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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 promotes Sec1p-SNARE complex association. (A) The effect of SEC1(658–724) (B3424) overexpression for growth in SEC1 wt (H2657) and sec1(1–657) mutant (H3659) strains. The growth of serial 10-fold dilutions of cells grown at the indicated temperatures is shown. (B) Sec1p tail overexpression enhances coimmunoprecipitation of SNARE complexes with Mso1p-HA. MSO1-HA cells (H2657) overexpressing SEC1(658–724) (B3424) were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, and -Sso1p/2p antibodies. (C) Quantification of three independent experiments of immunoprecipitations shown in (B). (D) Sec1p(658–724) promotes SNARE complex formation in vitro. Sso1p, Snc2p, Sec9p (30 μM each), and indicated amounts of His6-Sec1p(658–724) were mixed and incubated at room temperature for 2 h. The complex formation was analyzed by native gel. The 8% native gels were run from the – to the + pole. The black star indicates the position of ternary complexes, and the open star other complexes. (E) Kinetics of the Sec1p(658–724) effect on SNARE complex formation in vitro. Gel mobility assays were performed essentially as in (D) with the exception that proteins were incubated at 4°C for 1, 2, or 24 h. The arrowheads indicate the position of ternary complexes.
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Figure 4: The Sec1p-tail promotes Sec1p-SNARE complex association. (A) The effect of SEC1(658–724) (B3424) overexpression for growth in SEC1 wt (H2657) and sec1(1–657) mutant (H3659) strains. The growth of serial 10-fold dilutions of cells grown at the indicated temperatures is shown. (B) Sec1p tail overexpression enhances coimmunoprecipitation of SNARE complexes with Mso1p-HA. MSO1-HA cells (H2657) overexpressing SEC1(658–724) (B3424) were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, and -Sso1p/2p antibodies. (C) Quantification of three independent experiments of immunoprecipitations shown in (B). (D) Sec1p(658–724) promotes SNARE complex formation in vitro. Sso1p, Snc2p, Sec9p (30 μM each), and indicated amounts of His6-Sec1p(658–724) were mixed and incubated at room temperature for 2 h. The complex formation was analyzed by native gel. The 8% native gels were run from the – to the + pole. The black star indicates the position of ternary complexes, and the open star other complexes. (E) Kinetics of the Sec1p(658–724) effect on SNARE complex formation in vitro. Gel mobility assays were performed essentially as in (D) with the exception that proteins were incubated at 4°C for 1, 2, or 24 h. The arrowheads indicate the position of ternary complexes.

Mentions: To shed light on the Sec1p tail in vivo function we created a SEC1(658–724) overexpression construct and analyzed the contribution of the Sec1p tail on Sec1p-SNARE complex interaction in wt and sec1(1–657) mutant cells. Under the conditions tested, overexpression of SEC1(658–724) was clearly not harmful for cell growth (Figure 4A, top panel). Instead, compared to the vector control, overexpression of SEC1(658–724) was mildly beneficial for growth of the sec1(1–657) mutant strain at all temperatures (Figure 4A, bottom panel).FIGURE 4:


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 promotes Sec1p-SNARE complex association. (A) The effect of SEC1(658–724) (B3424) overexpression for growth in SEC1 wt (H2657) and sec1(1–657) mutant (H3659) strains. The growth of serial 10-fold dilutions of cells grown at the indicated temperatures is shown. (B) Sec1p tail overexpression enhances coimmunoprecipitation of SNARE complexes with Mso1p-HA. MSO1-HA cells (H2657) overexpressing SEC1(658–724) (B3424) were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, and -Sso1p/2p antibodies. (C) Quantification of three independent experiments of immunoprecipitations shown in (B). (D) Sec1p(658–724) promotes SNARE complex formation in vitro. Sso1p, Snc2p, Sec9p (30 μM each), and indicated amounts of His6-Sec1p(658–724) were mixed and incubated at room temperature for 2 h. The complex formation was analyzed by native gel. The 8% native gels were run from the – to the + pole. The black star indicates the position of ternary complexes, and the open star other complexes. (E) Kinetics of the Sec1p(658–724) effect on SNARE complex formation in vitro. Gel mobility assays were performed essentially as in (D) with the exception that proteins were incubated at 4°C for 1, 2, or 24 h. The arrowheads indicate the position of ternary complexes.
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Figure 4: The Sec1p-tail promotes Sec1p-SNARE complex association. (A) The effect of SEC1(658–724) (B3424) overexpression for growth in SEC1 wt (H2657) and sec1(1–657) mutant (H3659) strains. The growth of serial 10-fold dilutions of cells grown at the indicated temperatures is shown. (B) Sec1p tail overexpression enhances coimmunoprecipitation of SNARE complexes with Mso1p-HA. MSO1-HA cells (H2657) overexpressing SEC1(658–724) (B3424) were grown until OD600 = 1, lysed, and subjected to anti-HA immunoprecipitations. Immunoprecipitates were analyzed by Western blotting with anti-HA, -Sec1p, -Sec9p, and -Sso1p/2p antibodies. (C) Quantification of three independent experiments of immunoprecipitations shown in (B). (D) Sec1p(658–724) promotes SNARE complex formation in vitro. Sso1p, Snc2p, Sec9p (30 μM each), and indicated amounts of His6-Sec1p(658–724) were mixed and incubated at room temperature for 2 h. The complex formation was analyzed by native gel. The 8% native gels were run from the – to the + pole. The black star indicates the position of ternary complexes, and the open star other complexes. (E) Kinetics of the Sec1p(658–724) effect on SNARE complex formation in vitro. Gel mobility assays were performed essentially as in (D) with the exception that proteins were incubated at 4°C for 1, 2, or 24 h. The arrowheads indicate the position of ternary complexes.
Mentions: To shed light on the Sec1p tail in vivo function we created a SEC1(658–724) overexpression construct and analyzed the contribution of the Sec1p tail on Sec1p-SNARE complex interaction in wt and sec1(1–657) mutant cells. Under the conditions tested, overexpression of SEC1(658–724) was clearly not harmful for cell growth (Figure 4A, top panel). Instead, compared to the vector control, overexpression of SEC1(658–724) was mildly beneficial for growth of the sec1(1–657) mutant strain at all temperatures (Figure 4A, bottom panel).FIGURE 4:

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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