STE2 Gene Research Articles

Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes.

Cell type specialization in yeast haploids involves the mutually exclusive expression of one of two sets of genes, the a-specific and alpha-specific genes. We demonstrated that the products of the STE7, STE11, and STE12 genes were required for the expression of both gene sets. RNA levels transcribed from these gene sets were significantly decreased but not abolished in haploids containing a null mutation in the STE7, STE11, or STE12 gene. Transcript levels from the a- and alpha-specific gene sets were not further reduced in strains harboring mutations in all three STE genes, suggesting that STE7, STE11, and STE12 are required for the same aspect of transcription. We further showed that the requirement for these products was not the same for each member of a particular gene set. However, for any given a- or alpha-specific gene, the effect on RNA levels of any of the three ste mutations was similar.

Function of the STE4 and STE18 genes in mating pheromone signal transduction in Saccharomyces cerevisiae.

Function of the STE4 and STE18 genes in mating pheromone signal transduction in Saccharomyces cerevisiae.

Sequence-directed bends of DNA helix axis at the upstream activation sites of alpha-cell-specific genes in yeast.

The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-cell-specific gene whose expression is regulated by the mating-type locus, MAT. Two upstream activation sites (UASMF alpha 1s), which are binding sites for an activator protein, MAT alpha 1, mediate alpha-cell-specific expression of this gene. We show here that DNA fragments containing the UASMF alpha 1 region exhibited anomalous slow electrophoretic mobilities on gels at lower temperature, but not at higher temperature, that is characteristic of bent DNA. We confirmed the sequence-directed bend at the UASMF alpha 1 region by employing a circular permutation analysis and a DNA cyclization assay. Deletion analyses revealed the existence of two bends in this region, each of which overlaps each UASMF alpha 1 element. The two bends were almost in phase; they lie in a nearly same plane with a same direction. We also show the existence of sequence-directed bend at the UAS region of the STE3 gene, another alpha-specific gene in S. cerevisiae. These bent DNAs may be involved in transcriptional regulation of this set of genes.

Mapping of the Saccharomyces cerevisiae CDC3, CDC25, and CDC42 genes to chromosome XII by chromosome blotting and tetrad analysis.

CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome. The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5. The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.

Regulation by the yeast mating-type locus of STE12, a gene required for cell-type-specific expression.

The STE12 gene of the yeast Saccharomyces cerevisiae is necessary for RNA synthesis from two sets of cell-type-specific genes. We isolated a recombinant plasmid that carries the STE12 gene by complementation of the mating defect of ste12 cells. The DNA of the cloned gene was used to disrupt the chromosomal STE12 gene and to identify the STE12 transcript. We show that the STE12 transcript level is repressed 5- to 10-fold in a/alpha cells. The STE12 product thus acts to promote diploidy by activating expression of the two sets of genes necessary for mating, and then its synthesis is repressed by products unique to the diploid cell type.

Regulation by the Yeast Mating-Type Locus of STE12, a Gene Required for Cell-Type-Specific Expression

The STE12 gene of the yeast Saccharomyces cerevisiae is necessary for RNA synthesis from two sets of cell-type-specific genes. We isolated a recombinant plasmid that carries the STE12 gene by complementation of the mating defect of ste12 cells. The DNA of the cloned gene was used to disrupt the chromosomal STE12 gene and to identify the STE12 transcript. We show that the STE12 transcript level is repressed 5- to 10-fold in a/alpha cells. The STE12 product thus acts to promote diploidy by activating expression of the two sets of genes necessary for mating, and then its synthesis is repressed by products unique to the diploid cell type.

