane of the vacuole (Urban et al. 2007; Sturgill et al. sponse of yeast cells exposed to rapamycin, nutrient starva- 2008; Berchtold and Walther 2009; Binda et al. 2009). tion, or noxious stressors (Cardenas et al. 1999; Hardwick These observations are consistent with previous studies that et al. 1999; Komeili et al. 2000; Shamji et al. 2000; Gasch localized TORC1 via immunogold electron microscopy and and Werner-Washburne 2002). Although suggestive, these cellular fractionation (Chen and Kaiser 2003; Reinke et al. observations provided only correlative evidence that TORC1 2004). Artificial tethering of a TORC1 peptide substrate to activity is regulated in response to environmental cues. the vacuole demonstrates that vacuole-localized TORC1 is Characterization of a bona fide substrate of TORC1 allowed catalytically competent (Urban et al. 2007). This localization this model to be tested directly. appears to be constitutive (Binda et al. 2009), suggesting As detailed below, Sch9 presently remains the best- that changes in “geography” play no obvious role in regulat- characterized substrate of TORC1, and monitoring its phos- ing yeast TORC1-signaling output. The yeast vacuole is a ma- phorylation by Western blotting serves as a convenient proxy jor nutrient reservoir and TORC1 signaling is responsive to for TORC1 activity. In addition to exposure to rapamycin, nutrient cues (see below). Thus, vacuolar localization of Sch9 is rapidly dephosphorylated in cells experiencing acute TORC1 seems logical. Although convincing, these studies starvation of carbon, nitrogen, phosphate, or amino acids do not exclude the possibility that a fraction of TORC1 (Urban et al. 2007; Binda et al. 2009). These and other obser- may also be active elsewhere in the cell. Li et al. (2006), vations confirm that TORC1 is responsive to both the abun- for example, have reported that TOR1 dynamically associ- dance and the quality of nutrients in the environment; but, ates with the rDNA locus to regulate 35S rRNA with few exceptions (see The EGO complex), how nutrient transcription. cues are sensed and how this information is transduced to TORC1 remain unknown. Upstream of TORC1 TORC1 activity is also regulated in response to noxious Physiological regulators (carbon, nitrogen, phosphate, stressors. When cells are subjected to various stress con- stress, caffeine): A major breakthrough in the TOR field ditions, including high salt, redox stress, a shift to a higher came with the observation that rapamycin treatment alters temperature, or caffeine, Sch9 phosphorylation is reduced yeast physiology in much the same way as nutrient dramatically (Kuranda et al. 2006; Urban et al. 2007). With starvation (Barbet et al. 1996). Like starvation, exposure the exception of caffeine, which directly inhibits TORC1 Table 3 Salient features of EGO Complex components Protein Size Motifs/domains Potential function Gtr1 310 aa Ras-family GTPase GTP-bound form activates TORC1 Gtr2 341 aa Ras-family GTPase GDP-bound form activates TORC1 Ego1/Meh1/ Gse2 184 aa N-terminal palmitoylation/myristolation Vacuolar recruitment Ego3/Slm4/Nir1/Gse1 162 aa Transmembrane domain, PtdIns(3,5)P2 binding Vacuolar recruitment Described in Dubouloz et al. (2005), Hou et al. (2005), and references therein. TOR Function 1183 and the Rags mediate amino acid sufficiency signals to mTORC1 (reviewed in Kim and Guan 2011). Like its mammalian counterpart, the EGO complex resides on the vacuolar/lysosomal membrane and is thought to couple amino acid signals to TORC1 (Binda et al. 2009). Curiously, GTP GDP the Gtr1 Gtr2 combination activates TORC1 with the nucleotide-binding status of Gtr1 seemingly dominant over the nucleotide-binding status of Gtr2. TORC1 activity in both metazoans and yeast appears to be particularly responsive to glutamine (Crespo et al. 2002) and the branched-chain amino acid leucine (Binda et al. 2009; Cohen and Hall 2009). In yeast, leucine starvation destabilizes Gtr1-TORC1 association and causes a reduction Q65L in Sch9 phosphorylation whereas GTP-locked Gtr1 remains bound to TORC1 and Sch9 dephosphorylation is Figure 2 The EGO complex is a major regulator of TORC1. The EGO delayed in cells expressing this mutant (Binda et al. 2009). complex (EGOC) is composed of four proteins: the palmitoylated and Loss of Gtr1 results in reduced Sch9 phosphorylation and myristolated protein Ego1, the transmembrane protein Ego3, and two S20L slow growth whereas GDP-locked Gtr1 is dominant neg- Ras-family GTPases, Gtr1 and Gtr2. Like TORC1, the EGO complex is S20L ative. When Gtr1 is expressed as the sole version of Gtr1, localized to the vacuolar membrane where it appears to sense/respond cells are extremely sick. This near inviability is suppressed to intracellular leucine levels and potentially to intravacuolar amino acid levels. Vam6 has been identified as a guanine nucleotide exchange factor by deletion of the TCO89 gene encoding the TORC1 subunit for Gtr1 but no other GEFs or GAPS for this GTPase system have been Tco89. Collectively, these observations suggest that the EGO GTP GDP reported. In the Gtr1 and Gtr2 configuration, the EGO complex complex can both positively and negatively regulate TORC1 somehow activates TORC1; the reverse conformation inactivates TORC1. activity via Tco89. The fact that the EGO complex can neg- Activated TORC1, via its two main effector branches, the AGC kinase atively regulate TORC1 activity seems to be at odds with the Sch9 and the Tap42-PP2a and PP2a-like protein phosphatases, stimulates growth by favoring anabolic processes and by antagonizing catabolic current metazoan model according to which the EGO com- processes and stress-response programs. plex counterpart serves only to localize TORC1 to the vacu- ole. Indeed, in contrast to the results obtained in metazoans, kinase activity (Kuranda et al. 2006; Reinke et al. 2006; in yeast, TORC1 appears to stably localize to the vacuolar membrane regardless of nutrient conditions. Thus, how the Wanke et al. 2008), how environmental stress signals are EGO complex influences TORC1 activity remains a mystery transduced to TORC1 is also unclear. although the crystal structure of the Gtr1–Gtr2 complex, The EGO complex: When environmental conditions are in- reported very recently, provides some mechanistic insights appropriate for growth, cells stop dividing, slow their me- (Gong et al. 2011). tabolism, induce the expression of stress-responsive proteins, Also mysterious are the mechanisms by which amino acid and accumulate energy stores. This nondividing but meta- sufficiency modulates Gtr1/2 guanine nucleotide loading. bolically active state is known as quiescence (G0). How cells Given its localization, it is tempting to postulate that the enter into quiescence is relatively well characterized. In EGO complex responds to levels of intravacuolar amino contrast—and despite its medical relevance (inappropriate acids, possibly via the recently described Gtr1 guanine– exit from quiescence can lead to cancer or reactivation of nucleotide exchange factor (GEF) Vam6/Vps39 (Binda a latent infection)—how quiescent cells reinitiate growth is et al. 2009). It is equally plausible, however, that this signal poorly understood. To shed light on this process, a clever is mediated by an as-yet-unidentified GTPase-activating pro- screen was performed to identify mutants that are unable to tein (GAP) activity. Consistent with the conserved function escape from rapamycin-induced growth arrest (EGO) of the EGO/Ragulator complex, and like its yeast ortholog, mutants (Dubouloz et al. 2005). This and a follow-up study hVPS39 has been found to function positively upstream of (Binda et al. 2009) identified the EGO complex as an impor- mTORC1 (Flinn et al. 2010). tant regulator of TORC1. The EGO complex is composed of four proteins: Ego1, Feedback loop/ribosome biogenesis homeostasis: Although Ego3, Gtr1, and Gtr2 (Table 3 and Figure 2). Gtr1 and most recognized as a target of signals emanating from Gtr2 are Ras-family GTPases and orthologs of the metazoan extracellular nutrients and noxious stresses, it is becoming Rag GTPases (Kim et al. 2008; Sancak et al. 2008) (Table 1). increasingly apparent that TORC1 also responds to intracel- Although they lack obvious sequence homologies, Ego1 and lular cues. In addition to the sensing of intracellular amino Ego3 are likely the functional homologs of vertebrate p18 acids as described above, outputs from distal effectors also (LAMTOR1) and p14 + MP1 (LAMTOR2 + LAMTOR3), regulate TORC1 in apparent feedback loops. For example, respectively, which function together as the “Ragulator” in both yeast and mammalian cells, it is well documented complex (Kogan et al. 2010; Sancak et al. 2010). Ragulator that TORC1 activity stimulates