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The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER.

Morsomme P, Prescianotto-Baschong C, Riezman H - J. Cell Biol. (2003)

Bottom Line: Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM.Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature.Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum of the University of Basel, Basel, Switzerland.

ABSTRACT
Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

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ER v-SNAREs are necessary for protein sorting upon ER exit. (A) Vesicles were generated from sec18โ€“20 membranes and cytosol or from bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, or gos1ฮ” membranes and wild-type cytosol. The vesicles were immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellets (P) were processed for immunoprecipitation, and samples were analyzed by SDS-PAGE and quantified using a phosphorimager. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet. This experiment is representative of at least two independent experiments. (B) ER budding and fusion with the Golgi compartment from wild-type, bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, and sec18โ€“20 membranes. Purified vesicles were processed for two consecutive immunoprecipitations. Samples were immunoprecipitated first with antibodies against Gas1p, then reprecipitated with antibodies against Gas1p or ฮฑ-1,6 mannose, analyzed by SDS-PAGE, and quantified using a phosphor- imager. The percentage of fusion is the percentage of Gas1p after two immunoprecipitations that was recovered after precipitation with antibodies against ฮฑ-1,6 mannose.
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fig1: ER v-SNAREs are necessary for protein sorting upon ER exit. (A) Vesicles were generated from sec18โ€“20 membranes and cytosol or from bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, or gos1ฮ” membranes and wild-type cytosol. The vesicles were immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellets (P) were processed for immunoprecipitation, and samples were analyzed by SDS-PAGE and quantified using a phosphorimager. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet. This experiment is representative of at least two independent experiments. (B) ER budding and fusion with the Golgi compartment from wild-type, bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, and sec18โ€“20 membranes. Purified vesicles were processed for two consecutive immunoprecipitations. Samples were immunoprecipitated first with antibodies against Gas1p, then reprecipitated with antibodies against Gas1p or ฮฑ-1,6 mannose, analyzed by SDS-PAGE, and quantified using a phosphor- imager. The percentage of fusion is the percentage of Gas1p after two immunoprecipitations that was recovered after precipitation with antibodies against ฮฑ-1,6 mannose.

Mentions: To analyze cargo protein sorting upon exit from the ER, we used an in vitro assay that reconstitutes a single round of budding from the ER by incubation of permeabilized spheroplasts with exogenous cytosol and energy (Kuehn et al., 1996; Muniz et al., 2000). After the incubation, vesicles were purified by flotation into a Nycodenzยฎ gradient. To allow vesicle immunoisolation, we used a strain expressing an HA-tagged general amino acid permease (Gap1HA). With wild-type or sec18 extracts, we found 64% of Gap1pHA in the pellet after vesicle immunoisolation and only a small amount of the GPI-anchored protein, Gas1p, was coprecipitated (Fig. 1 A; Muniz et al., 2001). The immunoisolation required addition of HA antibody. This result shows that Gas1p is not incorporated into Gap1pHA-containing vesicles.


The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER.

Morsomme P, Prescianotto-Baschong C, Riezman H - J. Cell Biol. (2003)

ER v-SNAREs are necessary for protein sorting upon ER exit. (A) Vesicles were generated from sec18โ€“20 membranes and cytosol or from bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, or gos1ฮ” membranes and wild-type cytosol. The vesicles were immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellets (P) were processed for immunoprecipitation, and samples were analyzed by SDS-PAGE and quantified using a phosphorimager. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet. This experiment is representative of at least two independent experiments. (B) ER budding and fusion with the Golgi compartment from wild-type, bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, and sec18โ€“20 membranes. Purified vesicles were processed for two consecutive immunoprecipitations. Samples were immunoprecipitated first with antibodies against Gas1p, then reprecipitated with antibodies against Gas1p or ฮฑ-1,6 mannose, analyzed by SDS-PAGE, and quantified using a phosphor- imager. The percentage of fusion is the percentage of Gas1p after two immunoprecipitations that was recovered after precipitation with antibodies against ฮฑ-1,6 mannose.
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Related In: Results  -  Collection

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fig1: ER v-SNAREs are necessary for protein sorting upon ER exit. (A) Vesicles were generated from sec18โ€“20 membranes and cytosol or from bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, or gos1ฮ” membranes and wild-type cytosol. The vesicles were immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellets (P) were processed for immunoprecipitation, and samples were analyzed by SDS-PAGE and quantified using a phosphorimager. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet. This experiment is representative of at least two independent experiments. (B) ER budding and fusion with the Golgi compartment from wild-type, bos1โ€“1, bet1โ€“1, sec22โ€“3, sed5โ€“1, and sec18โ€“20 membranes. Purified vesicles were processed for two consecutive immunoprecipitations. Samples were immunoprecipitated first with antibodies against Gas1p, then reprecipitated with antibodies against Gas1p or ฮฑ-1,6 mannose, analyzed by SDS-PAGE, and quantified using a phosphor- imager. The percentage of fusion is the percentage of Gas1p after two immunoprecipitations that was recovered after precipitation with antibodies against ฮฑ-1,6 mannose.
Mentions: To analyze cargo protein sorting upon exit from the ER, we used an in vitro assay that reconstitutes a single round of budding from the ER by incubation of permeabilized spheroplasts with exogenous cytosol and energy (Kuehn et al., 1996; Muniz et al., 2000). After the incubation, vesicles were purified by flotation into a Nycodenzยฎ gradient. To allow vesicle immunoisolation, we used a strain expressing an HA-tagged general amino acid permease (Gap1HA). With wild-type or sec18 extracts, we found 64% of Gap1pHA in the pellet after vesicle immunoisolation and only a small amount of the GPI-anchored protein, Gas1p, was coprecipitated (Fig. 1 A; Muniz et al., 2001). The immunoisolation required addition of HA antibody. This result shows that Gas1p is not incorporated into Gap1pHA-containing vesicles.

Bottom Line: Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM.Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature.Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum of the University of Basel, Basel, Switzerland.

ABSTRACT
Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

Show MeSH
Related in: MedlinePlus