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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.016998
Research Article |
1 Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tübingen, Germany
2 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
3 Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany
Author for correspondence (e-mail: anne.spang{at}unibas.ch)
Accepted 28 January 2008
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Summary |
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F-HDEL (glycolylated pro--factor with an HDEL tag at the C-terminus) was blocked in 
ypt1/SLY1-20 membranes at the restrictive temperature in vitro. Moreover, Ypt1p and the ER-resident t-SNARE Ufe1p interact genetically and biochemically, indicating a role for Ypt1p in consumption of COPI vesicles at the ER. Ypt1p is also essential for the maintenance of the morphology and the protein composition of the Golgi. Interestingly, the concentrations of the Golgi enzymes Anp1p and Mnn1p, the cargo protein Emp47p and the v-SNARE Sec22p were all substantially reduced in Golgi from a ypt1/SLY1-20 strain as compared with wild-type Golgi, while the concentration of Arf1p and of coatomer were mildly affected. Finally, COPI vesicles generated from ypt1/SLY1-20 Golgi membranes in vitro were depleted of Emp47p and Sec22p. These data demonstrate that Ypt1p plays an essential role in retrograde transport from the Golgi to the ER.
Key words: ER-Golgi shuttle, YPT1, Rab, Retrograde transport, Yeast
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Introduction |
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Fusion of both COPII and COPI vesicles with their respective target membranes is tightly regulated. In principle, the general fusion process can be subdivided into tethering, docking and fusion (Bonifacino and Glick, 2004
). Tethering factors at the Golgi are Uso1p, TRAPPI and the COG complex, whereas at the ER membrane this function seems to be fulfilled by Dsl1p, Tip20p and probably other yet unspecified proteins (Andag et al., 2001; Kamena and Spang, 2004; Ram et al., 2002; Reilly et al., 2001). Docking of vesicles employs Rab/Ypt proteins. Ypt1p is the Rab protein required for anterograde ER-Golgi transport, whereas the Rab essential for retrograde transport remains elusive (Segev, 2001b). Finally, a recognition step mediated by the SNARE proteins leads to close opposition of the membranes of the vesicle and the target compartment and subsequently to fusion of the lipid bilayers. The precise mechanism by which fusion occurs is still a matter of debate (Peters et al., 2001; Weber et al., 1998).
Rab/Ypt proteins are small GTPases of the ras superfamily. They cycle between a GTP-bound (active) and a GDP-bound (inactive) form. The yeast genome encodes 11 YPTs. Ypt51/52/53p, Ypt10p and Ypt7p act in endocytosis en route to the vacuole, whereas Ypt1p, Ypt31p and Ypt32p function in exocytosis (Buvelot Frei et al., 2006; Segev, 2001a). Ypt6p is thought to be involved in both endocytosis and exocytosis (Li and Warner, 1996). Ypt11p seems to be involved in ER inheritance (Buvelot Frei et al., 2006). Ypt1p is crucial for the ER to Golgi transport and is also important for intra Golgi transport (Bacon et al., 1989; Baker et al., 1990; Jedd et al., 1995; Segev et al., 1988). Ypt31p and Ypt32p are partially functionally redundant and are both involved in protein exit from the trans-Golgi (Benli et al., 1996; Jedd et al., 1997). The switch between the GDP-bound and the GTP-bound state is mediated by specific guanine nucleotide exchange factors (GEFs) whereas GTPase activating proteins (GAPs) regulate the hydrolysis of GTP. GDI, the GDP dissociation inhibitor, is an additional regulator of Rab/Ypt proteins that sequesters the GDP-bound form of the GTPase and prevents the exchange of GDP for GTP, thereby inhibiting recruitment to the membrane (Araki et al., 1990; Garrett et al., 1994; Sasaki et al., 1990). A single GDP dissociation inhibitor, Gdi1p, has been identified in Saccharomyces cerevisiae. GDI1 is essential for cell viability and can probably act on all Ypt proteins (Dirac-Svejstrup et al., 1994; Haas et al., 1995; Peter et al., 1994; Ullrich et al., 1993).
Despite the importance of Rab/Ypt proteins and although vesicle fusion at the ER-Golgi interface has been subject of intensive research, it is still not known which Rab/Ypt protein functions at the fusion step of COPI vesicles with the ER. In this study, we identify Ypt1p as the Rab involved in the retrograde transport from the Golgi to the ER. In addition, we provide evidence for an additional function of Ypt1p in maintaining Golgi identity and integrity.
