Article |
Address correspondence to Charles Barlowe, Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel.: (603) 650-6516. Fax: (603) 650-1353. email: charles.barlowe{at}dartmouth.edu
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Abstract |
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Key Words: ER; Golgi; vesicles; coat proteins; trafficking
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Introduction |
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Transport between the ER and Golgi complex in the early secretory pathway also relies on GTPases and SNARE proteins. More specifically, the Rab GTPase Ypt1p is required for transport to the Golgi complex in yeast, and is localized primarily to Golgi membranes (Segev et al., 1988). To identify other cellular factors that bind to Ypt1p, yeast two-hybrid approaches uncovered a Ypt1p-interacting protein (Yip1p) that is essential for transport through the early secretory pathway (Yang et al., 1998). Yip1p is a 27-kD integral membrane protein predicted to span the membrane multiple times. The amino-terminal hydrophilic domain of Yip1p faces the cytosol and is sufficient for Ypt1p interaction. Yip1p also displays direct interactions with several other Rab GTPases in yeast including Ypt31p and Sec4p. Yip1p binding to Rabs depends on an intact carboxy-terminal CAAX motif and prenylation of these GTPases (Yang et al., 1998; Calero and Collins, 2002). The nucleotide status of the GTPase seems less critical for these associations.
Yip1p not only associates with Rab GTPases, but forms a heteromeric complex with a related integral membrane protein termed Yip1p-interacting factor (Yif1p). Yif1p shares a common domain topology with Yip1p, binds to Rab GTPases through a cytoplasmically exposed amino-terminal domain, and is required for transport through the early secretory pathway (Matern et al., 2000). Additional reports have shown that yeast cells contain an extended family of Yip1p related proteins that appear to form mixed heteromeric complexes with one another. This family displays some functional overlap and may act more broadly in intracellular transport (Calero and Collins, 2002; Calero et al., 2002). The subcellular distribution and molecular function of the Yip family of proteins remains to be determined.
In this report, we investigate the role of Yip1p in protein transport between the ER and Golgi complex. Using cell-free assays that recapitulate subreactions in this transport process, we observed that antibody inhibition of Yip1p activity and mutations within the amino-terminal cytosolic domain of Yip1p inhibit COPII-dependent vesicle budding from the ER. Moreover, these Yip1p-specific inhibitors do not directly interfere with the vesicle tethering or fusion stages of ER-derived vesicles with Golgi membranes. Our in vivo analyses also support a role for Yip1p in budding from the ER. These results are surprising in light of the fact that Ypt1p does not appear to be required for COPII-dependent budding.
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Results |
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The inhibition of budding by anti-Yip1p antibodies was surprising, as there is no apparent requirement for Ypt1p in the COPII-dependent vesicle-budding assay (Cao et al., 1998). Therefore, we considered the possibility that these anti-Yip1p antibodies interfered with budding in a nonspecific manner, perhaps by coating ER membranes and preventing COPII association. Alternatively, the antibodies could cross-link vesicles to ER membranes or to one another, producing an aggregate that would pellet under the conditions of our budding assay. These effects would then mask any later stage requirements for Yip1p in vesicle tethering or fusion. To address these possibilities, we performed an additional series of experiments. First, it should be noted that antibodies against other abundant vesicle proteins such as Sec22p, Sed5p, and Erv29p do not block budding nonspecifically (Cao et al., 1998; Belden and Barlowe, 2001b; Liu and Barlowe, 2002). Second, we prepared Fab fragments from the affinity-purified anti-Yip1p antibodies and observed specific inhibition of the vesicle-budding stage of cell-free transport (unpublished data). Finally, we generated COPII vesicles containing [35S]gpf for use in second-stage transport reactions to determine if anti-Yip1p antibodies influenced any of the post-budding assays. As seen in Fig. 2 A, addition of Uso1p to reactions containing isolated vesicles and wild-type acceptor membranes produced an
3.7-fold reduction in diffusible vesicles, indicating efficient tethering of vesicles to Golgi membranes (Fig. 2 A, compare column 1 with column 2). Vesicle tethering was unaffected by the addition of anti-Yip1p antibodies in amounts that effectively block vesicle budding. In columns 4 and 5 of Fig. 2 A, vesicles and acceptor membranes or vesicles alone were combined with anti-Yip1p antibodies in the absence of Uso1p. No reduction in diffusible vesicles was observed for either condition, indicating that an antibody cross-linking event was not responsible for the observed decrease in vesicle budding.
