Mating Pheromone Response Research Articles

The immunosuppressive drug leflunomide affects mating-pheromone response and sporulation by different mechanisms in Saccharomyces cerevisiae

Leflunomide (LFM) is a novel anti-inflammatory and immunosuppressive drug, and inhibits the growth of cytokine-stimulated lymphoid cells in vitro. The effect of LFM on haploid and diploid cells of Saccharomyces cerevisiae was investigated to elucidate the molecular mechanism of action of the drug. Using a halo assay, LFM was shown to enhance the cell cycle arrest of haploid cells induced by mating pheromone α-factor. LFM also inhibited sporulation of diploid cells completely. S. cerevisiae genes which were cloned to suppress the anti-proliferative effect when present in increased copy number were introduced and examined for their activity to suppress the effect of LFM. Out of them, MLF4/SSH4, was found to suppress the sporulation-inhibitory effect of LFM. However, MLF4 failed to suppress the enhancing effect of LFM on pheromone response. Thus, LFM is suggested to act on haploid and diploid cells by different mechanisms.

MAP kinase signaling induces nuclear reorganization in budding yeast.

During the mating pheromone response in budding yeast, activation of a mitogen-activated protein kinase (MAP kinase) cascade results in well-characterized changes in cytoskeletal organization and gene expression. Spatial reorganization of genes within the nucleus has been documented during cell-type differentiation in mammalian cells, but no information was previously available on the morphology of the yeast nucleus during the major transcriptional reprogramming that accompanies zygote formation. We find that in response to mating pheromone, budding yeast nuclei assume an unusual dumbbell shape, reflecting a spatial separation of chromosomal and nucleolar domains. Within the chromosomal domain, telomeric foci persist and maintain their associated complement of Sir proteins. The nucleolus, on the other hand, assumes a novel cup-shaped morphology and a position distal to the mating projection tip. Although microtubules are required for this orientation with respect to the projection tip, neither microtubules nor actin polymerization are necessary for the observed changes in nuclear shape. We find that activation of the pheromone-response MAP kinase pathway by ectopic expression of STE4 or STE11 leads to identical nuclear and nucleolar reorganization in the absence of pheromone. Mutation of downstream effector MAP kinases Fus3p and Kss1p, or of the transcriptional regulator Ste12p, blocks nuclear shape changes, whereas overexpression of Ste12p promotes dumbbell-shaped nuclei in the absence of pheromone. Nuclear remodeling occurs when the MAP kinase cascade is activated by yeast pheromone, but it is independent of the cytoskeletal reorganization regulated by the same signaling pathway. Activation of the Ste12p transcription factor is necessary, and may be sufficient, for the changes in nuclear structure that coincide with developmentally significant changes in gene expression.

The Transcriptional Response of Saccharomyces cerevisiae to Osmotic Shock: Hot1p AND Msn2p/Msn4p ARE REQUIRED FOR THE INDUCTION OF SUBSETS OF HIGH OSMOLARITY GLYCEROL PATHWAY-DEPENDENT GENES

We have analyzed the transcriptional response to osmotic shock in the yeast Saccharomyces cerevisiae. The mRNA level of 186 genes increased at least 3-fold after a shift to NaCl or sorbitol, whereas that of more than 100 genes was at least 1.5-fold diminished. Many induced genes encode proteins that presumably contribute to protection against different types of damage or encode enzymes in glycerol, trehalose, and glycogen metabolism. Several genes, which encode poorly expressed isoforms of enzymes in carbohydrate metabolism, were induced. The high osmolarity glycerol (HOG) pathway is required for full induction of many but not all genes. The recently characterized Hot1p transcription factor is required for normal expression of a subset of the HOG pathway-dependent responses. Stimulated expression of the genes that required the general stress-response transcription factors Msn2p and Msn4p was also reduced in a hog1 mutant, suggesting that Msn2p/Msn4p might be regulated by the HOG pathway. The expression of genes that are known to be controlled by the mating pheromone response pathway was stimulated by osmotic shock specifically in a hog1 mutant. Inappropriate activation of the mating response may contribute to the growth defect of a hog1 mutant in high osmolarity medium.

The Leu-132 of the Ste4(Gβ) subunit is essential for proper coupling of the G protein with the Ste2 α factor receptor during the mating pheromone response in yeast

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered α pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the β propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and α cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gβ and the Gα subunits.

Fus3 controls Ty1 transpositional dormancy through the invasive growth MAPK pathway.

Fus3, the mitogen-activated protein kinase (MAPK) of the mating pheromone response pathway, inhibits a post-translational step of Ty1 retrotransposition. Fus3 also inhibits haploid invasive growth by blocking cross-activation of invasive growth gene expression by the pheromone response signal cascade. Here, we show that Fus3 kinase activity and dosage co-ordinately regulate Ty1 transposition and invasive growth. A chromosomal copy of the kinase-defective fus3-K42R allele fails to inhibit either Ty1 transposition or invasive growth. When overexpressed, kinase-defective Fus3 weakly inhibits both Ty1 transposition and invasive growth, but is much less inhibitory than wild-type Fus3 expressed at the same level. Moreover, increasing the dosage of wild-type Fus3 intensifies the inhibition of both Ty1 transposition and invasive growth. To demonstrate that Fus3 regulates Ty1 transposition via its negative regulation of the invasive growth pathway, we show by epistatic analysis that the invasive growth pathway transcription factors Ste12 and Tec1 are both required for Fus3-mediated inhibition of Ty1 transposition. When haploid invasive growth is stimulated by high-copy expression of TEC1, by expression of the dominant hypermorphic allele STE11-4 or by deletion of HOG1, Ty1 transposition is concomitantly activated. In summary, these results demonstrate that the haploid invasive growth pathway activates Ty1 transposition at both transcriptional and post-transcriptional levels and that Fus3 inhibits Ty1 transposition by inhibiting the invasive growth pathway.