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Results |
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F-HDEL from the Golgi to ER in vitro). The round-trip assay has been used successfully to characterize several proteins involved in retrograde transport from the Golgi to the ER (Kamena and Spang, 2004; Poon et al., 1999; Spang et al., 2001). Essentially, the round-trip assay recapitulates the transport of a reporter protein from the ER to the Golgi complex and back to an acceptor ER (supplementary material Fig. S1). As a reporter we use radioactively labeled prepro--factor with an HDEL tag at the C-terminus ([35S]ppF-HDEL) (Dean and Pelham, 1990). A glucan trimming reaction provides the means to determine successful retrograde transport to the acceptor ER. Trimmed and untrimmed forms can be distinguished by their differential mobilities on SDS-PAGE, and the trimmed form of [35S]gpF-HDEL is produced only upon successful return to the ER. The untrimmed band represents [35S]gpF-HDEL present in the Golgi or in vesicles that have docked but not fused and cannot be correlated to the amount of trimmed [35S]gpF-HDEL.
We first asked, whether retrograde transport actually requires the action of a Rab protein. The GDP dissociation inhibitor Gdi1p binds the GDP-bound form of Rab proteins and can inhibit the exchange of GDP for GTP (Sasaki et al., 1990). We took advantage of the presence of only one Rab-specific GDI in yeast, which most probably acts on all yeast Rabs (Ypts). We performed a round-trip assay and compared the retrieval efficiency of [35S]gpF-HDEL to the ER in the presence and in the absence of exogenously added purified Gdi1p. The amount of [35S]gpF-HDEL that reached the ER in the presence of Gdi1p was significantly reduced compared with the control (Fig. 1A, compare lanes 1 and 2). This reduction of the signal in the presence of Gdi1p was independent of its ability to inhibit COPII vesicle fusion with the Golgi because purified Gdi1p was only added to the reaction after the COPII vesicles had fused with the Golgi. Furthermore, after the fusion of the COPII vesicles with the Golgi, the membranes were washed extensively prior to the addition of cytosol, in order to avoid any residual fusion activity of COPII vesicles with the Golgi complex. Therefore, Gdi1p blocks retrieval of [35S]gpF-HDEL to the ER in vitro, and therefore establishes a requirement for a Ypt protein in retrograde transport from the Golgi to the ER.
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Retrieval of [35S]gpF-HDEL is defective in ypt1/SLY1-20 in vitro
The result above indicates that retrograde transport depends on the action of at least one Ypt. We sought to determine which Ypt is involved in retrograde transport from the Golgi to the ER and decided to follow those Rabs involved in exocytosis, namely Ypt1p, Ypt31/32p and Ypt6p. YPT1 is essential, but the loss of YPT1 can be rescued by the expression of a mutation in SLY1, SLY1-20, which renders the strain temperature sensitive (ts) (Dascher et al., 1991; Ossig et al., 1991). Sly1p was isolated as suppressor of YPT1 loss of function. Sly1p is a member of the Sec1p family and may promote target SNARE complex formation at the Golgi (Dascher et al., 1991; Kosodo et al., 2002; Peng and Gallwitz, 2002). We also used a second ts mutant, ypt1-3, which has been used extensively for in vivo studies (Cao et al., 1998; Morsomme and Riezman, 2002). The ypt31/ypt32A141D is a temperature-sensitive strain bearing a point mutation in YPT32 in a ypt31 background (Jedd et al., 1997). YPT6 is a non-essential gene but deletion of the gene results in growth defects at 37°C (Li and Warner, 1996). The use of semi-intact cells derived from these strains in the round-trip assay combined with a temperature shift in the last step of the assay should enable us to determine, which Ypt is required for retrograde transport from the Golgi to the ER.
When the round-trip assay was performed at the permissive temperature, the retrieval of [35S]gpF-HDEL in the ypt mutant membranes was indistinguishable from that in the wild-type membranes (Fig. 1B). By contrast, when we shifted the incubation temperature in the last stage (retrieval from the Golgi to the ER) of the round-trip assay to 30°C, which should be at least a semi-restrictive temperature for these mutants, the amount of retrieved [35S]gpF-HDEL to the ER in ypt1/SLY1-20 membranes was strongly reduced (Fig. 1C, lane 2). The presence of the SLY1-20 plasmid itself did not exert any negative effect on the retrograde transport, because the retrieval efficiency of [35S]gpF-HDEL was not altered in a wild-type strain expressing SLY1-20 (Fig. 1D). All other mutants, including ypt1-3, showed retrieval efficiencies similar to that of the wild type (Fig. 1C,E). The temperature shift to 30°C in the last step of the assay might still be permissive for ypt1-3 in vitro. However, raising the temperature further is not possible in this in vitro system, because the membranes become leaky and make the interpretation of the assay impossible. In addition, Ballew et al. (Ballew et al., 2005) showed that Ypt6p becomes essential in ypt1/SLY1-20, indicating that it can take over part of the Ypt1p functions. Therefore, it is possible that Ypt6p compensates for the transport defect in ypt1-3 in vitro. Nonetheless, the results indicate that the defect in retrograde transport might be due to the loss of Ypt1p function in ypt1/SLY1-20.
