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Ordering the final events in yeast exocytosis.

Grote E, Carr CM, Novick PJ - J. Cell Biol. (2000)

Bottom Line: By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly.In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane.Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

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Binding of Sec1p to SNARE complexes in sec mutant strains. (a) Coimmunoprecipitation of Sec1p and Sncp with HA-Sso2p. Wild-type and sec mutant strains expressing HA-Sso2p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Sec1p and Sncp coprecipitating with HA-Sso2p in anti-HA immunoprecipitates were observed by Western blotting. Note that the strains in this experiment were shifted to 37°C rather than to 38°C as in Fig. 2. This lower temperature is partially permissive for SNARE complex assembly in the sec8-6 mutant strain. (b) Coimmunoprecipitation of Ssop with myc-Sec1p and Sncp. Wild-type and sec mutant strains expressing myc-Sec1p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Ssop coprecipitating with myc-Sec1p in anti-myc immunoprecipitates and coimmunoprecipitating with Sncp in anti-Sncp precipitates was observed by Western blotting.
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Figure 4: Binding of Sec1p to SNARE complexes in sec mutant strains. (a) Coimmunoprecipitation of Sec1p and Sncp with HA-Sso2p. Wild-type and sec mutant strains expressing HA-Sso2p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Sec1p and Sncp coprecipitating with HA-Sso2p in anti-HA immunoprecipitates were observed by Western blotting. Note that the strains in this experiment were shifted to 37°C rather than to 38°C as in Fig. 2. This lower temperature is partially permissive for SNARE complex assembly in the sec8-6 mutant strain. (b) Coimmunoprecipitation of Ssop with myc-Sec1p and Sncp. Wild-type and sec mutant strains expressing myc-Sec1p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Ssop coprecipitating with myc-Sec1p in anti-myc immunoprecipitates and coimmunoprecipitating with Sncp in anti-Sncp precipitates was observed by Western blotting.

Mentions: We have previously reported that Sec1p from yeast lysates binds to preassembled SNARE complexes but not to free Ssop (Carr et al. 1999). To address the possibility that other Sec proteins are required for the interaction between Sec1p and SNARE complexes, we used immunoprecipitation to examine this interaction in sec mutants incubated at 37°C for 10 min. For these experiments, epitope-tagged HA-Sso2 or myc-Sec1 proteins were immunoprecipitated with monoclonal anti-HA or anti-myc antibodies. To test for Sec1p binding to Ssop, we probed for Sec1p in the HA-Sso2p immunoprecipitates (Fig. 4 a) and probed for Ssop in the myc-Sec1 immunoprecipitates (Fig. 4 b). We also measured the abundance of SNARE complexes in each mutant by probing for Sncp in the HA-Sso2p immunoprecipitates and by probing for Ssop in an anti-Sncp immunoprecipitate from the myc-Sec1p lysates. Both precipitations were specific, because neither Ssop nor Sec1p was present in immunoprecipitations from untagged control strains. Except for the sec1-1 strain, there was a positive correlation between the amounts of Sec1p and Sncp bound to Ssop. Thus, the association of Sec1p with Ssop appears to be limited by the abundance of SNARE complexes in these sec mutants. The reduced binding of mutant Sec1-1p from the sec1-1 strain may result either from the reduced abundance of Sec1-1p in the lysate or because the mutant Sec1-1p is defective in SNARE binding.


Ordering the final events in yeast exocytosis.

Grote E, Carr CM, Novick PJ - J. Cell Biol. (2000)

Binding of Sec1p to SNARE complexes in sec mutant strains. (a) Coimmunoprecipitation of Sec1p and Sncp with HA-Sso2p. Wild-type and sec mutant strains expressing HA-Sso2p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Sec1p and Sncp coprecipitating with HA-Sso2p in anti-HA immunoprecipitates were observed by Western blotting. Note that the strains in this experiment were shifted to 37°C rather than to 38°C as in Fig. 2. This lower temperature is partially permissive for SNARE complex assembly in the sec8-6 mutant strain. (b) Coimmunoprecipitation of Ssop with myc-Sec1p and Sncp. Wild-type and sec mutant strains expressing myc-Sec1p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Ssop coprecipitating with myc-Sec1p in anti-myc immunoprecipitates and coimmunoprecipitating with Sncp in anti-Sncp precipitates was observed by Western blotting.
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Figure 4: Binding of Sec1p to SNARE complexes in sec mutant strains. (a) Coimmunoprecipitation of Sec1p and Sncp with HA-Sso2p. Wild-type and sec mutant strains expressing HA-Sso2p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Sec1p and Sncp coprecipitating with HA-Sso2p in anti-HA immunoprecipitates were observed by Western blotting. Note that the strains in this experiment were shifted to 37°C rather than to 38°C as in Fig. 2. This lower temperature is partially permissive for SNARE complex assembly in the sec8-6 mutant strain. (b) Coimmunoprecipitation of Ssop with myc-Sec1p and Sncp. Wild-type and sec mutant strains expressing myc-Sec1p and an untagged wild-type control were grown at 25°C and shifted to 37°C for 10 min. Ssop coprecipitating with myc-Sec1p in anti-myc immunoprecipitates and coimmunoprecipitating with Sncp in anti-Sncp precipitates was observed by Western blotting.
Mentions: We have previously reported that Sec1p from yeast lysates binds to preassembled SNARE complexes but not to free Ssop (Carr et al. 1999). To address the possibility that other Sec proteins are required for the interaction between Sec1p and SNARE complexes, we used immunoprecipitation to examine this interaction in sec mutants incubated at 37°C for 10 min. For these experiments, epitope-tagged HA-Sso2 or myc-Sec1 proteins were immunoprecipitated with monoclonal anti-HA or anti-myc antibodies. To test for Sec1p binding to Ssop, we probed for Sec1p in the HA-Sso2p immunoprecipitates (Fig. 4 a) and probed for Ssop in the myc-Sec1 immunoprecipitates (Fig. 4 b). We also measured the abundance of SNARE complexes in each mutant by probing for Sncp in the HA-Sso2p immunoprecipitates and by probing for Ssop in an anti-Sncp immunoprecipitate from the myc-Sec1p lysates. Both precipitations were specific, because neither Ssop nor Sec1p was present in immunoprecipitations from untagged control strains. Except for the sec1-1 strain, there was a positive correlation between the amounts of Sec1p and Sncp bound to Ssop. Thus, the association of Sec1p with Ssop appears to be limited by the abundance of SNARE complexes in these sec mutants. The reduced binding of mutant Sec1-1p from the sec1-1 strain may result either from the reduced abundance of Sec1-1p in the lysate or because the mutant Sec1-1p is defective in SNARE binding.

Bottom Line: By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly.In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane.Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

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Related in: MedlinePlus