translation initiation 1184 R. Loewith and M. N. Hall (Wullschleger et al. 2006). Interestingly, inhibition of trans- kinase activity, i.e., activity even in the absence of TORC1 lation with cycloheximide causes a pronounced increase in (Urban et al. 2007). Presumably, phosphorylation of the turn (m)TORC1 activity presumably by triggering an increase in motif helps to stabilize Sch9 while phosphorylation of the the concentration of free amino acids in the cytoplasm hydrophobic motif stabilizes Sch9 in an active conformation. (Beugnet et al. 2003; Urban et al. 2007; Binda et al. Curiously, although their in vivo functions are unknown, 2009). Ribosome biogenesis (described in more detail be- in vitro TORC1 preferentially phosphorylates Ser758 and low) is a second example. TORC1 regulates ribosome bio- Ser765 within the hydrophobic-like motifs (R. Loewith, un- genesis in part via two substrates, Sch9 and the transcription published results). That TORC1 can phosphorylate amino factor Sfp1. Reduced ribosome biogenesis resulting from de- acids found within such diverse sequence contexts, which letion of SCH9 or SFP1 results in a dramatic increase in is rather atypical for protein kinases, is also curious. TORC1 activity (Lempiainen et al. 2009). It is possible that Characterization of Tap42‐PP2A as a TORC1 effector: In blocking ribosome biogenesis, like translation inhibition, addition to Sch9, TORC1 also regulates type 2A (Pph21, causes an increase in free amino acids that subsequently Pph22, and Pph3—generically PP2Ac) and 2A-related phos- activates TORC1. Alternatively, other mechanisms could be phatases (Sit4, Ppg1). These partially redundant yet pleio- at play. Regardless of mechanism, such feedback loops pro- tropic enzymes are notoriously difficult to study. Analysis of vide an elegant means by which growth homeostasis can be Sit4 function, and therefore of TORC1 function, is further maintained by TORC1. complicated by strain-dependent polymorphisms at the SSD1 (Suppressor of SIT4 Deletion) locus (Reinke et al. Downstream of TORC1 2004). In general terms, when growth conditions permit, TORC1 A role for these phosphatases downstream of TORC1 was is active and its signals promote the accumulation of cel- first described by the Arndt lab (Di Como and Arndt 1996). lular mass. However, as both proximal and distal TORC1 In this work, a subpopulation of these enzymes was found to effectors continue to be described, the extent of this tem- interact in a TORC1-dependent manner with a regulatory poral regulation of growth control is only starting to be protein known as Tap42. Rrd1 and Rrd2, two peptidyl- appreciated. prolyl cis/trans isomerases, were subsequently also found to be present in these Tap42 complexes (Zheng and Jiang Proximal TORC1 effectors: Characterization of Sch9 as 2005; Jordens et al. 2006). Work, done in large part by the a TORC1 substrate: Arguably, the best-characterized sub- Jiang group, posits that when TORC1 is active, Tap42 is strates of both yeast and metazoan TOR complexes are the phosphorylated and bound tightly to the phosphatase–Rrd AGC family kinases. This rather well-studied family of complex (Di Como and Arndt 1996; Jiang and Broach 1999; kinases is so named on the basis of its mammalian members Zheng and Jiang 2005). Inactivation of TORC1 results in PKA, PKG, and PKC (Pearce et al. 2010). Typically, activation Tap42 dephosphorylation and a weakened association with of AGC family kinases requires phosphorylation of two con- phosphatases that presumably results in their activation served regulatory motifs, the “T,” or “activation,” loop lo- and/or change in substrate preference (Duvel et al. 2003; cated in the catalytic domain and the “hydrophobic” motif Yan et al. 2006). How TORC1 maintains Tap42 phosphory- found toward the C terminus. Phosphorylation of these lation is mechanistically unclear. It may phosphorylate motifs helps stabilize the kinase domain in an active confor- Tap42 directly (Jiang and Broach 1999), or it may act via mation. Several AGC family kinases additionally contain the Tap42 interacting phosphoprotein Tip41 (Jacinto et al. a “turn” motif located between the kinase domain and the 2001). Interestingly, Tip41 has been proposed to both an- hydrophobic motif, phosphorylation of which is thought to tagonize and cooperate with Tap42 in controlling TORC1 promote protein stability. While the T loop is phosphorylated signaling (Jacinto et al. 2001; Kuepfer et al. 2007). by PDK1 or its ortholog Pkh in mammalian or yeast cells, Although the mechanisms coupling TORC1 to Tap42– respectively, phosphorylation of the hydrophobic and possi- PPase complexes remain to be elucidated, genetic argu- bly the turn motifs is often mediated by TORC1 or TORC2. ments clearly position Tap42 as a prominent effector of Analogous to S6K for mTORC1, the AGC kinase Sch9 was TORC1. Specifically, several alleles of TAP42 (e.g., TAP42- recently found to be a direct substrate for yeast TORC1 11) that confer strong resistance to rapamycin by blocking (Powers 2007). Six target sites in the C terminus of Sch9 a subset of rapamycin-induced readouts have been identi- are phosphorylated by TORC1: Thr737 found in a classical fied (Di Como and Arndt 1996; Duvel et al. 2003). hydrophobic motif; Thr723 and Ser726, Ser/Thr-Pro sites Curiously, TAP42-11 does not provide rapamycin resis- found in what appears to be a turn motif; Ser758 and tance in all strain backgrounds. However, co-expression of Ser765 found in sequences that resemble the hydrophobic genetically activated Sch9 (described above) in rapamycin- motif; and Ser711 in a region that partially resembles a hy- sensitive TAP42-11 backgrounds results in a very strong syn- drophobic motif. TORC1-mediated phosphorylation is nec- thetic resistance to rapamycin (Urban et al. 2007). From this essary for Sch9 activity. Replacing the target amino acids observation, it appears that Sch9 and Tap42-PPase com- with alanine yields a nonfunctional Sch9, whereas replacing plexes are major effector branches downstream of TORC1 them with a phosphomimetic residue confers constitutive with each branch, at least in some backgrounds, performing TOR Function 1185 one or more essential function. The readouts mediated by lation of Ser577 and, consequently, an increase in Gcn2 these two TORC1 branches are discussed below. activity and a reduction in 59CAP-dependent translation Other TORC1 substrates: In addition to the regulation of (Cherkasova and Hinnebusch 2003). It is possible that one these two major effector branches, TORC1 has been or more Tap42-associated phosphatases directly dephos- reported to directly phosphorylate other substrates includ- phorylates Ser577, but this has not been formally demon- ing Sfp1(Lempiainen et al. 2009), Gln3 (Bertram et al. strated. The nature of the kinase that phosphorylates Gcn2 2000), and Atg13 (Kamada et al. 2010). The roles that these Ser577 is unknown other than it is not Sch9 (M. Stahl and proteins play downstream of TORC1 are discussed below. R. Loewith, unpublished results). Sch9 inhibition, however, Tyers and colleagues have recently defined a global also leads to eIF2a phosphorylation via an undefined path- protein kinase and phosphatase interaction network in yeast way (Urban et al. 2007). (Breitkreutz et al. 2010). This study, consisting of affinity Studies with rapamycin suggest that, in addition to purification followed by mass spectrometry, included TOR1 eIF2a, TORC1 may target additional translation factors and TOR2. They found and confirmed that TORC1 physi- such as the 59CAP-binding protein (eIF4E) interacting pro- cally interacts with the following proteins: Mks1, a protein teins Eap1 and/or the eIF4G scaffold (Berset et al. 1998; involved in retrograde (RTG) mitochondria-to-nucleus sig- Cosentino et al. 2000). Finally, recent phosphoproteomics naling (see below); curiously, FMP48, an uncharacterized studies (Huber et al. 2009; Loewith 2010; Soulard et al. protein presumed to localize to the mitochondria (Reinders 2010) have identified several translation-related proteins et al. 2006); Npr1, a protein kinase involved in the intracel- whose phosphorylation is altered by rapamycin treatment, lular sorting of nutrient permeases (see below); Ksp1, suggesting that these factors could also couple TORC1 to a protein kinase involved in nutrient-regulated haploid fila- protein synthesis. mentous growth (Bharucha et al. 2008); Nap1, a chromatin Ribosome biogenesis: In optimal conditions, yeast cells assembly factor and a mitotic factor involved in regulation of grow and divide approximately every 100 min. Such rapid bud formation (Calvert et al. 2008); Nnk1, the nitrogen growth requires robust protein synthesis, which of course network kinase presumably involved in intermediate