Ypt1-3 semi-intact cells are permissive for anterograde transport to the Golgi in vitro
To determine whether ypt1-3 might be functional at 30°C in vitro, we performed an anterograde ER-Golgi transport assay (Fig. 2). The role of Ypt1p in fusion of COPII vesicles with the Golgi is well established (Cao et al., 1998). Furthermore, a constitutive defect at the permissive temperature in the fusion of COPII vesicles with the Golgi could potentially abolish the round-trip and lead to a reduction of the amount of trimmed [35S]gpF-HDEL as was seen with the ypt1/SLY1-20 strain. To test this possibility, we measured the amount of anti--1,6-linked mannose-precipitable [35S]gpF-HDEL, which is an indicator of the arrival of the reporter in the Golgi. At the permissive temperature, the amount of precipitable [35S]gpF-HDEL was similar in all membranes tested (Fig. 2A). Additionally, as expected, at the restrictive temperature, the ypt1/SLY1-20 mutant strain displayed a drastic reduction in the transport of [35S]gpF-HDEL to the Golgi (Fig. 2B). This result confirms that there is no constitutive defect in anterograde transport in ypt1/SLY1-20 under the conditions of our assay. However, the ypt1-3 semi-intact cells were permissive for anterograde transport from the ER to the Golgi in vitro even at the restrictive temperature (Fig. 2B). We conclude, therefore, that the decrease in function of the ypt1-3 mutant is insufficient to be detectable in our assay. Taken together, these results suggest that Ypt1p is essential for retrograde transport form the Golgi to the ER.
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To substantiate our finding that Ypt1p is involved in retrograde transport, we scored the secretion of Kar2p. The chaperone Kar2p is an ER-resident protein, which can escape to the Golgi and is retrieved back to the ER by the HDEL-receptor Erd2p (Semenza et al., 1990). However, if retrograde transport is defective, Kar2p is secreted. We assessed secretion of Kar2p by colony blot after incubation at 30°C (Fig. 2C). We chose 30°C as semi-permissive temperature for the secretion assay, because all strains still grew at about the same rate at this temperature, so that unspecific effects caused by differences in cell number and growth behavior could be excluded. Although no Kar2p was secreted in the wild-type strain, both ypt1-3 and ypt1D136A secreted Kar2p efficiently, similar to that of tip20-5 and dsl1-22, two retrograde transport mutants with established Kar2p secretion phenotypes (Andag et al., 2001; Cosson et al., 1997). Hence ypt1-3 and ypt1D136A are defective in retrograde transport in vivo. This phenotype can probably be compensated for in our in vitro assays. The ypt1/SLY1-20 strain also secreted Kar2p, though to a lesser degree. This result was not unexpected, since ypt1/SLY1-20 is a much stronger mutant than the other ypt1 alleles in the assay and has defects at lower temperatures. Anterograde transport is likely to be more strongly affected in ypt1/SLY1-20 than in ypt1-3 and ypt1D136A. The ypt6 and ypt31/32 mutants also showed some Kar2p secretion, which is most likely due to an interference with Golgi function. However, a more direct effect is also possible at least for Ypt6 (Ballew et al., 2005). The Kar2p signal was not due to cell lysis, because the cytoplasmic protein Pgk1p was never detected for any of the strains grown at 30°C. Taken together, we find that multiple mutants in YPT1 secrete Kar2p, supporting a role for Ypt1p in retrograde transport from the Golgi to the ER.
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ypt1/SLY1-20 Golgi membranes are unable to bud functional COPI vesicles in vitro). Yet, recent investigations have shown that Ypt1p is also required for the sorting of GPI-anchored cargo molecules into ER-derived vesicles (Morsomme and Riezman, 2002). Furthermore, another recent study reports a role for Rab1, the mammalian orthologue of Ypt1p, in cargo uptake into COPI vesicles from the Golgi (Alvarez et al., 2003). If Ypt1p plays a similar role in yeast, then the defect observed in our round-trip assay might arise either from a failure in COPI vesicle generation at the Golgi or, alternatively, from defective consumption of COPI vesicles at the ER. Therefore, we investigated a possible role for Ypt1p in COPI vesicle formation in an in vitro Golgi budding assay (Spang and Schekman, 1998). Enriched Golgi membranes from wild-type or ypt1/SLY1-20 cells were incubated with coatomer, Arf1p and guanine nucleotide. The resulting COPI vesicles were purified, first over a sedimentation gradient and then by buoyant density centrifugation. As expected, the wild-type membranes formed normal COPI-coated vesicles as judged by the presence of the cargo Emp47p and the v-SNARE Sec22p in the vesicle fraction (Fig. 3A). By contrast, Golgi membranes from the ypt1/SLY1-20 mutant formed abnormal COPI-coated vesicles (Fig. 3B). Although, a coatomer signal was obtained in higher migrating fractions, which indicates a lower buoyant density, these fractions contained much less Emp47p and Sec22p (Fig. 3B). Thus, these vesicles might represent at least partially defective COPI vesicles because they did not contain normal amounts of Sec22p (Spang and Schekman, 1998). Together, these data support a requirement of Ypt1p in the formation of COPI-coated vesicles at the Golgi.
