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.     

The yeast STE12 product is required for expression of two sets of cell-type specific genes

Yeast alpha and a cells transcribe distinct sets of genes involved in mating behavior, alpha-specific genes and a-specific genes, respectively. The alpha 1 product of the alpha mating type locus (MAT alpha) has been the only known activator of either set of genes; it is required for synthesis of RNA from the alpha-specific genes, one of which is the major alpha-factor gene. By screening for mutants that are no longer able to express this gene, we have identified the STE12 gene product as another positive regulator of the alpha-factor gene. alpha ste12 cells are also defective in RNA production from the other known alpha-specific genes. Moreover, a ste12 cells fail to produce wild-type levels of RNA from the a-specific genes. The STE12 gene product is therefore an activator of two sets of genes involved in yeast cell type specialization.     

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.     

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.     

Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae: autocrine cell-cycle arrest results from forced expression of STE2

Induction of STE2 expression using the GAL1 promoter both in a wild-type MATalpha strain and in a MATalpha ste3 strain caused transient cell-cycle arrest and changes in morphology ('shmoo'-like phenotype) in a manner similar to alpha cells responding to alpha-factor. In addition, STE2 expressed in a MATalp[ha ste3 mutant allowed the cell to conjugate with alpha cells but at an efficiency lower than that of wil-type alpha cells. This result indicates that signal(s) generated by alpha-factor in alpha cells can be substituted by signal(s) generated by the interaction of alpha-factor with the expressed STE2 product. When STE2 or STE3 was expressed in a matalpha1 strain (insensitive to both alpha- and a-factors), the cell became sensitive to alpha- or a-factor, respectively, and resulted in morphological changes. These results suggest that STE2 and STE3 are the sole determinants for alpha-factor and a-factor sensitivity, respectively, in this strain. On the other hand, expression of STE2 in an a/alpha diploid cell did not affect the alpha-factor insensitive phenotype. Haploid-specific components may be necessary to transduce the alpha-factor signal. These results are consistent with the idea that STE2 encodes an alpha-factor receptor and STE3 encodes an a-factor receptor, and suggest that both alpha- and a-factors may generate an exchangeable signal(s) within haploid cells.     

Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.     

Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating.

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.     

Disruption of a Silencer Domain by a Retrotransposon

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.     

Down regulation of the alpha-factor pheromone receptor in S. cerevisiae

The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor. Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis. Pheromone-resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation. Our results suggest that alpha-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha-factor. We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor.     

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.