ABSTRACT
Cell polarization generally occurs along a single axis that is directed by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae undergo polarized growth and oriented cell division in a spatial pattern by selecting a specific bud site. Haploid a or α cells bud in the axial pattern in response to a transient landmark that includes Bud3, Bud4, Axl1, and Axl2. Septins, a family of filament-forming GTP-binding proteins, are also involved in axial budding and recruited to an incipient bud site, but the mechanism of recruitment remains unclear. Here, we show that Axl2 interacts with Bud3 and the Cdc42 GTPase in its GTP-bound state. Axl2 also interacts with Cdc10, a septin subunit, promoting efficient recruitment of septins near the cell division site. Furthermore, a cdc42 mutant defective in the axial budding pattern at a semi-permissive temperature had a reduced interaction with Axl2 and compromised septin recruitment in the G1 phase. We thus propose that active Cdc42 brings Axl2 to the Bud3-Bud4 complex and that Axl2 then interacts with Cdc10, linking septin recruitment to the axial landmark.
ABSTRACT
Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.
Subject(s)
Saccharomycetales , Cell Polarity/physiology , GTPase-Activating Proteins/metabolism , Longevity , Saccharomyces cerevisiae/metabolism , Saccharomycetales/metabolism , Up-Regulation , cdc42 GTP-Binding Protein/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
The Rho GTPase Cdc42 is a central regulator of cell polarity in diverse cell types. The activity of Cdc42 is dynamically controlled in time and space to enable distinct polarization events, which generally occur along a single axis in response to spatial cues. Our understanding of the mechanisms underlying Cdc42 polarization has benefited largely from studies of the budding yeast Saccharomyces cerevisiae, a genetically tractable model organism. In budding yeast, Cdc42 activation occurs in two temporal steps in the G1 phase of the cell cycle to establish a proper growth site. Here, we review findings in budding yeast that reveal an intricate crosstalk among polarity proteins for biphasic Cdc42 regulation. The first step of Cdc42 activation may determine the axis of cell polarity, while the second step ensures robust Cdc42 polarization for growth. Biphasic Cdc42 polarization is likely to ensure the proper timing of events including the assembly and recognition of spatial landmarks and stepwise assembly of a new ring of septins, cytoskeletal GTP-binding proteins, at the incipient bud site. Biphasic activation of GTPases has also been observed in mammalian cells, suggesting that biphasic activation could be a general mechanism for signal-responsive cell polarization. Cdc42 activity is necessary for polarity establishment during normal cell division and development, but its activity has also been implicated in the promotion of aging. We also discuss negative polarity signaling and emerging concepts of Cdc42 signaling in cellular aging.
ABSTRACT
Rho GTPases play critical roles in cell proliferation and cell death in many species. As in animal cells, cells of the budding yeast Saccharomyces cerevisiae undergo regulated cell death under various physiological conditions and upon exposure to external stress. The Rho5 GTPase is necessary for oxidant-induced cell death, and cells expressing a constitutively active GTP-locked Rho5 are hypersensitive to oxidants. Yet how Rho5 regulates yeast cell death has been poorly understood. To identify genes that are involved in the Rho5-mediated cell death program, we performed two complementary genome-wide screens: one screen for oxidant-resistant deletion mutants and another screen for Rho5-associated proteins. Functional enrichment and interaction network analysis revealed enrichment for genes in pathways related to metabolism, transport, and plasma membrane organization. In particular, we find that ATG21, which is known to be involved in the CVT (Cytoplasm-to-Vacuole Targeting) pathway and mitophagy, is necessary for cell death induced by oxidants. Cells lacking Atg21 exhibit little cell death upon exposure to oxidants even when the GTP-locked Rho5 is expressed. Moreover, Atg21 interacts with Rho5 preferentially in its GTP-bound state, suggesting that Atg21 is a downstream target of Rho5 in oxidant-induced cell death. Given the high degree of conservation of Rho GTPases and autophagy from yeast to human, this study may provide insight into regulated cell death in eukaryotes in general.
Subject(s)
Autophagy-Related Proteins/metabolism , Cell Death , Endopeptidases/metabolism , Oxidative Stress , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolism , Oxidants/toxicity , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/physiologyABSTRACT
The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel-one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.
Subject(s)
Cell Polarity , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Alleles , Cell Membrane/metabolism , G1 Phase , Green Fluorescent Proteins/metabolism , Models, Biological , Mutation/genetics , Protein Binding , Protein Domains , Protein Stability , Septins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
In yeast and animal cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate cell polarization. In budding yeast, selection of a bud site directs polarity establishment and subsequently determines the plane of cell division. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the division site by inhibiting Cdc42 repolarization. A protein complex including Nba1 and Nis1 is involved in preventing rebudding at old division sites, yet how these proteins and Rga1 might function in negative polarity signaling has been elusive. Here we show that Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. However, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and a new bud site is established prior to delocalization of Rga1. Indeed, our biphasic mathematical model of Cdc42 polarization predicts that premature delocalization of Rga1 leads to more frequent Cdc42 repolarization within the division site when the first temporal step in G1 is assumed to last longer. Spatial distribution of a Cdc42 GAP in coordination with G1 progression may thus be critical for fine-tuning the orientation of the polarity axis in yeast.