The Golgi is altered in ypt1/SLY1-20 mutant cells
Does the defect in COPI vesicle generation arise form a partially dysfunctional Golgi complex? To investigate this possibility, we first compared, by immunoblotting, the content of different Golgi proteins and Golgi-associated proteins in wild-type and ypt1/SLY1-20 Golgi membranes. As for the experiments above, the Golgi used for this analysis was enriched from cells that had been grown at the permissive temperature for ypt1/SLY1-20, 23°C. Surprisingly, the concentrations of the Golgi enzymes Anp1p and Mnn1p, the cargo Emp47p and the v-SNARE Sec22p were all dramatically reduced, whereas the concentration of coatomer remained constant and the level of Arf1p was increased (Fig. 4A,B). About half of the t-SNARE Sed5p and the v-SNARE Bos1p were lost in the ypt1/SLY1-20 Golgi. Expression of SLY1-20 in the wild-type background also caused a reduction of Anp1p, cargo and SNAREs, although this reduction was less dramatic. This loss of proteins from the SLY1-20 Golgi did not cause any obvious growth phenotype (data not shown) nor did it interfere with COPI vesicle formation in vitro.
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Sed5p-GFP and Emp47p-myc accumulate in the ER and Anp1p-GFP is dispersed in ypt1/SLY1-20 mutants
Next, we wanted to determine the fate of the proteins lost from ypt1/SLY1-20 Golgi membranes. They could either be degraded or accumulate in other organelles or structures in the cell. To distinguish between these two possibilities, we compared the protein levels in total yeast lysates derived from strains grown at 23°C or shifted for 1 hour to 37°C, which allows the induction of a comparable acute response in the different strains. Surprisingly, no big changes in the protein level of Golgi or Golgi-associated proteins were detected irrespective of the strain background or the temperature (Fig. 4C). Therefore, we next determined the subcellular localization of the Sed5p-GFP, Emp47p-myc, Anp1p-GFP and Sec7p-dsRed in wild-type, WT/SLY1-20 and ypt1/SLY1-20 at 23°C or after shifting to 37°C (Fig. 5). Consistent with the immunoblot data, all proteins were detected also at 37°C. Although no significant difference in protein localization was observed upon shift to 37°C in wild-type and WT/SLY1-20 cells, Sed5pGFP and Emp47p-myc accumulated in the ER and the Anp1p-GFP and the Sec7p-dsRed signal became diffuse in ypt1/SLY1-20. Moreover, a partial defect had been observed already for Emp47p, Anp1p and Sec7p at 23°C, indicating that this mutant has defects even at 23°C and these defects gradually increase with a rise in temperature. The effects observed could be due to a strong decrease in anterograde traffic, a defect in retrograde transport or a combination of both, because Ypt1p is required for the fusion of vesicles at the Golgi and the ER. Taken together our data suggest that Ypt1p plays a role in the maintenance of the Golgi complex.
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ypt1/SLY1-20 Golgi is at least partially functional at 23°Cypt1/SLY1-20 cells, we wondered whether efficient glycosylation could still take place. Surprisingly, the extent of glycosylation observed in the ypt1/SLY1-20 mutant strain was comparable to or even greater than that in the wild type, independent of the incubation temperature (Fig. 6A). This result confirms that, despite the loss of Anp1p and other proteins, the Golgi was still functional. A similar result was observed when we analyzed a mutant in the ARF-GEF SEC7, which possesses an abnormal Golgi morphology at the restrictive temperature (Achstetter et al., 1988). In addition, ypt1/SLY1-20 cells were not osmo-sensitive, which indicates that the extracellular matrix was functional. Taken together, our results suggest that despite the lack of at least one glycosylation enzyme, glycosylation in the Golgi occurred very efficiently.
