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The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells

The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized... JCB Article The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells Agnes Lee Ang, Heike Fölsch, Ulla-Maija Koivisto, Marc Pypaert, and Ira Mellman Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520 he AP-1B clathrin adaptor complex plays a key role that do not interact with AP-1B were targeted normally in the recognition and intracellular transport of many despite overexpression of mutant Rab8. Similar results T membrane proteins destined for the basolateral surface were obtained for a dominant-negative allele of the Rho of epithelial cells. However, little is known about other GTPase Cdc42, previously implicated in basolateral transport components that act in conjunction with AP-1B. We found but now shown to be selective for the AP-1B pathway. that the Rab8 GTPase is one such component. Expression of Rab8-GFP was localized to membranes in the TGN-recycling a constitutively activated GTP hydrolysis mutant selectively endosome, together with AP-1B complexes and the closely inhibited basolateral (but not apical) transport of newly related but ubiquitously expressed AP-1A complex. However, synthesized membrane proteins. Moreover, the effects were expression of active Rab8 caused a selective dissociation limited to AP-1B–dependent basolateral cargo; basolateral of AP-1B complexes, reflecting the specificity of Rab8 for transport of proteins containing dileucine targeting motifs AP-1B–dependent transport. Introduction Although epithelial cell polarity is initiated by spatial cues proteins internalized by endocytosis (Matter et al., 1993; Aroeti provided during cell–cell and cell–substrate contacts, the and Mostov, 1994; Odorizzi et al., 1996). The physiological subsequent generation of biochemically distinct apical and importance of this recognition system has been demonstrated basolateral plasma membrane domains is perhaps the most by human mutations causing familial hypercholesterolemia essential determinant of epithelial cell function (Drubin and due to a defective targeting signal in the LDL receptor Nelson, 1996; Mostov et al., 2000; Nelson, 2003). To a (LDLR; Koivisto et al., 2001). large extent, plasma membrane proteins are delivered to and Some of the components involved in basolateral transport maintained at the appropriate cell surface domain by distinct have recently been identified. Among these is an epithelial targeting signals that control polarized transport on the cell–specific form of the AP-1 clathrin adaptor complex, secretory and endocytic pathways (Mellman, 1996). This is AP-1B (Fölsch et al., 1999, 2003; Ohno et al., 1999). This best understood in the case of basolateral proteins, many of tetrameric complex is closely related to the ubiquitously which possess distinctive tyrosine- or dileucine-containing expressed AP-1A complex. The two differ only by substitution sequence motifs in their cytoplasmic domains (Hunziker et of the ubiquitous 50-kD 1A subunit (AP-1A) with a homol- al., 1991; Matter et al., 1992, 1994; Hunziker and Fumey, ogous 1B subunit (AP-1B), whose expression is limited to 1994; Matter and Mellman, 1994). Apical proteins are often epithelia. The presence of 1B confers the ability to recognize selected for transport by interactions involving carbohydrate and mediate basolateral transport of membrane proteins moieties in a protein’s lumenal domain or by the lipid-binding bearing tyrosine-dependent targeting signals (e.g., LDLR, properties of membrane anchors. Importantly, the same vesicular stomatitis virus G-protein [VSV-G], and asialoglyco- (or similar) sets of signals are decoded upon exit of newly protein receptor) and at least one tyrosine-independent signal synthesized proteins from the TGN and in endosomes for (transferrin [Tfn] receptor; Fölsch et al., 1999; Sugimoto et al., 2002). Interestingly, AP-1B does not program the Address correspondence to Ira Mellman, Department of Cell Biology, basolateral targeting of proteins bearing dileucine-type Ludwig Institute for Cancer Research, Yale University School of Medicine, 333 Cedar St., PO Box 208002, New Haven, CT 06520-8002. Tel.: Abbreviations used in this paper: FcR, Fc receptor; IF, immunofluorescence; (203) 785-4303. Fax: (203) 785-4301. email: ira.mellman@yale.edu LDLR, LDL receptor; Tfn, transferrin; VSV-G, vesicular stomatitis Key words: Rab8; AP-1; sorting; polarity; MDCK virus G-protein.  The Rockefeller University Press, 0021-9525/2003/10/339/12 $8.00 The Journal of Cell Biology, Volume 163, Number 2, October 27, 2003 339–350 http://www.jcb.org/cgi/doi/10.1083/jcb.200307046 339 The Journal of Cell Biology 340 The Journal of Cell Biology | Volume 163, Number 2, 2003 signals (e.g., FcRII-B2), as these reach the basolateral surface (Keller et al., 2001). After injection, the cells were incu- of 1B-negative LLC-PK1 cells (Roush et al., 1998; Fölsch bated at the nonpermissive temperature for ts045 VSV-G, et al., 1999). The precise site of AP-1B action is unknown, allowing it to accumulate in the ER while the expressed but it may act to control polarized sorting on both the en- Rab8 accumulated in the cytosol. Subsequent transfer to docytic and biosynthetic pathways, i.e., in endosomes and the permissive temperature (31C) in the presence of cyclo- the TGN (Gan et al., 2002). heximide initiated synchronous transport of VSV-G through Clearly, other components must play a role in the forma- the Golgi and to the surface. tion and delivery of basolateral transport carriers. The Rho As shown in Fig. 1 A, expression of the constitutively ac- family GTPase Cdc42 has been found to play an essential tive allele of Rab8 (Rab8Q67L) caused a great majority of role in basolateral targeting in MDCK cells (Kroschewski et VSV-G–GFP in any one cell to be expressed apically, in al., 1999; Cohen et al., 2001; Musch et al., 2001), although contrast to the lateral or basolateral expression obtained in it is unclear if this role is limited to AP-1B–dependent or cells injected with the dominant-negative Rab8 cDNA –independent pathways. Similarly, the multi-subunit exocyst (Rab8T22N) or with the VSV-G–GFP cDNA alone. Over- complex, first identified in yeast as being required for tether- expression of wild-type Rab8 elicited a phenotype similar ing secretory vesicles at the bud tip (TerBush et al., 1996), to Rab8Q67L (unpublished data). In both cases, the effects has also been implicated in basolateral transport. In MDCK were apparently selective for newly synthesized proteins cells, antibodies to two mammalian exocyst subunits (Sec6 and did not reflect an overall reorganization because the lo- and Sec8) partially inhibited the insertion of LDLR in the calization of the endogenous basolateral marker gp58 re- basolateral plasma membrane (Grindstaff et al., 1998; Yea- mained unchanged. Moreover, the expected apical local- man et al., 2001; Moskalenko et al., 2002). Because LDLR ization of VSV-G–G3 variant was not affected (Fig. 1 B), has been identified as AP-1B–dependent cargo (Fölsch et al., suggesting that Rab8 activation selectively affected basolat- 1999), it seems possible that the exocyst is involved in the eral transport. AP-1B pathway. Although total cell fluorescence showed VSV-G–GFP The small GTPases that regulate exocyst function might mostly at the apical side of MDCK cells in the presence of therefore have a role in AP-1B–dependent sorting by su- activated Rab8, it was ambiguous whether VSV-G had accu- pervising the organization of components required for mulated in vesicles near the cell apex or at the apical surface. sorting or vesicle delivery. In yeast, the Rab family member To distinguish these possibilities, we assayed surface appear- Sec4p is localized to transport vesicles and interacts geneti- ance using an antibody against the ectodomain of VSV-G cally with the exocyst to facilitate the targeting of secretory (generated by the late Thomas Kreis, and thus designated vesicles to the plasma membrane (Guo et al., 1997, 1999). TK-G) on nonpermeabilized cells. As shown in Fig. 1 C, Rab8 is among the closest mammalian homologues to VSV-G was clearly detected at the apical surface in the Sec4p and has, in fact, previously been associated with Rab8Q67L-expressing cells; basolateral VSV-G was only de- transport from the TGN to the plasma membrane in neu- tected in cells not injected with Rab8 (Fig. 1 C) or with the rons and MDCK cells (Huber et al., 1993, 1995; Moritz et dominant-negative allele (unpublished data). Thus, expres- al., 2001). A role in basolateral transport was suggested by sion of active Rab8 caused the missorting of VSV-G to the Rab8’s association with basolateral vesicles upon cell frac- apical plasma membrane. This phenotype was obtained in tionation, although the functional significance of this ob- 80% of the cells judged to be coexpressing VSV-G and servation has remained unclear; only a slight inhibition of Rab8Q67L (Rab8-expressing cells were visualized using an transport was seen in permeabilized MDCK cells using a antibody to T7 as in Fig. 1 B). peptide from the Rab8 hypervariable COOH-terminal do- Rab8Q67L expression may have caused ectopic expression main (Huber et al., 1993). of VSV-G, but it also might have had an effect on the overall efficiency of VSV-G transport, a feature that might have been missed by fluorescence microscopy alone. Therefore, Results ® we performed FACS analysis on nonpermeabilized cells to Activated Rab8 selectively affects basolateral transport compare the efficiency of VSV-G surface arrival in the pres- To better assess the role of Rab8 in polarized transport in ence or absence of activated Rab8, using the TK-G anti- epithelial cells, we adapted a microinjection assay for filter- body. To ensure that a substantial population of cells ex- grown MDCK cells previously used to illustrate the in- pressed VSV-G with or without Rab8, MDCK cells were volvement of Cdc42 in basolateral targeting (Kroschewski infected with recombinant adenoviruses encoding the VSV- et al., 1999). Cells were injected with cDNAs encoding G–GFP and Rab8Q67L genes described above. The FACS ts045 VSV-G–GFP as a plasma membrane reporter to- analysis revealed that the level of cell surface VSV-G expres- gether with constitutively active (Rab8Q67L, GTPase-defi- sion was unchanged by activated Rab8 expression (Fig. 1 D), cient) or dominant-negative (Rab8T22N, nucleotide bind- indicating that Rab8Q67L does not inhibit the delivery of ing–deficient) forms of Rab8. Rab8 alleles contained an VSV-G to the plasma membrane. Although only 50% of NH -terminal T7 tag to monitor expression (Peranen et al., the cells in the population expressed both markers, we noted 1996). Two forms of ts045 VSV-G–GFP were used, the no decrease in the level of TK-G binding. Together, these first being the wild-type protein, an AP-1B–dependent ba- data suggested that Rab8 has a selective role in the sorting or solateral marker (Fölsch et al., 2003), and the second being targeting of basolaterally directed cargo, and that expression an apical variant (VSV-G–G3) bearing a cytoplasmic do- of Rab8Q67L does not cause overall defects in polarity or main linker that appears to mask the AP-1B–binding site the secretory pathway. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 341 Figure 1. Activated Rab8 (but not dominant-negative Rab8) mislocalizes VSV-G to the apical surface. (A) Fully polarized MDCK cells were microinjected with cDNAs of ts045 VSV-G GFP and T7-tagged Rab8Q67L (dominant active) or Rab8T22N (dominant negative) at 200 ng/l, incubated at the nonpermissive temperature of 40C to accumulate VSV-G in the ER for 2 h, and chased for 2 h at the permissive temperature of 31C in the presence of cycloheximide. The cells were fixed, permeabilized, and processed for IF. Indirect IF of an endogenous basolateral protein using the anti-gp58 antibody (red, second row), followed by Alexa 568 secondary antibody. The cells were analyzed by confocal microscopy and a representative x-z section is shown. (B) Polarized MDCK cells were microinjected with the cDNAs of apical ts045 VSV-G–G3 with T7-Rab8Q67L or T7-Rab8T22N under the same pulse-chase conditions as above. Cells were processed for IF as above. Rab8 expression (red, second row) was monitored using a mouse anti-T7 tag antibody followed by anti–mouse Alexa 568 secondary antibody. (C) ts045 VSV-G–GFP was microinjected with T7-Rab8Q67L under the same pulse-chase conditions as in A, but the chase is followed by fixation without permeabilization. The cells are immunolabeled using an antibody, TK-G, against the ectodomain of VSV-G followed by Alexa 568 secondary antibody. (D) Confluent MDCK cells were infected overnight at 40C with adenoviruses encoding ts045 VSV-G–GFP and T7-Rab8Q67L. After a 2-h chase at 31C in the presence of cycloheximide, the cells were trypsinized, fixed without permeabilization, and immunolabeled using the TK-G antibody followed by an anti–mouse phytoerythrin secondary antibody. FACS analysis shows that the efficiency of surface expression of VSV-G is unchanged in the presence of Rab8Q67L. Newly synthesized VSV-G is directly missorted to the surface appearance of VSV-G was analyzed by pulse-chase apical surface in cells expressing activated Rab8 radiolabeling and surface biotinylation. As expected, in the absence of exogenous Rab8 expression, VSV-G appeared at We sought to determine if activated Rab8 acted to directly missort newly synthesized VSV-G on the secretory pathway the basolateral surface, reaching a plateau within 60–90 min and decreasing (in some experiments) thereafter (Fig. 2). or indirectly after initial basolateral insertion and missorting during endocytosis and recycling. For this purpose, filter- Importantly, little VSV-G was detected at the apical surface at early or late times of chase. However, in cells coinfected grown MDCK cells were doubly infected with adenoviruses encoding ts045 VSV-G–GFP or activated Rab8, and the with activated Rab8, VSV-G appeared simultaneously at The Journal of Cell Biology 342 The Journal of Cell Biology | Volume 163, Number 2, 2003 both the apical and basolateral surfaces at times as early as 30 was found to reach the basolateral surface of polarized pig kid- min of chase. The delivery of VSV-G to the apical surface ney epithelial (LLC-PK1) cells even in the absence of 1B ex- without prior appearance at the basolateral surface strongly pression (Roush et al., 1998; Fölsch et al., 1999). LDLR, on suggested that missorting occurred on the secretory pathway other hand, depends on tyrosine motifs for basolateral sorting upon exit from the TGN rather than in endosomes after en- (Matter et al., 1992) that physically interact with AP-1B adap- docytosis from the basolateral domain. tors and require 1B expression for basolateral targeting in Relative to the immunofluorescence (IF) results (Fig. 1), a LLC-PK1 cells (Ohno et al., 1998; Fölsch et al., 1999, 2001; significant fraction of VSV-G was, however, delivered to the Sugimoto et al., 2002). For these experiments, a Rab8-GFP basolateral surface in these experiments. This almost certainly was used to monitor expression while surface polarity of FcR reflected the fact that under the conditions of dual adeno- and LDLR was detected by staining nonpermeabilized cells virus infection used here (as opposed to microinjection), only with antibodies to the ectodomain of each receptor. 50% of the cells expressing VSV-G also expressed Rab8. It was necessary to alter the temperature shift protocol Thus, the observed effect of apical VSV-G expression caused used earlier in order to accommodate reporter proteins that, by Rab8 activation would be underestimated due to the ab- unlike ts045 VSV-G, could not be accumulated in the ER at sence of Rab8 expression in half the cells expressing VSV-G. 40C before assay. Thus, after microinjection, cells were maintained at 20C for 2.5 h to accumulate each reporter Expression of mutant Rab8 missorts only AP-1B cargo (i.e., FcR and LDLR) in the TGN before release by shifting To determine whether Rab8 caused the missorting of all baso- the temperature up to 31C. As a control, we first estab- lateral proteins regardless of their specific targeting determi- lished that this protocol resulted still in the missorting of nants, we analyzed basolateral reporters that have been shown ts045 VSV-G in mutant Rab8-expressing cells (unpublished to be AP-1B–dependent or –independent using the microin- data). After microinjection of wild-type or mutant Rab8- jection assay. The immunoglobulin Fc receptor (FcR) relies GFP together with LDLR or FcR cDNAs, the receptors on a dileucine motif for localization (Matter et al., 1994) and were accumulated in the Golgi complex by incubation at Figure 2. Activated Rab8 causes the missorting of VSV-G to the apical surface in the biosynthetic pathway. (A) Polarized MDCK cells were infected overnight with VSV-G–GFP and Rab8Q67L adenoviruses and pulse-labeled with [ S]Met/Cys for 15 min. Cells were then incubated at 37C in medium containing fivefold excess methionine and cysteine for the indicated times. After the chase, cells were placed in ice-cold PBS and biotinylated on either the apical (A) or basolateral (B) surfaces. VSV-G was immunoprecipitated using the P5D4 antibody, and the antibody complexes were pulled down with protein G–Sepharose. After spinning down the beads, 20% of the immunoprecipitated protein was set aside as the “total,” whereas the remaining 80% was applied to neutravidin beads in order to isolate the biotinylated membranes. All samples were run on 10% SDS-PAGE gels. The gels were dried and quantitative autoradiography was performed. (B) Graphical representation of the quantitation of the signal of total VSV-G at the surface in the absence or presence of activated Rab8 from A. VSV-G at the apical (AP) surface is in blue and VSV-G at the basolateral (BL) side is in red. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 343 20C (2.5 h) followed by release at 37C in the presence of expression. As shown in Fig. 3 A, expression of the nonpren- cycloheximide. As found for VSV-G, which also uses an AP- ylated activated Rab8 had no effect on LDLR surface local- 1B–dependent tyrosine-based sorting motif (Thomas and ization, suggesting that proper membrane localization of Roth, 1994; Fölsch et al., 2003), LDLR was missorted to Rab8 is important for regulating the basolateral pathway. the apical surface in the presence of activated Rab8 (Fig. 3 In a further control for the specificity of Rab8’s effect, we A). In striking contrast was the behavior of FcR. As shown microinjected wild-type or mutant GFP-tagged Rab11, a in Fig. 3 B, FcR remained at the basolateral surface despite GTPase that has an intracellular distribution similar to that the efficient expression of the active Rab8 mutant Rab8Q67L. of Rab8 (Fig. 3 A) and that is also a structural relative of Identical results for both receptors were obtained using non- Rab8 and Sec4p. However, unlike Rab8Q67L, activated GFP-tagged Rab8 (unpublished data). Thus, it appeared Rab11 (Rab11Q70L) had no effect on LDLR transport to that mutant Rab8 expression affected only the polarity of the basolateral plasma membrane (Fig. 3 A). Thus, whatever AP-1B–dependent cargo. its mechanism, the phenotype of missorting tyrosine motif– To demonstrate that the regulation by Rab8 was due to the containing basolateral proteins was specific to active Rab8, active, membrane-bound allele, cDNA encoding a nonpren- and not a general consequence of disrupting the function of ylated GFP-tagged activated Rab8 (Rab8C) was microin- Rab family proteins in the recycling endosome/TGN region jected with LDLR cDNA and analyzed for LDLR surface of the cytoplasm. Figure 3. Activated Rab8 missorts only AP-1B cargo. (A–C) Fully polarized MDCK cells were microinjected with the cDNAs encoding activated GFP-Rab8 (Rab8Q67L; A, second column), non- prenylated GFP-Rab8 (Rab8C; A, third column), activated GFP-Rab11 (Rab11Q70L; A, fourth column), or dominant-negative GFP-Cdc42 (Cdc42T17N) (C) with LDLR (A and C, red) or FcR (B and C, red). After injection, the cells were incubated at 37C for 1 h, at 20C for 2.5 h, and finally at 37C for 2 h in the presence of cyclohex- imide. Cells were fixed without permeabilization and stained for surface LDLR (C7) or FcR (24G2), followed with Alexa 568 secondary antibodies. GTPases are visualized by GFP fluorescence. Images are representative confocal z-sections. The Journal of Cell Biology 344 The Journal of Cell Biology | Volume 163, Number 2, 2003 Figure 4. Rab8 is localized to the peri- nuclear region, associating with recycling endosomes. (A and B) MDCKT (stably transfected with cDNA for Tfn receptor) cells grown on coverslips were microin- jected with 50 ng/l wild-type GFP-Rab8 and were incubated at 37C for 2 h. Cells were processed for IF and stained for the Golgi (A; GM130, red) and the TGN (B; -adaptin [red] arrows indicate colocalization of -adaptin with Rab8, and absence of colocalization with furin [blue]). (C) MDCKT cells were induced for 14 h with butyrate to express Tfn receptor, then microinjected with cDNA for 50 ng/l wild-type GFP-Rab8 and incu- bated at 37C for 2 h. After cold-binding Alexa 594–Tfn, cells were incubated for 22 min at 37C to accumulate Tfn in recycling endosomes. Colocalization of Tfn, Rab8, and -adaptin (arrow). Tfn, red; Rab8, green; -adaptin, blue. (D) Three-dimensional reconstruction of confocal serial sections, x-y plane of a representative cell as in C. (E) Same cell from D cut sagittally (white line in first panel of D) and rotated 45 to view colocalization of Tfn with Rab8. Expression of mutant Cdc42 also missorts does seem that they are restricted to a common basolateral- only AP-1B cargo sorting pathway. In previous work, we found that expression of mutant Rab8 is localized to the perinuclear region Cdc42, especially a Cdc42 dominant-negative allele, caused in recycling endosomes the missorting of basolateral but not apical cargo (Kro- schewski et al., 1999), a finding confirmed by others To understand the role of Rab8 in the AP-1B–dependent (Johnson, 1999; Joberty et al., 2000; Cohen et al., 2001; basolateral sorting pathway, we examined the localization of Musch et al., 2001). Because this effect was observed for Rab8 in MDCK cells. Although previous work had local- VSV-G, we next asked if Cdc42 might also selectively reg- ized Rab8 to the “Golgi region” (Huber et al., 1993; Per- ulate the AP-1B–sorting pathway. For these experiments, anen et al., 1996; Peranen and Furuhjelm, 2001), its dis- we again used the 20C temperature shift protocol to allow tribution remains unclear. cDNAs encoding GFP-tagged the use of LDLR and FcR as reporters in addition to VSV-G. Rab8, wild type, or Rab8Q67L were microinjected at low As shown previously, when Cdc42 was functionally de- concentration (to provide trace labeling) into MDCK cells leted by microinjection of a cDNA encoding a dominant- grown on coverslips. After fixation, the cells were colabeled negative allele (Cdc42T17N), VSV-G was missorted to the with antibodies against various compartments, particularly apical surface of filter-grown MDCK cells (Fig. 3 C). Sim- the TGN and recycling endosomes, thought to be sites of ilar results were obtained when the polarized expression of polarized sorting. As shown in Fig. 4, wild-type Rab8 was a second AP-1B cargo protein LDLR was monitored (Fig. localized to the perinuclear region, although in some cells 3 C). However, dominant-negative Cdc42 caused no de- labeling was occasionally in peripheral structures as well tectable alteration in the basolateral expression of FcR, an as possibly on plasma membrane. However, the perinu- AP-1B–independent basolateral protein (Fig. 3 C). To- clear Rab8-GFP was excluded from structures positive for gether, these observations strongly suggested that expres- GM130, a cis-Golgi marker (Fig. 4 A), as well as for gian- sion of Rab8 and dominant-negative Cdc42 selectively af- tin, a pan-Golgi marker (unpublished data), indicating that fected the localization of basolateral proteins that rely on this portion of its distribution did not reflect an association AP-1B adaptor. Even though the precise mechanism by with Golgi cisternae. Similar results were obtained for which either GTPase elicits its effects is not yet clear, it Rab8Q67L-GFP (unpublished data). The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 345 sent separate structures superimposed across different focal planes. To provide more direct evidence, immuno-EM was performed on cells prepared as in Fig. 4 D. As shown in Fig. 5 (A and B), at least a fraction of Rab8 (10-nm gold) was clearly localized to the Tfn-positive (5-nm gold) endosomes (arrows) and coated vesicle buds (arrowhead). As expected from the IF data, some Rab8 labeling was also found associ- ated with Golgi elements. In any event, the EM data suggest that recycling endosomes may be the site of action of Rab8, and thus a possible site for sorting of AP-1B–specific cargo. Activated Rab8 interferes with the localization of -adaptin Because Rab8 was localized to the same vicinity as -adaptin and selectively disrupted the basolateral transport of AP-1B– dependent cargo, we next determined if overexpression of ac- tivated Rab8 might also disrupt the distribution of AP-1B complexes. Coverslip-grown MDCK cells were injected with a high concentration of activated Rab8 cDNA (to induce rel- ative overexpression) and labeled for Rab8 and various Golgi or TGN markers. Although expression of activated Rab8 had no effect on the localization of Golgi- or TGN-resident proteins, Fig. 6 A illustrates that it did alter the distribution Figure 5. Rab8 colocalizes with Tfn in recycling endosomes. of -adaptin from a relatively compact perinuclear cluster to (A and B) Rab8 (10-nm gold) and Tfn (5-nm gold) are localized on one that is more diffuse (top panels, arrows; arrowheads endosomes (arrows) and vesicle bud (arrowhead) in MDCKT cells infected with GFP-Rab8 adenovirus. GFP-Rab8 and Alexa 488-Tfn show -adaptin in three noninjected cells). As a control, we were visualized using anti-GFP and anti-Alexa 488 antibodies, also expressed a nonprenylated Rab8 mutant (Rab8C) un- respectively, followed by IgG secondary antibodies and protein der the same conditions; there was no detectable effect on A–gold. e, endosome; g, Golgi. Bar, 100 nm. -adaptin localization (Fig. 6 A, bottom panels; arrow- heads). The distribution of TGN and cisternal Golgi resi- dents, furin and giantin (respectively), was not affected by A closer spatial relationship was observed between Rab8- activated Rab8 overexpression (Fig. 6 B), suggesting that GFP and the AP-1 adaptor subunit -adaptin, although the only -adaptin–containing membranes or -adaptin mem- frequency of actual colocalizing structures was very low (Fig. brane association were subject to disruption by Rab8. 4 B, arrows). This result was difficult to interpret because MDCK cells express two -adaptin–containing complexes, Activated Rab8 disrupts -adaptin on AP-1B complexes AP-1A and AP-1B. Because the two complexes may have -adaptin is present in both AP-1A and -1B complexes. Be- distinct distributions (Meyer et al., 2000), a preferential lo- calization of Rab8 with AP-1B might be obscured. How- cause active Rab8 expression might only interfere with AP- 1B, we next sought to determine if Rab8’s effects were man- ever, no colocalization was observed between Rab8-GFP and furin, an endogenous membrane protein that recycles be- ifested selectively. For this purpose, we took advantage of two cell lines, the porcine kidney epithelial cell line LLC- tween the TGN and endosomes as AP-1A–dependent cargo (Fig. 4 B; Teuchert et al., 1999). Conceivably, the slight co- PK1 that is deficient in 1B expression, and fibroblast cells isolated from a 1A knockout mouse that had been stably distribution between Rab8-GFP and -adaptin reflected a selective association of Rab8 with AP-1B (see Fig. 6). transfected with 1B (Fölsch et al., 1999; Zizioli et al., 1999; Meyer et al., 2000; Eskelinen et al., 2002). Thus, we At least a fraction of Rab8 appeared to associate with recy- cling endosomes, evidenced by the partial colocalization of could analyze cells with all three phenotypic combinations of 1A and 1B. GFP-Rab8 with internalized Tfn (Sheff et al., 1999; Fig. 4 C, arrow). This coregional distribution was particularly evi- As shown in Fig. 7 A (top panels), expression of Rab8Q67L in AP-1A/AP-1B LLC-PK1 cells did not dent in cells where the recycling endosomes were character- istically clustered in the perinuclear cytoplasm, although cause the dispersal of -adaptin seen in MDCK cells (which are AP-1A/AP-1B). Thus, it appeared that Rab8 activa- markers of cisternal Golgi and the TGN continued to be distinct from Rab8-GFP (unpublished data). Three-dimen- tion had no effect on AP-1A adaptor localization or mem- brane recruitment. -Adaptin staining remained similar to sional reconstruction of z-axis sections was used to further evaluate the spatial relationship of Rab8-GFP and Tfn (Fig. that of the AP-1A cargo protein TGN-38 (expressed from a cotransfected cDNA; Fig. 7 A, blue). In contrast, the local- 4 D). Upon rotating such images and sectioning them sagit- tally (Fig. 4 E), it was apparent that in areas of coregionaliza- ization of -adaptin was substantially altered in LLC-PK1 cells that had been transfected with a 1B cDNA and thus tion, the overlap between Rab8-GFP and Tfn was found throughout the volume of the structures observed (Fig. 4, D expressed functional AP-1B (Fig. 7 A, bottom, AP-1A/ AP-1B). Notably, some -adaptin staining did persist in and E, yellow). Thus, the areas of overlap did not repre- The Journal of Cell Biology 346 The Journal of Cell Biology | Volume 163, Number 2, 2003 Figure 6. Activated Rab8 disrupts -adaptin localization in MDCK cells, but does not disrupt Golgi- or furin- containing regions of the TGN. (A) Top: MDCK cells grown on coverslips were microinjected with the cDNA for T7- Rab8Q67L (200 ng/l), incubated at 37C for 2 h, fixed, permeabilized, and stained with anti--adaptin antibody 100/3 (red, second column) or anti-T7 (green, third column). Arrows, cells expressing Rab8Q67L where -adaptin localization is disrupted; arrowheads, cells not expressing Rab8 with normal -adaptin localization. Bottom: cells were injected with cDNA for nonprenylated, activated GFP-Rab8C (green, third column). Arrowheads, cells expressing Rab8C have normal -adaptin local- ization (red, second column). (B) MDCK cells were microinjected with cDNA of T7-Rab8Q67L, incubated at 37C for 2 h, and processed for IF with the TGN anti- body anti-furin (A; top, second column), for the Golgi with anti-giantin (B; bottom, second column), and for T7-Rab8 (A and B, third column). the perinuclear region of AP-1B–expressing LLC-PK1 cells. substitutes for 1A even in cell culture (Meyer et al., 2000; These structures most likely reflected the -adaptin subunits Fölsch et al., 2001; Eskelinen et al., 2002), it was not sur- of AP-1A adaptor complexes, as their staining pattern was prising to find that these cells were somewhat pleiomorphic similar to TGN-38. in shape. Nevertheless, expression of active Rab8-GFP in- We quantified (double-blinded) the number of cells ex- duced a disruption of the perinuclear -adaptin (AP-1B) hibiting -adaptin disruption in both AP-1A/AP-1B staining pattern (Fig. 7 B, top panels; arrowheads denote and AP-1A/AP-1B LLC-PK1 cells. As summarized in -adaptin in untransfected cells). Instead of being characteristi- Table I, activated Rab8 caused the dispersal or mislocaliza- cally clustered at one side of the nucleus, -adaptin appeared tion of -adaptin in 60% of the AP-1B–expressing LLC- more diffuse, and as a result, less intensely stained. Control PK1 cells. Literally none of the wild-type LLC-PK1 cells was experiments (transfection of a GFP vector alone) did not al- judged to exhibit a clear disruption in -adaptin localization ter the -adaptin staining pattern (Fig. 7 B, bottom panels). in the presence of activated Rab8. In each case, approxi- Quantitation was again performed, revealing that nearly mately one quarter of the transfected cells exhibited an am- 80% of the knockout cells containing AP-1B alone exhibited biguous phenotype. a dispersal of -adaptin upon Rab8Q67L expression (Table Finally, we examined the phenotype of -adaptin localiza- I). Less than 10% of those cells exhibited -adaptin disrup- tion as a function of activated Rab8 expression in the murine tion when GFP vector alone was expressed. These data AP-1A/AP-1B cells. Given that 1A expression causes strongly suggested that activated Rab8 disrupted only AP-1B early embryonic lethality and that 1B only incompletely complexes because -adaptin was mislocalized only in those Table I. Percentage of cells exhibiting dispersed -adaptin following expression of activated Rab8Q67L Cell type AP-1 phenotype Rab8 expressed TGN organization (percentage of transfected cells) Dispersed Not dispersed Other LLC-PK1 AP-1A/AP-1B Rab8Q67L 0 76 25 AP-1A/AP-1B Rab8Q67L 60 14 23 EFA AP-1A/AP-1B GFP alone 7 93 ND AP-1A/AP-1B GFP-Rab8Q67L 79 18 ND The numbers of cells with TGN disruption were counted in cells of each genotype, AP-1A only (AP-1A/AP-1B), AP-1A/AP-1B, and AP-1B only (AP- 1A/AP-1B) in the presence of GFP-Rab8Q67L or GFP vector alone. The SEM for Rab8Q67L expressed in LLC-PK1 AP-1A/AP-1B and AP-1A/AP- 1B was 13 and 20%, respectively. The SEM for the EFA cells expressing GFP alone or GFP-Rab8Q67L was 12 and 15%, respectively. The SEM was calculated as the square root of n (n number of cells). EFA, embryonic fibroblast cells; ND, not determined. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 347 Figure 7. Activated Rab8 causes the selective disruption of AP-1B complexes. (A) LLC-PK1 cells were transfected with cDNAs for GFP-Rab8Q67L and TGN-38, and then were processed for IF with -adaptin 100/3 (red, second column) and TGN-38 using anti-TGN-38 antibody (blue, fourth column). Top, wild-type LLC-PK1 cells (AP-1A/AP-1B); bottom, LLC-PK1 stably transfected with 1B gene (AP-1A/AP-1B). Arrows, colocalization of -adaptin with TGN-38. Notice there is no change in -adaptin localization in cells that express only AP-1A (top) even in the presence of Rab8Q67L expression. (B) Embryonic fibroblast cells expressing only AP-1B (AP-1A/AP-1B) were transfected as above with cDNA for GFP-Rab8Q67L or GFP vector alone (green). Mouse anti--adaptin antibody clone 88 (red). Arrowheads, nontransfected cells with normal -adaptin localization. cells expressing 1B. Thus, Rab8 may directly or indirectly remained unclear if the peptide’s effect applied to all forms regulate the assembly or function of AP-1B complexes. of basolateral transport or was selective for the AP-1B path- way (which had not yet been identified). Similarly, although Cdc42 had also been shown to be involved in basolateral Discussion transport, until now there was no indication that its effects Although the AP-1B complex plays an essential role in at were selective for the AP-1B pathway. The high degree of least a subset of basolateral-targeting events in vertebrate ep- specificity exhibited by both Rab8 and Cdc42 for inhibiting ithelia, there are many questions as to its mechanism of ac- AP-1B–dependent cargo strongly suggests that they func- tion. For example, the site (or sites) at which AP-1B acts re- tionally interact, even if indirectly, with the AP-1B complex, mains unclear. It is reasonable to presume that AP-1B and that they help define a common pathway. controls sorting of newly synthesized membrane proteins As a homologue of yeast Sec4p, it seems likely that Rab8 upon exit from the TGN. However, it is unknown if sorting might also function together with the mammalian exocyst occurs at the level of the TGN or at some post-TGN site complex. Although we have not demonstrated such an inter- (e.g., recycling endosomes). There is also evidence that AP- action directly, it is important to note that at least two exocyst 1B acts in addition, possibly even preferentially in endoso- components (Sec6 and Sec8) have been associated with the mal compartments, to mediate basolateral sorting after en- delivery of AP-1B cargo (e.g., LDLR) to the basolateral sur- docytosis (Gan et al., 2002). face of MDCK cells (Grindstaff et al., 1998). Moreover, ex- Understanding the pathway controlled by AP-1B will be pression of 1B in LLC-PK1 cells enhances the recruitment facilitated by identifying and characterizing the protein of exocyst subunits (Sec8 and Exo70) to the TGN/recycling components with which it must collaborate. We have identi- endosome region of AP-1B–negative LLC-PK1 cells (Fölsch fied two such components, Rab8 and Cdc42. Rab8 had et al., 2003). Rab8, too, was found in the same region. early on been implicated in basolateral transport in MDCK Our findings have one further implication for understand- cells. This suggestion came from work demonstrating a par- ing polarized transport in MDCK cells. They clearly indi- tial inhibition of VSV-G insertion into the basolateral cate that there are two distinct modes of reaching