nitro- requires ribosomes. Indeed, rapidly growing yeast cells gen metabolism (Breitkreutz et al. 2010); Sky1, an Ser/Arg contain 200,000 ribosomes, implying that each cell must domain kinase involved in pre-mRNA splicing (Shen and produce and assemble 2000 ribosomes per minute Green 2006); and Bck1 and Kdx1, which are involved in (Warner 1999). This is not a trivial feat as each ribosome MAPK signaling (Breitkreutz et al. 2010). Given their phys- contains 78 unique proteins (encoded by 137 RP genes) in ical interaction with TORC1, all of these proteins, in addi- addition to four rRNA molecules, three derived from the tion to multiple other, as-yet-unconfirmed interactors, are RNA Pol I-transcribed 35S pre-rRNA and one transcribed potential substrates (or regulators) of TORC1. These results by RNA Pol III. Fifty percent of RNA Pol II transcription is underscore the central role that TORC1 plays in cell growth. devoted to ribosomal proteins. In addition, numerous pro- tein and small RNA trans-acting factors, known as ribosome Distal readouts downstream of TORC1: TORC1 promotes biogenesis (RiBi) factors, are required for the correct pro- cell growth: When environmental conditions are favorable, cessing, folding, assembly, nuclear export of pre-ribosomal TORC1 coordinates the production and accumulation particles to the cytoplasm, and final maturation events into of cellular mass, i.e., growth, via regulation of several 40S and 60S particles. The production of all these abundant processes. molecules represents a huge energetic investment. Not sur- Protein synthesis: The first realization that TORC1 serves prisingly, yeast cells have developed elaborate measures to to couple environmental cues to the cell growth machinery coordinate the expression of rRNA, tRNA, RPs, and RiBi came with the observation that rapamycin treatment elicits factors in response to environmental conditions. Much of a marked drop in protein synthesis by blocking translation this regulation is mediated by TORC1 at the level of tran- initiation (Barbet et al. 1996). A major target for this regu- scription. As ribosome biogenesis has clear links to diseases lation appears to be the translation initiation factor eIF2. such as cancer, anemia, and aging, dissection of its regula- Upon amino acid starvation or rapamycin treatment, the tion will undoubtedly have clinical ramifications (Lempiainen a-subunit of eIF2 is phosphorylated and this dominantly and Shore 2009). interferes with 59CAP-dependent mRNA translation In S. cerevisiae, the rDNA locus consists of 150 tandemly (reviewed in Hinnebusch 2005). TORC1 signals to eIF2a repeated transcription units on chromosome XII, and yet via both the Sch9 and Tap42-PPase branches. The sole rRNA production is still limiting for cell growth (Warner eIF2a kinase is the conserved Gcn2 protein. Gcn2 binds 1999). Each of these rDNA units comprises the RNA poly- and is activated by uncharged tRNAs that accumulate when merase III transcribed 5S rRNA gene, the intergenic spacer cells are starved for an amino acid (detailed below). region, and the RNA Pol I-transcribed 35S rRNA gene, Gcn2 activity is also regulated by phosphorylation. encoding the 35S precursor of the mature 18S, 5.8S, and Gcn2 phosphorylation on Ser577 reduces tRNA binding 25S rRNAs. RNA Pol III also transcribes tRNA genes as well and, consequently, kinase activity. Treating cells with rapa- as several additional genes encoding small noncoding RNAs. mycin elicits a rapid, Tap42-PPase-dependent dephosphory- In the late 1990s, it was reported that rapamycin results in 1186 R. Loewith and M. N. Hall Figure 3 Control of RiBi and RP gene transcription by TORC1. RiBi factors are required for the proper expression, processing, assembly, export, and maturation of rRNA and RPs into ribosomes. This energetically costly procedure is under tight regulation, particularly at the transcription level. TORC1 regulates RiBi and RP gene transcription via multiple pathways: (1) TORC1 directly phosphorylates the Split Zn-finger transcription factor Sfp1, which presumably regulates its nuclear localization and/or binding to RP and possibly RiBi gene promoters to stimulate their expression. (2) Fhl1 and Rap1 bind constitutively to RP promoters. When TORC1 is active, phosphorylated Ifh1 binds to Fhl1 to stimulate transcription, possibly by recruiting the NuA4 histone acetyltransferase. When TORC1 is inactive, Yak1 phosphorylates Crf1, which subsequently outcom- petes Ifh1 for binding to Fhl1. (3) Sch9 phosphorylates and thus inhibits Stb3 and the paralogs Dot6 and Tod6. Inhibition of TORC1/Sch9 results in the dephosphorylation of these three transcription repressors, which subsequently bind to RRPE and PAC elements found in RiBi promoters. Stb3 additionally binds RP promoters. Bound to pro- moters, these repressors recruit the RPD3L histone deace- tylase complex to repress transcription. a rapid and pronounced drop in 5S, 35S, and tRNA produc- represses RNA Pol III in a Maf1-dependent fashion (Huber tion (Zaragoza et al. 1998; Powers and Walter 1999). Re- et al. 2009; Michels 2011). Maf1 is conserved and also func- cently, the relevant signaling pathways in this regulation tions downstream of mTORC1 to regulate RNA Pol III activ- have become clearer. ity. However, in mammalian cells, and perhaps in yeast cells TORC1 regulates the accumulation of RNA Pol I tran- too, Maf1 is directly phosphorylated by mTORC1 rather scripts at multiple levels. Processing of the 35S pre-rRNA than by the Sch9 ortholog S6K1 (Wei et al. 2009; Wei and occurs cotranscriptionally and is dependent on the presence Zheng 2010; Michels 2011). of ribosomal proteins (Tschochner and Hurt 2003). The fast A total of 137 genes encode the 78 proteins that make up drop in RNA Pol I-dependent transcripts observed upon a yeast ribosome (most RPs are encoded by two genes rapamycin treatment is apparently due to decreased trans- yielding nearly identical proteins). TORC1 coordinately lation (described above) of ribosomal proteins (Reiter et al. regulates the expression of these genes through several 2011). The majority of mRNAs being translated in a rapidly mechanisms (Figure 3) (Lempiainen and Shore 2009). A growing cell encode ribosomal proteins (Warner 1999), and central component of this regulation is the Fhl1 protein thus a drop in translation will rapidly reduce the levels of (Lee et al. 2002; Martin et al. 2004; Schawalder et al. free ribosomal proteins that are themselves needed stoichio- 2004; Wade et al. 2004; Rudra et al. 2005). Fhl1 has a fork- metrically for processing of rRNA into pre-ribosome par- head DNA-binding domain, and its constitutive association ticles. rRNA that is not efficiently processed is immediately to ribosomal protein gene (RP) promoters is facilitated by degraded, presumably to prevent imbalances in structural the DNA-binding protein Rap1 and the high mobility group components of the ribosome. At later time points following protein Hmo1 (Hall et al. 2006; Berger et al. 2007). TORC1 rapamycin treatment, RNA Pol I no longer associates with regulates RP transcription by determining the association the rDNA and transcription stops. This late effect could be between Fhl1 and either one of two FHB-containing pro- the result of rapamycin-induced degradation of the essential teins, Ifh1 and Crf1. Both Ifh1 and Crf1 are phosphopro- RNA Pol I transcription factor Rrn3 (Claypool et al. 2004; teins. When cells are growing and TORC1 is active, Ifh1 is Laferte et al. 2006; Reiter et al. 2011). phosphorylated and binds to Fhl1 to stimulate RP transcrip- TORC1 regulates RNA Pol III apparently exclusively via tion. Conversely, inhibition of TORC1 results in the phos- Sch9 and a repressor protein named Maf1 (Upadhya et al. phorylation of Crf1, which displaces Ifh1 to repress RP 2002; Oficjalska-Pham et al. 2006; Reina et al. 2006; Huber transcription. The signaling events upstream of Ifh1 are et al. 2009; Lee et al. 2009). Sch9 directly phosphorylates not known, whereas TORC1 seems to signal to Crf1 via seven sites in Maf1 that prevent it from interacting with and the Ras/PKA pathway target Yak1 (Martin et al. 2004). thus inhibiting RNA Pol III (Vannini et al. 2010). Phospho- However, it should be noted that the crosstalk between mimetic variants of Maf1 clearly fail to associate with RNA TORC1 signals and Ras/PKA signals has been debated. Pol III, but, curiously, Sch9 inhibition still causes a reduction While it is clear that hyperactivation of Ras/PKA can sup- in RNA Pol III activity in these strains but not in maf1D press many rapamycin-induced phenotypes (Schmelzle et al. strains. This and other observations suggest that an addi- 2004), suggesting that PKA is downstream of TORC1, it has tional Sch9 target exists that, when