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Anti-Yip1p antibodies block the COPII-dependent budding of vesicle proteins
Next, we examined whether the inhibition of vesicle budding by the anti-Yip1p antibodies was restricted to [35S]gpf monitored in the budding assay, or reflected a more complete block of vesicle biogenesis from ER membranes. Previous reports have shown that ER/Golgi SNARE proteins are efficiently packaged into COPII vesicles (Barlowe et al., 1994; Rexach et al., 1994). In addition, a group of conserved transmembrane proteins, termed ER vesicle (Erv) proteins, are efficiently incorporated into COPII vesicles (Rexach et al., 1994; Otte et al., 2001). We investigated whether anti-Yip1p antibodies affected the incorporation of these proteins into COPII vesicles. Washed semi-intact cells were incubated with COPII proteins and an energy regeneration system in the presence or absence of anti-Yip1p antibodies. The vesicles synthesized in each condition were then isolated and analyzed by immunoblotting. As seen in Fig. 3, COPII proteins catalyzed the efficient and specific incorporation of certain proteins into vesicles. The SNARE protein Sec22p, as well as Erv25p, Erv41p, Yif1p, and Yip1p, were efficiently packaged under these conditions (Fig. 3, lane 2). The ER resident proteins Sec12p and Sec61p were not efficiently packaged into COPII vesicles, demonstrating selective sorting in this budding assay. The presence of anti-Yip1p antibodies in the reaction effectively inhibited budding of all vesicle proteins examined, whereas preimmune IgGs at a comparable concentration had no effect (Fig. 3, lane 3 and lane 4). These results demonstrate the inhibition of vesicle budding by the anti-Yip1p antibodies occurs in a general manner, and is not restricted to [35S]gp
f.
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We also examined the distribution of an ER-localized protein in yip1-4 cells by fluorescence microscopy of GFP-KDEL. When yip1-4 mutants were shifted to a restrictive temperature for 60 min, striking elaborations of the ER were apparent (Fig. 5 A) while nuclear structures remained intact. Very similar elaborations of the ER were observed when GFP-KDEL was expressed in a sec12 strain under restrictive conditions (unpublished data). These observations are in accord with the images obtained by EM showing accumulation of ER membranes in yip1-4 mutants.
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In this set of experiments, GFP-tagged versions of Sed5p, Gos1p, and Sft2p were expressed in the yip1-4 mutant and monitored by fluorescence at permissive and restrictive temperatures (Fig. 5 B). GFP-Sed5p displayed a typical punctate pattern at a permissive temperature, but upon temperature shift, a distinct perinuclear localization emerged. Some GFP-Sed5p appeared to remain in spots and may represent Sed5p that functions in later Golgi compartments (Pelham, 2001). GFP-Gos1p also redistributed to the ER when ER export was blocked, consistent with its proposed role in transport through early Golgi compartments (McNew et al., 1998; Tsui et al., 2001). In contrast, GFP-Sft2p remained in a punctate pattern after temperature shift as expected for a protein that functions in later Golgi compartments (Wooding and Pelham, 1998). A very similar effect on the distribution of GFP-Gos1p and GFP-Sft2p was observed in sec12 strains (Fig. 5 C). These results provide further support for a Yip1p requirement in export from the ER.