the baso- plasma membrane of permeabilized cells treated with a Rab8 lateral plasma membrane. In LLC-PK1 cells (which do not hypervariable domain peptide (Huber et al., 1993). How- express 1B), dileucine-containing membrane proteins such ever, anti-Rab8 antibodies were without effect. Moreover, it as FcR are nevertheless targeted basolaterally, suggesting The Journal of Cell Biology 348 The Journal of Cell Biology | Volume 163, Number 2, 2003 the existence of a second, AP-1B–independent pathway or that polarized sorting may actually occur after exit of AP- mechanism of sorting in these cells. The fact that in 1B- 1B cargo from the TGN, perhaps in recycling endosomes positive MDCK cells, expression of mutant Rab8 and or a subcompartment closely apposed to these sites. A Cdc42 had no effect on FcR polarity suggests that a similar functional relationship between the TGN and recycling mechanism was simultaneously operative despite the pres- endosome has long been suspected. The finding that a Rab ence of AP-1B adaptors. Although this result suggests that protein controlling the transport of newly synthesized the AP-1B–independent pathway reflects the formation of a plasma membrane proteins in fact localizes to endocytic distinct class of transport carriers, it remains possible that structures supports the idea that the secretory and en- the dileucine-specific adaptor recruits its cargo to the same docytic pathways intersect (Futter et al., 1995; Harsay and carriers as does AP-1B. Such a mechanism would be similar Schekman, 2002). Because recycling endosomes in MDCK to what has recently been proposed for the selection of GGA cells may be associated with polarized sorting during en- and AP-1A cargo into clathrin-coated buds at the TGN dur- docytosis (Hedman et al., 1987; Stoorvogel et al., 1988; ing transport to lysosomes in mammalian cells (Hirst et al., Sheff et al., 1999), an intersection at this level would pro- 2000; Puertollano et al., 2001). However, it is unlikely that vide a common intracellular site at which polarity is gen- the AP-1B–independent pathway in MDCK cells (or LLC- erated. Although additional work will be required to PK1 cells) reflects the mechanism of basolateral targeting in establish this point functionally, our current experiments polarized cells such as hepatocytes or neurons. Such cells do suggest a number of testable hypotheses. not express 1B, yet they mediate the basolateral (or so- The final issue raised concerns the mechanism of Rab8 mato-dendritic) delivery of membrane proteins in a fashion action. The missorting phenotype was observed upon ex- strictly dependent on AP-1B signals (Jareb and Banker, pression of a constitutively active Rab8 GTPase allele or 1998; Koivisto et al., 2001). Similarly, in Drosophila eye disc overexpression of wild-type Rab8. No effect was observed epithelial cells, which do not express a second 1 gene, when a dominant-negative Rab8 allele was expressed. Con- mammalian AP-1B–dependent cargo is nevertheless targeted ceivably, the dominant-negative allele was simply inactive, accurately to the basolateral surface again in a signal-depen- as opposed to acting in a dominant-negative fashion. If dent fashion (unpublished data). true, only the active allele would be expected to interfere How and where does Rab8 work? The fact that Rab8-GFP with the normal cycle of nucleotide binding and hydroly- was localized to the perinuclear region of MDCK cells, as sug- sis, perhaps by sequestering one or more Rab8 effectors. gested previously for endogenous Rab8 (Huber et al., 1993), Such a mechanism might interfere with the proper sorting suggests that it exerts at least part of its function during sort- of AP-1B cargo into forming transport vesicles or might ing or vesicle formation. Indeed, expression of active Rab8 block transport vesicle formation itself. In turn, this would was found to cause missorting of newly synthesized VSV-G to be expected to cause VSV-G to “leak,” at least in part, into the apical surface. It also caused a selective disruption of AP- the apical pathway, as is seen for other basolateral proteins 1B’s association with membranes in the perinuclear region; when the AP-1B pathway is not available. Alternatively, ac- remarkably, AP-1A’s association with membranes in the same tive Rab8 might somehow enhance the apical pathway it- region was not affected by active Rab8 expression. Because self, rendering apical missorting of proteins with basolat- there are now clear examples (e.g., Rab9) of Rab proteins eral targeting signals quantitatively more efficient. Finally, playing an accessory role in sorting or transport vesicle forma- as is possible in the case of Cdc42, Rab8 may affect sorting tion (Carroll et al., 2001), such a function for Rab8 is indeed or traffic via the actin cytoskeleton. Rab8 is similar to Rho plausible. This is not inconsistent with early findings that family GTPases by the fact that its hypervariable domain Rab8 can be found on immunoisolated basolateral transport contains only a single prenyl group (Joberty et al., 1993). vesicles (Huber et al., 1993), or that it might also work (by Moreover, when greatly overexpressed, active Rab8 will analogy to Sec4p) in an exocyst-mediated tethering event be- cause the formation of dendritic extensions in neurons and fore fusion at the basolateral membrane. even MDCK cells (although not under the conditions used Similar considerations apply to Cdc42. It is found in the here; Peranen et al., 1996). In any event, we suspect that a Golgi region of MDCK cells (Erickson et al., 1996) and has search for Rab8-interacting proteins, as has been accom- also been associated with more rapid export of basolateral plished for other Rab proteins, will yield important in- cargo from the perinuclear zone (Musch et al., 2001). How- sights not only into Rab8 function in particular, but polar- ever, Cdc42 may interact with exocyst components and also ized sorting in general. is found in a ternary complex together with Par3, Par6, and PKD at junctional complexes in MDCK cells (Joberty et al., 2000; Zhang et al., 2001), all suggesting a possible role Materials and methods in plasma membrane tethering or fusion. Plasmid construction Our confocal microscopy results suggest that despite the Wild-type and mutant Rab8 cDNA were obtained from Johan Peranen (University of Helsinki, Helsinki, Finland; Peranen et al., 1996). T7-tagged close spatial apposition of Rab8-GFP to AP-1B adaptors Rab8 mutants were PCRed using primers 5 -CGGGATCCATGGCT- and recycling endosomes, the actual degree of “overlap” is AGCATGACTGGTGGACAGCAAATGGGTGAAGACCTACGATTACCT-3 limited. Based on our EM data, it would appear that Rab8 and 5 -GTCAAGCTTCACAGAAGAACACATCGG-3 into BamHI–HindIII localizes extensively with Tfn-containing recycling endo- sites of pRK5. EGFP-tagged Rab8 was made by ligating Rab8 to the EcoRI site of EGFP-C1 using 5 -GCGAATTCTGCGAAGACCTACGATTACCT-3 somes. Indeed, Sec8 and Exo70 also appear to exhibit a (EcoRI-Rab8) and 5 -CCGATGTGTTCTTCTGTGACTCTAGAG-3 (Rab8- similar recycling endosome-like pattern in AP-1B–express- XbaI; the reverse primer for Rab8Q67L was 5 -CCGCTCGAGTCACAGAA- ing cells (Fölsch et al., 2003). Such observations suggest GAACACATCGGAA-3 ). EGFP-Rab8 was cloned into pAdEasy™ shuttle The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 349 vector at KpnI–XbaI or KpnI–XhoI (for Rab8Q67L) using primers 5 and clone 100/3 (Sigma-Aldrich). Anti-VSV-G, for IF, TK-G (Thomas Kreis), GGGGTACCATGGTGAGCAAGGGCGAGGAGCTG-3 (KpnI-EGFP) and and for immunoprecipitation P5D4 (Thomas Kreis). -CTCTAGTCACAGAAGAACACATCGG-3 (Rab8-XbaI) or 5 -CCGCT- CGAGTCACAGAAGAACACATCGGAA-3 (Rab8-XhoI). Nonprenylated Pulse-chase biotinylation Rab8 (Rab8C) was made using the forward T7 or EGFP Rab8 primers Pulse-chase assays were performed as described previously (Matter et al., with 5 -GCTCTAGATCATCGGAAAAAGCTGCTCCTCTT-3 cloned into 1992). Samples were immunoprecipitated using P5D4 coupled to protein shuttle vector using KpnI–XbaI sites. G–Sepharose beads (Zymed Laboratories). Biotinylated surface proteins were pulled down using neutravidin beads (Pierce Chemical Co.). Samples were analyzed by SDS-PAGE followed by Western blot. The gels were Recombinant adenovirus construction dried and quantitative autoradiography was performed using a Phosphor- T7-tagged Rab8 was cloned into pAdEasy™ shuttle vector at KpnI–XhoI us- Imager (Storm 860; Molecular Dynamics). ing primers 5 -GGGGTACCATGGCTAGCATGACTGGTGGACAGCAAAT- GGGTGCGAAGACCTACGATTACCTG-3 and 5 -CCGCTCGAGTCACA- FACS analysis GAAGAACACATCGGAA-3 . Rab8C used primer 5 -CCCAAGCTTTCAT- Flow cytometry was performed with a FACSCalibur™ with CellQuest soft- CGGAAAAAGCTGCTCCTCTT-3 . Shuttle vector constructs were recom- bined with pAdEasy™-1 vector as described in the Qbiogene manual, ware (Becton Dickinson) for acquisition and with FlowJo (TreeStar) for anal- version 1.3. ysis. Secondary antibodies were anti–mouse phytoerythrin (Sigma-Aldrich). Tfn uptake Cell culture MDCKT cells grown on coverslips and induced with 10 mM butyrate for MDCK cells were cultured in MEM (10% FBS) and plated on clear perme- 5 2 14 h. Cells were preincubated in serum-free media for 30 min at 37C. cells/cm . able Transwell polycarbonate filters (Corning Costar) at 10 Coverslips were inverted on a droplet of 100 g/ml Tfn 594 or Tfn 488 Cells were grown 4 d and microinjected in Hepes-buffered media after ex- (Molecular Probes, Inc.) in PBS on ice for 30 min, followed by incubation cision from filter holders. The cDNA for Rab8, LDLR, and VSV-G–GFP at 37C for 22 min. Cells were processed for IF as described above. cDNAs (0.2 mg/ml) were injected into nuclei of 400 cells using an Ep- pendorf Transjector microinjection system mounted on an inverted micro- Immuno-EM scope (Axiovert; Carl Zeiss MicroImaging, Inc.) with a 40C heated stage. EM was performed as described previously (Fölsch et al., 2001) using anti- After injection, the filters were incubated at 40C for 2 h for ts045 VSV-G 488 (Molecular GFP expression and for retention in the ER. Cells injected with LDLR or GFP (CLONTECH Laboratories, Inc.) and anti-Alexa FcR were incubated at 37C for 1 h, at 20C for 2–2.5 h, and then returned Probes, Inc.). to 37C for 2 h with 0.1 mg/ml cycloheximide. Filters were fixed in 4% PFA and processed for IF. LLC-PK1 cells were cultured in -MEM, 10% We would like to thank J. Peranen for the Rab8 constructs, P. Keller for FBS, and 1.8 mg/ml geneticin. 1A/1B fibroblasts (Eskelinen et al., the VSV-G adenoviruses, and M.S. Robinson for the rabbit polyclonal 2002) were grown in Dulbecco’s minimum essential medium (DMEM), -adaptin antibody. Many thanks to G. Warren, H. Chang, E. Anderson, 10% FBS, and 200 g/ml hygromycin. For IF, cells were seeded on Alcian S. Maday, and D. Sheff for helpful discussions and comments; J. Lee for blue–coated coverslips and cultured for 3 d before transfection. MDCK-Tfn expert assistance; and to the entire Mellman/Warren laboratory for their receptor (MDCKT) stable cells were cultured in DMEM, 10% FBS, and 0.5 enthusiastic support. g/ml geneticin as described previously (Sheff et al., 1999.) This work was supported by the National Institutes of Health (grants GM29765 and CA46128 to I. Mellman) and by the Ludwig Institute for Recombinant adenoviruses and transfection Cancer Research. The ts045 VSV-G–GFP and apical variant were gifts from Patrick Keller Submitted: 8 July 2003 (European Molecular Biology Laboratory, Heidelberg, Germany; Keller et Accepted: 11 September 2003 al., 2001). Cells were infected 24 h before analysis by IF or pulse-chase biotinylation. Cells were infected at 4 plaque-forming units/cell. Propaga- tion and generation of recombinant adenoviruses were performed as de- scribed in the pAdEasy™ vector protocol (Qbiogene). 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The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells

The Journal of Cell Biology , Volume 163 (2) – Oct 27, 2003

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Copyright © 2003, The Rockefeller University Press
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10.1083/jcb.200307046
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Abstract

JCB Article The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells Agnes Lee Ang, Heike Fölsch, Ulla-Maija Koivisto, Marc Pypaert, and Ira Mellman Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520 he AP-1B clathrin adaptor complex plays a key role that do not interact with AP-1B were targeted normally in the recognition and intracellular transport of many despite overexpression of mutant Rab8. Similar results T membrane proteins destined for the basolateral surface were obtained for a dominant-negative allele of the Rho of epithelial cells. However, little is known about other GTPase Cdc42, previously implicated in basolateral transport components that act in conjunction with AP-1B. We found but now shown to be selective for the AP-1B pathway. that the Rab8 GTPase is one such component. Expression of Rab8-GFP was localized to membranes in the TGN-recycling a constitutively activated GTP hydrolysis mutant selectively endosome, together with AP-1B complexes and the closely inhibited basolateral (but not apical) transport of newly related but ubiquitously expressed AP-1A complex. However, synthesized membrane proteins. Moreover, the effects were expression of active Rab8 caused a selective dissociation limited to AP-1B–dependent basolateral cargo; basolateral of AP-1B complexes, reflecting the specificity of Rab8 for transport of proteins containing dileucine targeting motifs AP-1B–dependent transport. Introduction Although epithelial cell polarity is initiated by spatial cues proteins internalized by endocytosis (Matter et al., 1993; Aroeti provided during cell–cell and cell–substrate contacts, the and Mostov, 1994; Odorizzi et al., 1996). The physiological subsequent generation of biochemically distinct apical and importance of this recognition system has been demonstrated basolateral plasma membrane domains is perhaps the most by human mutations causing familial hypercholesterolemia essential determinant of epithelial cell function (Drubin and due to a defective targeting signal in the LDL receptor Nelson, 1996; Mostov et al., 2000; Nelson, 2003). To a (LDLR; Koivisto et al., 2001). large extent, plasma membrane proteins are delivered to and Some of the components involved in basolateral transport maintained at the appropriate cell surface domain by distinct have recently been identified. Among these is an epithelial targeting signals that control polarized transport on the cell–specific form of the AP-1 clathrin adaptor complex, secretory and endocytic pathways (Mellman, 1996). This is AP-1B (Fölsch et al., 1999, 2003; Ohno et al., 1999). This best understood in the case of basolateral proteins, many of tetrameric complex is closely related to the ubiquitously which possess distinctive tyrosine- or dileucine-containing expressed AP-1A complex. The two differ only by substitution sequence motifs in their cytoplasmic domains (Hunziker et of the ubiquitous 50-kD 1A subunit (AP-1A) with a homol- al., 1991; Matter et al., 1992, 1994; Hunziker and Fumey, ogous 1B subunit (AP-1B), whose expression is limited to 1994; Matter and Mellman, 1994). Apical proteins are often epithelia. The presence of 1B confers the ability to recognize selected for transport by interactions involving carbohydrate and mediate basolateral transport of membrane proteins moieties in a protein’s lumenal domain or by the lipid-binding bearing tyrosine-dependent targeting signals (e.g., LDLR, properties of membrane anchors. Importantly, the same vesicular stomatitis virus G-protein [VSV-G], and asialoglyco- (or similar) sets of signals are decoded upon exit of newly protein receptor) and at least one tyrosine-independent signal synthesized proteins from the TGN and in endosomes for (transferrin [Tfn] receptor; Fölsch et al., 1999; Sugimoto et al., 2002). Interestingly, AP-1B does not program the Address correspondence to Ira Mellman, Department of Cell Biology, basolateral targeting of proteins bearing dileucine-type Ludwig Institute for Cancer Research, Yale University School of Medicine, 333 Cedar St., PO Box 208002, New Haven, CT 06520-8002. Tel.: Abbreviations used in this paper: FcR, Fc receptor; IF, immunofluorescence; (203) 785-4303. Fax: (203) 785-4301. email: ira.mellman@yale.edu LDLR, LDL receptor; Tfn, transferrin; VSV-G, vesicular stomatitis Key words: Rab8; AP-1; sorting; polarity; MDCK virus G-protein.  The Rockefeller University Press, 0021-9525/2003/10/339/12 $8.00 The Journal of Cell Biology, Volume 163, Number 2, October 27, 2003 339–350 http://www.jcb.org/cgi/doi/10.1083/jcb.200307046 339 The Journal of Cell Biology 340 The Journal of Cell Biology | Volume 163, Number 2, 2003 signals (e.g., FcRII-B2), as these reach the basolateral surface (Keller et al., 2001). After injection, the cells were incu- of 1B-negative LLC-PK1 cells (Roush et al., 1998; Fölsch bated at the nonpermissive temperature for ts045 VSV-G, et al., 1999). The precise site of AP-1B action is unknown, allowing it to accumulate in the ER while the expressed but it may act to control polarized sorting on both the en- Rab8 accumulated in the cytosol. Subsequent transfer to docytic and biosynthetic pathways, i.e., in endosomes and the permissive temperature (31C) in the presence of cyclo- the TGN (Gan et al., 2002). heximide initiated synchronous transport of VSV-G through Clearly, other components must play a role in the forma- the Golgi and to the surface. tion and delivery of basolateral transport carriers. The Rho As shown in Fig. 1 A, expression of the constitutively ac- family GTPase Cdc42 has been found to play an essential tive allele of Rab8 (Rab8Q67L) caused a great majority of role in basolateral targeting in MDCK cells (Kroschewski et VSV-G–GFP in any one cell to be expressed apically, in al., 1999; Cohen et al., 2001; Musch et al., 2001), although contrast to the lateral or basolateral expression obtained in it is unclear if this role is limited to AP-1B–dependent or cells injected with the dominant-negative Rab8 cDNA –independent pathways. Similarly, the multi-subunit exocyst (Rab8T22N) or with the VSV-G–GFP cDNA alone. Over- complex, first identified in yeast as being required for tether- expression of wild-type Rab8 elicited a phenotype similar ing secretory vesicles at the bud tip (TerBush et al., 1996), to Rab8Q67L (unpublished data). In both cases, the effects has also been implicated in basolateral transport. In MDCK were apparently selective for newly synthesized proteins cells, antibodies to two mammalian exocyst subunits (Sec6 and did not reflect an overall reorganization because the lo- and Sec8) partially inhibited the insertion of LDLR in the calization of the endogenous basolateral marker gp58 re- basolateral plasma membrane (Grindstaff et al., 1998; Yea- mained unchanged. Moreover, the expected apical local- man et al., 2001; Moskalenko et al., 2002). Because LDLR ization of VSV-G–G3 variant was not affected (Fig. 1 B), has been identified as AP-1B–dependent cargo (Fölsch et al., suggesting that Rab8 activation selectively affected basolat- 1999), it seems possible that the exocyst is involved in the eral transport. AP-1B pathway. Although total cell fluorescence showed VSV-G–GFP The small GTPases that regulate exocyst function might mostly at the apical side of MDCK cells in the presence of therefore have a role in AP-1B–dependent sorting by su- activated Rab8, it was ambiguous whether VSV-G had accu- pervising the organization of components required for mulated in vesicles near the cell apex or at the apical surface. sorting or vesicle delivery. In yeast, the Rab family member To distinguish these possibilities, we assayed surface appear- Sec4p is localized to transport vesicles and interacts geneti- ance using an antibody against the ectodomain of VSV-G cally with the exocyst to facilitate the targeting of secretory (generated by the late Thomas Kreis, and thus designated vesicles to the plasma membrane (Guo et al., 1997, 1999). TK-G) on nonpermeabilized cells. As shown in Fig. 1 C, Rab8 is among the closest mammalian homologues to VSV-G was clearly detected at the apical surface in the Sec4p and has, in fact, previously been associated with Rab8Q67L-expressing cells; basolateral VSV-G was only de- transport from the TGN to the plasma membrane in neu- tected in cells not injected with Rab8 (Fig. 1 C) or with the rons and MDCK cells (Huber et al., 1993, 1995; Moritz et dominant-negative allele (unpublished data). Thus, expres- al., 2001). A role in basolateral transport was suggested by sion of active Rab8 caused the missorting of VSV-G to the Rab8’s association with basolateral vesicles upon cell frac- apical plasma membrane. This phenotype was obtained in tionation, although the functional significance of this ob- 80% of the cells judged to be coexpressing VSV-G and servation has remained unclear; only a slight inhibition of Rab8Q67L (Rab8-expressing cells were visualized using an transport was seen in permeabilized MDCK cells using a antibody to T7 as in Fig. 1 B). peptide from the Rab8 hypervariable COOH-terminal do- Rab8Q67L expression may have caused ectopic expression main (Huber et al., 1993). of VSV-G, but it also might have had an effect on the overall efficiency of VSV-G transport, a feature that might have been missed by fluorescence microscopy alone. Therefore, Results ® we performed FACS analysis on nonpermeabilized cells to Activated Rab8 selectively affects basolateral transport compare the efficiency of VSV-G surface arrival in the pres- To better assess the role of Rab8 in polarized transport in ence or absence of activated Rab8, using the TK-G anti- epithelial cells, we adapted a microinjection assay for filter- body. To ensure that a substantial population of cells ex- grown MDCK cells previously used to illustrate the in- pressed VSV-G with or without Rab8, MDCK cells were volvement of Cdc42 in basolateral targeting (Kroschewski infected with recombinant adenoviruses encoding the VSV- et al., 1999). Cells were injected with cDNAs encoding G–GFP and Rab8Q67L genes described above. The FACS ts045 VSV-G–GFP as a plasma membrane reporter to- analysis revealed that the level of cell surface VSV-G expres- gether with constitutively active (Rab8Q67L, GTPase-defi- sion was unchanged by activated Rab8 expression (Fig. 1 D), cient) or dominant-negative (Rab8T22N, nucleotide bind- indicating that Rab8Q67L does not inhibit the delivery of ing–deficient) forms of Rab8. Rab8 alleles contained an VSV-G to the plasma membrane. Although only 50% of NH -terminal T7 tag to monitor expression (Peranen et al., the cells in the population expressed both markers, we noted 1996). Two forms of ts045 VSV-G–GFP were used, the no decrease in the level of TK-G binding. Together, these first being the wild-type protein, an AP-1B–dependent ba- data suggested that Rab8 has a selective role in the sorting or solateral marker (Fölsch et al., 2003), and the second being targeting of basolaterally directed cargo, and that expression an apical variant (VSV-G–G3) bearing a cytoplasmic do- of Rab8Q67L does not cause overall defects in polarity or main linker that appears to mask the AP-1B–binding site the secretory pathway. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 341 Figure 1. Activated Rab8 (but not dominant-negative Rab8) mislocalizes VSV-G to the apical surface. (A) Fully polarized MDCK cells were microinjected with cDNAs of ts045 VSV-G GFP and T7-tagged Rab8Q67L (dominant active) or Rab8T22N (dominant negative) at 200 ng/l, incubated at the nonpermissive temperature of 40C to accumulate VSV-G in the ER for 2 h, and chased for 2 h at the permissive temperature of 31C in the presence of cycloheximide. The cells were fixed, permeabilized, and processed for IF. Indirect IF of an endogenous basolateral protein using the anti-gp58 antibody (red, second row), followed by Alexa 568 secondary antibody. The cells were analyzed by confocal microscopy and a representative x-z section is shown. (B) Polarized MDCK cells were microinjected with the cDNAs of apical ts045 VSV-G–G3 with T7-Rab8Q67L or T7-Rab8T22N under the same pulse-chase conditions as above. Cells were processed for IF as above. Rab8 expression (red, second row) was monitored using a mouse anti-T7 tag antibody followed by anti–mouse Alexa 568 secondary antibody. (C) ts045 VSV-G–GFP was microinjected with T7-Rab8Q67L under the same pulse-chase conditions as in A, but the chase is followed by fixation without permeabilization. The cells are immunolabeled using an antibody, TK-G, against the ectodomain of VSV-G followed by Alexa 568 secondary antibody. (D) Confluent MDCK cells were infected overnight at 40C with adenoviruses encoding ts045 VSV-G–GFP and T7-Rab8Q67L. After a 2-h chase at 31C in the presence of cycloheximide, the cells were trypsinized, fixed without permeabilization, and immunolabeled using the TK-G antibody followed by an anti–mouse phytoerythrin secondary antibody. FACS analysis shows that the efficiency of surface expression of VSV-G is unchanged in the presence of Rab8Q67L. Newly synthesized VSV-G is directly missorted to the surface appearance of VSV-G was analyzed by pulse-chase apical surface in cells expressing activated Rab8 radiolabeling and surface biotinylation. As expected, in the absence of exogenous Rab8 expression, VSV-G appeared at We sought to determine if activated Rab8 acted to directly missort newly synthesized VSV-G on the secretory pathway the basolateral surface, reaching a plateau within 60–90 min and decreasing (in some experiments) thereafter (Fig. 2). or indirectly after initial basolateral insertion and missorting during endocytosis and recycling. For this purpose, filter- Importantly, little VSV-G was detected at the apical surface at early or late times of chase. However, in cells coinfected grown MDCK cells were doubly infected with adenoviruses encoding ts045 VSV-G–GFP or activated Rab8, and the with activated Rab8, VSV-G appeared simultaneously at The Journal of Cell Biology 342 The Journal of Cell Biology | Volume 163, Number 2, 2003 both the apical and basolateral surfaces at times as early as 30 was found to reach the basolateral surface of polarized pig kid- min of chase. The delivery of VSV-G to the apical surface ney epithelial (LLC-PK1) cells even in the absence of 1B ex- without prior appearance at the basolateral surface strongly pression (Roush et al., 1998; Fölsch et al., 1999). LDLR, on suggested that missorting occurred on the secretory pathway other hand, depends on tyrosine motifs for basolateral sorting upon exit from the TGN rather than in endosomes after en- (Matter et al., 1992) that physically interact with AP-1B adap- docytosis from the basolateral domain. tors and require 1B expression for basolateral targeting in Relative to the immunofluorescence (IF) results (Fig. 1), a LLC-PK1 cells (Ohno et al., 1998; Fölsch et al., 1999, 2001; significant fraction of VSV-G was, however, delivered to the Sugimoto et al., 2002). For these experiments, a Rab8-GFP basolateral surface in these experiments. This almost certainly was used to monitor expression while surface polarity of FcR reflected the fact that under the conditions of dual adeno- and LDLR was detected by staining nonpermeabilized cells virus infection used here (as opposed to microinjection), only with antibodies to the ectodomain of each receptor. 50% of the cells expressing VSV-G also expressed Rab8. It was necessary to alter the temperature shift protocol Thus, the observed effect of apical VSV-G expression caused used earlier in order to accommodate reporter proteins that, by Rab8 activation would be underestimated due to the ab- unlike ts045 VSV-G, could not be accumulated in the ER at sence of Rab8 expression in half the cells expressing VSV-G. 40C before assay. Thus, after microinjection, cells were maintained at 20C for 2.5 h to accumulate each reporter Expression of mutant Rab8 missorts only AP-1B cargo (i.e., FcR and LDLR) in the TGN before release by shifting To determine whether Rab8 caused the missorting of all baso- the temperature up to 31C. As a control, we first estab- lateral proteins regardless of their specific targeting determi- lished that this protocol resulted still in the missorting of nants, we analyzed basolateral reporters that have been shown ts045 VSV-G in mutant Rab8-expressing cells (unpublished to be AP-1B–dependent or –independent using the microin- data). After microinjection of wild-type or mutant Rab8- jection assay. The immunoglobulin Fc receptor (FcR) relies GFP together with LDLR or FcR cDNAs, the receptors on a dileucine motif for localization (Matter et al., 1994) and were accumulated in the Golgi complex by incubation at Figure 2. Activated Rab8 causes the missorting of VSV-G to the apical surface in the biosynthetic pathway. (A) Polarized MDCK cells were infected overnight with VSV-G–GFP and Rab8Q67L adenoviruses and pulse-labeled with [ S]Met/Cys for 15 min. Cells were then incubated at 37C in medium containing fivefold excess methionine and cysteine for the indicated times. After the chase, cells were placed in ice-cold PBS and biotinylated on either the apical (A) or basolateral (B) surfaces. VSV-G was immunoprecipitated using the P5D4 antibody, and the antibody complexes were pulled down with protein G–Sepharose. After spinning down the beads, 20% of the immunoprecipitated protein was set aside as the “total,” whereas the remaining 80% was applied to neutravidin beads in order to isolate the biotinylated membranes. All samples were run on 10% SDS-PAGE gels. The gels were dried and quantitative autoradiography was performed. (B) Graphical representation of the quantitation of the signal of total VSV-G at the surface in the absence or presence of activated Rab8 from A. VSV-G at the apical (AP) surface is in blue and VSV-G at the basolateral (BL) side is in red. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 343 20C (2.5 h) followed by release at 37C in the presence of expression. As shown in Fig. 3 A, expression of the nonpren- cycloheximide. As found for VSV-G, which also uses an AP- ylated activated Rab8 had no effect on LDLR surface local- 1B–dependent tyrosine-based sorting motif (Thomas and ization, suggesting that proper membrane localization of Roth, 1994; Fölsch et al., 2003), LDLR was missorted to Rab8 is important for regulating the basolateral pathway. the apical surface in the presence of activated Rab8 (Fig. 3 In a further control for the specificity of Rab8’s effect, we A). In striking contrast was the behavior of FcR. As shown microinjected wild-type or mutant GFP-tagged Rab11, a in Fig. 3 B, FcR remained at the basolateral surface despite GTPase that has an intracellular distribution similar to that the efficient expression of the active Rab8 mutant Rab8Q67L. of Rab8 (Fig. 3 A) and that is also a structural relative of Identical results for both receptors were obtained using non- Rab8 and Sec4p. However, unlike Rab8Q67L, activated GFP-tagged Rab8 (unpublished data). Thus, it appeared Rab11 (Rab11Q70L) had no effect on LDLR transport to that mutant Rab8 expression affected only the polarity of the basolateral plasma membrane (Fig. 3 A). Thus, whatever AP-1B–dependent cargo. its mechanism, the phenotype of missorting tyrosine motif– To demonstrate that the regulation by Rab8 was due to the containing basolateral proteins was specific to active Rab8, active, membrane-bound allele, cDNA encoding a nonpren- and not a general consequence of disrupting the function of ylated GFP-tagged activated Rab8 (Rab8C) was microin- Rab family proteins in the recycling endosome/TGN region jected with LDLR cDNA and analyzed for LDLR surface of the cytoplasm. Figure 3. Activated Rab8 missorts only AP-1B cargo. (A–C) Fully polarized MDCK cells were microinjected with the cDNAs encoding activated GFP-Rab8 (Rab8Q67L; A, second column), non- prenylated GFP-Rab8 (Rab8C; A, third column), activated GFP-Rab11 (Rab11Q70L; A, fourth column), or dominant-negative GFP-Cdc42 (Cdc42T17N) (C) with LDLR (A and C, red) or FcR (B and C, red). After injection, the cells were incubated at 37C for 1 h, at 20C for 2.5 h, and finally at 37C for 2 h in the presence of cyclohex- imide. Cells were fixed without permeabilization and stained for surface LDLR (C7) or FcR (24G2), followed with Alexa 568 secondary antibodies. GTPases are visualized by GFP fluorescence. Images are representative confocal z-sections. The Journal of Cell Biology 344 The Journal of Cell Biology | Volume 163, Number 2, 2003 Figure 4. Rab8 is localized to the peri- nuclear region, associating with recycling endosomes. (A and B) MDCKT (stably transfected with cDNA for Tfn receptor) cells grown on coverslips were microin- jected with 50 ng/l wild-type GFP-Rab8 and were incubated at 37C for 2 h. Cells were processed for IF and stained for the Golgi (A; GM130, red) and the TGN (B; -adaptin [red] arrows indicate colocalization of -adaptin with Rab8, and absence of colocalization with furin [blue]). (C) MDCKT cells were induced for 14 h with butyrate to express Tfn receptor, then microinjected with cDNA for 50 ng/l wild-type GFP-Rab8 and incu- bated at 37C for 2 h. After cold-binding Alexa 594–Tfn, cells were incubated for 22 min at 37C to accumulate Tfn in recycling endosomes. Colocalization of Tfn, Rab8, and -adaptin (arrow). Tfn, red; Rab8, green; -adaptin, blue. (D) Three-dimensional reconstruction of confocal serial sections, x-y plane of a representative cell as in C. (E) Same cell from D cut sagittally (white line in first panel of D) and rotated 45 to view colocalization of Tfn with Rab8. Expression of mutant Cdc42 also missorts does seem that they are restricted to a common basolateral- only AP-1B cargo sorting pathway. In previous work, we found that expression of mutant Rab8 is localized to the perinuclear region Cdc42, especially a Cdc42 dominant-negative allele, caused in recycling endosomes the missorting of basolateral but not apical cargo (Kro- schewski et al., 1999), a finding confirmed by others To understand the role of Rab8 in the AP-1B–dependent (Johnson, 1999; Joberty et al., 2000; Cohen et al., 2001; basolateral sorting pathway, we examined the localization of Musch et al., 2001). Because this effect was observed for Rab8 in MDCK cells. Although previous work had local- VSV-G, we next asked if Cdc42 might also selectively reg- ized Rab8 to the “Golgi region” (Huber et al., 1993; Per- ulate the AP-1B–sorting pathway. For these experiments, anen et al., 1996; Peranen and Furuhjelm, 2001), its dis- we again used the 20C temperature shift protocol to allow tribution remains unclear. cDNAs encoding GFP-tagged the use of LDLR and FcR as reporters in addition to VSV-G. Rab8, wild type, or Rab8Q67L were microinjected at low As shown previously, when Cdc42 was functionally de- concentration (to provide trace labeling) into MDCK cells leted by microinjection of a cDNA encoding a dominant- grown on coverslips. After fixation, the cells were colabeled negative allele (Cdc42T17N), VSV-G was missorted to the with antibodies against various compartments, particularly apical surface of filter-grown MDCK cells (Fig. 3 C). Sim- the TGN and recycling endosomes, thought to be sites of ilar results were obtained when the polarized expression of polarized sorting. As shown in Fig. 4, wild-type Rab8 was a second AP-1B cargo protein LDLR was monitored (Fig. localized to the perinuclear region, although in some cells 3 C). However, dominant-negative Cdc42 caused no de- labeling was occasionally in peripheral structures as well tectable alteration in the basolateral expression of FcR, an as possibly on plasma membrane. However, the perinu- AP-1B–independent basolateral protein (Fig. 3 C). To- clear Rab8-GFP was excluded from structures positive for gether, these observations strongly suggested that expres- GM130, a cis-Golgi marker (Fig. 4 A), as well as for gian- sion of Rab8 and dominant-negative Cdc42 selectively af- tin, a pan-Golgi marker (unpublished data), indicating that fected the localization of basolateral proteins that rely on this portion of its distribution did not reflect an association AP-1B adaptor. Even though the precise mechanism by with Golgi cisternae. Similar results were obtained for which either GTPase elicits its effects is not yet clear, it Rab8Q67L-GFP (unpublished data). The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 345 sent separate structures superimposed across different focal planes. To provide more direct evidence, immuno-EM was performed on cells prepared as in Fig. 4 D. As shown in Fig. 5 (A and B), at least a fraction of Rab8 (10-nm gold) was clearly localized to the Tfn-positive (5-nm gold) endosomes (arrows) and coated vesicle buds (arrowhead). As expected from the IF data, some Rab8 labeling was also found associ- ated with Golgi elements. In any event, the EM data suggest that recycling endosomes may be the site of action of Rab8, and thus a possible site for sorting of AP-1B–specific cargo. Activated Rab8 interferes with the localization of -adaptin Because Rab8 was localized to the same vicinity as -adaptin and selectively disrupted the basolateral transport of AP-1B– dependent cargo, we next determined if overexpression of ac- tivated Rab8 might also disrupt the distribution of AP-1B complexes. Coverslip-grown MDCK cells were injected with a high concentration of activated Rab8 cDNA (to induce rel- ative overexpression) and labeled for Rab8 and various Golgi or TGN markers. Although expression of activated Rab8 had no effect on the localization of Golgi- or TGN-resident proteins, Fig. 6 A illustrates that it did alter the distribution Figure 5. Rab8 colocalizes with Tfn in recycling endosomes. of -adaptin from a relatively compact perinuclear cluster to (A and B) Rab8 (10-nm gold) and Tfn (5-nm gold) are localized on one that is more diffuse (top panels, arrows; arrowheads endosomes (arrows) and vesicle bud (arrowhead) in MDCKT cells infected with GFP-Rab8 adenovirus. GFP-Rab8 and Alexa 488-Tfn show -adaptin in three noninjected cells). As a control, we were visualized using anti-GFP and anti-Alexa 488 antibodies, also expressed a nonprenylated Rab8 mutant (Rab8C) un- respectively, followed by IgG secondary antibodies and protein der the same conditions; there was no detectable effect on A–gold. e, endosome; g, Golgi. Bar, 100 nm. -adaptin localization (Fig. 6 A, bottom panels; arrow- heads). The distribution of TGN and cisternal Golgi resi- dents, furin and giantin (respectively), was not affected by A closer spatial relationship was observed between Rab8- activated Rab8 overexpression (Fig. 6 B), suggesting that GFP and the AP-1 adaptor subunit -adaptin, although the only -adaptin–containing membranes or -adaptin mem- frequency of actual colocalizing structures was very low (Fig. brane association were subject to disruption by Rab8. 