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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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Anti–v-SNARE antibodies affect sorting. (A) Vesicles were generated from wild-type membranes and cytosol in the absence or presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies. 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 as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet from which the background was subtracted. The data are the mean ± SD of three independent experiments for anti-Bos1p, anti-Sed5p, and anti-Sec22p antibodies and two independent experiments for anti-Ste2p antibodies. (B) Immunoblot of floated vesicles generated from wild-type membranes and cytosol in absence or in presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies.
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fig3: Anti–v-SNARE antibodies affect sorting. (A) Vesicles were generated from wild-type membranes and cytosol in the absence or presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies. 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 as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet from which the background was subtracted. The data are the mean ± SD of three independent experiments for anti-Bos1p, anti-Sed5p, and anti-Sec22p antibodies and two independent experiments for anti-Ste2p antibodies. (B) Immunoblot of floated vesicles generated from wild-type membranes and cytosol in absence or in presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies.

Mentions: Another way to test the function of the SNAREs in sorting of GPI-anchored proteins from other secretory proteins is to block their function by adding antibodies specific for the different SNARE proteins during the in vitro budding assay. We thus performed the budding assay using wild-type membranes and cytosol in the presence of antibodies against Sed5p, Bos1p, or Sec22p (Fig. 3). The budding was still specific because it was cytosol and nucleotide dependent and was not the result of ER fragmentation (unpublished data). Budding was somewhat more efficient in the presence of antiserum, but the efficiency was improved even with control antibodies or antibodies against plasma membrane proteins (unpublished data). The presence of anti-Sed5p antibodies during budding did not perturb sorting because only 17% of Gas1p was coimmunoisolated with 72% of Gap1HA. In contrast, the presence of anti-Bos1p or anti-Sec22p antibodies during budding caused a significant sorting defect because 30 and 37% of Gas1p, respectively, was coimmunoisolated with Gap1HA. The presence of unrelated antibodies directed against the plasma membrane protein Ste2p did not affect sorting (Fig. 3 A). These data suggest a direct role of the v-SNAREs in sorting GPI-anchored proteins into distinct 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)

Anti–v-SNARE antibodies affect sorting. (A) Vesicles were generated from wild-type membranes and cytosol in the absence or presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies. 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 as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet from which the background was subtracted. The data are the mean ± SD of three independent experiments for anti-Bos1p, anti-Sed5p, and anti-Sec22p antibodies and two independent experiments for anti-Ste2p antibodies. (B) Immunoblot of floated vesicles generated from wild-type membranes and cytosol in absence or in presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172695&req=5

fig3: Anti–v-SNARE antibodies affect sorting. (A) Vesicles were generated from wild-type membranes and cytosol in the absence or presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies. 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 as described above. The total recovery, S plus P, was set to 100%. Numbers represent the percentage of recovery in the pellet from which the background was subtracted. The data are the mean ± SD of three independent experiments for anti-Bos1p, anti-Sed5p, and anti-Sec22p antibodies and two independent experiments for anti-Ste2p antibodies. (B) Immunoblot of floated vesicles generated from wild-type membranes and cytosol in absence or in presence of 5 μl of anti-Bos1p, anti-Sed5p, anti-Sec22p, or anti-Ste2p antibodies.
Mentions: Another way to test the function of the SNAREs in sorting of GPI-anchored proteins from other secretory proteins is to block their function by adding antibodies specific for the different SNARE proteins during the in vitro budding assay. We thus performed the budding assay using wild-type membranes and cytosol in the presence of antibodies against Sed5p, Bos1p, or Sec22p (Fig. 3). The budding was still specific because it was cytosol and nucleotide dependent and was not the result of ER fragmentation (unpublished data). Budding was somewhat more efficient in the presence of antiserum, but the efficiency was improved even with control antibodies or antibodies against plasma membrane proteins (unpublished data). The presence of anti-Sed5p antibodies during budding did not perturb sorting because only 17% of Gas1p was coimmunoisolated with 72% of Gap1HA. In contrast, the presence of anti-Bos1p or anti-Sec22p antibodies during budding caused a significant sorting defect because 30 and 37% of Gas1p, respectively, was coimmunoisolated with Gap1HA. The presence of unrelated antibodies directed against the plasma membrane protein Ste2p did not affect sorting (Fig. 3 A). These data suggest a direct role of the v-SNAREs in sorting GPI-anchored proteins into distinct 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