Conservation of a receptor/signal transduction system

The budding yeast Saccharomyces cerevisiae is a singlecell organism whose process of mating provides an excellent opportunity to study cell-cell interactions and response to diffusible factors. In this process, two partners of opposite type (a and a) fuse to produce a diploid cell. Each partner produces a peptide mating factor (a-factor or a-factor) that acts on the opposite cell type to prepare cells for mating by inducing expression of various genes whose products are necessary for cell and nuclear fusion (Tiueheart et al., MCB 7, 2316-2328, 1987; Rose et al., MCB 6, 3490~3497,1986) and by causing arrest in the Gl phase of the cell cycle. How do these mating factors trigger the multitude of cellular responses? The surprising answer, as the outlines of a molecular understanding become visible, seems to be that the machinery that yeast uses to respond to mating factors has similarity to the apparatus used in many other cells, including cells of the nervous system, for various signal transductions. The Receptors The receptor for yeast a-factor is coded by the STE2 gene, and the receptor for a-factor by the STE3 gene (Burkholder and Hartwell, NAR 73,8463-84751985; Nakayama et al., EMBO J. 4, 2643-2648, 1985; Hagen et al., PNAS 83, 1418-1422, 1986). The deduced amino acid sequence of the STf2 and STE3 products revealed that they are members of the rhodopsinlp-adrenergic receptor/muscarinic acetylcholine receptor family of integral membrane proteins. All of these proteins contain seven hydrophobic segments, each long enough to span a lipid bilayer membrane (see figure). These receptors are hypothesized to exhibit a transmembrane arrangement similar to that of bacteriorhodopsin, for which a partial three-dimensional structure is available. Amino acid sequence similarity among these receptors is modest; similarity between STf2 and STf3 polypeptides is virtually absent. The yeast receptors thus provide a striking example of gene products of similar function in which the topology of the protein (its arrangement with respect to the membrane), not primary sequence, has been strongly conserved (figure). The ligands for this receptor family are diverse: a skinny lipid chromophore (retinal) and small organic molecules (epinephrine and acetylcholine). The ligands for STE2 and STE3 proteins, a-factor and a-factor, respectively, are peptides of 13 and 12 amino acids (a-factor is probably a modified peptide). Intracellular Machinery Receptor switching experiments suggest that the response pathway in a and a cells may differ only in the type of receptor and that the intracellular machinery involved in subsequent transmission of the signal evoked by binding of the mating factors is the same in both cell types. It Minireview

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

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.

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.

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.

Sterility (ste) genes of Schizosaccharomyces pombe

AbstractIn previous experiments of Girgsdies (1982), eight sterile (ste) mutants of Schizosaccharomyces pombe did not sporulate when fused with h+ or h− protoplasts. We succeeded in achieving sporulation with these mutants. Two hitherto unknown ste genes, ste7 and ste8, were found.

The yeast repeated element sigma contains a hormone-inducible promoter.

A genomic clone (lambda ScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone alpha-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATrp gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to alpha-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following alpha-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MAT alpha cells that had been treated with a-factor, but were not present at significant levels in MATa/MAT alpha diploids. In MATa cells transformed with a plasmid in which the lambda ScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to alpha-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).

The yeast repeated element sigma contains a hormone-inducible promoter.

A genomic clone (lambda ScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone alpha-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATrp gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to alpha-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following alpha-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MAT alpha cells that had been treated with a-factor, but were not present at significant levels in MATa/MAT alpha diploids. In MATa cells transformed with a plasmid in which the lambda ScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to alpha-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).

Yeast peptide pheromones, a-factor and α-factor, activate a common response mechanism in their target cells

We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or α-factor receptor in matα1 mutants, which normally do not express either receptor or any other a- or α-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking α-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on α cells — increased transcription from the α-specific STE3 gene — can also be observed following α-factor treatment of pseudo-a cells (matα2 ste3 ste13), special mutants that respond to α-factor and also have an active STE3 promoter.

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

The peptide pheromone, α-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of α-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of teh STE2 gene were unable to internalize α-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 α-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of α-factor. We found no reduction of α-factor binding capacity in a cell cultures that had adapted to α-factor.

Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

Sequences upstream of the STE6 gene required for its expression and regulation by the mating type locus in Saccharomyces cerevisiae.

The STE6 gene of Saccharomyces cerevisiae is an a-specific gene; it is repressed in alpha cells by the alpha 2 product of the mating type locus. To study the role of sequences upstream of STE6 in its regulation and expression, we have determined the DNA sequence of the promoter region, identified the start sites for the STE6 transcript, and identified sequences governing its transcription. Deletions that remove DNA upstream of the STE6 gene were produced and assayed for effects on regulation and expression. The deletions defined two intervals upstream of the STE6 transcription initiation sites. One contains all or part of a negative element; the other contains all or part of a positive element. The negative element is required for repression of STE6 by alpha 2: deletions lacking this element express STE6 constitutively. Such deletions remove a 31-base-pair site, located 135 base pairs upstream of the first transcript start site, that is highly homologous to sites present in the upstream regions of four other genes repressed by alpha 2. These sites are presumably responsible for repression of the a-specific genes by alpha 2. The positive element (a putative upstream activation site) is required for expression of STE6. The deletions define the left boundary of the proposed upstream activation site. Sequence homologies between STE6 and other a-specific genes are found in this region and may mediate activation of this set of genes.