Genetic analysis of the bipolar pattern of bud site selection in the yeast Saccharomyces cerevisiae.

Previous analysis of the bipolar budding pattern of Saccharomyces cerevisiae has suggested that it depends on persistent positional signals that mark the region of the division site and the tip of the distal pole on a newborn daughter cell, as well as each previous division site on a mother cell. In an attempt to identify genes encoding components of these signals or proteins involved in positioning or responding to them, we identified 11 mutants with defects in bipolar but not in axial budding. Five mutants displaying a bipolar budding-specific randomization of budding pattern had mutations in four previously known genes (BUD2, BUD5, SPA2, and BNI1) and one novel gene (BUD6), respectively. As Bud2p and Bud5p are known to be required for both the axial and bipolar budding patterns, the alleles identified here probably encode proteins that have lost their ability to interact with the bipolar positional signals but have retained their ability to interact with the distinct positional signal used in axial budding. The function of Spa2p is not known, but previous work has shown that its intracellular localization is similar to that postulated for the bipolar positional signals. BNI1 was originally identified on the basis of genetic interaction with CDC12, which encodes one of the neck-filament-associated septin proteins, suggesting that these proteins may be involved in positioning the bipolar signals. One mutant with a heterogeneous budding pattern defines a second novel gene (BUD7). Two mutants budding almost exclusively from the proximal pole carry mutations in a fourth novel gene (BUD9). A bud8 bud9 double mutant also buds almost exclusively from the proximal pole, suggesting that Bud9p is involved in positioning the proximal pole signal rather than being itself a component of this signal.     

Functional cloning of BUD5, a CDC25-related gene from S. cerevisiae that can suppress a dominant-negative RAS2 mutant

By searching for genes that behave like CDC25 of S. cerevisiae in their ability to counteract a dominant-negative RAS2 mutant in a wild-type RAS-dependent manner, we have isolated a CDC25-like homolog, BUD5. BUD5 is tightly linked to the MAT locus. Although overexpressed BUD5 cannot substitute for CDC25 function, we present evidence that its gene product can bind to the guanine nucleotide binding-deficient RAS2val19ala22 gene product and thereby counteract its dominant-negative effect. We propose that BUD5 is a member of a family of CDC25-related genes that encode activators of RAS and RAS-like proteins.     

Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation.

Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.     

Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae

We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.     

Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1.

RHO3 and RHO4 are members of the ras superfamily genes of the yeast Saccharomyces cerevisiae and are related functionally to each other. Experiments using a conditionally expressed allele of RHO4 revealed that depletion of both the RHO3 and RHO4 gene products resulted in lysis of cells with a small bud, which could be prevented by the presence of osmotic stabilizing agents in the medium. rho3 rho4 cells incubated in medium containing an osmotic stabilizing agent were rounded and enlarged and displayed delocalized deposition of chitin and delocalization of actin patches, indicating that these cells lost cell polarity. Nine genes whose overexpression could suppress the defect of the RHO3 function were isolated (SRO genes). Two of them were identical with CDC42 and BEM1, bud site assembly genes involved in the process of bud emergence. A high dose of CDC42 complemented the rho3 defect, whereas overexpression of RHO3 had an inhibitory effect on the growth of mutants defective in the CDC24-CDC42 pathway. These results, along with comparison of cell morphology between rho3 rho4 cells and cdc24 (or cdc42) mutant cells kept under the restrictive conditions, strongly suggest that the functions of RHO3 and RHO4 are required after initiation of bud formation to maintain cell polarity during maturation of daughter cells.     

Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the STE and RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y. lipolytica morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of Y. lipolytica with a Tn3 derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to S. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5, and PPH21. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y. lipolytica and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.     

Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast.

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.     

Components required for cytokinesis are important for bud site selection in yeast

Polarized cell division is a fundamental process that occurs in a variety of organisms; it is responsible for the proper positioning of daughter cells and the correct segregation of cytoplasmic components. The SPA2 gene of yeast encodes a nonessential protein that localizes to sites of cell growth and to the site of cytokinesis. spa2 mutants exhibit slightly altered budding patterns. In this report, a genetic screen was used to isolate a novel ochre allele of CDC10, cdc10-10; strains containing this mutation require the SPA2 gene for growth. CDC10 encodes a conserved potential GTP-binding protein that previously has been shown to localize to the bud neck and to be important for cytokinesis. The genetic interaction of cdc10-10 and spa2 suggests a role for SPA2 in cytokinesis. Most importantly, strains that contain a cdc10-10 mutation and those containing mutations affecting other putative neck filament proteins do not form buds at their normal proximal location. The finding that a component involved in cytokinesis is also important in bud site selection provides strong evidence for the cytokinesis tag model; i.e., critical components at the site of cytokinesis are involved in determining the next site of polarized growth and division.     

Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site

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.     

RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.     

The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway

The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.     

The ras oncogene--an important regulatory element in lower eucaryotic organisms.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.     

Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway

Yeast cells choose bud sites on their surface in two distinct spatial patterns: axial for a and alpha cells and bipolar for a/alpha cells. We have identified four genes, BUD1-BUD4, necessary for the axial pattern by isolating mutants of alpha cells that do not exhibit this pattern. Mutations in BUD1 (which is the same as the previously identified gene RSR1) or BUD2 lead to a random budding pattern in all cell types; mutations in BUD3 or BUD4 lead to a bipolar pattern in all cell types. These observations indicate the existence of a basal budding pattern, requiring no BUD products, that is random; BUD1 and BUD2 act on this basal pattern to create the bipolar pattern; the further action of BUD3 and BUD4 leads to the axial pattern. These studies thus identify a set of gene products that directs cell morphogenesis to a genetically programmed site.     

Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1(G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins. Disruption of ROM2 resulted in a temperature-sensitive growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of deltarom1deltarom2 cells were similar to those of deltarho1 cells, including growth arrest with a small bud and cell lysis. Moreover, the temperature-sensitive growth phenotype of deltarom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rholp-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor. These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.