Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.
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.
Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.
Brenton L
BL
Scott
Jeffrey S
JS
Van Komen
Hassan
H
Irshad
Song
S
Liu
Kirilee A
KA
Wilson
James A
JA
McNew
D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008561:Membrane Fusion; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000378:metabolism* / Q000502:physiology*
D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; C497931:SEC1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase