Cell fusion during yeast mating requires high levels of a-factor mating pheromone

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.     

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.     

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.     

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.     

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.     

Genetic Analysis of Default Mating Behavior in Saccharomyces Cerevisiae

Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating.     

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.     

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.     

Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTRΔF508

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al . (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTRΔF508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of ΔF508, providing a yeast phenotype for this mutation. In human cells, the ΔF508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTRΔF508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTRΔF508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.     

Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells.

Saccharomyces cerevisiae MATa cells release a lipopeptide mating pheromone, a-factor. Radiolabeling and immunoprecipitation show that MATa ste6 mutants produce pro-a-factor and mature a-factor intracellularly, but little or no extracellular pheromone. Normal MATa cells carrying a multicopy plasmid containing both MFa1 (pro-a-factor structural gene) and the STE6 gene secrete a-factor at least five times faster than the same cells carrying only MFa1 in the same vector. The nucleotide sequence of the STE6 gene predicts a 1290 residue polypeptide with multiple membrane spanning segments and two hydrophilic domains, each strikingly homologous to a set of well-characterized prokaryotic permeases (including hlyB, oppD, hisP, malK and pstB) and sharing even greater identity with mammalian mdr (multiple drug resistance) transporters. These results suggest that the STE6 protein in yeast, and possibly mdr in animals, is a transmembrane translocator that exports polypeptides by a route independent of the classical secretory pathway.     

The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins.

Post-translational processing of a distinct group of proteins and polypeptides, including the a-factor mating pheromone and RAS proteins of Saccharomyces cerevisiae, results in the formation of a modified C-terminal cysteine that is S-isoprenylated and alpha-methyl esterified. We have shown previously that a membrane-associated enzymatic activity in yeast can mediate in vitro methylation of an isoprenylated peptide substrate and that this methyltransferase activity is absent in ste14 mutants. We demonstrate here that STE14 is the structural gene for this enzyme by expression of its product as a fusion protein in Escherichia coli, an organism in which this activity is lacking. We also show that a-factor, RAS1 and RAS2 are physiological methyl-accepting substrates for this enzyme by demonstrating that these proteins are not methylated in a ste14 null mutant. It is notable that cells lacking STE14 methyltransferase activity exhibit no detectable impairment of RAS function or cell viability. However, we did observe a kinetic delay in the rate of RAS2 maturation and a slight decrease in the amount of membrane localized RAS2. Thus, methylation does not appear to be essential for RAS2 maturation or localization, but the lack of methylation can have subtle effects on the efficiency of these processes.     

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.     

The exchange factor Cdc24 is required for cell fusion during yeast mating

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating.     

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 a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane- associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP- binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.     

Identification and Characterization of a Mutation Affecting the Division Arrest Signaling of the Pheromone Response Pathway in Saccharomyces Cerevisiae

Mating pheromones, a- and alpha-factors, arrest the division of cells of opposite mating types, alpha and a cells, respectively. I have isolated a sterile mutant of Saccharomyces cerevisiae that is defective in division arrest in response to alpha-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18 and dac1). Although dac2 cells had no phenotype in the absence of pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene (previously shown to encode a protein with similarity to the alpha subunit of mammalian G proteins). In addition, dac2 cells formed prezygotes with wild-type cells of opposite mating types, although they could not undergo cell fusion. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.     

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.