dephosphorylated, also been proposed that TORC1 and PKA signal in parallel TOR Function 1187 pathways that impinge on common targets (Zurita-Martinez active role in mRNA stability and, via its potential substrate and Cardenas 2005; Ramachandran and Herman 2011). Re- Sky1, in pre-mRNA splicing. This observation is significant cently, Soulard et al. (2010) have provided some clarifica- when one considers that 90% of all mRNA splicing events tion of this dilemma by proposing that TORC1 functions occur on RP transcripts (Warner 1999). Thus, TORC1 is well upstream of PKA but only for a subset of PKA targets. Thus, positioned to coordinate multiple aspects of ribosome biogen- TORC1 may be both upstream and parallel to PKA. esis in response to growth stimuli. As introduced above, TORC1-dependent regulation of RP gene transcription TORC1 activity is dramatically increased in sfp1 and sch9 cells still occurs in the absence of the Fhl1/Ifh1/Crf1 system, (Lempiainen et al. 2009), suggesting that some aspect of ribo- suggesting the existence of additional regulatory mecha- some biogenesis must also signal in a feedback loop to TORC1. nisms. One of these is the split zinc (Zn)-finger protein It will be interesting to see what steps of ribosome biogenesis Sfp1 (Fingerman et al. 2003; Jorgensen et al. 2004; Marion contribute to TORC1 regulation. et al. 2004; Lempiainen et al. 2009; Singh and Tyers 2009). Regulation of cell cycle/cell size: Although distinct pro- TORC1 binds and directly phosphorylates Sfp1 to promote cesses, cell growth and cell division are often intimately its binding to a subset of RP gene promoters. Curiously, un- linked. Yeast cells, for example, commit to a new round of like Sch9, TORC1-mediated Sfp1 phosphorylation appears cell division only after attaining a critical size. This cell-size to be insensitive to osmotic or nutritional stress, suggesting threshold is dictated in large part by environmental growth that TORC1 regulates these two substrates via very different conditions (Cook and Tyers 2007). How cells couple envi- mechanisms (Lempiainen et al. 2009). Sfp1 also interacts ronmental cues to the cell cycle machinery is fascinating but with the conserved Rab escort protein Mrs6, an essential poorly understood. Interestingly, sfp1 and sch9 were the top protein functioning in membrane sorting (Lempiainen two hits in a systematic search for mutations conferring et al. 2009; Singh and Tyers 2009). Sfp1-Mrs6 association small cell size (Jorgensen et al. 2002, 2004). This and fol- is important for the nuclear localization of Sfp1, but its low-up observations demonstrated that ribosome biogenesis functional implications are otherwise unclear. Intriguingly, plays a major role in cell-size determination. These results this association may underlie the presently unexplained further predict that environmental cues regulate the cell-size genetic and biochemical interactions between TORC1 and threshold via TORC1, i.e., that poor growth conditions re- vesicular transport machineries (Aronova et al. 2007; Zurita- duce the activity of TORC1 and subsequently the activities of Martinez et al. 2007). Although physical interaction with Sfp1 and Sch9. Consequently, this would decrease ribosome RiBi promoters has not been reported, overexpression of biogenesis, which, in mysterious ways, would lower the cell- Sfp1 causes a rapid upregulation of most RiBi genes, sug- size threshold required for cell division. In contrast, acute gesting that Sfp1 also regulates this important regulon inhibition of TORC1 with high concentrations of rapamycin (Jorgensen et al. 2004). Better understood is the regulation leads to an arrest in G1 due to reduced translation of the of RiBi gene expression downstream of Sch9. RiBi promoters cyclin Cln3 (Barbet et al. 1996) and a paradoxical increase typically possess polymerase A and C (PAC) and/or ribo- in cell size. This increase in cell size is actually due to swell- somal RNA processing element (RRPE) elements. PAC ele- ing of the vacuole as a consequence of increased autophagy ments are bound by the myb-family transcription factors (see below; sfp1 or sch9 deletions presumably do not induce Dot6 and Tod6 (Freckleton et al. 2009; Zhu et al. 2009) autophagy). whereas RRPE elements are bound by Stb3 (Liko et al. Although best appreciated for its role in G1 regulation, 2007). Stb3 seems to bind to T-rich elements in RP pro- TORC1 additionally regulates the transition through other moters as well (Huber et al. 2011). All three transcription phases of the cell cycle. TORC1 promotes S phase by factors are phosphorylated by Sch9 and thus are under maintaining deoxynucleoside triphosphate pools. Deoxy- TORC1 control (Lippman and Broach 2009; Liko et al. nucleoside triphosphates are the obligate building blocks 2010; Huber et al. 2011). When TORC1 is inactivated, for DNA synthesis, and a role for TORC1 in their synthesis Dot6, Tod6, and Stb3 are dephosphorylated, allowing becomes apparent under conditions of DNA replication them to bind to their cognate promoter elements and recruit stress or DNA damage when elevated deoxynucleoside the RPD3L histone acetyltransferase complex to repress triphosphate pools are necessary for error-prone trans- transcription. lesion DNA polymerases (Shen et al. 2007). Via the Tap42- In summary, TORC1 plays a central role in regulating PPase branch, TORC1 also influences the G2/M transition ribosome biogenesis, particularly at the transcriptional level. (Nakashima et al. 2008). Specifically, TORC1 regulates the However, it is now clear that TORC1 also influences ribosome subcellular localization of the polo-like kinase Cdc5. Cdc5 biogenesis post-transcriptionally. Phosphoproteomics as well as activity destabilizes Swe1, a kinase that phosphorylates and more directed studies suggest that TORC1 regulates various thus inactivates the mitotic cyclin-dependent kinase Cdc28. catalytic steps of ribosome assembly per se (Honma et al. 2006; Inhibition of TORC1 mislocalizes Cdc5, causing an inappro- Huber et al. 2009; Loewith 2010). Phosphoproteomics and priate stabilization of Swe1 and, consequently, inactivation biochemical studies (Albig and Decker 2001; Grigull et al. of Cdc28 and prolonged G2/M. Although TORC1 signals 2004; Huber et al. 2009; Breitkreutz et al. 2010; Loewith likely impinge upon additional nodes in the cell division 2010; Soulard et al. 2010) also suggest that TORC1 plays an cycle (Huber et al. 2009; Soulard et al. 2010), the above 1188 R. Loewith and M. N. Hall observations already exemplify the intricate connections be- ers, enabling them to selectively import only the desired tween cell growth signals and the cell division cycle. Recip- nutrients (Van Belle and Andre 2001). In general terms, rocal, but less well described, cues and/or outputs from the in good growth conditions, many high-affinity, substrate- cell division cycle regulate cell growth, likely in part via selective permeases are expressed and sorted to the plasma TORC1 (Goranov and Amon 2010). membrane to actively pump in nutrients that are used TORC1 inhibits stress responses: In addition to stimulating directly in ATP production and/or anabolism of nitrogenous anabolic processes, TORC1 also promotes growth by sup- compounds. Shift to poor growth conditions results in the pressing a variety of stress-response programs. Although replacement of high-affinity permeases, which are targeted essential for surviving environmental insults, activation of to the vacuole for degradation with few low-affinity, broad- stress-responsive pathways is incompatible with rapid specificity permeases that facilitate uptake of a wide range growth, and constitutive activation of these pathways of carbon and nitrogenous compounds that can be catabo- generally results in cell death. As described below, the lized by the cell. For example, in response to nitrogen star- best-characterized stress-response programs under the in- vation, the high-affinity tryptophan-specific permease, Tat2, fluence of TORC1 are transcriptional in nature. However, it localized to the plasma membrane, is ubiquitinated, endo- is clear that TORC1 also regulates post-transcriptional cytosed, and ultimately degraded. In contrast, the general aspects of stress responses such as mRNA stability, protein amino acid permease Gap1 is re-routed to the plasma mem- trafficking, and the activities of metabolic enzymes. brane instead of to the vacuole/endosomes. Although Environmental stress response: Exposure of yeast cells to details are still emerging, TORC1 appears to regulate such noxious stressors, including nutrient limitation and entry permease-sorting events primarily via Tap42-PPase and into stationary phase, elicits a stereotypic transcriptional its (potentially direct) effector Npr1 (Schmidt et al. 1998; response known as the environmental stress response Beck et al. 1999; De Craene et al. 2001; Jacinto et al. 2001; (ESR) (Gasch and Werner-Washburne 2002). This includes Soetens et al. 2001; Breitkreutz et al. 2010). Npr1 is a heavily 300 upregulated genes that encode activities such as pro- phosphorylated, seemingly fungal-specific, Ser/Thr kinase tein chaperones and oxygen radical scavengers that help that upon TORC1 inactivation is rapidly dephosphorylated cells endure stressful environments. The central compo- and activated (Gander et al. 2008). Although genetic studies nents of this pathway are the Zn-finger transcription fac- clearly imply a role for Npr1 in protein-sorting events, the tors Msn2/4 and Gis1, the LATS family kinase Rim15, and mechanisms of this regulation have remained elusive. It is the a-endosulfine family paralogs Igo1 and Igo2 (De Virgilio possible that the permeases themselves are Npr1 substrates. 