Article |
Address correspondence to Howard Riezman, Dept. of Biochemistry, University of Geneva, Sciences II, 30 quai E. Ansermet, CH-1211 Geneva, Switzerland. Tel.: 41-22-702-6469. Fax: 41-22-702-6465. email: Howard.Riezman{at}biochem.unige.ch
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Abstract |
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Key Words: membrane trafficking; SNARE; protein sorting; GPI; ER to Golgi
Abbreviations used in this paper: COG, conserved oligomeric Golgi; CPY, carboxypeptidase Y; GPI, glycosylphosphatidylinositol.
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Introduction |
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The mechanism involved in protein sorting upon ER exit is poorly understood. Recently, we showed that the Rab GTPase Ypt1p and the tethering factors Uso1, Sec34p, and Sec35p (two members of the conserved oligomeric Golgi [COG] complex) but not Bet3p (a member of the TRAPP complex) are necessary for sorting of GPI-anchored proteins upon ER exit (Morsomme and Riezman, 2002). Rab GTPases have also been proposed to facilitate cargo packaging on other donor compartments. The Rab9 GTPase seems to facilitate mannose-6-phosphate receptor recruitment for endosome to Golgi traffic (Carroll et al., 2001), and Rab5 seems to be required for ligand sequestration into clathrin-coated pits (McLauchlan et al., 1998). More recently, direct interactions between Rab GTPases and cargo were found, indicating that a cargo protein can bind to a specific Rab protein, which controls its trafficking (Seachrist et al., 2002; van IJzendoorn et al., 2002).
In addition to its function in sorting, the Rab-GTPase Ypt1p, in combination with the tethering factors Uso1p and Sec34/35p, is also involved in tethering of ER-derived vesicles to the Golgi membranes (Cao et al., 1998; VanRheenen et al., 1998, 1999), suggesting that Rab-mediated cargo sorting on donor membranes may be a general mechanism to couple protein sorting and packaging into vesicles to targeting, docking, and fusion. Consistent with this hypothesis, Rab GTPases were shown to recruit tethering factors and interact with SNARE molecules in order to ensure specificity of vesicle fusion with the acceptor membrane. For example, Rab5 was shown to regulate the recruitment of the tethering factor EEA1 into a complex with syntaxin13 (Simonsen et al., 1998; Christoforidis et al., 1999; McBride et al., 1999), and the mammalian homologue of Uso1p, p115, was shown to be recruited at the ER exit sites via Rab1 and to form a cis complex with v-SNAREs (Allan et al., 2000). These findings suggest that v-SNAREs can also participate in the same packaging and/or sorting function as Rab proteins.
Therefore, we decided to test the function of v-SNAREs in the sorting of GPI-anchored proteins using an in vitro assay that reconstitutes the ER-derived vesicle budding into distinct classes of vesicles upon exit from the ER. We found that sorting of GPI-anchored proteins from other secretory proteins was defective when we used membrane extracts from the v-SNARE mutants, bos11, bet11, or sec223, with the bos11 mutant membranes showing the strongest defect. In contrast, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Importantly, the packaging of cargo proteins into distinct vesicles budding from the ER and the Bos1p requirement for this process could be visualized by EM. Moreover, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in vivo in the bos11 mutant. Therefore, we propose that v-SNAREs are essential for protein sorting upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.
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Results |
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ER-derived vesicle fusion with the cis-Golgi compartment can be measured by following the -1,6 mannosylation of Gas1p. This fusion step is Sec18p dependent (Muniz et al., 2001). Here, we tested whether the vesicles produced from SNARE mutants were also deficient for fusion with the Golgi compartment. After a 1-h incubation of SNARE mutant extracts at 30°C, only a very low amount of
-1,6 mannose modification was detected on Gas1p, where >50% of it was modified with wild-type membranes and cytosol (Fig. 1 B). This result suggests that the vesicles we analyzed in our assay with the SNARE mutants are primary ER-derived vesicles because they were virtually incapable of fusion with the Golgi compartment.
We considered the possibility that sec223, bet11, or bos11 membranes are fragile and that the apparent lack of protein sorting was due to the isolation of ER fragments rather than bona fide vesicles that have budded from the ER. To test this, we determined the specificity and nucleotide dependence of vesicle formation using mutant membranes. The sorting defect observed with the sec223, bet11, or bos11 mutants was not due to a budding defect since the budding efficiency of Gap1p and Gas1p were similar to wild-type or sec18 membranes (Fig. 2 A). Packaging of Gas1p and Gap1p into ER-derived vesicles was dependent on cytosol (unpublished data) and on the presence of nucleotides in the assay (Fig. 2 A). Furthermore, resident ER proteins, like Sec61p, were not packaged into vesicles (Fig. 2 B). Finally, we treated the floated fraction of vesicles generated from bos11 membranes with proteinase K and observed that Gas1p was protected from protease digestion in absence of detergent but was digested when detergent was present (Fig. 2 C). This confirms that Gas1p exits the ER in vesicles in our in vitro assay even when using SNARE mutant extracts.
