A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37 degrees C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G(1) and G(2)/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.     

The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.     

Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling

In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.     

A defect of Kap104 alleviates the requirement of mitotic exit network gene functions in Saccharomyces cerevisiae

A subgroup of the karyopherin beta (also called importin beta) protein that includes budding yeast Kap104 and human transportin/karyopherin beta2 is reported to function as a receptor for the transport of mRNA-binding proteins into the nucleus. We identified KAP104 as a responsible gene for a suppressor mutation of cdc15-2. We found that the kap104-E604K mutation suppressed the temperature-sensitive growth of cdc15-2 cells by promoting the exit from mitosis and suppressed the temperature sensitivity of various mitotic-exit mutations. The cytokinesis defect of these mitotic-exit mutants was not suppressed by kap104-E604K. Furthermore, the kap104-E604K mutation delays entry into DNA synthesis even at a permissive temperature. In cdc15-2 kap104-E604K cells, SWI5 and SIC1, but not CDH1, became essential at a high temperature, suggesting that the kap104-E604K mutation promotes mitotic exit via the Swi5-Sic1 pathway. Interestingly, SPO12, which is involved in the release of Cdc14 from the nucleolus during early anaphase, also became essential in cdc15-2 kap104-E604K cells at a high temperature. The kap104-E604K mutation caused a partial delocalization of Cdc14 from the nucleolus during interphase. This delocalization of Cdc14 was suppressed by the deletion of SPO12. These results suggest that a mutation in Kap104 stimulates exit from mitosis through the activation of Cdc14 and implies a novel role for Kap104 in cell-cycle progression in budding yeast.     

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.     

Two fission yeast B-type cyclins, cig2 and Cdc13, have different functions in mitosis.

Cyclin B interacts with Cdc2 kinase to induce cell cycle events, particularly those of mitosis. The existence of cyclin B subtypes in several species has been known for some time, leading to speculation that key events of mitosis may be carried out by distinct functional classes of Cdc2/cyclin B. We report the discovery of cig2, a third B-type cyclin gene in Schizosaccharomyces pombe. Disruption of cig2 delays the onset of mitosis, to the degree that a cig2 null allele rescues mitotic catastrophe mutants, including those that are unable to carry out the inhibitory tyrosyl phosphorylation of Cdc2 kinase. Consistent with this, a cig2 null allele exhibits synthetic lethal interactions with cdc25ts and cdc2ts mutations. Mitotic phenotypes caused by disruption of cig2 are not reversed by increased production of Cdc13, the other fission yeast B-type cyclin that functions in mitosis. Likewise, a cdc13ts mutation is not rescued by increased gene dosage of cig2+. These data indicate that Cdc13 and Cig2 interact with Cdc2 to carry out different functions in mitosis. We suggest that some cyclin B subtypes found in other species, including humans, are also likely to have distinct, nonoverlapping functions in mitosis.     

Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae.

A genetic screen for GTPase-activating proteins (GAPs) or other negative regulators of the Rac/Rho family GTPase Cdc42p in Saccharomyces cerevisiae identified ZDS1, a gene encoding a protein of 915 amino acids. Sequence from the yeast genome project identified a homolog, ZDS2, whose predicted product of 942 amino acids is 38% identical in sequence to Zds1p. Zds1p and Zds2p have no detectable homology to known Rho-GAPs or to other known proteins. However, by several assays, it appears that overexpression of either Zds1p or Zds2p decreases the level of Cdc42p activity. Deletion analysis also suggests that Zds1p and Zds2p are at least partially overlapping in function. Deletion of ZDS2 produced no obvious phenotype, and deletion of ZDS1 produced no obvious phenotype other than a mild effect on cell shape. However, the zds1 zds2 double mutant grew slowly with an apparent mitotic delay and produced elongated cells and buds with other evidence of abnormal morphogenesis. A glutathione S-transferase-Zds1p fusion protein that fully complemented the double mutant localized to presumptive bud sites and the tips of small buds. The similarity of this localization to that of Cdc42p suggests that Zds1p may interact directly with Cdc42p. As ZDS1 and ZDS2 have recently been identified also by numerous other groups studying a wide range of biological phenomena, the roles of Cdc42p in intracellular signaling may be more diverse than has previously been appreciated.     

Cdc42 regulates multiple membrane traffic events in fission yeast

Fission yeast Cdc42 regulates polarized growth and is involved in For3 formin activation and actin cable assembly. We show here that a thermosensitive strain carrying the cdc42L160S allele has membrane traffic defects independent of the actin cable defects. This strain has decreased acid phosphatase (AP) secretion, intracellular accumulation of vesicles and fragmentation of vacuoles. In addition, the exocyst is not localized to the tips of these cells. Overproduction of the scaffold protein Pob1 suppressed cdc42L160S thermosensitive growth and restored exocyst localization and AP secretion. The GTPase Rho3 also suppressed cdc42L160S thermosensitivity, restored exocyst localization and AP secretion. However, Rho3 did not restore the actin cables in these cells as Pob1 does. Similarly, overexpression of psy1(+) , coding a syntaxin (t-SNARE) homolog, or of ypt2(+) , coding an SEC4 homolog in fission yeast, rescued growth at high temperature but did not restore actin cables, nor the exocyst-polarized localization. cdc42L160S cells also have defects in vacuole formation that were rescued by Pob1, Rho3 and Psy1. All together, we propose that Cdc42 and the scaffold Pob1 are required for membrane trafficking and fusion, contributing to polarized secretion, endosome recycling, vacuole formation and growth.     

Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication

Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms. In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication. Recent findings suggest that centrin also functions elsewhere in the cell. To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype. Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins. Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function. Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p. A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function. Mutations in the C-terminal region were also defective for interaction with Kic1p. Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.     

spo12 is a multicopy suppressor of mcs3 that is periodically expressed in fission yeast mitosis

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.     

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.     

Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G(1)/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4 (cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1 cells. However, the previously characterized mutation cdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G(2)/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G(2)/M transition when released from hydroxyurea-induced S-phase arrest. A second function for CDC4 in late S or G(2) phase was further confirmed by the observation that cells lacking the CDC4 gene are arrested both at G(1)/S and at G(2)/M. We subsequently isolated additional temperature-sensitive mutations in the CDC4 gene (such as cdc4-12) that render the mutant defective in both G(1)/S and G(2)/M transitions at the restrictive temperature. While the G(1)/S block in both cdc4-12 and cdc4Delta mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G(2)/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.     

Cdc28 and Ime2 possess redundant functions in promoting entry into premeiotic DNA replication in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2Delta diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2Delta diploids depends on a functional Cdc28. Ime2Delta cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.     

Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.     

Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone Cdc37

CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone Cdc37 on a conserved serine residue (Ser13) is essential for the function of Cdc37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829-2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117-38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065-4074, 2004]. We have recently produced an anti-[pSer13]-Cdc37 antibody which specifically recognizes Cdc37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690-5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-Cdc37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of Cdc37 on Ser13 by CK2 holoenzyme or CK2alpha, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous Cdc37 on Ser13, as revealed by anti-[pSer13]-Cdc37, and overexpression of either CK2alpha or CK2beta subunit enhanced the Cdc37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of Cdc37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of Cdc37 on Ser13. These results show that the anti-[pSer13]-Cdc37 antibody can be a promising new tool to evaluate in vivo CK2 activity.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.