2011). TORC1 via Sch9, and possibly also Tap42-PPase, Indeed, several nutrient and cation permeases have been promotes cytoplasmic anchoring of Rim15 to 14-3-3 pro- identified as rapamycin-sensitive phosphoproteins (Huber teins by maintaining Rim15 phosphorylated on Ser1061 et al. 2009; Soulard et al. 2010). Also identified in these and Thr1075 (Wanke et al. 2005, 2008). Inhibition of phosphoproteomics studies were several a-arrestin-related TORC1 results in nuclear localization of Rim15, which sub- proteins. These phosphoproteins function as clathrin adap- sequently triggers the activation, in a poorly understood tor molecules and have been implicated in mediating the fashion, of the expression of Msn2/4- and Gis1-dependent sorting fates of a number of different permeases; and, one, ESR genes. However, TORC1 inhibition results in a marked Aly2, has recently been reported to be an Npr1 substrate turnover of mRNAs (Albig and Decker 2001), and, as noted (Lin et al. 2008; Nikko et al. 2008; Nikko and Pelham above, in a dramatic drop in translation. Thus it would 2009; O’Donnell et al. 2010). Whether this observation is appear that increasing transcription of protein-coding genes indicative of a more general trend in Npr1-meditated per- in TORC1-inhibited cells would be futile as these mRNA mease trafficking remains to be seen. would likely be degraded before ever being translated. This TORC1 regulates permease activity by regulating not only appears not to be the case as Rim15 phosphorylates Igo1 permease localization but also expression. This was shown in and its paralog Igo2, allowing them to associate with newly early transcriptomics experiments, which clearly demon- transcribed Msn2/4- and Gis1-regulated mRNAs to protect strated that TORC1 regulates the expression of a large these transcripts from degradation via the 59-39 mRNA number of permeases and other factors required for the decay pathway (Talarek et al. 2010; Luo et al. 2011). assimilation of alternative nitrogenous sources (Cardenas Nutrient uptake and intermediary metabolism: To best et al. 1999; Hardwick et al. 1999; Komeili et al. 2000; Shamji compete with other microbes in their environment, yeast et al. 2000). TORC1 regulates the expression of nitrogen have optimized the use of available nutrients to accommo- catabolite repression (NCR)-sensitive genes via the Tap42- date fast growth (De Virgilio and Loewith 2006). Although PPase branch. The proteins encoded by these genes (e.g., a wide variety of compounds can be utilized as carbon or Gap1) enable cells to import and metabolize poor nitrogen nitrogen sources, yeast cells will exclusively assimilate pre- sources such as proline and allantoin. In the presence of ferred nutrient sources before using nonpreferred, subopti- preferred nitrogen sources such as glutamine, glutamate, or am- mal ones. To attain this dietary specificity, and to respond to monia, active TORC1 promotes the association of the GATA- nutritional stress, yeast cells carefully regulate the expres- family transcription factor Gln3 with its cytoplasmic anchor sion and sorting of their many (.270) membrane transport- Ure2. Mechanistically, this involves both TORC1-dependent TOR Function 1189 and TORC1-independent regulation of Gln3, and possibly of well as genetic studies have implicated TORC1 as a negative Ure2, phosphorylation (Beck and Hall 1999; Cardenas et al. regulator of Rtg1/3-dependent transcription (Komeili et al. 1999; Hardwick et al. 1999; Carvalho and Zheng 2003; 2000; Shamji et al. 2000; Chen and Kaiser 2003). However, Georis et al. 2009a; Tate et al. 2009, 2010). Two other it is presently unclear how TORC1 influences this pathway; less-characterized GATA factors, Gat1 and Dal81, also have TORC1 inhibition could indirectly influence retrograde re- roles in the regulation of NCR-sensitive genes (Georis et al. sponse signaling via alterations in metabolite levels. Alter- 2009b). natively, the direct association between TORC1 and Mks1 In addition to the NCR pathway, TORC1 also regulates observed by the Tyers group and described above and the the expression of amino acid permeases by modulating the fact that Mks1 is a rapamycin-sensitive phosphoprotein in- activity of the SPS-sensing pathway. This pathway consists of stead suggest that TORC1 could play a much more direct a plasma-membrane-localized sensor of external amino role in regulating this pathway (Liu et al. 2003; Breitkreutz acids, Ssy1, and two downstream factors, Ptr3 and Ssy5 et al. 2010). Finally, phosphoproteomics studies suggest that (Ljungdahl 2009). Upon activation of the pathway, Ssy5 TORC1 regulates intermediate metabolism by directly alter- catalyzes an endoproteolytic processing event that cleaves ing the activities of metabolic enzymes, particularly those and releases an N-terminal regulatory domain from two involved in the early steps of glycolysis (Loewith 2011). transcription factors, Stp1 and Stp2, the shortened forms Autophagy: As described above, starved cells express of which translocate to the nucleus and activate the tran- a suite of stress-responsive proteins to help them negotiate scription of a number of amino acid permease-encoding hostile environmental conditions. This new synthesis genes. TORC1 via Tap42-PPase modulates this pathway by requires both energy and amino acids that yeast cells obtain promoting the stability of Stp1 and thus the ability of cells to by inducing autophagy. Autophagy refers to a variety of utilize external amino acids (Shin et al. 2009). mechanisms by which cytosplasmic material, including In contrast to the SPS-sensing pathway that is activated proteins and lipids, is translocated to the vacuole and by amino acids, the Gcn4 transcription factor is activated catabolized. Amino acids and fatty acids thus acquired are, upon amino acid starvation (Hinnebusch 2005). As men- respectively, used to synthesize new proteins and oxidized tioned above, rapamycin treatment or amino acid starvation by mitochondria to produce ATP. Mechanistically, there are results in a rapid decline in translation initiation by trigger- two different modes of autophagy in yeast. One is micro- ing phosphorylation of the a-subunit of eIF2. Although autophagy, which involves the direct transfer of cytoplasm eIF2a phosphorylation results in the repression of bulk into the vacuole via invaginations of the vacuolar mem- translation, due to the presence of four short upstream open brane. The other is macroautophagy, which involves the de reading frames in its leader sequence, the mRNA encoding novo formation of double-membrane vesicles called auto- Gcn4 is, in contrast, preferentially translated. Subsequent phagosomes. Autophagosomes encapsulate cytoplasm and accumulation of Gcn4 protein leads to the transcriptional then fuse with the vacuole. Both forms of autophagy are induction of nearly all genes encoding amino acid biosyn- regulated by TORC1 (De Virgilio and Loewith 2006) thetic enzymes. although, mechanistically, macroautophagy is better under- TORC1 also regulates amino acid biosynthesis, in partic- stood (reviewed in Cebollero and Reggiori 2009; Nakatogawa ular glutamine and glutamate homeostasis, via the retro- et al. 2009; Inoue and Klionsky 2010). grade response pathway (Komeili et al. 2000; Crespo and Autophagy is conserved across eukarya, and there is Hall 2002; Crespo et al. 2002; Liu and Butow 2006). This much interest in understanding how macroautophagy is signaling pathway serves to communicate mitochondrial regulated as it has been linked to several pathologies dysfunction to the nucleus to induce an appropriate tran- including cancer, neurological disorders, and longevity scriptional response. In addition to hosting the aerobic en- (Yang and Klionsky 2010). In yeast, many autophagy- ergy production machinery, mitochondria are