Yip1p cycles between the ER and Golgi compartments
Although Yip1p was detected in ER-derived vesicles, previous reports indicated that the protein was largely Golgi localized (Yang et al., 1998). We generated a GFP-Yip1p fusion protein under its native promoter to monitor the location of Yip1p in live cells. Strains expressing this GFP-Yip1p fusion as the sole source of Yip1p activity displayed growth rates that were comparable to wild-type strains (Fig. 6 A). Fluorescence imaging of GFP-Yip1p cells at various cell cycle stages (Fig. 6 B) revealed both a punctate pattern typical of Golgi-localized proteins and ring-like perinuclear structures indicative of ER localization. We also examined the fate of GFP-Yip1p in a sec12 mutant shifted to 37°C (Fig. 6 C). If GFP-Yip1p traffics through the ER, we would expect it to accumulate in the ER when exit from this compartment is blocked. Indeed, GFP-Yip1p redistributed to ER structures under this condition. Finally, we compared the distribution of endogenous Yip1p under wild-type conditions or under a sec12 block using subcellular fractionation schemes. Larger ER membranes pellet at a lower g-force (13,000 g) than Golgi membranes (100,000 g) when cell lysates are prepared under specified conditions (Wooding and Pelham, 1998). As seen in Fig. 6 D, Yip1p was found in both ER and Golgi fractions in wild-type strains, but shifts to the ER fraction under a sec12 block. Together with our budding experiments showing Yip1p is efficiently packaged into ER-derived vesicles, these results indicate Yip1p cycles between the ER and Golgi compartments and is dynamically localized to the early secretory pathway.
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The yip1-4 cells were also examined for their ability to package proteins other than [35S]gpf into COPII vesicles. COPII vesicles were generated from equivalent amounts of wild-type or yip1-4 semi-intact cells and analyzed by immunoblot. As shown in Fig. 8, wild-type membranes efficiently packaged Sec22p, Erv25p, Erv46p, Yif1p, and Yip1p into vesicles in a COPII-dependent fashion. In contrast, yip1-4 membranes packaged these vesicle proteins at significantly lower efficiencies upon reconstitution of COPII budding. This result mirrors the decrease observed in packaging of [35S]gp
f, indicating an overall defect in COPII vesicle biogenesis. In summary, the in vitro budding defects caused by the yip1-4 mutation are in accord with the phenotypes observed by microscopic inspection of the yip1-4 strain and with our observation that anti-Yip1p antibodies block the production of COPII vesicles in vitro.
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Discussion |
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Genetic analyses provided an independent line of evidence for Yip1p function in vesicle budding. The thermosensitive yip1-4 allele caused an accumulation of ER membranes with no apparent accumulation of 5060-nm transport vesicle intermediates. When the yip1-4 mutation was combined with the vesicle accumulating sec18-1 mutation, the double mutant accumulated ER structures when shifted to restrictive temperatures. This result demonstrates a requirement for YIP1 in the production of vesicles that accumulate in sec18-1 cells. Furthermore, pair-wise combinations of yip1-4 with other ER/Golgi transport mutants revealed specific interactions with genes (SEC12, SEC13, and SEC23) involved in formation of COPII vesicles. Mutations that influence COPI vesicle biogenesis (SEC21) and Golgi tethering (USO1) also displayed synthetic lethal relationships with yip1-4, revealing potential roles for Yip1p in Golgi structure or function. Finally, in vitro analysis of the yip1-4 allele revealed a significant defect in COPII-dependent vesicle budding, a result that is in good accord with the antibody inhibition analysis. Based on these observations, we propose that Yip1p acts in vesicle biogenesis and may be required for the assembly of coat structures, or possibly in the scission of coated vesicles from the ER.
Our findings are unexpected, given that much of the current data regarding Yip1p suggest a role for this protein in regulating aspects of Rab GTPase function. Indeed, Yip1p was originally identified as a Ypt1p- and Ypt31p-binding protein using yeast two-hybrid screens (Yang et al., 1998). Subsequent reports identified Yif1p as a Yip1p-binding partner, and it has been proposed that a Golgi-localized Yif1pYip1p complex acts to bind Ypt1p and Ypt31p to facilitate vesicle docking and fusion (Matern et al., 2000). More recent data demonstrate the presence of an extended family of Yip1-related proteins that form mixed heteromeric complexes with one another (Calero et al., 2002). Yip1p and the Yip1-related proteins interact with multiple Rabs and in a manner that depends on carboxy-terminal prenylation of the Rab protein. Based on these observations, the Yip1 family of proteins has been proposed to act in the pathway by which Rab proteins are recruited to membrane compartments (Calero et al., 2002). Analyses on the mammalian homologue of yeast Yip1p have been undertaken as well. The mammalian protein Yip1A shares 31% identity with yeast Yip1p and functionally complements the loss of yeast YIP1 (Calero et al., 2003), indicating a functional conservation of mechanism. Interestingly, Yip1A localizes to vesicular structures composing ER export sites, and interacts with the Sec23/24 subunit of the mammalian COPII complex (Tang et al., 2001). The authors of this report suggest that Yip1A is involved in the regulation of ER/Golgi traffic at the level of ER exit sites. This conclusion is consistent with our findings that inhibitors of yeast Yip1p function block COPII-dependent budding from the ER and provides support for the hypothesis that Yip1p acts in COPII vesicle biogenesis.