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The presence of anti-SNARE antibodies also reduced the fusion competence of ER-derived vesicles. When the budding reaction was performed with wild-type membranes and cytosol, 62% of Gas1p received -1,6 mannose modification. In contrast, in presence of anti-Bos1p, anti-Sec22p, or anti-Sed5p antibodies only 6 to 15% of Gas1p received
-1,6 mannose modification (unpublished data). This indicates that the anti-SNARE antibodies also blocked fusion of Gas1p-containing vesicles with the Golgi, even in the case of Sed5p where there was no effect on protein sorting.
Bos1p requirement for Gas1p sorting upon ER exit can be visualized by EM
Thus far, there is strong genetic and biochemical evidence that GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins (Sütterlin et al., 1997; Muniz et al., 2001; Morsomme and Riezman, 2002). Although this evidence is solid, we sought to strengthen these findings by visualizing the sorting process using EM. We performed an in vitro budding experiment using sec18 mutant membranes and cytosol as described before and fixed the reactions directly after a 10-min incubation with cytosol and energy. The fixed material was embedded, and thin sections were prepared as described (Prescianotto-Baschong and Riezman, 2002). We performed a double immunolabeling on these sections with antibodies against Gas1p and Gap1p followed by IgG colloidal gold detection. The large gold particles were used to localize Gas1p, and the small gold particles were used to localize Gap1p. In a first set of experiments, we used 10- and 5-nm gold particles for Gas1p and Gap1p detection, respectively. Then, we used 15- and 10-nm gold particles for Gas1p and Gap1p, respectively. Since we wanted to analyze protein sorting upon ER exit, we focused on vesicles in the process of budding from the ER membranes. ER membranes were easily distinguished from other membranes by the high density of attached ribosomes. We could clearly observe vesicles budding from these ER membranes. The majority of these vesicles contained gold particles of only one size, meaning one type of cargo (Fig. 4, A and B). Quantitative analysis revealed that only 14% of the labeled budding vesicles were labeled for both Gas1p and Gap1p. These results are consistent with the biochemical findings shown above and visualize the segregation of Gas1p from Gap1p upon ER exit.
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Sorting of GPI-anchored proteins from other secretory proteins can be measured in vivo and requires functional Bos1p
Sorting of GPI-anchored proteins from other secretory proteins can also be measured in vivo (Morsomme and Riezman, 2002). The recovery of ER-derived vesicles generated in vivo was only possible in mutants defective in tethering of ER-derived vesicles to the Golgi compartment (Sütterlin et al., 1997; Morsomme and Riezman, 2002). As previously, ER-derived vesicles were immunoisolated from radiolabeled Bet3p-depleted cells. 60% of Gap1p signal present in the vesicle fraction was immunoisolated after incubation with anti-HA antibodies, and only 8% of Gas1p signal was coimmunoisolated showing efficient sorting in vivo (Fig. 5 A). Next, ER-derived vesicles were generated in vivo at 37°C, conditions in which ER to Golgi transport is blocked and purified from the bos11 mutant. 66% of the Gap1p signal was immunoisolated from bos11-generated vesicles, and 41% of Gas1p signal was coimmunoisolated, indicating a strong sorting defect in vivo (Fig. 5 A). The vesicle fractions isolated from the different strains did not contain Sec61p, showing that the signals were not generated as a result of ER fragmentation (unpublished data). Moreover, Gas1p was protected from protease digestion in absence of detergent but sensitive to degradation in presence of detergent (Fig. 5 B), demonstrating its presence in a closed membrane compartment.
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Discussion |
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In addition, we provide the first morphological evidence for sorting of cargo proteins upon ER exit. When we used sec18 membranes for the in vitro budding assay and fixed the membranes just after the budding reaction started, we could clearly discriminate between the two different pathways. To ensure that we analyzed the sorting event upon ER exit, we restricted our analysis to vesicles in the process of budding from the ER. The ER membranes were clearly identified by their electron-dense pattern due to the presence of ribosomes. Isolated vesicles containing separate cargoes were also detected, but since their origin was not certain, they were not included in our analysis. The role of Bos1p in sorting GPI-anchored proteins into distinct vesicles was also confirmed by EM. The analysis of budding profiles revealed that the proportion of ER-derived vesicles containing both Gap1p and Gas1p was 52% using bos11 mutant membranes and 14% using wild-type membranes. These numbers correlate well with the in vitro data and confirm the role of Bos1p in sorting of GPI-anchored proteins into distinct vesicles.
