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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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Gas1p reaches the cell surface in the bos1–1 mutant. Mutant bos1–1 cells were pulse labeled for 3 min and chased for 60 min. Cell walls were mildly digested and incubated with or without proteinase K as described. Proteinase K was inactivated and Gas1p and Tdh1p were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and quantified using a phosphorimager.
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fig7: Gas1p reaches the cell surface in the bos1–1 mutant. Mutant bos1–1 cells were pulse labeled for 3 min and chased for 60 min. Cell walls were mildly digested and incubated with or without proteinase K as described. Proteinase K was inactivated and Gas1p and Tdh1p were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and quantified using a phosphorimager.

Mentions: The localization of radiolabeled Gas1p in the bos1–1 mutant after 60-min chase could be informative about the reasons for this defective glycosylation. To see if the radiolabeled Gas1p had arrived to the cell surface, we used a modification of a protease shaving technique (Nuoffer et al., 1991). We pulse labeled bos1–1 cells at 30°C, chased for 60 min, digested the cell wall, then incubated with or without proteinase K. At 24 and 30°C, 74 and 70% of Gas1p, respectively, was digested by proteinase K, indicating that the majority of the enzyme reached the plasma membrane (Fig. 7). In contrast, Gas1p was completely protected from protease digestion at 37°C, indicating that Gas1p did not reach the plasma membrane at restrictive temperature. As a control, the cytosolic glyceraldehyde-3-phosphate dehydrogenase 1, Tdh1p, was protected at the different temperatures (Fig. 7). These results show that, although Gas1p shows abnormal modification in bos1–1 at 30°C, it is transported to the cell surface.


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)

Gas1p reaches the cell surface in the bos1–1 mutant. Mutant bos1–1 cells were pulse labeled for 3 min and chased for 60 min. Cell walls were mildly digested and incubated with or without proteinase K as described. Proteinase K was inactivated and Gas1p and Tdh1p were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and quantified using a phosphorimager.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172695&req=5

fig7: Gas1p reaches the cell surface in the bos1–1 mutant. Mutant bos1–1 cells were pulse labeled for 3 min and chased for 60 min. Cell walls were mildly digested and incubated with or without proteinase K as described. Proteinase K was inactivated and Gas1p and Tdh1p were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and quantified using a phosphorimager.
Mentions: The localization of radiolabeled Gas1p in the bos1–1 mutant after 60-min chase could be informative about the reasons for this defective glycosylation. To see if the radiolabeled Gas1p had arrived to the cell surface, we used a modification of a protease shaving technique (Nuoffer et al., 1991). We pulse labeled bos1–1 cells at 30°C, chased for 60 min, digested the cell wall, then incubated with or without proteinase K. At 24 and 30°C, 74 and 70% of Gas1p, respectively, was digested by proteinase K, indicating that the majority of the enzyme reached the plasma membrane (Fig. 7). In contrast, Gas1p was completely protected from protease digestion at 37°C, indicating that Gas1p did not reach the plasma membrane at restrictive temperature. As a control, the cytosolic glyceraldehyde-3-phosphate dehydrogenase 1, Tdh1p, was protected at the different temperatures (Fig. 7). These results show that, although Gas1p shows abnormal modification in bos1–1 at 30°C, it is transported to the cell surface.

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