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Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis.

Weber-Boyvat M, Aro N, Chernov KG, Nyman T, Jäntti J - Mol. Biol. Cell (2010)

Bottom Line: The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting.The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud.Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Biology Program Research Program in Structural Biology and Biophysics, Institute of Biotechnology, FI-0001 University of Helsinki, Finland.

ABSTRACT
The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

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The Sec1p tail is important for Sec1p interaction with SNARE complexes and membrane association in vivo. (A) The Sec1p C-terminal tail is needed for Sec1p coimmunoprecipitation with SNARE components. MSO1-HA cells expressing wt SEC1 or mutant sec1(1–657) cells were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, -Sso1p/2p, and -Snc1p/2p antibodies. (B) Sec1p(1–657) has reduced membrane association. Membranes were fractionated by differential centrifugation and analyzed by Western blotting and detection with anti-HA, -Sec1p, and -Sso1p/2p antibodies. S2, the supernatant after centrifugation at 10,000 × g, P3, the pellet after centrifugation at 100,000 × g and S3, the supernatant after centrifugation at 100,000 × g. The Sec1p (wt and 1–657) signal from three independent experiments was quantified and normalized to the amount of Sec1p in the S2 fraction. (C) Localization of the Mso1p interaction site with Sec1p and Sec1p(1–657) in vivo. Haploid, vegetatively grown cells (H304) expressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and Sec1p-Venus(N) (CEN, ADH1 promoter, B2930) or Sec1p(1–657)-Venus(N) (CEN, ADH1 promoter, B3279) were investigated by fluorescence microscopy. The distribution of the BiFC signal was analyzed in a minimum of 70 cells per interaction mode.
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Figure 2: The Sec1p tail is important for Sec1p interaction with SNARE complexes and membrane association in vivo. (A) The Sec1p C-terminal tail is needed for Sec1p coimmunoprecipitation with SNARE components. MSO1-HA cells expressing wt SEC1 or mutant sec1(1–657) cells were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, -Sso1p/2p, and -Snc1p/2p antibodies. (B) Sec1p(1–657) has reduced membrane association. Membranes were fractionated by differential centrifugation and analyzed by Western blotting and detection with anti-HA, -Sec1p, and -Sso1p/2p antibodies. S2, the supernatant after centrifugation at 10,000 × g, P3, the pellet after centrifugation at 100,000 × g and S3, the supernatant after centrifugation at 100,000 × g. The Sec1p (wt and 1–657) signal from three independent experiments was quantified and normalized to the amount of Sec1p in the S2 fraction. (C) Localization of the Mso1p interaction site with Sec1p and Sec1p(1–657) in vivo. Haploid, vegetatively grown cells (H304) expressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and Sec1p-Venus(N) (CEN, ADH1 promoter, B2930) or Sec1p(1–657)-Venus(N) (CEN, ADH1 promoter, B3279) were investigated by fluorescence microscopy. The distribution of the BiFC signal was analyzed in a minimum of 70 cells per interaction mode.

Mentions: The Sec1p(1–657) and wt Sec1p are expressed at similar levels (Figure 2A, input) indicating that the observed temperature sensitivity or Bgl2p secretion defect in sec1(1–657) cells is not due to reduced levels of the mutant protein. At the same time, expression of the Sec1p(1–657) as the sole copy of Sec1p in cells did not affect the expression levels of Sec9p, Sso1/2p, or Snc1/2p (Figure 2A, input). To understand the reason for the sec1(1–657) cell in vivo phenotype, immunoprecipitations were performed. It has been previously shown that Mso1p coimmunoprecipitates with Sec1p and SNARE components Sso1/2p-Sec9p-Snc1/2p (Castillo-Flores et al., 2005; Knop et al., 2005; Weber et al., 2010). To assess the contribution of the Sec1p C-terminal tail for Mso1p and SNARE interactions, Mso1p-HA (expressed at the MSO1 locus from its endogenous promoter) was immunoprecipitated with anti-HA antibodies and the coimmunoprecipitation of Sec1p and SNARE components was analyzed by SDS–PAGE and Western blotting. Compared to the wt Sec1p, there was no notable difference in the efficiency of Sec1p(1–657) coprecipitation with Mso1p (Figure 2A). At the same time, even at the permissive temperature, deletion of the Sec1p tail caused loss of SNARE complex coprecipitation. Similar results were obtained when immunoprecipitations were performed with Sec1p-HA or Sec1p(1–657)-HA tagged at the genomic SEC1 locus (unpublished data). These results suggest a positive role for the C-terminal tail in Sec1p-SNARE component interactions.FIGURE 2:


Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis.