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ypt1/SLY1-20 at 23°C. However, the processing of CPY seemed to occur normally, indicating that the Golgi must be at least partially functional. As expected, the p1 of CPY accumulated in the ypt1/SLY1-20 cells at 37°C as the transport to the Golgi was blocked.
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ypt1/SLY1-20 cells even at 23°C. In addition the vacuole seemed fragmented (compare Fig. 7A,B with C,D). Upon shift of ypt1/SLY1-20 cells for 1 hour to 37°C, no normal Golgi cisternae could be identified, the ER was dilated and vesicle accumulations were frequently observed, consistent with a block in the ER-Golgi transport shuttle (Fig. 7G). Furthermore, lipid-rich structures were present (Fig. 7G,H). Therefore, ypt1/SLY1-20 cells have a vesicle transport and a Golgi morphology defect.
Ypt1p interact physically and genetically with Ufe1p
Our results suggest a role for Ypt1p in Golgi maintenance and in the generation of functional COPI vesicles. Is Ypt1p also required for COPI vesicle consumption? If so, we should be able to establish a direct interaction between Ypt1p and an ER t-SNARE. Ypt1p binds to the t-SNARE Sed5p at the Golgi (Lupashin and Waters, 1997). Similarly, Ypt1p should bind the t-SNARE on the ER membrane protein Ufe1p. We performed GST-pull down assays using GST-SNARE fusion proteins and purified His6-Ypt1p. GST-Ufe1p as well as GST-Sed5p could specifically recruit His6-Ypt1p (Fig. 8, lanes 2 and 6). By contrast, the GST fusion protein of Snc1p, which functions in post Golgi transport, bound only background amounts of His6-Ypt1p (Fig. 8, compare lanes 8 and 10). GST-Sec22p binding was slightly over the background (Fig. 8, lane 4). Binding of His6-Ypt1p to GST-Ufe1p was specific because when we used His6-Ypt7p instead of His6-Ypt1p no association with GST-Ufe1p was detected (data not shown). Ypt7p is the Rab protein required for all fusion steps with the vacuole. These results demonstrate a direct physical interaction between the Rab Ypt1p and the ER t-SNARE Ufe1p. To corroborate these findings, we aimed to establish a genetic interaction between YPT1 and UFE1. For that we crossed the temperature-sensitive ypt1-3 strain with a strain in which UFE1 was chromosomally deleted and the temperature-sensitive ufe1-1 mutation was present on a CEN plasmid (Fig. 9A). Sixty-eight tetrads were dissected, 21 of which had only two viable spores at 23°C and 37°C, indicating that they carried the wild-type alleles of YPT1 and UFE1. Because ufe1-1 was only present on a plasmid, we determined the transmission rate during sporulation (Fig. 9B). The plasmid was transmitted in the wild-type spores with 36% efficiency. Given this transmission rate, we should have been able to recover tetrads with three or four spores growing at 23°C and only two spores growing at 37°C, reflecting the temperature-sensitive phenotype of pufe1-1 ufe1 and ypt1-3 (Fig. 9A). However, this combination was never obtained, demonstrating that UFE1 and YPT1 interact genetically. By contrast crossing ypt1-3 with tip20-8, a mutant that was recently shown to allow back fusion of COPII vesicles with the ER (Kamena and Spang, 2004), we could isolate viable double mutant haploids, indicating that the genetic interaction between YPT1 and UFE1 is indeed specific. These results demonstrate that Ypt1p interacts with Ufe1p both physically and genetically, consistent with an involvement of Ypt1p in the fusion process of COPI vesicles with the ER.
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Discussion |
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Ypt1p is required for sorting of GPI-anchored proteins into COPII vesicles at the ER (Morsomme and Riezman, 2002) and the role of Ypt1p in fusion of COPII vesicles with the Golgi is well established (Cao et al., 1998; Cao and Barlowe, 2000). However, no requirement of Ypt1p in retrograde transport from the Golgi to the ER had been demonstrated. Using an in vitro assay that recapitulates retrograde transport from the Golgi to the ER, we were able to show that Ypt1p is involved in this pathway. Consistent with data by Morsomme and Riezman (Morsomme and Riezman, 2002) on the role of Ypt1p in cargo inclusion we found that, in the absence of Ypt1p, COPI vesicles were still formed at the Golgi, yet these vesicles contained less of the cargo Emp47p and of the v-SNARE Sec22p than did vesicles from wild-type cells. These COPI vesicles might only contain minor amounts of cargo, because the protein to lipid ratio was significantly decreased compared with COPI vesicles derived from wild-type Golgi membranes. In mammalian cells, the overexpression of a dominant-negative form of Rab1 causes displacement of coatomer from Golgi membranes and a BFA phenotype, indicating that Rab1 is required in a Arf1-dependent recruitment step of coatomer at the Golgi (Alvarez et al., 2003). In yeast, we could not detect a loss of coatomer from Golgi membranes in the ypt1/SLY1-20 mutant. However, the expression of SLY1-20 might prevent the dissociation of coatomer from the Golgi. Yet, the protein composition of the mutant Golgi was altered. Surprisingly, the cells still performed glycosylation efficiently, and the cells were still inert against osmotic stress. In addition, we observed a change in morphology of the Golgi or a loss of Golgi cisternae by electron microscopy, which indicates that neither the absolute concentration of some Golgi proteins or peripheral Golgi proteins nor the Golgi morphology are important for survival in yeast.