If Yip1p functions in the process of vesicle budding, how are Yip1p interactions with Rab GTPases integrated into this model? Rab proteins have been traditionally thought to act after vesicle formation to regulate the subsequent targeting and fusion of vesicles to their acceptor membranes (Segev, 2001). However, recent evidence has also suggested a role for Rab proteins in the process of vesicle formation. For example, experiments in mammalian cells indicate that Rab1 acts during budding to program COPII vesicles for docking and fusion competency. Here, Rab1 activity was proposed to recruit the vesicle tethering factor p115 into COPII vesicles to promote targeting to the Golgi apparatus (Allan et al., 2000). Other reports implicate Rab9 in coordinating cargo selection with vesicle formation. Specifically, the authors of this paper suggest that activated Rab9 binds directly to the protein TIP47, which in turn facilitates the recruitment of the mannose-6-phosphate receptor into a forming vesicle (Carroll et al., 2001). Taking these observations into account when considering Yip1p function, a model can be envisioned in which Yip1p acts to recruit Rabs/Ypts into forming transport vesicles. In this way, vesicle biogenesis would be coupled to the incorporation of cargos necessary for the subsequent docking and fusion of the vesicle. If the ability of Yip1p to recruit Rabs into forming vesicles is inhibited, this might then cause overall vesicle formation to become blocked as well. However, it should be noted that other vesicle proteins required for subsequent fusion, such as the SNARE proteins Sec22p, Bos1p, Bet1p, rbet1, and membrin, can be efficiently depleted from forming COPII vesicles, yet vesicle formation is not compromised (Allan et al., 2000; Liu and Barlowe, 2002; Miller et al., 2002).
Although some of the data support a role for Rab proteins in vesicle formation, additional reports suggest that certain Rabs are not required for budding, but are required for the docking and fusion of transport vesicles. For example, sec4 and ypt1 thermosensitive mutant strains accumulate transport vesicles at nonpermissive temperatures (Novick et al., 1980; Becker et al., 1991). These observations indicate that Sec4p and Ypt1p are not required for the formation of vesicles that accumulate, but instead act to fuse vesicles to the correct target membrane. In vitro experiments also demonstrate that inhibition of Ypt1p activity does not block formation of COPII vesicles, but inhibits post-budding stages of transport to the Golgi (Rexach and Schekman, 1991; Segev, 1991; Cao and Barlowe, 2000). Together, these results argue against a requirement for Ypt1p in COPII vesicle formation and suggest Yip1p does not rely on Ypt1p in this stage of transport.
During the course of our investigation, a similar analysis of Yip1p and Yif1p function in ER/Golgi transport was reported (Barrowman et al., 2003). This report showed that antibodies directed against Yip1p or Yif1p blocked transport to the Golgi complex, but only reduced budding efficiencies by one half. Interestingly, the vesicles formed in the presence of their inhibitory antibodies failed to fuse with Golgi membranes. In agreement with our findings, their anti-Yip1p antibodies did not inhibit vesicle tethering or fusion when added after vesicle production. The authors concluded that the Yip1pYif1p complex is required during vesicle formation to produce fusion competent vesicles (Barrowman et al., 2003). Although we cannot easily explain the differential effects of anti-Yip1p antibodies on the level of vesicle budding, both reports indicate a role for Yip1p function during COPII vesicle biogenesis. Additional experiments will be needed to clarify the role of Yip1p in post-budding transport stages.