also the sites related (ATG) genes encode proteins that participate in the of amino acid precursor, nucleotide, and lipid production. induction of autophagy, the nucleation of the autophagosome, Signals, possibly changes in glutamate or glutamine levels, elongation and completion of the autophagosome, and, emanating from dysfunctional mitochondria impinge upon finally, in fusion of the autophagosome with the vacuole to a cytosolic regulatory protein, Rtg2. Thus activated, Rtg2 release the autolysosome into the vacuolar lumen (Chen and antagonizes the ability of Mks1 to sequester the heterodi- Klionsky 2011; Reiter et al. 2011). TORC1 regulates macro- meric bZip/HLH transcription factor complex composed of autophagy by signaling to the Atg1 kinase complex that is Rtg1 and Rtg3 in the cytoplasm. Allowed to enter the nu- required for the induction of macroautophagy. Specifically, cleus, Rtg1/3 activates genes encoding enzymes required for when TORC1 is active, Atg13 is hyperphosphorylated, pre- anaplerotic reactions that resupply tri-carboxylic acid cycle sumably directly by TORC1 (although Tap42-PPase has also intermediates that have been extracted for biosynthetic been implicated in this regulation), and this prevents the reactions. Key among these intermediates is a-ketoglutarate, association of Atg13 with Atg1, Atg17, Atg31,and Atg29 the precursor of glutamate and glutamine from which (Yorimitsu et al. 2009; Kamada et al. 2010). Inhibition of all nitrogen-containing metabolites evolve (Magasanik and TORC1 results in dephosphorylation of Atg13, assembly Kaiser 2002). Both transcriptome-profiling experiments as of the Atg1 protein kinase complex, phosphorylation and 1190 R. Loewith and M. N. Hall activation of Atg1 (Kijanska et al. 2010; Yeh et al. 2010), and, 2009; Bjedov et al. 2010). These observations have created subsequently, macroautophagy mediated by as-yet-unidenti- much excitement in that aging is now thought of as a dis- fied Atg1 substrates. Although metazoan homologs exist for ease, which, like other diseases, can be ameliorated through many of the Atg1 kinase complex components, a unifying pharmaceutical intervention. These observations have also model of how TORC1 regulates this complex in different spe- raised the important question, what are the downstream cies has yet to emerge (Chen and Klionsky 2011; Reiter et al. function(s) of TORC1 that modulate life span? The answer 2011). to this question is presently unclear, and it is very likely that Cell-wall integrity pathway: The cell wall is essential for multiple TORC1 effector pathways contribute (Blagosklonny yeast cells to survive hostile environments and, more and Hall 2009). Studies in many model systems are pres- importantly, to prevent internal turgor pressure from rup- ently underway to address this issue. Below are some of the turing the plasma membrane. Although a thickening of the highlights from studies in yeast. cell wall helps protect stressed or stationary-phase cells, this Yeast life span is assayed in one of two ways. Replicative rigid structure must also be remodelled to accommodate cell life span (RLS) is a measure of the number of progeny that growth. Homeostasis of this structure is maintained by the a single mother cell can produce before senescence. Chro- cell-wall integrity (CWI) pathway (Levin 2005). Cell-wall nological life span (CLS) is a measure of the length of time integrity is monitored by WSC (cell-wall integrity and stress a population of yeast cells can remain in stationary phase response component) family proteins. WSCs, which are in- before they lose the ability to restart growth following re- tegral plasma membrane proteins, function upstream of the inoculation into fresh media. RLS is thought to be a para- Rho1 GTPase by modulating the activity of the GEFs Rom1 digm for aging of mitotic cells while CLS is thought to be GTP and Rom2. Rho1 has several effectors including the yeast a paradigm for aging of quiescent cells. Consistent with protein kinase C homolog, Pkc1. The best-characterized bigger eukaryotes, where newborns are obviously born Pkc1 effector is a mitogen-activated protein kinase (MAPK) young, gametogenesis (i.e., cells derived from meiotic cell cascade composed of Bck1 (a MAPKKK), Mkk1 and -2 divisions) resets RLS in yeast (Unal et al. 2011). (redundant MAPKKs), and Slt2/Mpk1 (a MAPK). Activation Kaeberlein et al. (2005) have recently attempted labor- of this pathway leads to the expression of many cell-wall intensive approaches to identify genes involved in both rep- biosynthetic enzymes, which helps to remodel the cell wall licative and chronological life span. A random screen of 564 both during normal growth and in response to stress. yeast strains, each lacking a single nonessential gene, impli- Both TORC1 and TORC2 (discussed below) appear to cated both TOR1 and SCH9 in RLS downstream of caloric impinge upon the CWI pathway. Entry into stationary phase, restriction. Also identified in this screen were a number of carbon starvation, nitrogen starvation, and rapamycin treat- genes encoding ribosomal proteins. Further analyses of RP ment all elicit activation of the CWI pathway, demonstrating genes subsequently demonstrated that specific depletion of that TORC1 negatively regulates the CWI pathway (Ai et al. 60S ribosomal protein subunits extends RLS (Steffen et al. 2002; Krause and Gray 2002; Torres et al. 2002; Reinke 2008). Curiously, RLS extension observed upon TORC1 inhi- et al. 2004; Araki et al. 2005; Soulard et al. 2010). Further- bition and 60S subunit depletion seems to be mediated by more, pkc1, bck1, and mpk1 mutants rapidly lose viability Gcn4, the TORC1-dependent transcription factor that regu- upon carbon or nitrogen starvation, demonstrating that the lates the expression of amino acid biosynthetic genes as de- CWI pathway is required for viability in G0. Mechanistically, scribed above. The relevant Gcn4 target genes/processes how TORC1 signals impinge on the CWI pathway is not involved in RLS are not yet known, but an interesting candi- clear. Soulard et al. (2010) have implicated the Sch9 effector date could be macroautophagy. Induction of macroautophagy, branch while Torres et al. (2002) have postulated that sig- like TORC1 and Sch9 inhibition, increases both RLS and CLS nals through the Tap42-PPase branch causes membrane (Madeo et al. 2010a,b; Morselli et al. 2011; and see below), stress that, via WSC family members, activates downstream and Gcn4 is required for amino acid-starvation-induced mac- components of the CWI pathway. roautophagy (Ecker et al. 2010). Furthermore, spermidine, TORC1 accelerates aging: Arguably one of the most a potent inducer of macroautophagy, potentially via Gcn4 interesting functions of TORC1 is its involvement in the (Teixeira et al. 2010), appears to promote longevity not only regulation of life span. It is well established that, in virtually in yeast but also in several other model organisms (Eisenberg every biological system, aging, i.e., the progressive deterio- et al. 2009). Since TORC1, Sch9,and Gcn4 homologs are ration of cell, tissue, and organ function, can be delayed found in most eukaryotes, this appears to represent a con- through calorie or dietary restriction. Epistasis studies have served aging pathway (Kaeberlein and Kennedy 2011). led many to believe that this is due to reduced TORC1 Sch9 was one of the first genes to be implicated in CLS signaling (reviewed in Weindruch and Walford 1988; (Fabrizio et al. 2001). A subsequent high-throughput assay Kapahi et al. 2010; Zoncu et al. 2010, 2011; Kaeberlein involving 4800 viable single-gene yeast mutants further impli- and Kennedy 2011). Indeed, genetic or chemical targeting cated TORC1 in CLS (Powers et al. 2006). These and other of TORC1 has been demonstrated to increase life span in studies (Wanke et al. 2008; Wei et al. 2008) provided evidence yeast, worms, flies, and mice (Vellai et al. 2003; Jia et al. that reduced TORC1-Sch9-signaling activity promotes life span 2004; Kapahi et al. 2004; Wanke et al. 2008; Harrison et al. by increasing the Rim15-dependent expression of environmental TOR Function 1191 stress-response genes (described above). Later, Burtner et al. et al. 2003; Reinke et al. 2004; Zinzalla et al. 2010) (Figure (2009) demonstrated that acetic acid-induced mortality is the 1C). The names of mammalian and invertebrate orthologs of primary mechanism of chronological aging in yeast under stan- TORC2 subunits and the salient features of S. cerevisiae dard conditions and that this toxicity is better tolerated when TORC2 subunits are summarized in Table 1 and Table 4, environmental stress-response genes are artificially induced, respectively. The highly conserved, essential core subunits for example, upon