Finally, to complete the cycle, we propose that Ypt1p is required for fusion of COPI vesicles with the ER. This hypothesis is based on our demonstration of a direct interaction between Ypt1p and the ER t-SNARE as well as on genetic analysis. Collectively, these data suggest that Ypt1p is not only involved in vesicle consumption but also vesicle generation at each step in the ER-Golgi shuttle. This rather surprising mechanism might be re-used at different places in the cell by different Rabs/Ypts. Recently, Vonderheit and Helenius (Vonderheit and Helenius, 2005) suggested a role for Rab7 in cargo sorting at the early endosome and in the formation of late-endosome-targeted transport vesicles. Rab7 has been implicated previously in fusion of transport intermediates with the late endosome and the lysosome (Bucci et al., 2000; Meresse et al., 1995) and the conversion from early to late endosomes (Poteryaev et al., 2007; Rink et al., 2005). Whether Rab7 is necessary for the generation of vesicles from the late endosomes that might be targeted to the TGN to recycle SNAREs and other transport factors remains to be determined. Yet, a common picture seems to emerge where the role of Ypts/Rabs is not limited to docking and fusion of vesicles and organelles, but also extends to cargo recruitment and transport carrier formation. However, this also implies that the regulation of the Ypts/Rabs must be more complicated than thus far anticipated. A focus of research in the near future must be to identify new upstream regulators of Rab proteins, in order to understand their regulation, which can no longer be limited to cycles of activation and inactivation.
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). Yeast strains used in this study are listed in Table 1.
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Polyclonal rabbit antibodies directed against coatomer, Arf1p, Sec22p, Bos1p, Sed5p, Emp47p, Kar2p, -1,6-linked mannose residues, Anp1p, Mnn1p, and Ypt1p and mouse monoclonal anti-myc antibodies were used in this study. His6-Ypt7p was detected using the Super Signal West HisProbe Kit (Pierce Biotechnology Inc., Bonn, Germany)
Preparation of perforated yeast spheroplasts and cytosol
Perforated yeast spheroplasts (semi-intact cells) were prepared as described previously (Spang and Schekman, 1998).
To obtain cytosol, yeast cells were grown to early- to mid-log phase in YPD medium at either 23°C or 30°C. Cells were harvested by centrifugation and washed twice with water. The cell pellet was resuspended in a minimal volume of buffer B88 (20 mM Hepes pH 6.8, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate) and pipetted into liquid nitrogen. The cell beads were ground up under liquid nitrogen in a blender (Worthington Biochemical, Lakewood, NJ) for large-scale preparations or in a mortar for small-scale preparations. The cell powder was thawed in an ice-water bath, and was then diluted 1:1 with B88, and 1 mM DTT (dithiothreitol) and protease inhibitors were added. The lysate was centrifuged (5 minutes at 3000 g, 15 minutes at 20,000 g, 1 hour at 100,000 g). The 100,000 g supernatant was collected, carefully avoiding the pellet and the lipids that floated to the top.
Purification of coatomer, Sec23/24p, Sec13/31p, Sar1p, Lma1p, Uso1p, Sec18p, Ypt1p, Ypt7p and N-myristoylated Arf1p
The purifications of Sar1p, Sec23/24p, Sec13/31p, coatomer, myc-tagged Uso1p, Sec18-His6, N-myr-yArf1p and the Lma1p complex were performed as described previously (Barlowe, 1997; Barlowe et al., 1994; Hosobuchi et al., 1992; Kahn et al., 1995; Salama et al., 1993; Xu et al., 1997). His6-Ypt1p and His6-Ypt7p were prepared following the N17-Arf1p-His6 purification protocol (Rein et al., 2002).
In vitro round-trip assay
Stage I
Translocation. The translocation reaction using [35S]ppF-HDEL and gls1-1 as donor membranes was performed as described previously (Spang and Schekman, 1998).
Stage II
Budding. To the membranes of the stage I reaction, we added 25 µg/ml Sar1p, 25 µg/ml Sec23/24p complex, 75 µg/ml Sec13/31p complex, 50 µM GTP and an ATP regeneration system (Baker et al., 1988). The reaction mixture was incubated for 30 minutes at 20°C, chilled for 5 minutes on ice, and subjected to a medium speed centrifugation (12,000 g, 30 seconds), which retained COPII vesicles in the supernatant fraction (MSS). An aliquot of the MSS was saved to determine the efficiency of the retrograde transport.
Stage III
Fusion. The MSS from stage II was supplemented with an ATP regeneration system, 50 µM GTP, 1 µM Lma1p, 1 µg/ml Sec18p, 1.5 µg/ml Uso1p and 600 µg/ml perforated spheroplast membranes from the GLS1 strain. Fusion was allowed to take place for 20 minutes at 20°C.
Stage IV
Retrieval. Cytosol was added to a final concentration of 2 mg/ml or the same volume of B88 was added to the non-cytosol control. Reactions were incubated for 30 minutes at either 23°C (permissive temperature for ts strains) or 30°C (semi-permissive temperature for ts strains). The reaction mixture was chilled on ice for 5 minutes and the acceptor ER sedimented by centrifugation at 12,000 g for 30 seconds. The pellet was washed once with 2.5 M urea in B88 for 10 minutes on ice and twice with B88. Fusion with the acceptor ER was measured by precipitation of protease-protected [35S]gpF-HDEL with concanavalin A-Sepharose followed by separation of untrimmed [35S]gpF-HDEL from trimmed [35S]gpF-HDEL by SDS-PAGE. The read out takes advantage of the different glycosylation patterns of the reporter in the donor ER and the acceptor ER. The donor ER is defective in glucosidase I, the enzyme, which together with glucosidase II, is responsible for trimming the N-glycans of glucosylated proteins prior to their ER exit. The acceptor membranes always contained a functional glucosidase I, so that successful retrieval of the reporter to the acceptor ER is monitored by trimming of the N-glycans. Trimmed and untrimmed forms of [35S]gpF-HDEL have different electrophoretic mobilities and can be discriminated by SDS-PAGE. In the experiments using Gdi1p, the membranes were washed twice with B88 after stage III and retrieval was performed either in presence of purified Gdi1p (1.72 µg/ml) or complemented with the same volume of B88.
In vitro forward-transport assay
Stage I
Translocation. The translocation reaction using either [35S]ppF-HDEL or [35S]ppF was performed as described above.
Stage II
Transport. An aliquot (10 µl) of the stage I membranes was incubated with an ATP regeneration system, 50 µM GTP, and 2.5 mg/ml cytosol for 1 hour at 30°C. Each reaction was performed in quadruplicate, and the final volume of each reaction was 50 µl. After the incubation, the samples were chilled for 5 minutes at 4°C and centrifuged for 30 seconds at 12,000 g. A 30 µl aliquot of the supernatant collected from the meniscus was treated with trypsin followed by trypsin inhibitor as described previously (Rexach and Schekman, 1991). The membranes were solubilized with 1% SDS and heated for 5 minutes at 95°C. For each set of experiments, two reactions were precipitated with concanavalin A-Sepharose or antibodies directed against -1,6-linked mannose modifications and protein A-Sepharose. Washed immunoprecipitates were quantified in a liquid scintillation counter (Beckman Instruments, Krefeld, Germany). The budding efficiency was determined by comparing the amount of protease-protected [35S]gpF-HDEL in the supernatant to the total amount of [35S]gpF-HDEL translocated into the ER in the stage I reaction precipitated with concanavalin A-Sepharose. The efficiency of the fusion of COPII vesicles with the Golgi is given as the ratio of anti--1,6-linked mannose precipitated counts over concanavalin A-Sepharose precipitated counts.
Golgi-budding assay
The in vitro Golgi-budding assay was performed as described by Spang and Schekman (Spang and Schekman, 1998) with modifications. Enriched Golgi membranes were incubated with 0.1 mM GTP, coatomer (100 µg/ml) and Arf1p (80 µg/ml) for 30 minutes at 30°C in a volume of 200 µl. After chilling on ice, samples were loaded on top of a Ficoll-sucrose gradient consisting of 0.4 ml 60% (wt/wt) sucrose, 0.8 ml 7.5% (wt/wt) Ficoll, 1 ml 5%, 4%, 3% and 0.8 ml 2% (wt/wt) Ficoll in 15% sucrose, 20 mM Hepes pH 6.8, and 5 mM magnesium acetate. Vesicles were separated from the Golgi by centrifugation for 2 hours at 35,000 rpm (SW55 rotor; Beckman Instruments). Fractions (400 µl) were collected from the top of the gradient. Fractions 5-7 were pooled, mixed with an equal volume of 80% Nycodenz in 20 mM Hepes pH 6.8, 150 mM potassium acetate, 5 mM magnesium acetate (B150), and overlaid with 600 µl 30%, 25%, 20% and 15%, and 400 µl 10% Nycodenz in B150. The gradient was centrifuged for 16 hours at 40,000 rpm (SW55 rotor). Fractions (300 µl) were collected from the top, TCA-precipitated, and analyzed by immunoblotting.
