Negative regulation of STE6 gene expression by the alpha 2 product of Saccharomyces cerevisiae.

The alpha 2 product of the alpha mating type locus of Saccharomyces cerevisiae is proposed to be a negative regulator of a set of dispersed genes concerned with specialized properties of a cells. This set of genes includes those, termed a-specific STE genes (STE2, STE6, and STE14), which are required for mating by a cells but not by alpha cells. We cloned the STE6 gene to determine whether its expression is limited to a cells and, if so, whether its expression is inhibited in alpha cells by the alpha 2 product. Expression of STE6 was assayed in two ways: by blot hybridization, RNA and by beta-galactosidase activity in strains carrying a STE6-lacZ hybrid gene. We found that STE6 expression was limited to a cells and was negatively regulated by the alpha 2 product. STE6 RNA was not detectable in strains containing the wild-type alpha 2 gene product. Expression of STE6 was at least 150-fold lower in alpha cells than in a cells, based on beta-galactosidase activities in a and alpha cells carrying the STE6-lacZ gene. These results confirmed that the alpha 2 product is a negative regulator of gene expression and showed that it acts at the level of RNA production. We also examined the phenotype of a mutant carrying an insertion mutation of the STE6 gene, the ste6::lacZ allele. In addition, an a-specific defect in mating, this mutant was greatly reduced (but not completely deficient) in a-factor production. Other phenotypes characteristic of a cells--Barrier activity, agglutination, and response to alpha-factor--were normal. STE6 thus appears to be necessary for biosynthesis of a-factor.     

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.     

Induction of the yeast alpha-specific STE3 gene by the peptide pheromone a-factor

The yeast Saccharomyces cerevisiae exhibits two mating types, a and alpha. Efficient mating of a and alpha cells requires the action of peptide pheromones secreted by each cell type. For example, a cells secrete a-factor, which alters the physiology of alpha cells, thereby preparing those cells for mating. To investigate the mechanism by which the pheromones act on the target cells, we have examined the effect of a-factor on expression of the STE3 gene, a gene which is required for mating by alpha cells and which is expressed only in alpha cells. We have monitored STE3 expression by two assays: RNA production from the chromosomal STE3 locus and beta-galactosidase activity produced from a plasmid-borne STE3-lacZ gene fusion. By both assays we show that a-factor induces a rapid increase in STE3 expression. Induction of STE3 RNA occurs even if protein synthesis is blocked by cycloheximide. Using temperature-sensitive cell division cycle mutants, we have also shown that induction occurs in cells arrested at several discrete positions in the cell cycle. These results demonstrate (1) that induction of STE3 expression by a-factor is a primary response to the pheromone, and (2) that alpha cells are capable of responding to a-factor regardless of their position in the cell cycle.     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.     

STE16, a New Gene Required for Pheromone Production by a Cells of Saccharomyces cerevisiae

Genes required for mating by a and alpha cells of Saccharomyces cerevisiae (STE, "sterile," genes) encode products such as peptide pheromones, pheromone receptors, and proteins responsible for pheromone processing. a-specific STE genes are those required for mating by a cells but not by alpha cells. To identify new a-specific STE genes, we have employed a novel strategy that enabled us to determine if a ste mutant defective in mating as a is also defective in mating as alpha without the need to do crosses. This technique involved a strain (K12-14b) of genotype mata1 HML alpha HMR alpha sir3ts, which mates as a at 25 degrees and as alpha at 34 degrees. We screened over 40,000 mutagenized colonies derived from K12-14b and obtained 28 a-specific ste mutants. These strains contained mutations in three known a-specific genes--STE2, STE6 and STE14--and in a new gene, STE16. ste16 mutants are defective in the production of the pheromone, a-factor, and exhibit slow growth. Based on the distribution of a-specific ste mutants described here, we infer that we have identified most if not all nonessential genes that can give rise to a-specific mating defects.     

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.     

