title: The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.
abstract: The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion.
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Miner
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Starr
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Logan R
authors.initials: LR
authors.lastname: Hurst
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Sparks
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Mark
authors.initials: M
authors.lastname: Padolina
authors.affiliation: From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 rfratti@illinois.edu.
authors.forename: Rutilio A
authors.initials: RA
authors.lastname: Fratti
mesh_terms: D019943:Amino Acid Substitution; D020125:Mutation, Missense; D018129:Phosphatidylinositol Phosphates / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*
chemical_list: D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae; C055525:phosphatidylinositol 3-phosphate
keywords: HOPS; SNARE proteins; calcium transport; diacylglycerol; membrane fusion; phosphatidic acid; phosphatidylinositol; ycf1