Importantly, sorting of GPI-anchored proteins from other secretory proteins upon exit of the ER can also be measured in vivo. The sorting defect observed in vitro for the bos11 mutant was reproduced in vivo. The physiological consequence of missorting can be illustrated by the maturation defect observed for Gas1p in the bos11 mutant at 30°C. Although transport of CPY was slightly delayed, Gas1p was slowly and continuously matured. In theory, this could be due to the slow transport kinetics. However, in the sec223 mutant the kinetics of CPY export was similar to that of bos11 mutant at 30°C, but the maturation of Gas1p was apparently normal albeit with a small delay. The maturation defect of Gas1p is thus specific for the bos11 mutant at 30°C, conditions where sorting is defective. This specific bos11 allele may be more appropriate to observe the predominance of the sorting defect on the fusion defect compared with other mutants such as sec223 or bet11. Unfortunately, we were not able to measure sorting in vivo for sec223 or bet11 mutants because recovery of Gas1p-containing vesicles was too low (unpublished data). One possible explanation for the low recovery could be that tethering of ER-derived vesicles is not defective in these mutants but is defective in the bos11 mutant used in this study. Previously, we have proposed that recovery of ER-derived vesicles from the in vivo sorting assay is efficient only when the vesicles are in an untethered state (Morsomme and Riezman, 2002). The maturation defect of Gas1p observed in the bos11 mutant at 30°C is not due to a general glycosylation defect since both CPY and invertase are glycosylated to the same extent at 24 and 30°C. Moreover, Gas1p travels through the Golgi apparatus because it receives -1,6 mannose modification and reaches the plasma membrane where it can be digested by proteinase K. The fact that Gas1p-containing vesicles did not fuse in vitro at 30°C while Gas1p was transported to the cell surface in vivo at the same temperature indicates that the temperature sensitivity is more stringent in vitro than in vivo. Interestingly, it was shown recently that a soluble form of Gas1p is inefficiently matured in wild-type cells (Watanabe et al., 2002). Indeed, the mutant form of Gas1p (L526R), which is not GPI anchored, is released into the media and shows a slow maturation kinetic. Although there is no data concerning the sorting of the soluble form of Gas1p, we could speculate that the maturation defect reflects a sorting defect. Previous experiments suggested that the two ER-derived vesicle populations could reach two different Golgi domains or membranes (Muniz et al., 2001; Morsomme and Riezman, 2002). According to this hypothesis, the maturation defect of Gas1p could reflect the mistargeting of Gas1p to the wrong Golgi domain, leading to a specific glycosylation defect for Gas1p.
Although we identified several components necessary for protein sorting in the ER, the molecular mechanisms triggering this process are still unknown. Ypt1p was shown to activate v-SNARES on carrier vesicles, conferring them fusion competence (Lian and Ferro-Novick, 1993; Lian et al., 1994) and p115, the mammalian homologue of Uso1p, forms a cis-SNARE complex on the ER exit sites and is recruited to the membrane by the mammalian homologue of Ypt1p, Rab1 (Allan et al., 2000). It was reported recently that SNARE proteins can be concentrated into lipid microdomains (Lafont et al., 1999; Chamberlain et al., 2001; Lang et al., 2001), which might be relevant to GPI-anchored protein transport (Sütterlin et al., 1997; Bagnat et al., 2000) . However, if the formation of lipid rafts in the ER could possibly contribute to the sorting mechanism of GPI-anchored proteins in the ER, proteins are also certainly involved in this process. The SNARE proteins may constitute the link between the cytosolic factors involved in sorting (Ypt1p, Uso1p, and the COG complex) (Morsomme and Riezman, 2002) and the GPI-anchored proteins that are exclusively lumenal. On the other hand, v-SNAREs were shown to interact with COPII coat proteins (Matsuoka et al., 1998; Springer and Schekman, 1998), which are themselves responsible for efficient packaging of secreted proteins into the vesicles (Kuehn et al., 1998). It is thus possible that Ypt1p recruits the tethering factors onto the ER membrane where they would interact with specific SNAREs and coat proteins ensuring specific packaging and thus sorting of cargo molecules into ER-derived vesicles. These findings suggest that Rab-mediated recruitment of tethering factors to membranes and interaction of these proteins with v-SNAREs may be a general mechanism to couple protein sorting and packaging into vesicles to targeting, docking, and fusion.