Barrowman and colleagues also reported that depletion of cellular Yip1p did not affect membrane binding or localization of Ypt1p (Barrowman et al., 2003). Similarly, we have observed that thermosensitive mutations in Yip1p and Yif1p as well as antibodies directed against Yip1p did not inhibit Ypt1p membrane association in vitro (unpublished data). Although it is possible that other Yip family members may drive membrane association of Ypt1p in the absence of Yip1p and Yif1p, it may be useful to consider a model for Yip1p function in budding that is independent of Ypt1p or Rab protein activity. It is also possible that Yip1p serves as a regulatory checkpoint in vesicle budding to ensure that ER-derived vesicles can ultimately interact with Rab GTPases. In such a model, the Rab protein, per se, may not be required for vesicle budding, but a Rab-binding activity would be required.
The production of COPII-coated vesicles has been extensively studied in reconstituted budding reactions that use purified COPII proteins and ER microsomes or defined synthetic liposomes as the source of donor membrane (Matsuoka et al., 1998). In contrast to microsomes, budding from synthetic liposomes requires addition of nonhydrolyzable analogs of GTP to prolong the Sar1p-GTP state until COPII coats assemble. In addition, synthetic liposomes with an acidic phospholipid content that approximates the composition of ER membranes must be used in the minimal budding reaction. Given these distinct requirements, Yip1p could serve a role in stabilizing Sar1p-GTP during vesicle formation from microsomal membranes, or it could influence local lipid concentrations at transitional ER sites. Further investigations are required to fully define the mechanism of Yip1p in COPII-dependent budding from the ER. One approach may be to accumulate arrested budding intermediates in the presence of anti-Yip1p antibodies. Such intermediates could then be characterized biochemically and morphologically to provide further insights into Yip1p function.
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Materials and methods |
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In vitro vesicle budding, tethering, and transport assays
Yeast semi-intact cells from wild-type and mutant strains were prepared as described previously (Baker et al., 1988). Vesicle budding, tethering, and fusion assays following [35S]gpf were published previously (Barlowe, 1997; Cao et al., 1998). For two-stage fusion assays with wild-type and yip1-4 acceptor membranes, wild-type vesicles containing [35S]gp
f were first isolated from density gradients (Barlowe, 1997). Vesicles were then added to acceptor membranes to measure fusion (Cao and Barlowe, 2000). Experiments to assay packaging of proteins into vesicles by Western blot were performed as described previously (Liu and Barlowe, 2002) using semi-intact cell membranes. For ordering experiments, wild-type semi-intact cells containing translocated [35S]gp
f were first incubated with either COPII proteins or anti-Yip1p antibodies (40 µg/ml) for 5 min at 15°C. Secondary factors were then added, and cells were incubated at 23°C for an additional 25 min. Reactions were then processed to measure the level of freely diffusible vesicles (Cao et al., 1998). For in vitro assays, data points are the average of duplicate determinations and the error bars represent the range.
Microscopy
For EM experiments, cells were grown overnight to a final cell density of 0.40.7 A600. After shift to the restrictive temperature, cells were washed once with buffer (0.1 M Pipes, 0.1 M sorbitol, and 50 mM KPi, pH 7.3) and then fixed with fixative (2% glutaraldehyde, 2% PFA, 0.1 M Pipes, pH 6.8, 0.1 M sorbitol, 1 mM MgCl2, 1 mM CaCl2, and 10 µM CuCl2) for 1 h at RT and then overnight at 4°C. The cell walls were removed by treatment with 0.2 mg/ml zymolyase 100T in KPi buffer, pH 7.3 (1 ml/5 OD unit cells). An aliquot of the cells was collected in a microfuge tube and the pellet was incubated with 2% OsO4 for 1 h followed by incubation with 1% uranyl acetate (aqueous) at 4°C for 30 min. The pellets were dehydrated with sequential ethanol washes and incubated with 50% ethanol/50% SPURR resin (Electron Microscopy Sciences), then changed to 100% SPURR, and the sample was transferred to beem capsules (Electron Microscopy Sciences) and baked at 70°C for at least 24 h. Thin sections were cut onto 3-mm-diam 75/300-type mesh copper specimen grids (Veco), contrasted with lead citrate and uranyl acetate, and examined in an electron microscope (model 201; Philips) at 80 kV.
