title: Prm1 functions as a disulfide-linked complex in yeast mating.
abstract: Prm1 is a pheromone-induced membrane glycoprotein that promotes plasma membrane fusion in yeast mating pairs. HA-Prm1 migrates at twice its expected molecular weight on non-reducing SDS-PAGE gels and coprecipitates with Prm1-TAP, indicating that Prm1 is a disulfide-linked homodimer. The N terminus of a plasma membrane-localized GFP-Prm1 endocytic mutant projects into the cytoplasm, where it is protected from low pH quenching in live cells and from external protease in spheroplasts. In a revised topological map, Prm1 has four transmembrane domains and two large extracellular loops. Mutation of all four cysteines in the extracellular loops blocked disulfide bond formation and destabilized the Prm1 homodimer without preventing Prm1 transport to contact sites in mating pairs. Cys(120) in loop 1 and Cys(545) in loop 2 form disulfide cross-links in the Prm1 homodimer and are required for fusion activity. Cys(120) lies between a hydrophobic segment formerly thought to be a transmembrane domain and an amphipathic helix. An interaction between either of these regions and the opposing membrane could promote fusion.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.
authors.forename: Valerie N
authors.initials: VN
authors.lastname: Olmo
authors.forename: Eric
authors.initials: E
authors.lastname: Grote
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D008565:Membrane Proteins; C499613:PRM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D003545:Cysteine