Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

doi:10.1083/jcb.200405018

Published online 4 October 2004. doi:10.1083/jcb.200405018
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 167, Number 1, 75-85

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Article

Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

Brenton L. Scott, Jeffrey S. Van Komen, Hassan Irshad, Song Liu, Kirilee A. Wilson, and James A. McNew

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251

Correspondence to James A. McNew: mcnew{at}rice.edu

Sec1 proteins are critical players in membrane trafficking,yet their precise role remains unknown. We have examined therole of Sec1p in the regulation of post-Golgi secretion in Saccharomycescerevisiae. Indirect immunofluorescence shows that endogenousSec1p is found primarily at the bud neck in newly budded cellsand in patches broadly distributed within the plasma membranein unbudded cells. Recombinant Sec1p binds strongly to the t-SNAREcomplex (Sso1p/Sec9c) as well as to the fully assembled ternarySNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly tofree Sso1p. We used recombinant Sec1p to test Sec1p functionusing a well-characterized SNARE-mediated membrane fusion assay.The addition of Sec1p to a traditional in vitro fusion assaymoderately stimulates fusion; however, when Sec1p is allowedto bind to SNAREs before reconstitution, significantly moreSec1p binding is detected and fusion is stimulated in a concentration-dependentmanner. These data strongly argue that Sec1p directly stimulatesSNARE-mediated membrane fusion.

Abbreviations used in this paper: ß-ME, ß-mercaptoethanol;GSH, reduced glutathione; His₆-Sec1p, His₆-tagged Sec1p; OG,n-octyl-ß-D-glucopyranoside; SM, Sec1/Munc18; t-SNARE,target membrane SNARE; v-SNARE, vesicle SNARE.

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