Ca2+ signal is generated only once in the mating pheromone response pathway in Saccharomyces cerevisiae.

The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating. In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling. Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response. Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration. In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone. These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway. Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway [Iida et al., (1990) J. Biol. Chem., 265: 13391-13399] Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.

Functional characterization of the interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

Mot3, a Zn finger transcription factor that modulates gene expression and attenuates mating pheromone signaling in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein beta subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5' flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

Osmotin, a Plant Antifungal Protein, Subverts Signal Transduction to Enhance Fungal Cell Susceptibility

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein α subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of Gα proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Posttranslational regulation of Ty1 retrotransposition by mitogen-activated protein kinase Fus3.

Ty1 retrotransposons in Saccharomyces cerevisiae are maintained in a state of transpositional dormancy. We isolated a mutation, rtt100-1, that increases the transposition of genomic Ty1 elements 18- to 56-fold but has little effect on the transposition of related Ty2 elements. rtt100-1 was shown to be a null allele of the FUS3 gene, which encodes a haploid-specific mitogen-activated protein kinase. In fus3 mutants, the levels of Ty1 RNA, protein synthesis, and proteolytic processing were not altered relative to those in FUS3 strains but steady-state levels of TyA, integrase, and reverse transcriptase proteins and Ty1 cDNA were all increased. These findings suggest that Fus3 suppresses Ty1 transposition by destabilizing viruslike particle-associated proteins. The Fus3 kinase is activated through the mating-pheromone response pathway by phosphorylation at basal levels in naive cells and at enhanced levels in pheromone-treated cells. We demonstrate that suppression of Ty1 transposition in naive cells requires basal levels of Fus3 activation. Substitution of conserved amino acids required for activation of Fus3 derepressed Ty1 transposition. Moreover, epistasis analyses revealed that components of the pheromone response pathway that act upstream of Fus3, including Ste4, Ste5, Ste7, and Ste11, are required for the posttranslational suppression of Ty1 transposition by Fus3. The regulation of Ty1 transposition by Fus3 provides a haploid-specific mechanism through which environmental signals can modulate the levels of retrotransposition.

Signal transduction by MAP kinase cascades in budding yeast

Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.

MAP Kinases with Distinct Inhibitory Functions Impart Signaling Specificity during Yeast Differentiation

Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK.

Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions.

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein beta subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5 delta cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

Genetic evidence for a tyrosine kinase cascade preceding the mitogen-activated protein kinase cascade in vertebrate G protein signaling.

The signal transduction pathway from heterotrimeric G proteins to the mitogen-activated protein kinase (MAPK) cascade is best understood in the yeast mating pheromone response, in which a serine/threonine protein kinase (STE20) serves as the critical linking component. Little is known in metazoans on how G proteins and the MAPK cascade are coupled. Here we provide genetic and biochemical evidence that a tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Targeted deletion of tyrosine kinase Csk in avian B lymphoma cells blocks the stimulation of MAPK by Gq-, but not Gi-, coupled receptors. In cells deficient in Bruton's tyrosine kinase (Btk), Gi-coupled receptors failed to activate MAPK, while Gq-coupled receptor-mediated stimulation is unaffected. Taken together with our previous data on tyrosine kinases Lyn and Syk, the Gq-coupled pathway requires tyrosine kinases Csk, Lyn, and Syk, while the Gi-coupled pathway requires tyrosine kinases Btk and Syk to feed into the MAPK cascade in these cells. The central role of Syk is further strengthened by data showing that Syk can bind to purified Lyn, Csk, or Btk.

Differential regulation of FUS3 MAP kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase MSG5 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae mating pheromone response is mediated by activation of a MAP kinase (Fus3p and Kss1p) signaling pathway. Pheromone stimulation causes cell cycle arrest. Therefore, inactivation of the Fus3p and Kss1p MAP kinases is required during recovery phase for the resumption of cell growth. We have isolated a novel protein tyrosine phosphatase gene, PTP3, as a negative regulator of this pathway. Ptp3p directly dephosphorylates and inactivates Fus3p MAP kinase in vitro. Multicopy PTP3 represses pheromone-induced transcription and promotes recovery. In contrast, disruption of PTP3 in combination with its homolog PTP2 results in constitutive tyrosine phosphorylation, enhanced kinase activity of Fus3p MAP kinase on stimulation, and delayed recovery from the cell cycle arrest. Both tyrosine phosphorylation and kinase activity of Fus3p are further increased by disruption of PTP3 and PTP2 in combination with MSG5, which encodes a dual-specificity phosphatase. Cells deleted for all three of the phosphatases (ptp2delta ptp3delta msg5delta) are hypersensitive to pheromone and exhibit a severe defect in recovery from pheromone-induced growth arrest. Our data indicate that Ptp3p is the major phosphatase responsible for tyrosine dephosphorylation of Fus3p to maintain a low basal activity; it also has important roles, along with Msg5p, in inactivation of Fus3p following pheromone stimulation. These data present the first evidence for a coordinated regulation of MAP kinase function through differential actions of protein tyrosine phosphatases and a dual-specificity phosphatase.

Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis.

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.

The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli. MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types. SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis. Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases. CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression. The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway. First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis. Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA. Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall. These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development.

The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.

Isolation of a gene encoding a G protein alpha subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis.

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0.5 kb (P-1) and 0.8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the G alpha subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein alpha subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this G alpha subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian G alpha s family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence.

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeastKluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).