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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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Bos1p is necessary for sorting in vivo. (A) After pulse–chase labeling, vesicles were purified through a Nycodenz® step gradient directly from Bet3p-depleted cells or bos1–1 mutant cells and then immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellet (P) were processed for immunoprecipitation, and samples were analyzed as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage recovery in the pellet. (B) The integrity of bos1–1-generated vesicles was tested as described above.
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fig5: Bos1p is necessary for sorting in vivo. (A) After pulse–chase labeling, vesicles were purified through a Nycodenz® step gradient directly from Bet3p-depleted cells or bos1–1 mutant cells and then immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellet (P) were processed for immunoprecipitation, and samples were analyzed as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage recovery in the pellet. (B) The integrity of bos1–1-generated vesicles was tested as described above.

Mentions: 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 bos1–1 mutant. 66% of the Gap1p signal was immunoisolated from bos1–1-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.


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)

Bos1p is necessary for sorting in vivo. (A) After pulse–chase labeling, vesicles were purified through a Nycodenz® step gradient directly from Bet3p-depleted cells or bos1–1 mutant cells and then immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellet (P) were processed for immunoprecipitation, and samples were analyzed as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage recovery in the pellet. (B) The integrity of bos1–1-generated vesicles was tested as described above.
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Related In: Results  -  Collection

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fig5: Bos1p is necessary for sorting in vivo. (A) After pulse–chase labeling, vesicles were purified through a Nycodenz® step gradient directly from Bet3p-depleted cells or bos1–1 mutant cells and then immunoisolated with or without monoclonal anti-HA antibody. The supernatants (S) and pellet (P) were processed for immunoprecipitation, and samples were analyzed as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage recovery in the pellet. (B) The integrity of bos1–1-generated vesicles was tested as described above.
Mentions: 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 bos1–1 mutant. 66% of the Gap1p signal was immunoisolated from bos1–1-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.

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