Microinjection of recombinant p21rho induces rapid changes in cell morphology

The rho proteins, p21rho, are ubiquitously expressed guanine nucleotide binding proteins with approximately 30% amino acid homology to p21ras, but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Val14rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Val14rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Micro-injection of ribosylated normal rho, on the other hand, has a similar effect of injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Val14rho protein. The effects induced by activation or inactivation of p21rho described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization.     

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.     

Rho4 GTPase is involved in secretion of glucanases during fission yeast cytokinesis

Rho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+ or agn1+ overexpression suppressed the rho4delta multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4delta cells have a lower level of (1,3)-beta-D-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4delta cells grown at 37 degrees C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4delta cells at 37 degrees C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.     

Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.     

Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI)

Evidence is accumulating that the rho family, a member of the ras p21- related small GTP-binding protein superfamily, regulates cell morphology, cell motility, and smooth muscle contraction through the actomyosin system. The actomyosin system is also known to be essential for cytoplasmic division of cells (cytokinesis). In this study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein, named rho GDI, its stimulatory GDP/GTP exchange protein, named smg GDS, and botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in the cytoplasmic division using Xenopus embryos. The sperm-induced cytoplasmic division of Xenopus embryos was not affected by microinjection into the embryos of either smg GDS or the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of rhoA p21, one member of the rho family, but completely inhibited by microinjection of rho GDI or C3. Under these conditions, nuclear division occurred normally but the furrow formation, which was induced by the contractile ring consisting of actomyosin just beneath the plasma membrane, was impaired. Comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 prevented the rho GDI action. Moreover, the sperm-induced cytoplasmic division of Xenopus embryos was inhibited by microinjection into the embryos of the rhoA p21 pre-ADP- ribosylated by C3 which might serve as a dominant negative inhibitor of endogenous rho p21. These results indicate that rho p21 together with its regulatory proteins regulates the cytoplasmic division through the actomyosin system.     

ZIP3, a new splice variant of the PKC-zeta-interacting protein family,binds to GABAC receptors,PKC-zeta,and Kv beta 2

The correct targeting of modifying enzymes to ion channels and neurotransmitter receptors represents an important biological mechanism to control neuronal excitability. The recent cloning of protein kinase C-zeta interacting proteins (ZIP1, ZIP2) identified new scaffolds linking the atypical protein kinase PKC-zeta to target proteins. GABA(C) receptors are composed of three rho subunits (rho 1-3) that are highly expressed in the retina, where they are clustered at synaptic terminals of bipolar cells. A yeast two-hybrid screen for the GABA(C) receptor rho 3 subunit identified ZIP3, a new C-terminal splice variant of the ZIP protein family. ZIP3 was ubiquitously expressed in non-neuronal and neuronal tissues, including the retina. The rho 3-binding region of ZIP3 contained a ZZ-zinc finger domain, which interacted with 10 amino acids conserved in rho 1-3 but not in GABA(A) receptors. Consistently, only rho 1-3 subunits bound to ZIP3. ZIP3 formed dimers with ZIP1-3 and interacted with PKC-zeta and the shaker-type potassium channel subunit Kv beta 2. Different domains of ZIP3 interacted with PKC-zeta and the rho 3 subunit, and simultaneous assembly of ZIP3, PKC-zeta and rho 3 was demonstrated in vitro. Subcellular co-expression of ZIP3 binding partners in the retina supported the proposed protein interactions. Our results indicate the formation of a ternary postsynaptic complex containing PKC-zeta, ZIP3, and GABA(C) receptors.     

Studies on the function of Rho A protein in cardiac myofibrillogenesis

The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, β-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and α-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes. J. Cell. Biochem. 66:43–53, 1997. © 1997 Wiley-Liss, Inc.     

ADP-ribosylation of the rho/rac gene products by botulinum ADP-ribosyltransferase: identity of the enzyme and effects on protein and cell functions

1. Botulinum C1 toxin and C3 exoenzyme were purified from the culture filtrate of type C Clostridium botulinum strain 003-9, and specific antibodies were raised against each protein. Immunochemical analysis using these antibodies revealed the presence of minute amount of a C3-like molecule in C1 toxin preparation which tightly binds to the toxin component(s). This enzyme complex was separated from the major neurotoxin. Thus, the ADP-ribosyltransferases in C1 and D toxins and C3 exoenzyme appear to come from the same origin, and should be called together botulinum C3 enzyme. 2. Botulinum C3 enzyme ADP-ribosylates the rho and rac gene products, a family of small molecular weight GTP-binding proteins homologous to ras p21s. This ADP-ribosylation occurs at Asn41 of the rho products which is located in their putative effector domain, suggesting that it interferes interaction of these GTP binding proteins with their effector molecules. 3. When incubated with PC-12 cells, the enzyme inhibits cell growth and induces neurites and acetylcholine esterase. Several lines of evidence suggest that the ADP-ribosylation of the rho/rac proteins is responsible for these changes.     

Plasma membrane NADH-oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (rho0)

The mammalian plasma membrane (PM) NADH-oxidoreductase (PMOR) system is a multi-enzyme complex located in the plasma membrane of all eukaryotic cells, harboring at least two distinct activities, the plasma membrane NADH-ferricyanide reductase and the NADH-oxidase. To assess the behaviour of the two activities of the PMOR system, we measured the NADH-ferricyanide reductase and NADH-oxidase activities in fibroblast cell lines derived from patients carrying a mitochondrial DNA (mtDNA) G11778A mutation. We also measured the two activities in other cell lines, the HL-60 and HeLa (S3) lines, as well as in rho0 cells (cells devoid of mtDNA) generated from those lines and the fibroblast cells. These rho0 cells consequently lack oxidative phosphorylation and rely on anaerobic glycolysis for their ATP need. We have proposed that in rho0 cells, at least in part, up-regulation of the PMOR is a necessity to maintain the NAD+/NADH ratio, and a pre-requisite for cell growth and viability. We show here that the PM NADH-ferricyanide reductase activity was up-regulated in HL-AV2 (HL-60 rho0) cell lines, but not in the other rho0 and mtDNA mutant lines. The plasma membrane NADH oxidase activity was found to be up-regulated in both HL-AV2 and HeLa rho0 cell lines, but not significantly in the fibroblast rho0 and G11778A lines.     

Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho

Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras- related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP- ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.     

Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.

smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins). rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins. We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate. smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP. The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS. The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence. Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells. On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21. This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS. These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS. Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other.     

NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.     

rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.

Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.     

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.     

A new method of identifying the site of tyrosyl radicals in proteins

Protein-bound tyrosyl radicals catalyze many important enzymatic reactions. They can also initiate oxidative damage to cells. Here we report a new method of computer simulation of tyrosyl radical electron paramagnetic resonance spectra. The method enables the determination of the rotational conformation of the phenoxyl ring in a radical with unprecedented accuracy (approximately 2 degrees ). When coupled with a new online database, all tyrosine residues in a protein can be screened for that particular conformation. For the first time we show relationships between the spin density on atom C1 (rho(C1)) and the principal g-factors measured by electron paramagnetic resonance spectroscopy (rho(C1) on g(x) is shown to be linear). The new method enables the accurate determination of rho(C1) in all known tyrosyl radicals, evaluates the likelihood of a hydrogen bond, and determines the possibility of a rho(C1) distribution in the radicals. This information, together with the accurately determined rotational conformation, is frequently sufficient to allow for an unambiguous identification of the site of radical formation. The possibility of a similar relationship between rho(C) and g(x) in other radicals, e.g., tryptophanyl, is discussed.     

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.     

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.     

Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.     

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.