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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.

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Effect of Sec1p on SNARE complex formation. (A) Binary t-SNARE complex and ternary SNARE complex formation is unaffected by Sec1p when all components are added simultaneously. GST-Sec9c was bound to resin and equimolar amounts of His8-Sso1p, Snc2p-His6, and increasing amounts of His6-Sec1p (0, 0.1, 0.2, and 0.4 nmol) were added at the same time and incubated at 4°C for ∼16 h. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue. (B) Snc2p can efficiently bind to Sec1p bound t-SNARE complexes without significant displacement of Sec1p. Increasing amounts of Snc2p (0, lane 1 and 5, 0.4 nmol lane 2 and 6, 0.8 nmol, lanes 3 and 7 and 1.6 nmol, lanes 4 and 8) were allowed to associate with preformed t-SNARE complex (Sso1p/Sec9c, lanes 1–4) or Sec1p bound t-SNARE complexes (Sec1p:Sso1p/Sec9c, lanes 5–8) for ∼16 h at 4°C. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue.
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fig4: Effect of Sec1p on SNARE complex formation. (A) Binary t-SNARE complex and ternary SNARE complex formation is unaffected by Sec1p when all components are added simultaneously. GST-Sec9c was bound to resin and equimolar amounts of His8-Sso1p, Snc2p-His6, and increasing amounts of His6-Sec1p (0, 0.1, 0.2, and 0.4 nmol) were added at the same time and incubated at 4°C for ∼16 h. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue. (B) Snc2p can efficiently bind to Sec1p bound t-SNARE complexes without significant displacement of Sec1p. Increasing amounts of Snc2p (0, lane 1 and 5, 0.4 nmol lane 2 and 6, 0.8 nmol, lanes 3 and 7 and 1.6 nmol, lanes 4 and 8) were allowed to associate with preformed t-SNARE complex (Sso1p/Sec9c, lanes 1–4) or Sec1p bound t-SNARE complexes (Sec1p:Sso1p/Sec9c, lanes 5–8) for ∼16 h at 4°C. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue.

Mentions: Binding of Sec1p to the ternary SNARE complex was consistently reduced compared with t-SNARE complex binding (Fig. 3, compare lane 5 vs. lane 6 and lane 8 vs. lane 9) suggesting that Snc2p may influence Sec1p binding when SNARE complexes are preassembled. To address this issue, we analyzed Sec1p (and Snc2p) binding under different experimental conditions. First, we conducted a binding experiment where all of the protein components were added simultaneously to GST-Sec9c bound resin (Fig. 4 A). Binary t-SNARE complexes (Fig. 4 A, lanes 1–4) or ternary SNARE complexes (Fig. 4 A, lanes 5–8) were formed in the presence of increasing concentrations (0, 0.25, 0.5, and 1.0 μM) of Sec1p. These results show that the presence of Sec1p does not affect the extent of t-SNARE complex formation or the extent of ternary SNARE complex formation. The overall degree of Sec1p binding is similar when SNARE complexes are preformed and Sec1p is in excess (Fig. 3, lanes 8 and 9) or when equal molar amounts of SNARE complexes form in the presence of Sec1p (Fig. 4 A, lanes 4 and 8).


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)

Effect of Sec1p on SNARE complex formation. (A) Binary t-SNARE complex and ternary SNARE complex formation is unaffected by Sec1p when all components are added simultaneously. GST-Sec9c was bound to resin and equimolar amounts of His8-Sso1p, Snc2p-His6, and increasing amounts of His6-Sec1p (0, 0.1, 0.2, and 0.4 nmol) were added at the same time and incubated at 4°C for ∼16 h. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue. (B) Snc2p can efficiently bind to Sec1p bound t-SNARE complexes without significant displacement of Sec1p. Increasing amounts of Snc2p (0, lane 1 and 5, 0.4 nmol lane 2 and 6, 0.8 nmol, lanes 3 and 7 and 1.6 nmol, lanes 4 and 8) were allowed to associate with preformed t-SNARE complex (Sso1p/Sec9c, lanes 1–4) or Sec1p bound t-SNARE complexes (Sec1p:Sso1p/Sec9c, lanes 5–8) for ∼16 h at 4°C. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue.
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fig4: Effect of Sec1p on SNARE complex formation. (A) Binary t-SNARE complex and ternary SNARE complex formation is unaffected by Sec1p when all components are added simultaneously. GST-Sec9c was bound to resin and equimolar amounts of His8-Sso1p, Snc2p-His6, and increasing amounts of His6-Sec1p (0, 0.1, 0.2, and 0.4 nmol) were added at the same time and incubated at 4°C for ∼16 h. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue. (B) Snc2p can efficiently bind to Sec1p bound t-SNARE complexes without significant displacement of Sec1p. Increasing amounts of Snc2p (0, lane 1 and 5, 0.4 nmol lane 2 and 6, 0.8 nmol, lanes 3 and 7 and 1.6 nmol, lanes 4 and 8) were allowed to associate with preformed t-SNARE complex (Sso1p/Sec9c, lanes 1–4) or Sec1p bound t-SNARE complexes (Sec1p:Sso1p/Sec9c, lanes 5–8) for ∼16 h at 4°C. Bound complexes were resolved by SDS-PAGE and stained with Coomassie blue.
Mentions: Binding of Sec1p to the ternary SNARE complex was consistently reduced compared with t-SNARE complex binding (Fig. 3, compare lane 5 vs. lane 6 and lane 8 vs. lane 9) suggesting that Snc2p may influence Sec1p binding when SNARE complexes are preassembled. To address this issue, we analyzed Sec1p (and Snc2p) binding under different experimental conditions. First, we conducted a binding experiment where all of the protein components were added simultaneously to GST-Sec9c bound resin (Fig. 4 A). Binary t-SNARE complexes (Fig. 4 A, lanes 1–4) or ternary SNARE complexes (Fig. 4 A, lanes 5–8) were formed in the presence of increasing concentrations (0, 0.25, 0.5, and 1.0 μM) of Sec1p. These results show that the presence of Sec1p does not affect the extent of t-SNARE complex formation or the extent of ternary SNARE complex formation. The overall degree of Sec1p binding is similar when SNARE complexes are preformed and Sec1p is in excess (Fig. 3, lanes 8 and 9) or when equal molar amounts of SNARE complexes form in the presence of Sec1p (Fig. 4 A, lanes 4 and 8).

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