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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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Mso1p-Sec4p colocalization is depended on Sec2p and independent of a functional SNARE complex. (A) wt, (B) sso2–1 Δsso1, and (C) sec2–41 cells coexpressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and YFP(N)-Sec4p (2μ, ADH1 promoter, B3316) were grown to OD600 = 0.8–1 at 24°C, and the cultures were split and either left at 24°C or shifted to the indicated restrictive temperature for 1 h prior to investigation. For each condition, the signal localization was quantified in at least 50 cells. The numbers in the figures indicate the percentage of cells showing Mso1p-Sec4p localization in the bud (top panels) or septum (bottom panels), respectively.
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Figure 7: Mso1p-Sec4p colocalization is depended on Sec2p and independent of a functional SNARE complex. (A) wt, (B) sso2–1 Δsso1, and (C) sec2–41 cells coexpressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and YFP(N)-Sec4p (2μ, ADH1 promoter, B3316) were grown to OD600 = 0.8–1 at 24°C, and the cultures were split and either left at 24°C or shifted to the indicated restrictive temperature for 1 h prior to investigation. For each condition, the signal localization was quantified in at least 50 cells. The numbers in the figures indicate the percentage of cells showing Mso1p-Sec4p localization in the bud (top panels) or septum (bottom panels), respectively.

Mentions: To position the Mso1p-Sec4p interaction in the cascade leading to vesicle fusion at the plasma membrane, the Mso1p-Sec4p BiFC signal was analyzed in different temperature-sensitive mutants. In wt cells, the Mso1p-Sec4p BiFC signal localized similarly to the bud and the septum area both at the permissive temperature 24°C and the restrictive temperature 37°C (Figure 7A). This polarized localization of the Mso1p-Sec4p BiFC signal was also observed in sso2–1 Δsso1 cells grown at the restrictive temperature 30°C (Figure 7B). This temperature completely inhibits growth through inactivation of the exocytic SNARE complexes (Jantti et al., 2002). The result suggests that in a situation where SNARE complexes are functionally compromised, the Mso1p-Sec4p proximity is still maintained. Sec2p is a GEF for Sec4p required for GTP loading in Sec4p. In sec2–41 cells, a 34% reduction of the Mso1p-Sec4p BiFC signal was observed in the bud at the permissive temperature (25% versus 38%, Figure 7C). This effect became more pronounced at the restrictive temperature (74% less, 10% versus 38%). This result suggests that fully functional GTP loading for Sec4p is important for the Mso1p-Sec4p association. This result, together with the BiFC results with Sec4p(Q79L), suggests that in vivo Mso1p cooperates preferentially with the GTP-bound Sec4p.FIGURE 7:


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)

Mso1p-Sec4p colocalization is depended on Sec2p and independent of a functional SNARE complex. (A) wt, (B) sso2–1 Δsso1, and (C) sec2–41 cells coexpressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and YFP(N)-Sec4p (2μ, ADH1 promoter, B3316) were grown to OD600 = 0.8–1 at 24°C, and the cultures were split and either left at 24°C or shifted to the indicated restrictive temperature for 1 h prior to investigation. For each condition, the signal localization was quantified in at least 50 cells. The numbers in the figures indicate the percentage of cells showing Mso1p-Sec4p localization in the bud (top panels) or septum (bottom panels), respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Mso1p-Sec4p colocalization is depended on Sec2p and independent of a functional SNARE complex. (A) wt, (B) sso2–1 Δsso1, and (C) sec2–41 cells coexpressing YFP(C)-Mso1p (CEN, MET25 promoter, B3044) and YFP(N)-Sec4p (2μ, ADH1 promoter, B3316) were grown to OD600 = 0.8–1 at 24°C, and the cultures were split and either left at 24°C or shifted to the indicated restrictive temperature for 1 h prior to investigation. For each condition, the signal localization was quantified in at least 50 cells. The numbers in the figures indicate the percentage of cells showing Mso1p-Sec4p localization in the bud (top panels) or septum (bottom panels), respectively.
Mentions: To position the Mso1p-Sec4p interaction in the cascade leading to vesicle fusion at the plasma membrane, the Mso1p-Sec4p BiFC signal was analyzed in different temperature-sensitive mutants. In wt cells, the Mso1p-Sec4p BiFC signal localized similarly to the bud and the septum area both at the permissive temperature 24°C and the restrictive temperature 37°C (Figure 7A). This polarized localization of the Mso1p-Sec4p BiFC signal was also observed in sso2–1 Δsso1 cells grown at the restrictive temperature 30°C (Figure 7B). This temperature completely inhibits growth through inactivation of the exocytic SNARE complexes (Jantti et al., 2002). The result suggests that in a situation where SNARE complexes are functionally compromised, the Mso1p-Sec4p proximity is still maintained. Sec2p is a GEF for Sec4p required for GTP loading in Sec4p. In sec2–41 cells, a 34% reduction of the Mso1p-Sec4p BiFC signal was observed in the bud at the permissive temperature (25% versus 38%, Figure 7C). This effect became more pronounced at the restrictive temperature (74% less, 10% versus 38%). This result suggests that fully functional GTP loading for Sec4p is important for the Mso1p-Sec4p association. This result, together with the BiFC results with Sec4p(Q79L), suggests that in vivo Mso1p cooperates preferentially with the GTP-bound Sec4p.FIGURE 7:

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.

Show MeSH