inhibition of TORC1 or Sch9 activities. How- are TOR2, Avo1, Avo3, and Lst8. Avo1 and Avo3 bind co- ever, this model is not universally accepted. Pan et al. (2011) operatively to the N-terminal HEAT repeat region in TOR2 have proposed that TORC1 inhibition leads to increased mito- and are required for TORC2 integrity (Wullschleger et al. chondrial function and a consequent increase in reactive oxy- 2005). TORC2 autophosphorylates sites in Avo1 and Avo3, gen species that elicit a Rim15-independent pro-survival signal. but the purpose of this phosphorylation is unknown. Avo1 Furthermore, acetic acid accumulation appears not to be a con- has a C-terminal PH-like domain that mediates binding tributing factor in CLS in this study. Given its apparent conser- to the plasma membrane (Berchtold and Walther 2009). vation across eukarya (Blagosklonny and Hall 2009), Avo3 has a RasGEFN domain, a subdomain often found in elucidation of the mechanisms by which TORC1 regulates life the N-terminal part of a larger GDP/GTP exchange domain span is eagerly awaited. of exchange factors for Ras-like GTPases, but the function of Less-characterized effectors identified in phosphoproteomic the RasGEFN domain is unknown. Lst8 binds to the kinase studies: As alluded to above, large-scale mass spectrometry- domain in TOR2 and is required for TOR2 kinase activity based phosphoproteomic studies have recently been per- (Wullschleger et al. 2005). Lst8 is a Gb-like propeller pro- formed to identify the rapamycin-sensitive phosphoproteome tein consisting of seven WD40 motifs. TORC2 is rapamycin (Huber et al. 2009; Soulard et al. 2010). The major limitation insensitive whereas TORC1 is rapamycin sensitive because of these studies was their poor coverage as evidenced by their FKBP-rapamycin binds only TORC1 (Loewith et al. 2002). rather modest overlap, although this could be partly explained This selective FKBP-rapamycin binding is presumably due to by the different growth conditions and technical approaches Avo1 masking the FRB domain in TOR2 in TORC2. Finally, employed. Rapamycin exposure times were chosen such that co-immunoprecipitation and gel filtration experiments sug- layers of signaling events (e.g., kinase/phosphatase cascades) gest that TORC2 is a multimer, likely a TORC2-TORC2 di- would be observed. These events should have been triggered mer (Wullschleger et al. 2005). as a direct consequence of TORC1 inhibition and not as a sec- The cellular localization of TORC2 has been studied by ondary consequence of cell cycle delays or changes in tran- subcellular fractionation, indirect immunofluorescence, scription. Hundreds of rapamycin-sensitive phosphorylation immunogold electron microscopy, and visualization of GFP- sites were mapped, the majority of which are in proteins tagged TORC2 components (Kunz et al. 2000; Wedaman not previously implicated in TORC1 signaling. However, as et al. 2003; Aronova et al. 2007; Sturgill et al. 2008; Berchtold sufficient time elapsed to activate entire signaling cascades, and Walther 2009). In considering these studies, it is impor- a potential TORC1 consensus target motif was not evident tant to realize that the vast majority of TOR2 (90%) is in from the data analyses. Still, the data from these studies TORC2 (vs. TORC1), and thus TOR2 localization studies pre- will be instrumental in both elucidating how TORC1 signals sumably detect mainly, if not exclusively, TORC2. All studies to its known distal readouts and discovering new TORC1 indicate that TORC2 is at or near the plasma membrane. functions. Berchtold and Walther (2009) suggest that TORC2 is dynam- ically localized to a previously unrecognized plasma mem- TOR Complex 2 brane domain termed the MCT (membrane compartment containing TORC2). Furthermore, they conclude that TORC2 Composition and localization of TOR complex 2 plasma membrane localization is essential for viability and is TOR complex 2 (TORC2) is rapamycin insensitive and mediated by the C-terminal PH domain in Avo1.Mostof the consists of TOR2, Avo1, Avo2, Avo3, Bit61 (and/or its localization studies have found that TORC2 is also at another, paralog Bit2), and Lst8 (Loewith et al. 2002; Wedaman ill-defined cellular location(s). For example, Kunz et al. Table 4 Salient features of TORC2 components Protein Size Motifs/domains Potential function Tor2 2470 aa HEAT repeats, FAT domain, FRB domain, Protein kinase, scaffold kinase domain, and FATC domain Avo1 1176 aa PH Recruit TORC2 to plasma membrane Avo2 426 aa None obvious Unknown Avo3/Tsc11 1430 aa RasGEFN Scaffold Bit61 543 aa None obvious Paralogs with unknown function Bit2 545 aa None obvious Paralogs with unknown function Lst8 303 aa 7 WD-40 repeats Stabilize kinase domain Data for this table were obtained from Cybulski and Hall (2009). 1192 R. Loewith and M. N. Hall some. As ribosomes determine the growth capacity of a cell, this mechanism ensures that TORC2 is active only in growing cells. There are also indications that environmental stress inhibits TORC2 signaling, possibly to prevent growth in unfavorable conditions. The mechanism of this regulation and the level at which it intersects with the TORC2 pathway are poorly defined, but it may involve the Slm proteins (see below) and the stress-activated phosphatase calcineurin (Bultynck et al. 2006; Mulet et al. 2006). TORC2 substrates Figure 4 Signaling by TORC2. TORC2 directly phosphorylates the AGC kinase family member Ypk (Ypk1 and 2) and the PH domain containing The best-characterized and possibly the major TORC2 sub- protein Slm (Slm1 and -2). Downstream effectors include the phospha- strate is the protein kinase Ypk. Ypk1 and Ypk2 are an es- tase calcineurin, the transcription factor Crz1, and Pkc1. TORC2 controls organization of the actin cytoskeleton, endocytosis, sphingolipid biosyn- sential pair of homologous kinases and members of the AGC thesis, and stress-related transcription. The effector pathways by which kinase family (Roelants et al. 2004) (Figure 4). Kamada TORC2 controls these processes are incompletely understood (see Distal et al. (2005) linked Ypk to TORC2 signaling upon isolating readouts downstream of TORC2 for further details). YPK2 as a multicopy suppressor of a TORC2 defect. They then showed that immunopurified TOR2 directly phosphor- ylates Ypk2 at Ser641 in the turn motif and Thr659 in the (2000) report that part of TOR2 is also in an unknown sub- hydrophobic motif. TORC2 phosphorylates and activates cellular membrane fraction distinct from Golgi, vacuoles, Gad8 and SGK1, the S. pombe and mammalian orthologs mitochondria, and the nucleus. Wedaman et al. (2003) of Ypk, respectively, in a similar manner (Matsuo et al. showed that TOR2 can be in the cell interior often in asso- 2003; Garcia-Martinez and Alessi 2008). It is well estab- ciation with membrane tracks. Sturgill et al. (2008) detected lished that TORC1 or TORC2 phosphorylates the turn and a cytoplasmic fluorescent signal in cells expressing GFP- hydrophobic motifs in several kinases as a conserved mech- tagged TOR2. In conclusion, TORC2 appears to be at mul- anism of activation of AGC kinase family members (see tiple cellular locations, the plasma membrane, and one or above) (Jacinto and Lorberg 2008). Ypk/Gad8/SGK1 possibly more other sites. A plasma membrane location is appears to be a major TORC2 substrate as an ypk, gad8,or consistent with the role of TORC2 in controlling the actin sgk1 mutation phenocopies a TORC2 defect, and overex- cytoskeleton and endocytosis (see below). pression of Ypk2, Gad8, or SGK1 is sufficient to suppress Upstream of TORC2 a TORC2 defect in S. cerevisiae, S. pombe,or Caenorhabditis elegans, respectively (Matsuo et al. 2003; Kamada et al. The upstream regulation of TORC2 is poorly characterized 2005; Jones et al. 2009; Soukas et al. 2009). The two ho- (Cybulski and Hall 2009). Several lines of evidence in many mologous, TORC2- and phosphoinositide (PI4,5P )-binding different organisms indicate that nutrients regulate TORC1 proteins Slm1 and Slm2 have also been reported to be phos- (see above). On the other hand, there is no reported evidence phorylated in a TORC2-dependent manner both in vivo and supporting the notion that TORC2 is controlled by nutrients. in vitro (Audhya et al. 2004; Fadri et al. 2005). However, the Knockout of TORC2 does not confer a starvation-like pheno- physiological relevance of Slm phosphorylation is unknown type, and the nutrient-sensitive EGO complex appears not to other than that it appears to be required for localization of be upstream of TORC2. Zinzalla et al. (2011) recently devised Slm to the plasma membrane (Audhya et al. 2004; Fadri a “reverse” suppressor screen to identify upstream regulators et al. 2005). of TORC2. This screen was based on the observation that constitutively