Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement.
Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.
Tedrick
Kelly
K
Department of Cell Biology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada.
Trischuk
Tim
T
Lehner
Richard
R
Eitzen
Gary
G
D000199:Actins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004875:Ergosterol / Q000737:chemistry / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry* / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein
D000199:Actins; D003598:Cytoskeletal Proteins; C480361:LAS17 protein, S cerevisiae; D008563:Membrane Lipids; D008840:Microfilament Proteins; D029701:Saccharomyces cerevisiae Proteins; C090350:VRP1 protein, S cerevisiae; D051316:Wiskott-Aldrich Syndrome Protein; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D005947:Glucose; D005690:Galactose; D004875:Ergosterol