4 B, arrows). This result was difficult to interpret because MDCK cells express two -adaptin–containing complexes, Activated Rab8 disrupts -adaptin on AP-1B complexes AP-1A and AP-1B. Because the two complexes may have -adaptin is present in both AP-1A and -1B complexes. Be- distinct distributions (Meyer et al., 2000), a preferential lo- calization of Rab8 with AP-1B might be obscured. How- cause active Rab8 expression might only interfere with AP- 1B, we next sought to determine if Rab8’s effects were man- ever, no colocalization was observed between Rab8-GFP and furin, an endogenous membrane protein that recycles be- ifested selectively. For this purpose, we took advantage of two cell lines, the porcine kidney epithelial cell line LLC- tween the TGN and endosomes as AP-1A–dependent cargo (Fig. 4 B; Teuchert et al., 1999). Conceivably, the slight co- PK1 that is deficient in 1B expression, and fibroblast cells isolated from a 1A knockout mouse that had been stably distribution between Rab8-GFP and -adaptin reflected a selective association of Rab8 with AP-1B (see Fig. 6). transfected with 1B (Fölsch et al., 1999; Zizioli et al., 1999; Meyer et al., 2000; Eskelinen et al., 2002). Thus, we At least a fraction of Rab8 appeared to associate with recy- cling endosomes, evidenced by the partial colocalization of could analyze cells with all three phenotypic combinations of 1A and 1B. GFP-Rab8 with internalized Tfn (Sheff et al., 1999; Fig. 4 C, arrow). This coregional distribution was particularly evi- As shown in Fig. 7 A (top panels), expression of Rab8Q67L in AP-1A/AP-1B LLC-PK1 cells did not dent in cells where the recycling endosomes were character- istically clustered in the perinuclear cytoplasm, although cause the dispersal of -adaptin seen in MDCK cells (which are AP-1A/AP-1B). Thus, it appeared that Rab8 activa- markers of cisternal Golgi and the TGN continued to be distinct from Rab8-GFP (unpublished data). Three-dimen- tion had no effect on AP-1A adaptor localization or mem- brane recruitment. -Adaptin staining remained similar to sional reconstruction of z-axis sections was used to further evaluate the spatial relationship of Rab8-GFP and Tfn (Fig. that of the AP-1A cargo protein TGN-38 (expressed from a cotransfected cDNA; Fig. 7 A, blue). In contrast, the local- 4 D). Upon rotating such images and sectioning them sagit- tally (Fig. 4 E), it was apparent that in areas of coregionaliza- ization of -adaptin was substantially altered in LLC-PK1 cells that had been transfected with a 1B cDNA and thus tion, the overlap between Rab8-GFP and Tfn was found throughout the volume of the structures observed (Fig. 4, D expressed functional AP-1B (Fig. 7 A, bottom, AP-1A/ AP-1B). Notably, some -adaptin staining did persist in and E, yellow). Thus, the areas of overlap did not repre- The Journal of Cell Biology 346 The Journal of Cell Biology | Volume 163, Number 2, 2003 Figure 6. Activated Rab8 disrupts -adaptin localization in MDCK cells, but does not disrupt Golgi- or furin- containing regions of the TGN. (A) Top: MDCK cells grown on coverslips were microinjected with the cDNA for T7- Rab8Q67L (200 ng/l), incubated at 37C for 2 h, fixed, permeabilized, and stained with anti--adaptin antibody 100/3 (red, second column) or anti-T7 (green, third column). Arrows, cells expressing Rab8Q67L where -adaptin localization is disrupted; arrowheads, cells not expressing Rab8 with normal -adaptin localization. Bottom: cells were injected with cDNA for nonprenylated, activated GFP-Rab8C (green, third column). Arrowheads, cells expressing Rab8C have normal -adaptin local- ization (red, second column). (B) MDCK cells were microinjected with cDNA of T7-Rab8Q67L, incubated at 37C for 2 h, and processed for IF with the TGN anti- body anti-furin (A; top, second column), for the Golgi with anti-giantin (B; bottom, second column), and for T7-Rab8 (A and B, third column). the perinuclear region of AP-1B–expressing LLC-PK1 cells. substitutes for 1A even in cell culture (Meyer et al., 2000; These structures most likely reflected the -adaptin subunits Fölsch et al., 2001; Eskelinen et al., 2002), it was not sur- of AP-1A adaptor complexes, as their staining pattern was prising to find that these cells were somewhat pleiomorphic similar to TGN-38. in shape. Nevertheless, expression of active Rab8-GFP in- We quantified (double-blinded) the number of cells ex- duced a disruption of the perinuclear -adaptin (AP-1B) hibiting -adaptin disruption in both AP-1A/AP-1B staining pattern (Fig. 7 B, top panels; arrowheads denote and AP-1A/AP-1B LLC-PK1 cells. As summarized in -adaptin in untransfected cells). Instead of being characteristi- Table I, activated Rab8 caused the dispersal or mislocaliza- cally clustered at one side of the nucleus, -adaptin appeared tion of -adaptin in 60% of the AP-1B–expressing LLC- more diffuse, and as a result, less intensely stained. Control PK1 cells. Literally none of the wild-type LLC-PK1 cells was experiments (transfection of a GFP vector alone) did not al- judged to exhibit a clear disruption in -adaptin localization ter the -adaptin staining pattern (Fig. 7 B, bottom panels). in the presence of activated Rab8. In each case, approxi- Quantitation was again performed, revealing that nearly mately one quarter of the transfected cells exhibited an am- 80% of the knockout cells containing AP-1B alone exhibited biguous phenotype. a dispersal of -adaptin upon Rab8Q67L expression (Table Finally, we examined the phenotype of -adaptin localiza- I). Less than 10% of those cells exhibited -adaptin disrup- tion as a function of activated Rab8 expression in the murine tion when GFP vector alone was expressed. These data AP-1A/AP-1B cells. Given that 1A expression causes strongly suggested that activated Rab8 disrupted only AP-1B early embryonic lethality and that 1B only incompletely complexes because -adaptin was mislocalized only in those Table I. Percentage of cells exhibiting dispersed -adaptin following expression of activated Rab8Q67L Cell type AP-1 phenotype Rab8 expressed TGN organization (percentage of transfected cells) Dispersed Not dispersed Other LLC-PK1 AP-1A/AP-1B Rab8Q67L 0 76 25 AP-1A/AP-1B Rab8Q67L 60 14 23 EFA AP-1A/AP-1B GFP alone 7 93 ND AP-1A/AP-1B GFP-Rab8Q67L 79 18 ND The numbers of cells with TGN disruption were counted in cells of each genotype, AP-1A only (AP-1A/AP-1B), AP-1A/AP-1B, and AP-1B only (AP- 1A/AP-1B) in the presence of GFP-Rab8Q67L or GFP vector alone. The SEM for Rab8Q67L expressed in LLC-PK1 AP-1A/AP-1B and AP-1A/AP- 1B was 13 and 20%, respectively. The SEM for the EFA cells expressing GFP alone or GFP-Rab8Q67L was 12 and 15%, respectively. The SEM was calculated as the square root of n (n number of cells). EFA, embryonic fibroblast cells; ND, not determined. The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 347 Figure 7. Activated Rab8 causes the selective disruption of AP-1B complexes. (A) LLC-PK1 cells were transfected with cDNAs for GFP-Rab8Q67L and TGN-38, and then were processed for IF with -adaptin 100/3 (red, second column) and TGN-38 using anti-TGN-38 antibody (blue, fourth column). Top, wild-type LLC-PK1 cells (AP-1A/AP-1B); bottom, LLC-PK1 stably transfected with 1B gene (AP-1A/AP-1B). Arrows, colocalization of -adaptin with TGN-38. Notice there is no change in -adaptin localization in cells that express only AP-1A (top) even in the presence of Rab8Q67L expression. (B) Embryonic fibroblast cells expressing only AP-1B (AP-1A/AP-1B) were transfected as above with cDNA for GFP-Rab8Q67L or GFP vector alone (green). Mouse anti--adaptin antibody clone 88 (red). Arrowheads, nontransfected cells with normal -adaptin localization. cells expressing 1B. Thus, Rab8 may directly or indirectly remained unclear if the peptide’s effect applied to all forms regulate the assembly or function of AP-1B complexes. of basolateral transport or was selective for the AP-1B path- way (which had not yet been identified). Similarly, although Cdc42 had also been shown to be involved in basolateral Discussion transport, until now there was no indication that its effects Although the AP-1B complex plays an essential role in at were selective for the AP-1B pathway. The high degree of least a subset of basolateral-targeting events in vertebrate ep- specificity exhibited by both Rab8 and Cdc42 for inhibiting ithelia, there are many questions as to its mechanism of ac- AP-1B–dependent cargo strongly suggests that they func- tion. For example, the site (or sites) at which AP-1B acts re- tionally interact, even if indirectly, with the AP-1B complex, mains unclear. It is reasonable to presume that AP-1B and that they help define a common pathway. controls sorting of newly synthesized membrane proteins As a homologue of yeast Sec4p, it seems likely that Rab8 upon exit from the TGN. However, it is unknown if sorting might also function together with the mammalian exocyst occurs at the level of the TGN or at some post-TGN site complex. Although we have not demonstrated such an inter- (e.g., recycling endosomes). There is also evidence that AP- action directly, it is important to note that at least two exocyst 1B acts in addition, possibly even preferentially in endoso- components (Sec6 and Sec8) have been associated with the mal compartments, to mediate basolateral sorting after en- delivery of AP-1B cargo (e.g., LDLR) to the basolateral sur- docytosis (Gan et al., 2002). face of MDCK cells (Grindstaff et al., 1998). Moreover, ex- Understanding the pathway controlled by AP-1B will be pression of 1B in LLC-PK1 cells enhances the recruitment facilitated by identifying and characterizing the protein of exocyst subunits (Sec8 and Exo70) to the TGN/recycling components with which it must collaborate. We have identi- endosome region of AP-1B–negative LLC-PK1 cells (Fölsch fied two such components, Rab8 and Cdc42. Rab8 had et al., 2003). Rab8, too, was found in the same region. early on been implicated in basolateral transport in MDCK Our findings have one further implication for understand- cells. This suggestion came from work demonstrating a par- ing polarized transport in MDCK cells. They clearly indi- tial inhibition of VSV-G insertion into the basolateral cate that there are two distinct modes of reaching the baso- plasma membrane of permeabilized cells treated with a Rab8 lateral plasma membrane. In LLC-PK1 cells (which do not hypervariable domain peptide (Huber et al., 1993). How- express 1B), dileucine-containing membrane proteins such ever, anti-Rab8 antibodies were without effect. Moreover, it as FcR are nevertheless targeted basolaterally, suggesting The Journal of Cell Biology 348 