Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo.

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.

Interaction of the Saccharomyces cerevisiae cortical actin patch protein Rvs167p with proteins involved in ER to Golgi vesicle trafficking.

We have used affinity chromatography to identify two proteins that bind to the SH3 domain of the actin cytoskeleton protein Rvs167p: Gyp5p and Gyl1p. Gyp5p has been shown to be a GTPase activating protein (GAP) for Ypt1p, a Rab GTPase involved in ER to Golgi trafficking; Gyl1p is a protein that resembles Gyp5p and has recently been shown to colocalize with and belong to the same protein complex as Gyp5p. We show that Gyl1p and Gyp5p interact directly with each other, likely through their carboxy-terminal coiled-coil regions. In assays of GAP activity, Gyp5p had GAP activity toward Ypt1p and we found that this activity was stimulated by the addition of Gyl1p. Gyl1p had no GAP activity toward Ypt1p. Genetic experiments suggest a role for Gyp5p and Gyl1p in ER to Golgi trafficking, consistent with their biochemical role. Since Rvs167p has a previously characterized role in endocytosis and we have shown here that it interacts with proteins involved in Golgi vesicle trafficking, we suggest that Rvs167p may have a general role in vesicle trafficking.

CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.

Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.

G1 transcription factors are differentially regulated in Saccharomyces cerevisiae by the Swi6-binding protein Stb1.

Stage-specific transcriptional programs are an integral feature of cell cycle regulation. In the budding yeast Saccharomyces cerevisiae, over 120 genes are coordinately induced in late G(1) phase by two heterodimeric transcription factors called SBF and MBF. Activation of SBF and MBF is an upstream initiator of key cell cycle events, including budding and DNA replication. SBF and MBF regulation is complex and genetically redundant, and the precise mechanism of G(1) transcriptional activation is unclear. Assays using SBF- and MBF-specific reporter genes revealed that the STB1 gene specifically affected MBF-dependent transcription. STB1 encodes a known Swi6-binding protein, but an MBF-specific function had not been previously suspected. Consistent with a specific role in regulating MBF, a STB1 deletion strain requires SBF for viability and microarray studies show a decrease in MBF-regulated transcripts in a swi4Delta mutant following depletion of Stb1. Chromatin immunoprecipitation experiments confirm that Stb1 localizes to promoters of MBF-regulated genes. Our data indicate that, contrary to previous models, MBF and SBF have unique components and might be distinctly regulated.