CPY pulse-chase analysis
Cells were grown to an OD600 of 1, and the cell equivalent of an OD600 of 2.5 was resuspended in 200 µl HC-MET-D. Cells were preincubated for 15 minutes, incubated at 23°C or 37°C for 30 minutes and then labeled for 10 minutes with 100 µCi/ml [35S]methionine (GE Healthcare, Freiburg, Germany). The radioactive product was chased with an excess of methionine and cysteine for the indicated time points. Cells were lysed and carboxypeptidase C (CPY) was immunoprecipitated using anti-CPY antibodies and protein A-Sepharose (GE Healthcare). The immunoprecipitates were resolved by SDS-PAGE, and the bands were visualized with a PhosphoImager (GE Healthcare).
GST pull-down assay
SNARE-GST fusion proteins (5 µg) were immobilized onto 25 µl 50% glutathione-agarose slurry (GE Healthcare) for 30 minutes at 4°C. Unbound proteins were removed by three washes with BBP (25 mM Hepes, pH 6.8, 1 mM DTT, 0.5 mM MgCl2, 300 mM potassium acetate, 0.2% Triton X-100). Beads were incubated with His6-Ypt1p (100 pmol) or His6-Ypt7p (100 pmol) in BBP for 1 hour at 4°C. The total reaction volume was 500 µl. After binding, beads were washed three times with BBP, transferred to a fresh tube, washed once with 20 mM Hepes, pH 6.8, and then heated to 65°C for 10 minutes in sample buffer. Eluted proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining or immunoblot.
Kar2p secretion assay and glycostain
Secretion of the ER-resident Kar2p was assessed as described by Andag et al. (Andag et al., 2001). The detection was performed with antibodies directed against Kar2p or Pgk1p.
For the detection of glycosylated proteins, equal amounts of total yeast lysates were separated by SDS-PAGE and visualized using a Pro-Q Emerald 300 Kit from Molecular Probes (Mobitec, Göttingen, Germany) according to the manufacturer's recommendations. The yeast lysates were prepared according to Spang et al. (Spang et al., 2001).
Fluorescence microscopy and electron microscopy
Yeast cells were grown to early log phase and where indicated shifted for 1 hour to 37°C. The cells were immobilized on ConA-coated slides, mounted with Citifluor and GFP fluorescence was detected using an Axioplan microscope equipped with an Axiocam (Zeiss, Oberkochen, Germany). The detection of Emp47-myc by immunofluorescence was performed as described previously (Spang et al., 2001). For electron microscopy analysis, the cells were frozen under high pressure and treated as described in Sandmann et al. (Sandmann et al., 2003).
Genetic interaction analysis between YPT1 and UFE1
To test for genetic interaction between YPT1 and UFE1, ypt1-3 was crossed to a deletion of UFE1, which is kept alive by the ts-sensitive mutation ufe1-1 on a CEN plasmid. For sporulations, diploids were grown overnight at 37°C as a patch on GNA pre-sporulation plates (5% glucose, 1% yeast extract, 3% nutrient broth, 2% agar). Cells from the patch were incubated for 5 days at 30°C in liquid sporulation medium (1% potassium acetate, 0.005% zinc acetate) on a roller wheel. Tetrads were dissected on YPD plates and incubated at 23°C. Tetrads with two living and two dead spores were analyzed further. The two living spores always carried the wild-type copy of both YPT6 and UFE1. Deletion of UFE1 is lethal by itself. Therefore, we had to determine the inheritance rate of the ufe1-1 ts plasmid in the surviving wild-type spores. Plasmid DNA was isolated and retransformed into E. coli. Thirty-six percent of the wild-type spores contained the ufe1-1 plasmid. However, we never recovered a spore with theypt6 ufe1 pufe1-1 genotype in our tetrad analysis.
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* These authors contributed equally to this work 
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-S, to generate COPI vesicles. The vesicles were separated by velocity sedimentation centrifugation. The vesicle peak was collected and floated on a buoyant density gradient. Fractions were collected from the top, precipitated, and analyzed by immunoblotting. Sec22p is a v-SNARE in the ER-Golgi shuttle and Emp47p is a vesicle cargo. The arrows indicate the movement of the lipid particles in the gradient.