Subject(s)
GTPase-Activating Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Cell Cycle/physiology , Cell Division/genetics , Cell Division/physiology , Cell Polarity/physiology , G1 Phase/physiology , GTPase-Activating Proteins/genetics , Orientation, Spatial , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Signal TransductionABSTRACT
Cell polarization occurs along a single axis that is generally determined by a spatial cue, yet the underlying mechanism is poorly understood. Using biochemical assays and live-cell imaging, we show that cell polarization to a proper growth site requires activation of Cdc42 by Bud3 in haploid budding yeast. Bud3 catalyzes the release of guanosine diphosphate (GDP) from Cdc42 and elevates intracellular Cdc42-guanosine triphosphate (GTP) levels in cells with inactive Cdc24, which has as of yet been the sole GDP-GTP exchange factor for Cdc42. Cdc42 is activated in two temporal steps in the G1 phase: the first depends on Bud3, whereas subsequent activation depends on Cdc24. Mutational analyses suggest that biphasic activation of Cdc42 in G1 is necessary for assembly of a proper bud site. Biphasic activation of Cdc42 or Rac GTPases may be a general mechanism for spatial cue-directed cell polarization in eukaryotes.
Subject(s)
Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/enzymology , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cell Division , Cell Polarity , Enzyme Activation , G1 Phase Cell Cycle Checkpoints , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Time-Lapse ImagingABSTRACT
Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and α cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure-function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.
Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Septins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytokinesis/genetics , Cytokinesis/physiology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Immunoblotting , Immunoprecipitation , Microscopy, Fluorescence , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Septins/geneticsABSTRACT
Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and α cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here, we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1 phase. Whereas Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP-binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and α cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.
Subject(s)
Cell Cycle Proteins/metabolism , GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomycetales/metabolism , Amino Acid Sequence , Cell Cycle , Cell Cycle Proteins/genetics , Cell Division/physiology , Cell Polarity/physiology , Cytokinesis/physiology , GTP-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Triphosphate/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomycetales/geneticsABSTRACT
Cell polarization occurs along a single axis that is generally determined in response to spatial cues. In budding yeast, the Rsr1 GTPase and its regulators direct the establishment of cell polarity at the proper cortical location in response to cell type-specific cues. Here we use a combination of in vivo and in vitro approaches to understand how Rsr1 polarization is established. We find that Rsr1 associates with itself in a spatially and temporally controlled manner. The homotypic interaction and localization of Rsr1 to the mother-bud neck and to the subsequent division site are dependent on its GDP-GTP exchange factor Bud5. Analyses of rsr1 mutants suggest that Bud5 recruits Rsr1 to these sites and promotes the homodimer formation. Rsr1 also exhibits heterotypic interaction with the Cdc42 GTPase in vivo. We show that the polybasic region of Rsr1 is necessary for the efficient homotypic and heterotypic interactions, selection of a proper growth site, and polarity establishment. Our findings thus suggest that dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.
Subject(s)
Cell Division , Cell Polarity , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , rab GTP-Binding Proteins/metabolism , Amino Acid Sequence , Cell Nucleus/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Multimerization , Protein Transport , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , rab GTP-Binding Proteins/chemistryABSTRACT
In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.
Subject(s)
Apoptosis , Oxidants/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Thioredoxin-Disulfide Reductase/metabolism , rho GTP-Binding Proteins/metabolism , Gene Deletion , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Guanosine Triphosphate/metabolism , Hydrogen Peroxide/pharmacology , Oxidative Stress , Reactive Oxygen Species , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/genetics , rho GTP-Binding Proteins/analysis , rho GTP-Binding Proteins/geneticsABSTRACT
Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.
Subject(s)
Bacterial Proteins/metabolism , Cyclic AMP Receptor Protein/metabolism , Fimbriae, Bacterial/physiology , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/pathogenicity , Transcription Factors/metabolism , Cyclic AMP/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Fimbriae Proteins/chemistry , Fimbriae Proteins/genetics , Fimbriae Proteins/metabolism , Operon , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Virulence/physiologyABSTRACT
In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.
Subject(s)
Glycoproteins/metabolism , Membrane Glycoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Binding Sites , Cell Division , Cell Membrane/metabolism , Cell Polarity , Cell Proliferation , Cloning, Molecular , Cytoplasm/metabolism , Fungal Proteins/physiology , Gene Deletion , Green Fluorescent Proteins/metabolism , Membrane Proteins , Mutation , Plasmids/metabolism , Protein Binding , Protein Structure, TertiaryABSTRACT
Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.
Subject(s)
Saccharomyces cerevisiae Proteins/genetics , Saccharomycetales/physiology , Alanine/chemistry , Alleles , Binding Sites , Codon , Codon, Initiator , Gene Deletion , Glutathione Transferase/metabolism , Green Fluorescent Proteins , Guanine Nucleotide Exchange Factors , Haploidy , Immunoblotting , Luminescent Proteins/metabolism , Mutagenesis , Mutagenesis, Site-Directed , Mutation , Open Reading Frames , Peptides/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomycetales/metabolismABSTRACT
Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.