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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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In vitro budding of ER-derived vesicles containing secretory proteins from SNARE mutants. (A) In vitro budding reactions were performed using sec18–20 membranes and cytosol or from bos1–1, bet1–1, sec22–3, sed5–1, or gos1Δ membranes and wild-type cytosol. The nucleotide requirement was tested in the absence of exogenous ATP and GTP and in presence of apyrase (1.5 U/reaction). Budding efficiencies were calculated as the percentage of the total input that was recovered in the purified vesicles for each individual protein. (B) Budding efficiencies determined for Sec61p packaging into ER-derived vesicles generated from bos1–1, bet1–1, sec22–3, and sed5–1 extracts. Numbers represent the percentage of the total input (T) that was recovered in the purified vesicles (V). (C) Integrity of vesicles generated from bos1–1 membranes. Purified vesicles produced from bos1–1 membranes were incubated on ice for 30 min with or without 0.5 mg/ml proteinase K in the presence or absence of 1% Triton X-100. Samples were processed for Gas1p immunoprecipitation, analyzed by SDS-PAGE, and visualized using a phosphorimager.
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fig2: In vitro budding of ER-derived vesicles containing secretory proteins from SNARE mutants. (A) In vitro budding reactions were performed using sec18–20 membranes and cytosol or from bos1–1, bet1–1, sec22–3, sed5–1, or gos1Δ membranes and wild-type cytosol. The nucleotide requirement was tested in the absence of exogenous ATP and GTP and in presence of apyrase (1.5 U/reaction). Budding efficiencies were calculated as the percentage of the total input that was recovered in the purified vesicles for each individual protein. (B) Budding efficiencies determined for Sec61p packaging into ER-derived vesicles generated from bos1–1, bet1–1, sec22–3, and sed5–1 extracts. Numbers represent the percentage of the total input (T) that was recovered in the purified vesicles (V). (C) Integrity of vesicles generated from bos1–1 membranes. Purified vesicles produced from bos1–1 membranes were incubated on ice for 30 min with or without 0.5 mg/ml proteinase K in the presence or absence of 1% Triton X-100. Samples were processed for Gas1p immunoprecipitation, analyzed by SDS-PAGE, and visualized using a phosphorimager.

Mentions: We considered the possibility that sec22–3, bet1–1, or bos1–1 membranes are fragile and that the apparent lack of protein sorting was due to the isolation of ER fragments rather than bona fide vesicles that have budded from the ER. To test this, we determined the specificity and nucleotide dependence of vesicle formation using mutant membranes. The sorting defect observed with the sec22–3, bet1–1, or bos1–1 mutants was not due to a budding defect since the budding efficiency of Gap1p and Gas1p were similar to wild-type or sec18 membranes (Fig. 2 A). Packaging of Gas1p and Gap1p into ER-derived vesicles was dependent on cytosol (unpublished data) and on the presence of nucleotides in the assay (Fig. 2 A). Furthermore, resident ER proteins, like Sec61p, were not packaged into vesicles (Fig. 2 B). Finally, we treated the floated fraction of vesicles generated from bos1–1 membranes with proteinase K and observed that Gas1p was protected from protease digestion in absence of detergent but was digested when detergent was present (Fig. 2 C). This confirms that Gas1p exits the ER in vesicles in our in vitro assay even when using SNARE mutant extracts.


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)

In vitro budding of ER-derived vesicles containing secretory proteins from SNARE mutants. (A) In vitro budding reactions were performed using sec18–20 membranes and cytosol or from bos1–1, bet1–1, sec22–3, sed5–1, or gos1Δ membranes and wild-type cytosol. The nucleotide requirement was tested in the absence of exogenous ATP and GTP and in presence of apyrase (1.5 U/reaction). Budding efficiencies were calculated as the percentage of the total input that was recovered in the purified vesicles for each individual protein. (B) Budding efficiencies determined for Sec61p packaging into ER-derived vesicles generated from bos1–1, bet1–1, sec22–3, and sed5–1 extracts. Numbers represent the percentage of the total input (T) that was recovered in the purified vesicles (V). (C) Integrity of vesicles generated from bos1–1 membranes. Purified vesicles produced from bos1–1 membranes were incubated on ice for 30 min with or without 0.5 mg/ml proteinase K in the presence or absence of 1% Triton X-100. Samples were processed for Gas1p immunoprecipitation, analyzed by SDS-PAGE, and visualized using a phosphorimager.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: In vitro budding of ER-derived vesicles containing secretory proteins from SNARE mutants. (A) In vitro budding reactions were performed using sec18–20 membranes and cytosol or from bos1–1, bet1–1, sec22–3, sed5–1, or gos1Δ membranes and wild-type cytosol. The nucleotide requirement was tested in the absence of exogenous ATP and GTP and in presence of apyrase (1.5 U/reaction). Budding efficiencies were calculated as the percentage of the total input that was recovered in the purified vesicles for each individual protein. (B) Budding efficiencies determined for Sec61p packaging into ER-derived vesicles generated from bos1–1, bet1–1, sec22–3, and sed5–1 extracts. Numbers represent the percentage of the total input (T) that was recovered in the purified vesicles (V). (C) Integrity of vesicles generated from bos1–1 membranes. Purified vesicles produced from bos1–1 membranes were incubated on ice for 30 min with or without 0.5 mg/ml proteinase K in the presence or absence of 1% Triton X-100. Samples were processed for Gas1p immunoprecipitation, analyzed by SDS-PAGE, and visualized using a phosphorimager.
Mentions: We considered the possibility that sec22–3, bet1–1, or bos1–1 membranes are fragile and that the apparent lack of protein sorting was due to the isolation of ER fragments rather than bona fide vesicles that have budded from the ER. To test this, we determined the specificity and nucleotide dependence of vesicle formation using mutant membranes. The sorting defect observed with the sec22–3, bet1–1, or bos1–1 mutants was not due to a budding defect since the budding efficiency of Gap1p and Gas1p were similar to wild-type or sec18 membranes (Fig. 2 A). Packaging of Gas1p and Gap1p into ER-derived vesicles was dependent on cytosol (unpublished data) and on the presence of nucleotides in the assay (Fig. 2 A). Furthermore, resident ER proteins, like Sec61p, were not packaged into vesicles (Fig. 2 B). Finally, we treated the floated fraction of vesicles generated from bos1–1 membranes with proteinase K and observed that Gas1p was protected from protease digestion in absence of detergent but was digested when detergent was present (Fig. 2 C). This confirms that Gas1p exits the ER in vesicles in our in vitro assay even when using SNARE mutant extracts.

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