title: An Amphiphysin-Like Domain in Fus2p Is Required for Rvs161p Interaction and Cortical Localization.
abstract: Cell-cell fusion fulfils essential roles in fertilization, development and tissue repair. In the budding yeast, Saccharomyces cerevisiae, fusion between two haploid cells of opposite mating type generates the diploid zygote. Fus2p is a pheromone-induced protein that regulates cell wall removal during mating. Fus2p shuttles from the nucleus to localize at the shmoo tip, bound to Rvs161p, an amphiphysin. However, Rvs161p independently binds a second amphiphysin, Rvs167p, playing an essential role in endocytosis. To understand the basis of the Fus2p-Rvs161p interaction, we analyzed Fus2p structural domains. A previously described N-terminal domain (NTD) is necessary and sufficient to regulate nuclear/cytoplasmic trafficking of Fus2p. The Dbl homology domain (DBH) binds GTP-bound Cdc42p; binding is required for cell fusion, but not localization. We identified an approximately 200 amino acid region of Fus2p that is both necessary and sufficient for Rvs161p binding. The Rvs161p binding domain (RBD) contains three predicted alpha-helices; structural modeling suggests that the RBD adopts an amphiphysin-like structure. The RBD contains a 13-amino-acid region, conserved with Rvs161p and other amphiphysins, which is essential for binding. Mutations in the RBD, predicted to affect membrane binding, abolish cell fusion without affecting Rvs161p binding. We propose that Fus2p/Rvs161p form a novel heterodimeric amphiphysin required for cell fusion. Rvs161p binding is required but not sufficient for Fus2p localization. Mutations in the C-terminal domain (CTD) of Fus2p block localization, but not Rvs161p binding, causing a significant defect in cell fusion. We conclude that the Fus2p CTD mediates an additional, Rvs161p-independent interaction at the shmoo tip.
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stein
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544.
authors.forename: Jean A
authors.initials: JA
authors.lastname: Smith
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544 mdrose@princeton.edu.
authors.forename: Mark D
authors.identifier: http://orcid.org/0000-0003-1112-4765
authors.initials: MD
authors.lastname: Rose
mesh_terms: D000595:Amino Acid Sequence; D003227:Conjugation, Genetic; D017124:Conserved Sequence; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010675:Pheromones / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D003598:Cytoskeletal Proteins; C095898:FUS2 protein, S cerevisiae; D008565:Membrane Proteins; D010675:Pheromones; C089181:RVS161 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: BAR domain; conjugation; karyogamy