ABSTRACT
The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it an ideal system for studying the control of cellular dimensions. In this study, we carried out a near-genome-wide screen for mutants wider than wild-type cells. We found 11 deletion mutants that were wider; seven of the deleted genes are implicated in the control of the small GTPase Cdc42, including the Cdc42 guanine nucleotide exchange factor (GEF) Scd1 and the Cdc42 GTPase-activating protein (GAP) Rga4. Deletions of rga4 and scd1 had additive effects on cell width, and the proteins localized independently of one another, with Rga4 located at the cell sides and Scd1 at the cell tips. Activated Cdc42 localization is altered in rga4Δ, scd1Δ, and scd2Δ mutants. Delocalization and ectopic retargeting experiments showed that the localizations of Rga4 and Scd1 are crucial for their roles in determining cell width. We propose that the GAP Rga4 and the GEF Scd1 establish a gradient of activated Cdc42 within the cellular tip plasma membrane, and it is this gradient that determines cell growth-zone size and normal cell width.
Subject(s)
Cytoskeleton/metabolism , GTPase-Activating Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Organisms, Genetically Modified/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/metabolism , Signal Transduction/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , Cell Division/genetics , Cell Enlargement , Cell Shape , Cell Size , Cytoskeleton/genetics , GTPase-Activating Proteins/deficiency , Gene Deletion , Gene Expression Regulation, Fungal , Guanine Nucleotide Exchange Factors/deficiency , Microscopy, Fluorescence , Microscopy, Interference , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/growth & development , Plasmids , Protein Binding , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Transduction, Genetic , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/deficiencyABSTRACT
The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.
