Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone

Temperature-sensitive mutations that produce insensitivity to division arrest by alpha-factor, a mating pheromone, were isolated in an MATa strain of Saccharomyces cerevisiae and shown by complementation studies to difine eight genes. All of these mutations (designated ste) produce sterility at the restrictive temperature in MATa cells, and mutations in seven of the genes produce sterility in MAT alpha cells. In no case was the sterility associated with these mutations coorectible by including wild-type cells of the same mating type in the mating test nor did nay of the mutants inhibit mating of the wild-type cells; the defect appears to be intrinsic to the cell for mutations in each of the genes. Apparently, none of the mutants is defective exclusively in division arrest by alpha-factor, as the sterility of none is suppressed by a temperature-sensitive cdc 28 mutation (the latter imposes division arrest at the correct cell cycle stage for mating). The mutants were examined for features that are inducible in MATa cells by alpha-factor (agglutinin synthesis as well as division arrest) and for the characteristics that constitutively distinguish MATa from MAT alpha cells (a-factor production, alpha-factor destruction). ste2 Mutants are defective specifically in the two inducible properties, whereas ste4, 5, 7, 8, 9, 11, and 12 mutants are defective, to varying degrees, in constitutive as well as inducible aspects. Mutations in ste8 and 9 assume a polar budding pattern unlike either MATa or MAT alpha cells but characteristic of MATa/alpha cells. This study defines seven genes that function in two cell types (MATa and alpha) to control the differentiation of cell type and one gene, ste2, that functions exclusively in MATa cells to mediate responsiveness to polypeptide hormone.     

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.     

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.     

Suppressors of a Gpa1 Mutation Cause Sterility in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the α subunit of mammalian G proteins and is essential for haploid cell growth. We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3). Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major α-factor gene (MFα1) as judged by the ability to express a MFα1-lacZ fusion gene. The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus. The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12). sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function. While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of α-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ. These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G(1) phase and induction of genes necessary for mating such as FUS1.     

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.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

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.     

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.     

Role of the ABC transporter Ste6 in cell fusion during yeast conjugation

Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals. Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen. The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor. The ste6(cefl-1) allele was recovered and sequenced. The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall. Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 play more complex roles in cell fusion.     

Mating Pheromones of Saccharomyces kluyveri: Pheromone Interactions Between Saccharomyces kluyveri and Saccharomyces cerevisiae

Saccharomyces kluyveri is a heterothallic yeast with two allelic mating types denoted as a-k and alpha-k by analogy with Saccharomyces cerevisiae and from the work described here. S. kluyveri produces mating pheromones analogous to those of S. cerevisiae, but which appear to have different specificity. S. kluyveri thus differs from S. cerevisiae, Hansenula wingei, and Schizosaccharomyces pombe in that it exhibits both strong constitutive agglutination and mating pheromones. alpha-k cells produce a pheromone ("alpha-k-factor") which causes a-k cells to arrest in the G1 phase of the cell cycle and to undergo a morphological change. After a period of time dependent on the concentration of alpha-k-factor, cells exposed to the factor resume cell division. alpha-k-factor has no effect on a-k/alpha-k diploids or on alpha-k cells, but at high concentration does induce G1 arrest of S. cerevisiaea cells (a-c). a-k cells produce a pheromone ("a-k-factor") which causes alpha-k cells to exhibit a morphological change. In addition, a-k cells exhibit the Bar phenotype with respect to alpha-k-factor. Partially purified preparations of S. cerevisiae alpha-factor are more active in inducing G1 arrest of a-k cells than of a-c cells. A more purified preparation of alpha-c-factor is less active against a-k cells than a-c cells, suggesting that an additional factor (KRE, kluyveri response enhancer) may be lost during purification. Attempts to mate S. kluyveri and S. cerevisiae cells by prototroph selection and by cell-to-cell mating have been unsuccessful with all combinations of mating types. Thus, S. cerevisiae and S. kluyveri are incompatible for mating even though their pheromones exhibit some physiological cross-reaction.     

Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").     

Mating-Type Differentiation by Transposition of Controlling Elements in SACCHAROMYCES CEREVISIAE

The nonfunctional mutation of the homothallic gene HML alpha, designated hml alpha, produced two mutant alleles, hml alpha-1 and hml alpha-2. Both mutant clones were mixed cultures consisting of a mating-type cells and nonmating haploid cells. The frequencies of the two cell types were different, and a few diploid cells able to sporulate were found in the hml alpha-2 mutant. Conversions of an a mating-type cell to nonmater, and vice versa, were observed in both mutants. The conversion of an a mating phenotype to nonmating is postulated to occur by alteration of the a mating type to the sterile mating-type allele in the hml alpha-1 mutant. In tetrad dissection of prototrophic diploids that were obtained by rare mating of hml alpha-1 mutants with a heterothallic strain having the MATa ho HMRa HMLa genotype, many mating-deficient haploid segregants were found, while alpha mating-type segregants were observed in a similar diploid using an hml alpha-2 mutant. The mating-type-deficient haploid segregants were supposed to have the sterile alpha mating-type allele because the nonmating genetic trait always segregated with the mating-type locus. Sporogenous diploid cells obtained in the hml alpha-2 mutant clone had the MATa/MAT alpha HO/HO HMRa/HMRa hml alpha-2/hml alpha-2 genotype. These observations suggested that the hml alpha-1 allele produces a transposable element that gives rise to the sterile alpha mating type by transposition into the mating-type locus, and that the hml alpha-2 allele produces an element that provides alpha mating-type information, but is defective in the structure for transposition.     

Saccharomyces cerevisiae Ste5 is important for induction and substrate specificity of Fus3 MAP kinase in the pheromone signaling pathway

The pheromone pathway is one of the mitogen activated protein kinase (MAPK) signaling pathways identified in Saccharomyces cerevisiae and is involved in both G1 cell cycle arrest and mating of cells. Fus3 functions at a branching point for G1 cell cycle arrest and mating responses in the signaling cascade, and the Fus3 MAPK uses components of both G1 arrest and mating routes as substrates. The Ste5 is a scaffold protein of the MAPK module and is essential for the activation of Fus3. However, it is not known how Ste5 is involved in the specific activation of Fus3 in G1 arrest and mating. In this study, we characterized several G1 arrest defective Ste5 mutants to better understand the roles of Ste5 in the regulation of Fus3. The level of Fus3 increased by treatment with alpha-factor. However, the alpha-factor effects were not readily apparent in the observation of yeast cells containing G1 arrest defective ste5 mutant. This suggests that Ste5 plays an essential role in Fus3 induction. Fus3 immune kinase assay of G1 arrest defective ste5 transformants revealed that Ste5 is important for substrate specificity of Fus3 for G1 arrest and/or mating.     

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.