The Pheromone Receptors Inhibit the Pheromone Response Pathway in Saccharomyces Cerevisiae by a Process That Is Independent of Their Associated Gα Protein

Dominant mutations at the DAF2 locus confer resistance to the cell-cycle arrest that normally occurs in MATa cells exposed to α-factor. One of these alleles, DAF2-2, has also been shown to suppress the constitutive signaling phenotype of null alleles of the gene encoding the α subunit of the G protein involved in pheromone signaling. These observations indicate that DAF2-2 inhibits transmission of the pheromone response signal. The DAF2-2 mutation has two effects on the expression of a pheromone inducible gene, FUS1. In DAF2-2 cells, FUS1 RNA is present at an increased basal level but is no longer fully inducible by pheromone. Cloning of DAF2-2 revealed that it is an allele of STE3, the gene encoding the a-factor receptor. STE3 is normally an α-specific gene, but is inappropriately expressed in a cells carrying a STE3(DAF2-2) allele. The two effects of STE3(DAF2-2) alleles on the pheromone response pathway are the result of different functions of the receptor. The increased basal level of FUS1 RNA is probably due to stimulation of the pathway by an autocrine mechanism, because it required at least one of the genes encoding a-factor. Suppression of a null allele of the G(α) subunit gene, the phenotype associated with the inhibitory function of STE3, was independent of a-factor. This suppression was also observed when the wild-type STE3 gene was expressed in a cells under the control of an inducible promoter. Inappropriate expression of STE2 in α cells was able to suppress a point mutation, but not a null allele, of the G(α) subunit gene. The ability of the pheromone receptors to block the pheromone response signal in the absence of the G(α) subunit indicates that these receptors interact with another component of the signal transduction pathway.     

Constitutive mutants in the yeast pheromone response: ordered function of the gene products

The alpha factor pheromone inhibits the division of yeast a cells. A general method was developed for isolating mutants that exhibit constitutive activation of the pheromone response pathway. A dominant allele of the STE4 locus was recovered in addition to recessive mutations in the SCG1 gene. SCG1 and STE4 are known to encode G alpha and G beta homologs, respectively. Analysis of double mutants suggests that the STE4 gene product functions after the SCG1 product but before the STE5 product.     

Regulation of the yeast pheromone response pathway by G protein subunits.

The yeast GPA1, STE4, and STE18 genes encode proteins homologous to the respective alpha, beta and gamma subunits of the mammalian G protein complex which appears to mediate the response to mating pheromones. Overexpression of the STE4 protein by the galactose-inducible GAL1 promoter caused activation of the pheromone response pathway which resulted in cell-cycle arrest in late G1 phase and induction of the FUS1 gene expression, thereby suppressing the sterility of the receptor-less mutant delta ste2. Disruption of STE18, in turn, suppressed activation of the pheromone response induced by overexpression of STE4, suggesting that the STE18 product is required for the STE4 action. However, overexpression of both the STE4 and STE18 proteins did not generate a stronger pheromone response than overexpression of STE4 in the presence of wild-type levels of STE18. These results suggest that the beta subunit is the limiting component for the pheromone response and support the idea that beta and gamma subunits act as a positive regulator. Furthermore, overexpression of GPA1 prevented cell-cycle arrest but not FUS1 induction mediated by overexpression of STE4. This implies that the alpha subunit acts as a negative regulator presumably through interacting with beta and gamma subunits in the mating pheromone signaling pathway.     

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase

Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cerevisiae alpha cells. Nonmating ("sterile," or ste) alpha-cell mutants bearing defects in the STE13 gene do not produce normal alpha factor, but release a collection of incompletely processed forms (alpha factor) that have a markedly reduced specific biological activity. The major alpha-factor peptides have the structures H2N-GluAlaGluAla-alpha factor and H2N-AspAlaGluAla-alpha factor. The ste13 mutants lack a membrane-bound heat-stable dipeptidyl aminopeptidase (DPAPase A) that specifically cleaves on the carboxyl side of repeating -X-Ala- sequences. Absence of DPAPase A and the other phenotypes of a ste13 lesion cosegregate in genetic crosses. The cloned STE13 gene on a plasmid causes yeast cells to overproduce DPAPase A severalfold. A different cloned DNA segment, which weakly suppresses the ste13 defects, causes overproduction of a heat-labile activity (DPAPase B) by about tenfold. Other experiments indicate that DPAPase A action may be rate-limiting for alpha-factor maturation in normal alpha cells.