Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

Abstract

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response. INTRODUCTIONRVS161 and RVS167, which encode closely related proteins in Saccharomyces cerevisiae, were first identified in a screen for mutants that exhibited reduced viability upon starvation (Bauer et al., 1993). Mutation of RVS161 or RVS167 causes a phenotype consistent with a role for the Rvs proteins in cortical actin cytoskeleton organization and endocytosis: loss of viability and unusual cell morphology in poor growth medium or salt-containing medium, delocalized actin distribution under suboptimal growth conditions, abnormal (random) budding in diploids, and defects in endocytosis and sporulation (Bauer et al., 1993). Ultrastructural studies have revealed that rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction (Breton et al., 2001), suggesting an additional role for Rvs161p and Rvs167p in vesicle trafficking.Rvs161p and Rvs167p are members of the BAR-domain family of proteins, which includes Bin1, Amphiphysin, and Rvs proteins (Sivadon et al., 1997). Amphiphysins are enriched in the mammalian brain and seem to function in synaptic vesicle endocytosis (for review, see Zhang and Zelhof, 2002). Bin1 is a splice isoform of amphiphysin 2 that has features of a tumor suppressor (Sakamuro et al., 1996). Proteins in this family are characterized by the presence of a conserved BAR domain, and it is through their BAR domains that Rvs161p interacts with Rvs167p (Navarro et al., 1997;Sivadon et al., 1997;Colwill et al., 1999). The crystal structure of the BAR domain of Drosophila amphiphysin has recently been solved (Peter et al., 2004). It is a crescentshaped dimer, in which each monomer forms a coiled coil. The curved shape is only revealed upon dimerization of the two slightly kinked ...

Identification and Characterization of Genes Required for Cell-to-Cell Fusion in Neurospora crassa

“…1 and 3) and ham-10 (NCU02833) genes affect CAT formation, and the proteins encoded may be associated with the actin cytoskeleton, which has been shown to be required for CAT formation (43). The amph-1 gene is homologous to the S. cerevisiae RVS161 and RVS167 (amphiphysin) genes, known to be required for cell fusion events in yeast (14,15). The ham-10 The first step involved screening each of the 10,000 mutants in the single gene deletion library to identify those with a cell fusion phenotype.…”

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

“…PtdIns(4,5)P 2 at the plasma membrane is required for invagination of clathrin-coated pits (Doherty and McMahon, 2009; Haucke, 2005), whereas PtdIns(4,5)P 2 turnover is known to play a pivotal role in the assembly and disassembly of endocytic patches in yeast (Sun et al, 2007). This presumably explains why a number of endocytic proteins possess PtdIns(4,5)P 2 -binding domains (Ford et al, 2001;Friesen et al, 2006;Gaidarov and Keen, 1999;Itoh et al, 2001;Jost et al, 1998;Sun et al, 2005;Takei et al, 1999;Vallis et al, 1999) or associate with enzymes involved in PtdIns(4,5)P 2 synthesis and breakdown (Bairstow et al, 2006;Haffner et al, 1997;McPherson et al, 1996;Padron et al, 2003;Stefan et al, 2005). Fission yeast Pik1p localizes uniformly throughout the cell and concentrates in the Golgi apparatus (Park et al, 2009) irrespective of whether Cam2p is deleted (supplementary material Fig.…”

Kel1p Mediates Yeast Cell Fusion Through a Fus2p- and Cdc42p-Dependent Mechanism

“…FUS1, FUS2, and PRM1 are all pheromone-induced genes and are required in at least one of two mating cells to produce a diploid. Rvs161p is a BAR domain protein related to amphiphysin that plays a role in endocytosis in mitotic cells by stabilizing curved membranes (Crouzet et al 1991;Friesen et al 2006). Therefore, RVS161 is expressed in mitotic cells, but strongly induced by pheromone .…”