Weber-Boyvat M, Aro N, Chernov KG, Nyman T, Jäntti J - Mol. Biol. Cell (2010)

The Sec1p tail is important for Sec1p interaction with SNARE complexes and membrane association in vivo. (A) The Sec1p C-terminal tail is needed for Sec1p coimmunoprecipitation with SNARE components. MSO1-HA cells expressing wt SEC1 or mutant sec1(1–657) cells were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, -Sso1p/2p, and -Snc1p/2p antibodies. (B) Sec1p(1–657) has reduced membrane association. Membranes were fractionated by differential centrifugation and analyzed by Western blotting and detection with anti-HA, -Sec1p, and -Sso1p/2p antibodies. S2, the supernatant after centrifugation at 10,000 × g, P3, the pellet after centrifugation at 100,000 × g and S3, the supernatant after centrifugation at 100,000 × g. The Sec1p (wt and 1–657) signal from three independent experiments was quantified and normalized to the amount of Sec1p in the S2 fraction. (C) Localization of the Mso1p interaction site with Sec1p and Sec1p(1–657) in vivo. Haploid, vegetatively grown cells (H304) expressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and Sec1p-Venus(N) (CEN, ADH1 promoter, B2930) or Sec1p(1–657)-Venus(N) (CEN, ADH1 promoter, B3279) were investigated by fluorescence microscopy. The distribution of the BiFC signal was analyzed in a minimum of 70 cells per interaction mode.
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Figure 2: The Sec1p tail is important for Sec1p interaction with SNARE complexes and membrane association in vivo. (A) The Sec1p C-terminal tail is needed for Sec1p coimmunoprecipitation with SNARE components. MSO1-HA cells expressing wt SEC1 or mutant sec1(1–657) cells were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, -Sso1p/2p, and -Snc1p/2p antibodies. (B) Sec1p(1–657) has reduced membrane association. Membranes were fractionated by differential centrifugation and analyzed by Western blotting and detection with anti-HA, -Sec1p, and -Sso1p/2p antibodies. S2, the supernatant after centrifugation at 10,000 × g, P3, the pellet after centrifugation at 100,000 × g and S3, the supernatant after centrifugation at 100,000 × g. The Sec1p (wt and 1–657) signal from three independent experiments was quantified and normalized to the amount of Sec1p in the S2 fraction. (C) Localization of the Mso1p interaction site with Sec1p and Sec1p(1–657) in vivo. Haploid, vegetatively grown cells (H304) expressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and Sec1p-Venus(N) (CEN, ADH1 promoter, B2930) or Sec1p(1–657)-Venus(N) (CEN, ADH1 promoter, B3279) were investigated by fluorescence microscopy. The distribution of the BiFC signal was analyzed in a minimum of 70 cells per interaction mode.
Mentions: The Sec1p(1–657) and wt Sec1p are expressed at similar levels (Figure 2A, input) indicating that the observed temperature sensitivity or Bgl2p secretion defect in sec1(1–657) cells is not due to reduced levels of the mutant protein. At the same time, expression of the Sec1p(1–657) as the sole copy of Sec1p in cells did not affect the expression levels of Sec9p, Sso1/2p, or Snc1/2p (Figure 2A, input). To understand the reason for the sec1(1–657) cell in vivo phenotype, immunoprecipitations were performed. It has been previously shown that Mso1p coimmunoprecipitates with Sec1p and SNARE components Sso1/2p-Sec9p-Snc1/2p (Castillo-Flores et al., 2005; Knop et al., 2005; Weber et al., 2010). To assess the contribution of the Sec1p C-terminal tail for Mso1p and SNARE interactions, Mso1p-HA (expressed at the MSO1 locus from its endogenous promoter) was immunoprecipitated with anti-HA antibodies and the coimmunoprecipitation of Sec1p and SNARE components was analyzed by SDS–PAGE and Western blotting. Compared to the wt Sec1p, there was no notable difference in the efficiency of Sec1p(1–657) coprecipitation with Mso1p (Figure 2A). At the same time, even at the permissive temperature, deletion of the Sec1p tail caused loss of SNARE complex coprecipitation. Similar results were obtained when immunoprecipitations were performed with Sec1p-HA or Sec1p(1–657)-HA tagged at the genomic SEC1 locus (unpublished data). These results suggest a positive role for the C-terminal tail in Sec1p-SNARE component interactions.FIGURE 2:

Bottom Line: The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting.The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud.Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Biology Program Research Program in Structural Biology and Biophysics, Institute of Biotechnology, FI-0001 University of Helsinki, Finland.

ABSTRACT
The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

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