active Ypk2 (Ypk2*) suppresses the loss of via- Distal readouts downstream of TORC2 bility due to a TORC2 defect. Ypk2 is a protein kinase nor- mally phosphorylated and activated by TORC2 (see below). The first described and best-characterized TORC2 readout is Zinzalla et al. (2011) screened for mutants that require Ypk2* the actin cytoskeleton (Figure 4). TORC2 controls the cell for viability. As predicted, this screen isolated several mutants cycle-dependent polarization of the actin cytoskeleton. As defective in genes encoding essential TORC2 components, the polarized actin cytoskeleton directs the secretory path- but also in the gene NIP7. Subsequent experiments confirmed way and thereby newly made protein and lipid to the grow- that Nip7, a ribosome maturation factor, is required for ing daughter bud, this is a mechanism by which TORC2 TORC2 kinase activity. The role of Nip7 in the activation of mediates spatial control of cell growth. The first indication yeast TORC2 has so far not been pursued further, but experi- that TOR2 is linked to the actin cytoskeleton came from the ments in mammalian cells suggest that mNip7 is required for isolation of TCP20, which encodes an actin-specific chaper- mTORC2 activation indirectly via its role in ribosome matu- one, as a dosage suppressor of a dominant-negative TOR2 ration. In mammalian cells, and presumably also in yeast “kinase-dead” mutation (Schmidt et al. 1996). This, in turn, cells, TORC2 is activated by direct association with the ribo- led to the discovery that tor2 mutants display an actin TOR Function 1193 organization defect (Schmidt et al. 1996). The subsequent mutations in genes encoding components of the sphingolipid isolation of sac7, which encodes a Rho-GAP (GTPase- biosynthetic pathway, suppress a csg2 mutation. Sur1/Csg1 activating protein), as a second-site suppressor of a tor2- and Csg2 are subunits, probably the catalytic and regulatory temperature-sensitive (ts) mutation suggested that TOR2 subunits, respectively, of mannosylinositol phosphorylcera- is linked to the actin cytoskeleton via a signaling pathway mide synthase that mediates a late step in sphingolipid bio- containing a Rho GTPase. It was later demonstrated that synthesis. The Slm proteins were subsequently also linked to Sac7 is indeed a GAP for Rho1 and that TOR2 activates sphingolipid metabolism (Tabuchi et al. 2006; Daquinag et al. the Rho1 GTPase switch via the Rho1-GEF Rom2 (Schmidt 2007). Most recently, Aronova et al. (2008) profiled sphingo- et al. 1997; Bickle et al. 1998). Rom2 GEF activity is reduced lipids in a conditional avo3 mutant and thereby confirmed in extracts from a tor2-ts mutant (Schmidt et al. 1997; Bickle that TORC2 plays a positive role in sphingolipid biosynthesis. et al. 1998). The finding that overexpression of Rom2 sup- Aronova et al. (2008) also investigated the molecular mech- presses a tor2-ts mutation, whereas overexpression of cata- anism by which TORC2 controls sphingolipids. They found lytically active Rom2 lacking its lipid-binding PH domain that TORC2 regulates sphingolipid production via Ypk2 and does not suppress, suggested that TOR2 signals to Rom2 suggest a model wherein TORC2 signaling is coupled to via the PH domain. It was subsequently shown that TOR2 sphingoid long-chain bases (early intermediates in sphingoli- signals to the actin cytoskeleton mainly, if not exclusively, pid synthesis) to control Ypk2 and late steps in sphingolipid via the Rho1 effector Pkc1 (protein kinase C) and the Pkc1- synthesis. Furthermore, the biosynthetic step controlled controlled cell-wall integrity MAP kinase cascade (Helliwell by TORC2 and Ypk2 is antagonized by the phosphatase cal- et al. 1998b). cineurin that is functionally linked to the Slm proteins How might TORC2 signal to Rom2 to activate the Rho1 (Bultynck et al. 2006; Mulet et al. 2006; Aronova et al. GTPase switch? The PH domain in Rom2 suggests that it 2008). Another potential target for the regulation of sphingo- may involve a lipid intermediate. This possibility is sup- lipid biosynthesis by TOR are the Orm1 and Orm2 proteins. ported by the observation that overexpression of the PI-4- The conserved Orm proteins, identified as a potential risk P 5-kinase Mss4 suppresses a tor2-ts mutation (Desrivieres factor for childhood asthma, form a complex that negatively et al. 1998; Helliwell et al. 1998a) and that PI4,5P at the regulates the first and rate-limiting step in sphingolipid bio- plasma membrane is required to recruit/activate Rom2 synthesis (Breslow et al. 2010; Han et al. 2010). Both Orm1 (Audhya and Emr 2002). The mechanism by which TORC2 and Orm2 are phosphoproteins and at least Orm1 phosphor- mayactivatePI4,5P signaling or possibly a parallel path- ylation changes upon rapamycin treatment (Huber et al. wayconverging on thecell-wall integritypathway is un- 2009; Soulard et al. 2010). Furthermore, loss of Orm2 sup- known, but likely involves the well-established TORC2 presses a Ypk deficiency (Roelants et al. 2002; Schmelzle et al. substrate Ypk (Roelants et al. 2002; Schmelzle et al. 2002; Kamada et al. 2005; Mulet et al. 2006). These findings 2002; Kamada et al. 2005; Mulet et al. 2006). The phos- suggest that both TORC1 and TORC2 may control sphingoli- phoinositide-binding Slm proteins and sphingolipids may pid synthesis via Orm proteins. also be functionally related to TORC2-mediated control of the actin cytoskeleton (Sun et al. 2000; Friant et al. Future Directions 2001; Roelants et al. 2002; Audhya et al. 2004; Fadri et al. 2005; Liu et al. 2005; Tabuchi et al. 2006; Daquinag What is upstream of the two complexes? et al. 2007). How TORC activities are altered in response to environ- A second downstream process controlled by TORC2 is mental cues remains a major void in our understanding of endocytosis. Efficient internalization of cell-surface compo- the TOR-signaling network. The TOR complexes are regu- nents is an important aspect of cell growth control. deHart lated by nutrients, stress, or ribosomes, but the mechanisms et al. (2003) identified a tor2 mutation in a screen for by which these inputs are sensed and how this information mutants defective in ligand-stimulated internalization of is transduced, with the notable exceptions discussed above, a cell-surface receptor. TORC2 appears to control endocyto- to ultimately regulate kinase activity remain largely un- sis via Rho1, Ypk1, and possibly the Slm proteins, but how known. Genetic screens, such as the reverse suppressor Rho1, Ypk1, and the Slm proteins are functionally related in screen described above, should help to further elucidate mediating TORC2-controlled endocytosis is unknown these signaling pathways. Unlike growth factor-signaling (deHart et al. 2002, 2003; Bultynck et al. 2006). pathways, which are present only in metazoans, nutrient A third TORC2-regulated process is sphingolipid biosyn- and stress-responsive pathways are found in all eukaryotic thesis (Powers et al. 2010). Sphingolipids serve as essential cells, and thus their characterization in model organisms structural components in lipid bilayers and as signaling mol- would have far-reaching implications. ecules. The first indication that TORC2 controls sphingolipid synthesis was the finding that overexpression of SUR1 sup- What is downstream of the TORCs? presses a temperature-sensitive tor2 mutation (Helliwell et al. 1998a). In a parallel study, Beeler et al. (1998) reported that The TORCs play a central role in the regulation of cell a mutation in TOR2 or AVO3 (also known as TSC11), or growth by signaling to a staggering number of distal 1194 R. Loewith and M. N. Hall Audhya, A., and S. D. Emr, 2002 Stt4 PI 4-kinase localizes to the downstream processes. Recent phosphoproteomics studies plasma membrane and functions in the Pkc1-mediated MAP have begun to illuminate the relevant phosphorylation kinase cascade. Dev. Cell 2: 593–605. cascades and, in addition, have suggested the existence of Audhya, A., R. Loewith, A. B. Parsons, L. Gao, M. Tabuchi et al., novel growth-related effectors downstream of TORC1. Sim- 2004 Genome-wide lethality screen identifies new PI4,5P2 ilar studies describing the TORC2-dependent phosphopro- effectors that regulate the actin cytoskeleton. EMBO J. 23: 3747–3757. teome are eagerly anticipated. Elucidating these downstream Barbet, N. C., U. Schneider, S. B. Helliwell, I. Stansfield, M. F. Tuite signaling events is both academically interesting and medi- et al., 1996 TOR controls translation initiation and early G1 cally important; cell growth, like cell birth (division) and cell progression in yeast. Mol. Biol. 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GeneticsPubmed Central

Published: Dec 1, 2011

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