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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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Immuno-EM of Gas1p and Gap1p upon exit from the ER in sec18–20 and bos1–1 mutants. In vitro budding reactions were performed using sec18–20 membranes and cytosol (A and B) or from bos1–1 membranes and wild-type cytosol (C). Extracts were fixed 10 min after addition of cytosol and incubation at 30°C. Immunodetection and quantification were performed as described in Materials and methods. For the quantification, >60 sections were counted for each mutant. In A, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively. In B, Gas1p was labeled with either 10-nm (three left) or 15-nm (three right) gold particles (top); Gap1p was labeled with either 5-nm (three left) or 10-nm (three right) gold particles (bottom). In C, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively (top) or with 10- and 15-nm gold particles, respectively (bottom).
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fig4: Immuno-EM of Gas1p and Gap1p upon exit from the ER in sec18–20 and bos1–1 mutants. In vitro budding reactions were performed using sec18–20 membranes and cytosol (A and B) or from bos1–1 membranes and wild-type cytosol (C). Extracts were fixed 10 min after addition of cytosol and incubation at 30°C. Immunodetection and quantification were performed as described in Materials and methods. For the quantification, >60 sections were counted for each mutant. In A, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively. In B, Gas1p was labeled with either 10-nm (three left) or 15-nm (three right) gold particles (top); Gap1p was labeled with either 5-nm (three left) or 10-nm (three right) gold particles (bottom). In C, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively (top) or with 10- and 15-nm gold particles, respectively (bottom).

Mentions: 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.


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)

Immuno-EM of Gas1p and Gap1p upon exit from the ER in sec18–20 and bos1–1 mutants. In vitro budding reactions were performed using sec18–20 membranes and cytosol (A and B) or from bos1–1 membranes and wild-type cytosol (C). Extracts were fixed 10 min after addition of cytosol and incubation at 30°C. Immunodetection and quantification were performed as described in Materials and methods. For the quantification, >60 sections were counted for each mutant. In A, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively. In B, Gas1p was labeled with either 10-nm (three left) or 15-nm (three right) gold particles (top); Gap1p was labeled with either 5-nm (three left) or 10-nm (three right) gold particles (bottom). In C, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively (top) or with 10- and 15-nm gold particles, respectively (bottom).
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Related In: Results  -  Collection

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fig4: Immuno-EM of Gas1p and Gap1p upon exit from the ER in sec18–20 and bos1–1 mutants. In vitro budding reactions were performed using sec18–20 membranes and cytosol (A and B) or from bos1–1 membranes and wild-type cytosol (C). Extracts were fixed 10 min after addition of cytosol and incubation at 30°C. Immunodetection and quantification were performed as described in Materials and methods. For the quantification, >60 sections were counted for each mutant. In A, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively. In B, Gas1p was labeled with either 10-nm (three left) or 15-nm (three right) gold particles (top); Gap1p was labeled with either 5-nm (three left) or 10-nm (three right) gold particles (bottom). In C, Gap1p and Gas1p were labeled with 5- and 10-nm gold particles, respectively (top) or with 10- and 15-nm gold particles, respectively (bottom).
Mentions: 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.

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