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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 vivo Gas1p maturation is specifically affected in bos1–1 mutant at 30°C. Protein maturation in bos1–1, sec22–3, and sed5–1 mutants at 24 and 30°C. (A) Wild-type and mutant cells were pulse labeled and chased at the indicated temperatures for the indicated times. Gas1p and CPY were immunoprecipitated from cell lysates and separated by SDS-PAGE and quantified using a phosphorimager. (B) Analysis of invertase secretion in bos1–1 mutant. Cells were pulse–chase labeled, and then the cells and medium were separated and analyzed separately for invertase by immunoprecipitation, SDS-PAGE, and visualization using a phosphorimager.
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fig6: In vivo Gas1p maturation is specifically affected in bos1–1 mutant at 30°C. Protein maturation in bos1–1, sec22–3, and sed5–1 mutants at 24 and 30°C. (A) Wild-type and mutant cells were pulse labeled and chased at the indicated temperatures for the indicated times. Gas1p and CPY were immunoprecipitated from cell lysates and separated by SDS-PAGE and quantified using a phosphorimager. (B) Analysis of invertase secretion in bos1–1 mutant. Cells were pulse–chase labeled, and then the cells and medium were separated and analyzed separately for invertase by immunoprecipitation, SDS-PAGE, and visualization using a phosphorimager.

Mentions: The ER to Golgi transport of Gas1p can be followed in vivo by measuring the modification of the protein after its delivery to the Golgi complex (Doering and Schekman, 1996; Sütterlin et al., 1997). The ER form of Gas1p shows an apparent molecular weight of 105 kD. Gas1p is then modified in the Golgi compartment to its mature form of 125 kD. The other pathway of ER to Golgi transport can be followed by the maturation of carboxypeptidase Y (CPY), which is present in the ER in its p1 form, then modified in the Golgi complex to its p2 form, which is then cleaved into its mature form (m) in the vacuole. Transport of the two proteins to the Golgi compartment is reliably measured by analysis of the addition of α-1,6 mannose residues using antiserum against this carbohydrate modification. Gas1p transport to the Golgi compartment was shown previously to have specific requirements (Sütterlin et al., 1997). Therefore, we examined whether v-SNARE mutants could affect Gas1p transport in a specific manner. v-SNAREs apparently have at least two functions: a cargo-sorting function upon ER exit and a fusion function of ER-derived vesicles with the Golgi compartment. At 37°C, the transport of Gas1p and CPY was completely blocked in bos1–1, sec22–3, bet1–1, and sed5–1 mutant cells (unpublished data). This defect at restrictive temperature was probably due to the loss of fusion competence of ER-derived vesicles to the Golgi compartment. At 24°C, the transport of Gas1p and CPY from the ER to the Golgi compartment was similar in the bos1–1, sec22–3, and sed5–1 strains (Fig. 6 A). At 30°C, transport of both Gas1p and CPY was slightly delayed in bos1–1 and sec22–3 mutants and more severely affected in the sed5–1 mutant. However, at 30°C the maturation of Gas1p was affected in a particular manner in the bos1–1 strain compared with sec22–3 (Fig. 6 A). The molecular size of Gas1p increased continuously, but no clear shift from the 105- to 125-kD form was observed. The continuous increase of molecular shift was nevertheless due to addition of α-1,6 mannose residues because these forms were precipitated by anti–α1,6 mannose antibodies. Invertase was normally secreted and glycosylated at 24°C and at 30°C in the bos1–1 mutant (Fig. 6 B).


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 vivo Gas1p maturation is specifically affected in bos1–1 mutant at 30°C. Protein maturation in bos1–1, sec22–3, and sed5–1 mutants at 24 and 30°C. (A) Wild-type and mutant cells were pulse labeled and chased at the indicated temperatures for the indicated times. Gas1p and CPY were immunoprecipitated from cell lysates and separated by SDS-PAGE and quantified using a phosphorimager. (B) Analysis of invertase secretion in bos1–1 mutant. Cells were pulse–chase labeled, and then the cells and medium were separated and analyzed separately for invertase by immunoprecipitation, SDS-PAGE, and visualization using a phosphorimager.
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Related In: Results  -  Collection

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fig6: In vivo Gas1p maturation is specifically affected in bos1–1 mutant at 30°C. Protein maturation in bos1–1, sec22–3, and sed5–1 mutants at 24 and 30°C. (A) Wild-type and mutant cells were pulse labeled and chased at the indicated temperatures for the indicated times. Gas1p and CPY were immunoprecipitated from cell lysates and separated by SDS-PAGE and quantified using a phosphorimager. (B) Analysis of invertase secretion in bos1–1 mutant. Cells were pulse–chase labeled, and then the cells and medium were separated and analyzed separately for invertase by immunoprecipitation, SDS-PAGE, and visualization using a phosphorimager.
Mentions: The ER to Golgi transport of Gas1p can be followed in vivo by measuring the modification of the protein after its delivery to the Golgi complex (Doering and Schekman, 1996; Sütterlin et al., 1997). The ER form of Gas1p shows an apparent molecular weight of 105 kD. Gas1p is then modified in the Golgi compartment to its mature form of 125 kD. The other pathway of ER to Golgi transport can be followed by the maturation of carboxypeptidase Y (CPY), which is present in the ER in its p1 form, then modified in the Golgi complex to its p2 form, which is then cleaved into its mature form (m) in the vacuole. Transport of the two proteins to the Golgi compartment is reliably measured by analysis of the addition of α-1,6 mannose residues using antiserum against this carbohydrate modification. Gas1p transport to the Golgi compartment was shown previously to have specific requirements (Sütterlin et al., 1997). Therefore, we examined whether v-SNARE mutants could affect Gas1p transport in a specific manner. v-SNAREs apparently have at least two functions: a cargo-sorting function upon ER exit and a fusion function of ER-derived vesicles with the Golgi compartment. At 37°C, the transport of Gas1p and CPY was completely blocked in bos1–1, sec22–3, bet1–1, and sed5–1 mutant cells (unpublished data). This defect at restrictive temperature was probably due to the loss of fusion competence of ER-derived vesicles to the Golgi compartment. At 24°C, the transport of Gas1p and CPY from the ER to the Golgi compartment was similar in the bos1–1, sec22–3, and sed5–1 strains (Fig. 6 A). At 30°C, transport of both Gas1p and CPY was slightly delayed in bos1–1 and sec22–3 mutants and more severely affected in the sed5–1 mutant. However, at 30°C the maturation of Gas1p was affected in a particular manner in the bos1–1 strain compared with sec22–3 (Fig. 6 A). The molecular size of Gas1p increased continuously, but no clear shift from the 105- to 125-kD form was observed. The continuous increase of molecular shift was nevertheless due to addition of α-1,6 mannose residues because these forms were precipitated by anti–α1,6 mannose antibodies. Invertase was normally secreted and glycosylated at 24°C and at 30°C in the bos1–1 mutant (Fig. 6 B).

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