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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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Production of Sec1p and characterization of Sec1p antisera. (A) Recombinant His6-Sec1p production. Size exclusion chromatogram. His6-Sec1p migrates as a single species and elutes slightly slower than BSA (67,000 D, middle arrowhead) on a Superose 12 (HR 10/30) column. Left arrowhead is 200,000 D (β-amylase) and the right arrowhead is 12,400 D (Cytochrome c). (Inset) Coomassie blue–stained gel of purified Sec1p. Lane 1 is a pooled fraction from metal chelate chromatography. This pool was further purified by ion-exchange chromatography, shown in lane 2. (B) Sec1p antibody production and yeast overexpression. The specificity of the polyclonal antisera is shown by detection of endogenous Sec1p in cytosolic extracts of the BY4741pep4Δ strain carrying the empty parent vector pYX223, and the detection of overexpressed Sec1p (2Xmyc-His6-Sec1p, pJM255). The degree of overproduction is also measured with the Sec1p antisera in comparison to a dilution series of recombinant Sec1p from E. coli.
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fig1: Production of Sec1p and characterization of Sec1p antisera. (A) Recombinant His6-Sec1p production. Size exclusion chromatogram. His6-Sec1p migrates as a single species and elutes slightly slower than BSA (67,000 D, middle arrowhead) on a Superose 12 (HR 10/30) column. Left arrowhead is 200,000 D (β-amylase) and the right arrowhead is 12,400 D (Cytochrome c). (Inset) Coomassie blue–stained gel of purified Sec1p. Lane 1 is a pooled fraction from metal chelate chromatography. This pool was further purified by ion-exchange chromatography, shown in lane 2. (B) Sec1p antibody production and yeast overexpression. The specificity of the polyclonal antisera is shown by detection of endogenous Sec1p in cytosolic extracts of the BY4741pep4Δ strain carrying the empty parent vector pYX223, and the detection of overexpressed Sec1p (2Xmyc-His6-Sec1p, pJM255). The degree of overproduction is also measured with the Sec1p antisera in comparison to a dilution series of recombinant Sec1p from E. coli.

Mentions: One of the primary goals of this work was to determine the effect of adding Sec1p to an in vitro fusion assay containing reconstituted SNARE proteins in synthetic phospholipids. Sec1 family members have been notoriously difficult to prepare in recombinant form; however, we succeeded in producing soluble recombinant Sec1p in E. coli. A full-length NH2-terminal His6-tagged Sec1p (His6-Sec1p) resulted in the best yield of soluble pure protein. Optimal conditions included coexpression with the E. coli chaperones GroEL and GroES (Yasukawa et al., 1995) and a 12-h induction at 25°C with low (0.2 mM) IPTG. Nickel affinity chromatography followed by ion exchange with Q-Sepharose resulted largely in a single protein by SDS-PAGE analysis (Fig. 1 A, inset). Recombinant Sec1p migrated as a single peak on size exclusion chromatography with a molecular size slightly more compact than the predicted 85,600 D molecular mass (Fig. 1 A). The purified protein has a tendency to aggregate and precipitate upon storage and repeated freeze-thaw cycles. Maintaining Sec1p concentrations below ∼0.2 mg/ml minimized this problem.


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)

Production of Sec1p and characterization of Sec1p antisera. (A) Recombinant His6-Sec1p production. Size exclusion chromatogram. His6-Sec1p migrates as a single species and elutes slightly slower than BSA (67,000 D, middle arrowhead) on a Superose 12 (HR 10/30) column. Left arrowhead is 200,000 D (β-amylase) and the right arrowhead is 12,400 D (Cytochrome c). (Inset) Coomassie blue–stained gel of purified Sec1p. Lane 1 is a pooled fraction from metal chelate chromatography. This pool was further purified by ion-exchange chromatography, shown in lane 2. (B) Sec1p antibody production and yeast overexpression. The specificity of the polyclonal antisera is shown by detection of endogenous Sec1p in cytosolic extracts of the BY4741pep4Δ strain carrying the empty parent vector pYX223, and the detection of overexpressed Sec1p (2Xmyc-His6-Sec1p, pJM255). The degree of overproduction is also measured with the Sec1p antisera in comparison to a dilution series of recombinant Sec1p from E. coli.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172507&req=5

fig1: Production of Sec1p and characterization of Sec1p antisera. (A) Recombinant His6-Sec1p production. Size exclusion chromatogram. His6-Sec1p migrates as a single species and elutes slightly slower than BSA (67,000 D, middle arrowhead) on a Superose 12 (HR 10/30) column. Left arrowhead is 200,000 D (β-amylase) and the right arrowhead is 12,400 D (Cytochrome c). (Inset) Coomassie blue–stained gel of purified Sec1p. Lane 1 is a pooled fraction from metal chelate chromatography. This pool was further purified by ion-exchange chromatography, shown in lane 2. (B) Sec1p antibody production and yeast overexpression. The specificity of the polyclonal antisera is shown by detection of endogenous Sec1p in cytosolic extracts of the BY4741pep4Δ strain carrying the empty parent vector pYX223, and the detection of overexpressed Sec1p (2Xmyc-His6-Sec1p, pJM255). The degree of overproduction is also measured with the Sec1p antisera in comparison to a dilution series of recombinant Sec1p from E. coli.
Mentions: One of the primary goals of this work was to determine the effect of adding Sec1p to an in vitro fusion assay containing reconstituted SNARE proteins in synthetic phospholipids. Sec1 family members have been notoriously difficult to prepare in recombinant form; however, we succeeded in producing soluble recombinant Sec1p in E. coli. A full-length NH2-terminal His6-tagged Sec1p (His6-Sec1p) resulted in the best yield of soluble pure protein. Optimal conditions included coexpression with the E. coli chaperones GroEL and GroES (Yasukawa et al., 1995) and a 12-h induction at 25°C with low (0.2 mM) IPTG. Nickel affinity chromatography followed by ion exchange with Q-Sepharose resulted largely in a single protein by SDS-PAGE analysis (Fig. 1 A, inset). Recombinant Sec1p migrated as a single peak on size exclusion chromatography with a molecular size slightly more compact than the predicted 85,600 D molecular mass (Fig. 1 A). The purified protein has a tendency to aggregate and precipitate upon storage and repeated freeze-thaw cycles. Maintaining Sec1p concentrations below ∼0.2 mg/ml minimized this problem.

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
Related in: MedlinePlus