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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.

Scott BL, Van Komen JS, Irshad H, Liu S, Wilson KA, McNew JA - J. Cell Biol. (2004)

Bottom Line: We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae.Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells.Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.

ABSTRACT
Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Show MeSH
Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) and the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p). GST pull-down experiments were used to assess the degree of Sec1p binding to various SNAREs and SNARE complexes. The level of nonspecific binding was determined by incubation of recombinant Sec1p with protein-free GSH beads (lane 1), GST (lane 2), or GST-Sed5p (lane 3). The level of Sec1p binding to GST-Sed5p (which was approximately four times higher than either protein free beads or GST alone) was used as the background value for quantitative analysis. Sec1p binding to three SNARE species was analyzed: free t-SNARE (lanes 4 and 7), the binary t-SNARE complex (lanes 5 and 8) or the ternary SNARE complex (lanes 6 and 9). The three species were attached to glutathione beads via GST-Sso1p (lanes 4–6) or GST-Sec9c (lanes 7–9). Purified recombinant Sec1p (lane 10) is also shown (∼1.4 μg, ∼16 pmol). Sec1p binding was quantified by densitometry and is represented as fold above the GST-Sed5p background. Sec1p binds most significantly to the t-SNARE complex (lanes 5 and 8) as well as the ternary SNARE complex (lanes 6 and 9). A weaker association was also seen with the free monomeric SNAREs GST-Sso1p (lane 4). No binding to free GST-Sec9c (lane 7) was detected above background.
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fig3: Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) and the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p). GST pull-down experiments were used to assess the degree of Sec1p binding to various SNAREs and SNARE complexes. The level of nonspecific binding was determined by incubation of recombinant Sec1p with protein-free GSH beads (lane 1), GST (lane 2), or GST-Sed5p (lane 3). The level of Sec1p binding to GST-Sed5p (which was approximately four times higher than either protein free beads or GST alone) was used as the background value for quantitative analysis. Sec1p binding to three SNARE species was analyzed: free t-SNARE (lanes 4 and 7), the binary t-SNARE complex (lanes 5 and 8) or the ternary SNARE complex (lanes 6 and 9). The three species were attached to glutathione beads via GST-Sso1p (lanes 4–6) or GST-Sec9c (lanes 7–9). Purified recombinant Sec1p (lane 10) is also shown (∼1.4 μg, ∼16 pmol). Sec1p binding was quantified by densitometry and is represented as fold above the GST-Sed5p background. Sec1p binds most significantly to the t-SNARE complex (lanes 5 and 8) as well as the ternary SNARE complex (lanes 6 and 9). A weaker association was also seen with the free monomeric SNAREs GST-Sso1p (lane 4). No binding to free GST-Sec9c (lane 7) was detected above background.

Mentions: Recombinant neuronal Sec1 binds to the closed conformation of Syntaxin1A (Misura et al., 2000; Yang et al., 2000), whereas Sec1p from a yeast cytosol extract has been reported to bind to the fully assembled ternary SNARE complex (Carr et al., 1999). To resolve these differences, we examined the binding characteristics of recombinant yeast Sec1p to various SNAREs and SNARE complexes. We used well-characterized GST pull-down assays where individual SNAREs or SNARE complexes were bound to glutathione agarose beads and roughly twofold molar excess amounts of recombinant Sec1p was added. Sec1p was allowed to bind for ∼16 h at 4°C, and after extensive washing, the bound complexes were eluted by SDS sample buffer, resolved by SDS-PAGE and visualized by Coomassie blue staining. We examined complexes assembled on GST-Sso1p and GST-Sec9c (containing only the SNAP25 homologous portion of Sec9p). Three conditions were examined for Sec1p binding: free GST-SNARE, the t-SNARE complex, and the fully assembled ternary SNARE complex. Fig. 3 illustrates the results of a representative binding assay. Minimal amounts of Sec1p nonspecifically associated with reduced glutathione (GSH) resin (lane 1), GST (lane 2), or the Golgi SNARE Sed5p (lane 3). Specific Sec1p binding was detected to free GST-Sso1p (Fig. 3, 1.9-fold above the highest background, lane 4 vs. lane 3), but not to free GST-Sec9c (Fig. 3, lane 7). Binding of Sec1p to the t-SNARE complex (Fig. 3, lanes 5 and 8) was 4–10-fold more than background values. Significant binding of Sec1p to the ternary SNARE complex (Fig. 3, lanes 6 and 9) was also observed, strengthening a previous observation that an immunodetectable amount of Sec1p from cytosol associates with the fully assembled SNARE complex (Carr et al., 1999). We have now shown binding to the ternary SNARE complex at levels detected by Coomassie blue staining. Our data extend that observation to include detectable binding of Sec1p to the uncomplexed t-SNARE protein GST-Sso1p. Importantly, maximum Sec1p binding was detected to Sso1p/Sec9c t-SNARE complexes, suggesting that this is the preferred partner.


Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.

Scott BL, Van Komen JS, Irshad H, Liu S, Wilson KA, McNew JA - J. Cell Biol. (2004)

Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) and the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p). GST pull-down experiments were used to assess the degree of Sec1p binding to various SNAREs and SNARE complexes. The level of nonspecific binding was determined by incubation of recombinant Sec1p with protein-free GSH beads (lane 1), GST (lane 2), or GST-Sed5p (lane 3). The level of Sec1p binding to GST-Sed5p (which was approximately four times higher than either protein free beads or GST alone) was used as the background value for quantitative analysis. Sec1p binding to three SNARE species was analyzed: free t-SNARE (lanes 4 and 7), the binary t-SNARE complex (lanes 5 and 8) or the ternary SNARE complex (lanes 6 and 9). The three species were attached to glutathione beads via GST-Sso1p (lanes 4–6) or GST-Sec9c (lanes 7–9). Purified recombinant Sec1p (lane 10) is also shown (∼1.4 μg, ∼16 pmol). Sec1p binding was quantified by densitometry and is represented as fold above the GST-Sed5p background. Sec1p binds most significantly to the t-SNARE complex (lanes 5 and 8) as well as the ternary SNARE complex (lanes 6 and 9). A weaker association was also seen with the free monomeric SNAREs GST-Sso1p (lane 4). No binding to free GST-Sec9c (lane 7) was detected above background.
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fig3: Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) and the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p). GST pull-down experiments were used to assess the degree of Sec1p binding to various SNAREs and SNARE complexes. The level of nonspecific binding was determined by incubation of recombinant Sec1p with protein-free GSH beads (lane 1), GST (lane 2), or GST-Sed5p (lane 3). The level of Sec1p binding to GST-Sed5p (which was approximately four times higher than either protein free beads or GST alone) was used as the background value for quantitative analysis. Sec1p binding to three SNARE species was analyzed: free t-SNARE (lanes 4 and 7), the binary t-SNARE complex (lanes 5 and 8) or the ternary SNARE complex (lanes 6 and 9). The three species were attached to glutathione beads via GST-Sso1p (lanes 4–6) or GST-Sec9c (lanes 7–9). Purified recombinant Sec1p (lane 10) is also shown (∼1.4 μg, ∼16 pmol). Sec1p binding was quantified by densitometry and is represented as fold above the GST-Sed5p background. Sec1p binds most significantly to the t-SNARE complex (lanes 5 and 8) as well as the ternary SNARE complex (lanes 6 and 9). A weaker association was also seen with the free monomeric SNAREs GST-Sso1p (lane 4). No binding to free GST-Sec9c (lane 7) was detected above background.
Mentions: Recombinant neuronal Sec1 binds to the closed conformation of Syntaxin1A (Misura et al., 2000; Yang et al., 2000), whereas Sec1p from a yeast cytosol extract has been reported to bind to the fully assembled ternary SNARE complex (Carr et al., 1999). To resolve these differences, we examined the binding characteristics of recombinant yeast Sec1p to various SNAREs and SNARE complexes. We used well-characterized GST pull-down assays where individual SNAREs or SNARE complexes were bound to glutathione agarose beads and roughly twofold molar excess amounts of recombinant Sec1p was added. Sec1p was allowed to bind for ∼16 h at 4°C, and after extensive washing, the bound complexes were eluted by SDS sample buffer, resolved by SDS-PAGE and visualized by Coomassie blue staining. We examined complexes assembled on GST-Sso1p and GST-Sec9c (containing only the SNAP25 homologous portion of Sec9p). Three conditions were examined for Sec1p binding: free GST-SNARE, the t-SNARE complex, and the fully assembled ternary SNARE complex. Fig. 3 illustrates the results of a representative binding assay. Minimal amounts of Sec1p nonspecifically associated with reduced glutathione (GSH) resin (lane 1), GST (lane 2), or the Golgi SNARE Sed5p (lane 3). Specific Sec1p binding was detected to free GST-Sso1p (Fig. 3, 1.9-fold above the highest background, lane 4 vs. lane 3), but not to free GST-Sec9c (Fig. 3, lane 7). Binding of Sec1p to the t-SNARE complex (Fig. 3, lanes 5 and 8) was 4–10-fold more than background values. Significant binding of Sec1p to the ternary SNARE complex (Fig. 3, lanes 6 and 9) was also observed, strengthening a previous observation that an immunodetectable amount of Sec1p from cytosol associates with the fully assembled SNARE complex (Carr et al., 1999). We have now shown binding to the ternary SNARE complex at levels detected by Coomassie blue staining. Our data extend that observation to include detectable binding of Sec1p to the uncomplexed t-SNARE protein GST-Sso1p. Importantly, maximum Sec1p binding was detected to Sso1p/Sec9c t-SNARE complexes, suggesting that this is the preferred partner.

Bottom Line: We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae.Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells.Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.

ABSTRACT
Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Show MeSH