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Materials and methods |
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The in vitro ER-budding assay was performed in a total volume of 300 µl in the presence of 25 x 107 permeabilized cells, 600 µg cytosol, 1 mM ATP, 1 mM creatine phosphate, 1 mM phosphocreatine kinase, 0.2 mM GTP, 0.1 mM GDP-mannose, 6 µg Sar1p, 1 mM protease inhibitor mix (leupeptin, pepstatin, and antipain) for 1 h at the indicated temperature. Vesicle immunoisolation was performed as described previously (Muniz et al., 2001).
In vivo sorting assay
Yeast cells were grown, harvested, and washed as described for in vitro assays. For the labeling step, cells were preincubated at 37°C for 5 min, pulse labeled for 6 min at 37°C with Trans-label mix (NEN), and chased for 10 min at 37°C by adding 1/100 volume of chase cocktail (0.3% methionine, 0.3% cysteine, 0.3 M [NH4]2SO4). The labeling reaction was stopped by the addition of 20 mM NaN3 and incubation on ice for 15 min. Permeabilized spheroplasts were prepared as described previously (Kuehn et al., 1996) and directly sedimented (14000 rpm for 10 min at 4°C). The supernatant was subjected to flotation on a Nycodenz® step gradient, and the ER-derived vesicle-containing fractions were processed as described (Muniz et al., 2001).
EM
The permeabilized spheroplasts were washed and incubated with cytosol and energy exactly as described for the in vitro budding/sorting assay. After 10-min incubation at 30°C, the spheroplasts were fixed overnight at 4°C by addition of glutaraldehyde (0.2% final concentration) and formaldehyde (3% final concentration), washed in 50 mM Hepes, pH 7.0, 3 mM KCl, and free aldehyde groups were quenched for 30 min as described (van Tuinen and Riezman, 1987). Dehydration, infiltration, and polymerization in LRGold resin (London Resin Company, Ltd.) were done according to the supplier's instructions. 50- to 60-nm sections were cut and mounted on 200-mesh nickel grids. Gas1p and Gap1p antibodies were raised in rabbits and used for immunolabeling at dilutions determined empirically. Grids were placed upside down on 50-µl droplets of blocking solution (150 mM NaCl, 10 mM potassium phosphate, pH 7.5, 0.1% Tween 20, 2% fatty acidfree BSA [Sigma-Aldrich]) for 20 min. The grids were then transferred to droplets containing appropriate dilutions of the primary antibodies in blocking buffer and incubated for 4 h at room temperature. The grids were then washed three times for 5 min with PBS solution and 0.2% BSA and three times for 5 min in PBS solution. The grids were incubated for 10 min in blocking buffer before transferring to droplets with secondary antibody gold conjugates and incubation for 2 h. After washing as above, they were fixed for 5 min in 1% glutaraldehyde in PBS solution to preserve the immunolabeling and then washed (by dipping 10 times in a 100-ml beaker) in distilled water. Free aldehydes were again quenched with 50 mM NH4Cl and washed with distilled water. Labeling with the second primary antibodies was done essentially as described for the first primary antibodies except the blocking solution contained 0.5% Tween 20 in PBS solution. After washing in water, the grids were stained in 6% uranyl acetate for 10 min and in Reynold's lead citrate for 3060 s.
For quantification, immunolabeled vesicles were counted when (a) they were labeled with more than one IgG colloidal gold particle and (b) they were still attached to the ER membrane. More than 60 vesicles were counted per experiment. ER membranes were identified by their electron-dense pattern due to the presence of ribosomes.
Pulsechase experiments
Pulsechase experiments were performed as described previously (Sütterlin et al., 1997). Invertase was induced by preincubating the cells at 24°C for 30 min in a medium containing 0.1% glucose.
Plasma membrane proteinase K shaving
The cells were grown, pulsed labeled, and chased for 60 min as described for the pulsechase experiments. Cell walls were mildly digested by a 10-min incubation in 10 mM Mesna followed by a 20-min incubation with lyticase at 24°C. Spheroplasts were incubated for 30 min with 200 µg/ml proteinase K. Digestion was stopped by addition of 1 mM PMSF and 0.5 N NaOH followed by incubation on ice for 10 min. Proteins were TCA precipitated and analyzed by immunoprecipitation, SDS-PAGE, and quantitation using a phosphorimager.
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Acknowledgments |
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This work was supported by a grant from the Swiss National Science Foundation (to H. Riezman), the Fonds National de la Recherche Scientifique (Belgium) and a Human Frontier Science Program long-term fellowship (to P. Morsomme).
Submitted: 18 December 2002
Accepted: 4 June 2003
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