For fluorescence microscopy, the GFP-KDEL construct was generated in plasmid pRS316 (Sikorski and Hieter, 1989) using standard molecular biology techniques. In brief, the construct comprises 363 bp of the KAR2 promoter with 45 amino acids of the Kar2p presequence fused to a linker (GGPGG) followed by yeast-enhanced GFP (yEGFP; Cormack et al., 1997), which in turn is followed by second linker (GGPGG) and the sequence HDEL. The ADH1 3' region (572 bp) was added after the stop codon to provide transcription termination. The GFP amino-terminal fusions of GOS1, SED5, and SFT2 were also constructed in plasmid pRS316. Each construct contains 238 amino acids of yEGFP fused to the start methionine of the tagged protein preceded by a linker (GGPGG). The fusions are driven by 452 bp of the YOP1 promoter and contain the endogenous gene terminator (441 bp, 378 bp, and 553 bp of the noncoding 3' region for SED5, GOS1, and SFT2, respectively). GFP-YIP1 was constructed by inserting yEGFP after the initiator methionine in pRS315-YIP1, leaving the endogenous promoter intact. This construct (pRC693) was expressed at wild-type levels in haploid cells as the only source of YIP1. Cells containing GFP fusion plasmids were examined with a microscope (Eclipse E600; Nikon) equipped with a 60x objective and 2x optovar. A Spot-RT monochrome CCD camera (Diagnostic Instruments)with version 3.5 software was used for image capture. All images shown are representative images from cells during logarithmic phase growth in minimal media supplemented as necessary.
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Acknowledgments |
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This work was supported by grants from the American Heart Association (0030316T) and the National Science Foundation (MCB0079045) to R.S. Collins, and from the National Institutes of Health (GM52549) to C. Barlowe.
Submitted: 23 June 2003
Accepted: 26 August 2003
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References |
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---|
Allan, B.A., B.D. Moyer, and W.E. Balch. 2000. Rab1 recruitment of p115 into a cis-SNARE complex: programming budding of COPII vesicles for fusion. Science. 289:444448.
Ausubel, R.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1987. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-InterScience, New York. 3.0.13.14.3.
Baker, D., L. Hicke, M. Rexach, M. Schleyer, and R. Schekman. 1988. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell. 54:335344.[Medline]
Barlowe, C. 1997. Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. J. Cell Biol. 139:10971108.
Barlowe, C., L. Orci, T. Yeung, M. Hosobuchi, S. Hamamoto, N. Salama, M. Rexach, M. Ravazzola, M. Amherdt, and R. Schekman. 1994. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the ER. Cell. 77:895907.[Medline]
Barrowman, J., W. Wang, Y. Zhang, and S. Ferro-Novick. 2003. The Yip1p/Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles. J. Biol. Chem. 278:1987819884.
Becker, J., T.J. Tan, H.-H. Trepte, and D. Gallwitz. 1991. Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity. EMBO J. 10:785792.[Abstract]
Belden, W.J., and C. Barlowe. 2001a. Deletion of yeast p24 genes activates the unfolded protein response. Mol. Biol. Cell. 12:957969.
Belden, W.J., and C. Barlowe. 2001b. Role of Erv29p in collecting soluble secretory proteins into ER-derived transport vesicles. Science. 294:15281531.
Calero, M., and R.N. Collins. 2002. Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins. Biochem. Biophys. Res. Commun. 290:676681.[CrossRef][Medline]
Calero, M., N.J. Winand, and R.N. Collins. 2002. Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors. FEBS Lett. 515:8998.[CrossRef][Medline]
Calero, M., C.Z. Chen, W. Zhu, N. Winand, K.A. Havas, P.M. Gilbert, C.G. Burd, and R.N. Collins. 2003. Dual prenylation is required for Rab protein localization and function. Mol. Biol. Cell. 14:18521867.
Cao, X., and C. Barlowe. 2000. Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes. J. Cell Biol. 149:5565.
Cao, X., N. Ballew, and C. Barlowe. 1998. Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J. 17:21562165.
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. 292:13731376.
Cormack, B.P., G. Bertram, M. Egerton, N.A. Gow, S. Falkow, and A.J. Brown. 1997. Yeast-enhanced green fluorescent protein (yEGFP) a reporter of gene expression in Candida albicans. Microbiol. 143:303311.[Abstract]
Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 312319.
