Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.     

The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone

STE2 encodes a component of the S. cerevisiae alpha-pheromone receptor that is essential for induction of physiological changes associated with mating. Analysis of C-terminal truncation mutants of STE2 demonstrated that the essential sequences for ligand binding and signal transduction are included within a region containing seven putative transmembrane domains. However, truncation of the C-terminal 105 amino acids of the receptor resulted in a 4- to 5-fold increase in cell-surface pheromone binding sites, a 10-fold increase in pheromone sensitivity, a defect in recovery of cell division after pheromone treatment, and a defect in pheromone-induced morphogenesis. Overproduction of STE2 resulted in about a 6-fold increase in alpha-pheromone binding capacity but did not produce the other phenotypes associated with the ste2-T326 mutant receptor. We conclude that the C-terminus of the receptor is responsible for one aspect of cellular adaptation to pheromone that is distinct from adaptation controlled by the SST2 gene, for decreasing the stability of the receptor, and for some aspect of cellular morphogenesis.     

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.     

Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii

The genome of Ashbya gossypii contains homologs of most of the genes that are part of the Saccharomyces cerevisiae pheromone-signal transduction cascade. However, we currently lack understanding of a potential sexual cycle for this pre-whole genome duplication hemiascomycete. The sequenced strain bears three identical copies encoding MATa. We show that the syntenic A. gossypii homolog of MFα1 (AFL062w) does not encode a mature α-factor peptide, but identified another gene, AAR163c, which encodes a candidate α-specific mating pheromone and is thus reannotated as AgMFα2. The expression of the AgSTE2α-factor receptor in an Scste2 S. cerevisiae MATa strain resulted in dosage-dependent growth arrest upon exposure to A. gossypiiα-factor, which indicated that the pheromone response was effectively coupled to the S. cerevisiae signal transduction cascade. Comparison of α-pheromones and α-pheromone receptors showed greater conservation between Eremothecium cymbalariae and S. cerevisiae than between A. gossypii and E. cymbalariae. We constructed A. gossypii strains deleted for the STE2 and STE3 pheromone receptors. These strains showed no phenotypic abnormalities and an ste2, ste3 double mutant is still able to sporulate. The deletion of STE12 as the downstream target of pheromone signalling, however, led to a hypersporulation phenotype.     

The yeast G-protein homolog is involved in the mating pheromone signal transduction system.

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

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.