The Journal of Cell Biology | Volume 163, Number 2, 2003 the existence of a second, AP-1B–independent pathway or that polarized sorting may actually occur after exit of AP- mechanism of sorting in these cells. The fact that in 1B- 1B cargo from the TGN, perhaps in recycling endosomes positive MDCK cells, expression of mutant Rab8 and or a subcompartment closely apposed to these sites. A Cdc42 had no effect on FcR polarity suggests that a similar functional relationship between the TGN and recycling mechanism was simultaneously operative despite the pres- endosome has long been suspected. The finding that a Rab ence of AP-1B adaptors. Although this result suggests that protein controlling the transport of newly synthesized the AP-1B–independent pathway reflects the formation of a plasma membrane proteins in fact localizes to endocytic distinct class of transport carriers, it remains possible that structures supports the idea that the secretory and en- the dileucine-specific adaptor recruits its cargo to the same docytic pathways intersect (Futter et al., 1995; Harsay and carriers as does AP-1B. Such a mechanism would be similar Schekman, 2002). Because recycling endosomes in MDCK to what has recently been proposed for the selection of GGA cells may be associated with polarized sorting during en- and AP-1A cargo into clathrin-coated buds at the TGN dur- docytosis (Hedman et al., 1987; Stoorvogel et al., 1988; ing transport to lysosomes in mammalian cells (Hirst et al., Sheff et al., 1999), an intersection at this level would pro- 2000; Puertollano et al., 2001). However, it is unlikely that vide a common intracellular site at which polarity is gen- the AP-1B–independent pathway in MDCK cells (or LLC- erated. Although additional work will be required to PK1 cells) reflects the mechanism of basolateral targeting in establish this point functionally, our current experiments polarized cells such as hepatocytes or neurons. Such cells do suggest a number of testable hypotheses. not express 1B, yet they mediate the basolateral (or so- The final issue raised concerns the mechanism of Rab8 mato-dendritic) delivery of membrane proteins in a fashion action. The missorting phenotype was observed upon ex- strictly dependent on AP-1B signals (Jareb and Banker, pression of a constitutively active Rab8 GTPase allele or 1998; Koivisto et al., 2001). Similarly, in Drosophila eye disc overexpression of wild-type Rab8. No effect was observed epithelial cells, which do not express a second 1 gene, when a dominant-negative Rab8 allele was expressed. Con- mammalian AP-1B–dependent cargo is nevertheless targeted ceivably, the dominant-negative allele was simply inactive, accurately to the basolateral surface again in a signal-depen- as opposed to acting in a dominant-negative fashion. If dent fashion (unpublished data). true, only the active allele would be expected to interfere How and where does Rab8 work? The fact that Rab8-GFP with the normal cycle of nucleotide binding and hydroly- was localized to the perinuclear region of MDCK cells, as sug- sis, perhaps by sequestering one or more Rab8 effectors. gested previously for endogenous Rab8 (Huber et al., 1993), Such a mechanism might interfere with the proper sorting suggests that it exerts at least part of its function during sort- of AP-1B cargo into forming transport vesicles or might ing or vesicle formation. Indeed, expression of active Rab8 block transport vesicle formation itself. In turn, this would was found to cause missorting of newly synthesized VSV-G to be expected to cause VSV-G to “leak,” at least in part, into the apical surface. It also caused a selective disruption of AP- the apical pathway, as is seen for other basolateral proteins 1B’s association with membranes in the perinuclear region; when the AP-1B pathway is not available. Alternatively, ac- remarkably, AP-1A’s association with membranes in the same tive Rab8 might somehow enhance the apical pathway it- region was not affected by active Rab8 expression. Because self, rendering apical missorting of proteins with basolat- there are now clear examples (e.g., Rab9) of Rab proteins eral targeting signals quantitatively more efficient. Finally, playing an accessory role in sorting or transport vesicle forma- as is possible in the case of Cdc42, Rab8 may affect sorting tion (Carroll et al., 2001), such a function for Rab8 is indeed or traffic via the actin cytoskeleton. Rab8 is similar to Rho plausible. This is not inconsistent with early findings that family GTPases by the fact that its hypervariable domain Rab8 can be found on immunoisolated basolateral transport contains only a single prenyl group (Joberty et al., 1993). vesicles (Huber et al., 1993), or that it might also work (by Moreover, when greatly overexpressed, active Rab8 will analogy to Sec4p) in an exocyst-mediated tethering event be- cause the formation of dendritic extensions in neurons and fore fusion at the basolateral membrane. even MDCK cells (although not under the conditions used Similar considerations apply to Cdc42. It is found in the here; Peranen et al., 1996). In any event, we suspect that a Golgi region of MDCK cells (Erickson et al., 1996) and has search for Rab8-interacting proteins, as has been accom- also been associated with more rapid export of basolateral plished for other Rab proteins, will yield important in- cargo from the perinuclear zone (Musch et al., 2001). How- sights not only into Rab8 function in particular, but polar- ever, Cdc42 may interact with exocyst components and also ized sorting in general. is found in a ternary complex together with Par3, Par6, and PKD at junctional complexes in MDCK cells (Joberty et al., 2000; Zhang et al., 2001), all suggesting a possible role Materials and methods in plasma membrane tethering or fusion. Plasmid construction Our confocal microscopy results suggest that despite the Wild-type and mutant Rab8 cDNA were obtained from Johan Peranen (University of Helsinki, Helsinki, Finland; Peranen et al., 1996). T7-tagged close spatial apposition of Rab8-GFP to AP-1B adaptors Rab8 mutants were PCRed using primers 5 -CGGGATCCATGGCT- and recycling endosomes, the actual degree of “overlap” is AGCATGACTGGTGGACAGCAAATGGGTGAAGACCTACGATTACCT-3 limited. Based on our EM data, it would appear that Rab8 and 5 -GTCAAGCTTCACAGAAGAACACATCGG-3 into BamHI–HindIII localizes extensively with Tfn-containing recycling endo- sites of pRK5. EGFP-tagged Rab8 was made by ligating Rab8 to the EcoRI site of EGFP-C1 using 5 -GCGAATTCTGCGAAGACCTACGATTACCT-3 somes. Indeed, Sec8 and Exo70 also appear to exhibit a (EcoRI-Rab8) and 5 -CCGATGTGTTCTTCTGTGACTCTAGAG-3 (Rab8- similar recycling endosome-like pattern in AP-1B–express- XbaI; the reverse primer for Rab8Q67L was 5 -CCGCTCGAGTCACAGAA- ing cells (Fölsch et al., 2003). Such observations suggest GAACACATCGGAA-3 ). EGFP-Rab8 was cloned into pAdEasy™ shuttle The Journal of Cell Biology Rab8 regulates AP-1B–dependent transport | Ang et al. 349 vector at KpnI–XbaI or KpnI–XhoI (for Rab8Q67L) using primers 5 and clone 100/3 (Sigma-Aldrich). Anti-VSV-G, for IF, TK-G (Thomas Kreis), GGGGTACCATGGTGAGCAAGGGCGAGGAGCTG-3 (KpnI-EGFP) and and for immunoprecipitation P5D4 (Thomas Kreis). -CTCTAGTCACAGAAGAACACATCGG-3 (Rab8-XbaI) or 5 -CCGCT- CGAGTCACAGAAGAACACATCGGAA-3 (Rab8-XhoI). Nonprenylated Pulse-chase biotinylation Rab8 (Rab8C) was made using the forward T7 or EGFP Rab8 primers Pulse-chase assays were performed as described previously (Matter et al., with 5 -GCTCTAGATCATCGGAAAAAGCTGCTCCTCTT-3 cloned into 1992). Samples were immunoprecipitated using P5D4 coupled to protein shuttle vector using KpnI–XbaI sites. G–Sepharose beads (Zymed Laboratories). Biotinylated surface proteins were pulled down using neutravidin beads (Pierce Chemical Co.). Samples were analyzed by SDS-PAGE followed by Western blot. The gels were Recombinant adenovirus construction dried and quantitative autoradiography was performed using a Phosphor- T7-tagged Rab8 was cloned into pAdEasy™ shuttle vector at KpnI–XhoI us- Imager (Storm 860; Molecular Dynamics). ing primers 5 -GGGGTACCATGGCTAGCATGACTGGTGGACAGCAAAT- GGGTGCGAAGACCTACGATTACCTG-3 and 5 -CCGCTCGAGTCACA- FACS analysis GAAGAACACATCGGAA-3 . Rab8C used primer 5 -CCCAAGCTTTCAT- Flow cytometry was performed with a FACSCalibur™ with CellQuest soft- CGGAAAAAGCTGCTCCTCTT-3 . Shuttle vector constructs were recom- bined with pAdEasy™-1 vector as described in the Qbiogene manual, ware (Becton Dickinson) for acquisition and with FlowJo (TreeStar) for anal- version 1.3. ysis. Secondary antibodies were anti–mouse phytoerythrin (Sigma-Aldrich). Tfn uptake Cell culture MDCKT cells grown on coverslips and induced with 10 mM butyrate for MDCK cells were cultured in MEM (10% FBS) and plated on clear perme- 5 2 14 h. Cells were preincubated in serum-free media for 30 min at 37C. cells/cm . able Transwell polycarbonate filters (Corning Costar) at 10 Coverslips were inverted on a droplet of 100 g/ml Tfn 594 or Tfn 488 Cells were grown 4 d and microinjected in Hepes-buffered media after ex- (Molecular Probes, Inc.) in PBS on ice for 30 min, followed by incubation cision from filter holders. The cDNA for Rab8, LDLR, and VSV-G–GFP at 37C for 22 min. Cells were processed for IF as described above. cDNAs (0.2 mg/ml) were injected into nuclei of 400 cells using an Ep- pendorf Transjector microinjection system mounted on an inverted micro- Immuno-EM scope (Axiovert; Carl Zeiss MicroImaging, Inc.) with a 40C heated stage. EM was performed as described previously (Fölsch et al., 2001) using anti- After injection, the filters were incubated at 40C for 2 h for ts045 VSV-G 488 (Molecular GFP expression and for retention in the ER. Cells injected with LDLR or GFP (CLONTECH Laboratories, Inc.) and anti-Alexa FcR were incubated at 37C for 1 h, at 20C for 2–2.5 h, and then returned Probes, Inc.). to 37C for 2 h with 0.1 mg/ml cycloheximide. Filters were fixed in 4% PFA and processed for IF. LLC-PK1 cells were cultured in -MEM, 10% We would like to thank J. Peranen for the Rab8 constructs, P. Keller for FBS, and 1.8 mg/ml geneticin. 1A/1B fibroblasts (Eskelinen et al., the VSV-G adenoviruses, and M.S. Robinson for the rabbit polyclonal 2002) were grown in Dulbecco’s minimum essential medium (DMEM), -adaptin antibody. Many thanks to G. Warren, H. Chang, E. Anderson, 10% FBS, and 200 g/ml hygromycin. For IF, cells were seeded on Alcian S. Maday, and D. Sheff for helpful discussions and comments; J. Lee for blue–coated coverslips and cultured for 3 d before transfection. MDCK-Tfn expert assistance; and to the entire Mellman/Warren laboratory for their receptor (MDCKT) stable cells were cultured in DMEM, 10% FBS, and 0.5 enthusiastic support. g/ml geneticin as described previously (Sheff et al., 1999.) This work was supported by the National Institutes of Health (grants GM29765 and CA46128 to I. Mellman) and by the Ludwig Institute for Recombinant adenoviruses and transfection Cancer Research. The ts045 VSV-G–GFP and apical variant were gifts from Patrick Keller Submitted: 8 July 2003 (European Molecular Biology Laboratory, Heidelberg, Germany; Keller et Accepted: 11 September 2003 al., 2001). Cells were infected 24 h before analysis by IF or pulse-chase biotinylation. Cells were infected at 4 plaque-forming units/cell. Propaga- tion and generation of recombinant adenoviruses were performed as de- scribed in the pAdEasy™ vector protocol (Qbiogene). LipofectAMINE™ References (Invitrogen) was used for transient transfection in LLC-PK1 and embryonic fibroblast cells. Aroeti, B., and K.E. Mostov. 1994. Polarized sorting of the polymeric immuno- globulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids. EMBO J. 13:2297–2304. IF microscopy For total cell staining, cells were fixed in 4% PFA for 15 min followed by Carroll, K.S., J. Hanna, I. Simon, J. Krise, P. Barbero, and S.R. Pfeffer. 2001. Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science. 292: 5–10 min of blocking/permeabilization in PBS with 10% goat serum (GS) and 0.25% saponin. Cells were incubated for 1 h with primary antibody 1373–1376. diluted in blocking solution (BS). The cells were washed three times for 10 Cohen, D., A. Musch, and E. Rodriguez-Boulan. 2001. Selective control of baso- min and incubated for 30 min in secondary antibody (with appropriate Al- lateral membrane protein polarity by cdc42. 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