title: The lipid composition and physical properties of the yeast vacuole affect the hemifusion-fusion transition.
abstract: Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane-anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion-incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion-fusion transition. The hemifusion-fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7p(Q283R) . Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion-stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE-generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild-type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion-fusion transition.
authors: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors: Surya
authors: S
authors: Karunakaran
authors: Rutilio A
authors: RA
authors: Fratti
mesh_terms: D008055:Lipids / Q000737:chemistry*; D008561:Membrane Fusion; D009154:Mutation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008055:Lipids; D050765:Qa-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C107706:VAM3 protein, S cerevisiae; C494205:VAM7 protein, S cerevisiae