Hosobuchi, M., T. Kreis, and R. Schekman. 1992. SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of the yeast coatomer. Nature. 360:603605.[CrossRef][Medline]
Kaiser, C., and R. Schekman. 1990. Distinct sets of SEC genes govern transport vesicle formation and fusion in the early secretory pathway. Cell. 61:723733.[Medline]
Liu, Y., and C. Barlowe. 2002. Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function. Mol. Biol. Cell. 13:33143324.
Matern, H., X. Yang, E. Andrulis, R. Sternglanz, H.-H. Trepte, and D. Gallwitz. 2000. A novel Golgi membrane protein is part of a GTPase-binding protein complex involved in vesicle targeting. EMBO J. 19:44854492.
Matsuoka, K., L. Orci, M. Amherdt, S.Y. Bednarek, S. Hamamoto, R. Schekman, and T. Yeung. 1998. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell. 93:263275.[Medline]
McNew, J.A., J.G. Coe, M. Sogaard, B.V. Zemelman, C. Wimmer, W. Hong, and T.H. Sollner. 1998. Gos1p, a Saccharomyces SNARE protein involved in Golgi transport. FEBS Lett. 435:8995.[CrossRef][Medline]
Mellman, I., and G. Warren. 2000. The road taken: past and future foundations of membrane traffic. Cell. 100:99112.[Medline]
Miller, E., B. Antonny, S. Hamamoto, and R. Schekman. 2002. Cargo selection into COPII vesicles is driven by the Sec24p subunit. EMBO J. 21:61056113.
Novick, P., C. Field, and R. Schekman. 1980. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 21:205215.[Medline]
Otte, S., W.J. Belden, M. Heidtman, J. Liu, O.N. Jensen, and C. Barlowe. 2001. Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and the Golgi complex. J. Cell Biol. 152:503518.
Pelham, H.R.B. 2001. SNAREs and the specificity of membrane fusion. Trends Cell Biol. 11:99101.[CrossRef][Medline]
Rexach, M.F., and R. Schekman. 1991. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J. Cell Biol. 114:219229.[Abstract]
Rexach, M.F., M. Latterich, and R.W. Schekman. 1994. Characteristics of endoplasmic reticulum-derived transport vesicles. J. Cell Biol. 126:11331148.[Abstract]
Schroder, S., F. Schimmoler, B. Singer-Kruger, and H. Riezman. 1995. The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in -COP. J. Cell Biol. 131:895912.[Abstract]
Segev, N. 1991. Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein. Science. 252:15531556.[Medline]
Segev, N. 2001. Ypt and Rab GTPases: insight into functions through novel interactions. Curr. Opin. Cell Biol. 13:500511.[CrossRef][Medline]
Segev, N., J. Mulholland, and D. Botstein. 1988. The yeast GTP-binding Ypt1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 52:915924.[Medline]
Sherman, F. 1991. Getting started with yeast. Methods Enzymol. 194:320.[Medline]
Sikorski, R.S., and P.A. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Tang, B.L., Y.S. Ong, B. Huang, S. Wei, E.S. Wong, R. Qi, H. Horstmann, and W. Hong. 2001. A membrane protein enriched in endoplasmic reticulum exit sites interacts with COPII. J. Biol. Chem. 276:4000840017.
Tsui, M.M.K., W.C.S. Tai, and D.K. Banfield. 2001. Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions. Mol. Biol. Cell. 12:521538.
Ward, T.H., R.S. Polishchuk, S. Caplan, K. Hirschberg, and J. Lippincott-Schwartz. 2001. Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol. 155:557570.
Wilson, D.W., C.A. Wilcox, G.C. Flynn, E. Chen, W. Kuang, W.J. Henzel, M.R. Block, A. Ullrich, and J.E. Rothman. 1989. A fusion protein is required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 339:355359.[CrossRef][Medline]
Winston, F., C. Dollard, and L.L. Ricupero-Hovasse. 1995. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 11:5355.[Medline]
Wooding, S., and H.R.B. Pelham. 1998. The dynamics of Golgi protein traffic visualized in living yeast cells. Mol. Biol. Cell. 9:26672680.
Wright, R., M. Basson, L. D'Ari, and J. Rine. 1988. Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus. J. Cell Biol. 107:101114.[Abstract]
Yang, X., H.T. Matern, and D. Gallwitz. 1998. Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p. EMBO J. 17:49544963.