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PMID: 15466482 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.

The Journal of cell biology ·Vol. 167 ·No. 1 ·2004-10-11 ·Pages 75-85

Scott BL, Van Komen JS, Irshad H, Liu S, Wilson KA, McNew JA

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.

MeSH Terms
Cell Membrane/metabolism Dose-Response Relationship, Drug Escherichia coli/metabolism Fluorescent Antibody Technique, Indirect Glutathione Transferase/metabolism Golgi Apparatus/metabolism Membrane Fusion Munc18 Proteins Nerve Tissue Proteins/metabolism,physiology Oligonucleotides/chemistry Plasmids/metabolism Protein Binding Recombinant Proteins/chemistry,metabolism SNARE Proteins Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins Temperature Time Factors Vesicular Transport Proteins/metabolism,physiology
Chemicals
Munc18 Proteins Nerve Tissue Proteins Oligonucleotides Recombinant Proteins SEC1 protein, S cerevisiae SNARE Proteins Saccharomyces cerevisiae Proteins Vesicular Transport Proteins Glutathione Transferase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Scott Brenton L
Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.
Van Komen Jeffrey S
Irshad Hassan
Liu Song
Wilson Kirilee A
McNew James A
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.200405018
Published
2004-10-11
Epub
2004-00-04
Pages
75-85
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2172507
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.200405018 PMC Full Text View journal details
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