STE2 Gene Research Articles

Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype.

The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L194Q mutation, which leads to the substitution of glutamine for leucine-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial constitutive activation of the response pathway. Moreover, other amino acid substitutions at the 194 position and several deletion mutations that collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the third cytoplasmic loop is to function as a negative regulatory domain involved in the maintenance of a nonsignaling state of the receptor. The constitutive activity and the pheromone hypersensitivity of ste3L194Q cells were recessive, suggesting that the wild-type receptor can antagonize the signal associated with the activated receptor. The ste3 delta 306 mutation, which results in truncation of most of the C-terminal domain of the receptor, led to a 20-fold increase in pheromone sensitivity, indicating that this domain also mediates negative regulation of the receptor. The ste3L194Q and ste3 delta 306 mutations appear to affect receptor activity independently, because the double mutant was associated with a 400-fold increase in pheromone sensitivity.

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

Myristic acid (C14:0) is added to the N-terminal glycine residue of the alpha subunits of certain receptor-coupled guanine nucleotide-binding regulatory proteins (G proteins). The G alpha subunit (GPA1 gene product) coupled to yeast pheromone receptors exists as a pool of both myristoylated and unmyristolyated species. After treatment of MATa cells with alpha factor, the myristoylated form of Gpa1p increases dramatically, and the unmyristoylated form decreases concomitantly. This pheromone-stimulated shift depends on the function of STE2 (alpha-factor receptor), STE11 (a protein kinase in the response pathway), and NMT1 (myristoyl-CoA:protein N-myristoyltransferase) genes and uses the existing pool of fatty acids (is not blocked by cerulenin). Myristoylated Gpa1p persists long after pheromone is removed. Because myristoylation is essential for proper G alpha-G beta gamma association and receptor coupling, pheromone-dependent stimulation of Gpa1p myristoylation may be an important contributing factor in adaptation after signal transmission.

NPK1, a tobacco gene that encodes a protein with a domain homologous to yeast BCK1, STE11, and Byr2 protein kinases.

We have isolated a cDNA (cNPK1) that encodes a predicted protein kinase of 690 amino acids from suspension cultures of tobacco cells. The deduced sequence is closely related to those of the protein kinases encoded by the STE11 and BCK1 genes of Saccharomyces cerevisiae and the byr2 gene of Schizosaccharomyces pombe. STE11 and Byr2 function in the yeast mating pheromone response pathways, and BCK1 acts downstream of the yeast protein kinase C homolog encoded by the PKC1 gene, which is essential for normal growth and division of yeast cells. Overexpression in yeast cells of a truncated form of cNPK1, which encodes only the putative catalytic domain, replaced the growth control functions of BCK1 and PKC1 but not the mating pheromone response function of STE11. Thus, the catalytic domain of NPK1 specifically activates the signal transduction pathway mediated by BCK1 in yeast. In tobacco cells in suspension culture, the NPK1 gene is transcribed during logarithmic phase and early stationary phase but not during late stationary phase. In a tobacco plant, it is also transcribed in stems and roots but not in mature leaves, which rarely contain growing cells. The present results suggest that a signal transduction pathway mediated by this BCK1- and STE11-related protein kinase is also conserved in plants and that a function of NPK1 is controlled at least in part at a transcriptional level.

Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro.

The AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in the development of stamens and carpels. This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF. To examine the DNA-binding activity of the AGAMOUS protein, double-stranded oligonucleotides with random sequences of 40 bp in the central region were synthesized and mixed with the AGAMOUS MADS domain overproduced in Escherichia coli. Oligonucleotides which bound to the MADS domain were recovered by repeated immunoprecipitation with an antibody which recognizes the overproduced protein. From a comparison of the recovered DNA sequences, the consensus sequence of the high-affinity binding-sites for the AGAMOUS MADS domain was determined to be 5'-TT(A/T/G) CC(A/T)6GG(A/T/C)AA-3'. DNase I footprinting and methylation interference experiments showed that the MADS domain binds to this motif. Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCM1 binding-sites in a-mating type-specific promoters of Saccharomyces cerevisiae show similarities with the binding-site sequence of the AGAMOUS MADS domain. A synthetic MCM1 binding-site in the upstream region of the STE2 gene is recognized by the AGAMOUS MADS domain.

Mating of the fission yeast occurs independently of pmd1+ gene product, a structural homologue of budding yeast STE6 and mammalian P-glycoproteins.

The pmd1+, a multidrug resistance gene of the fission yeast Schizosaccharomyces pombe, encodes a protein similar to the budding yeast Saccharomyces cerevisiae STE6 gene product and mammalian P-glycoproteins. The STE6 protein is a membrane transporter of a-factor, a mating pheromone of a-type S. cerevisiae, which is structurally related to M-factor of the fission yeast. However, heterothallic or homothallic pmd1 null mutant cells of S. pombe, which were constructed by means of gene disruption, showed no significant decrease in the mating abilities. On the other hand, the multidrug resistance conferred by the pmd1+ was overcome by the treatment with verapamil, a typical inhibitor of mammalian P-glycoproteins. These results indicate that the pmd1+ gene product is functionally similar to mammalian P-glycoproteins, rather than to the budding yeast STE6.

Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway.

In the yeast Saccharomyces cerevisiae, a G protein-linked signal transduction pathway mediates response to the oligopeptide mating pheromones a-factor and alpha-factor. Because cellular responses, including G1 arrest, occur transiently, cells can adapt or desensitize and resume growth. To address whether the balance between response and adaptation is influenced by the efficiency of receptor-G protein interaction, we introduced random point mutations in sequences that encode the third cytoplasmic loop of the alpha-factor receptor (STE2 gene product). Three mutations were identified that confer alpha-factor-resistant phenotypes, yet preserve normal cell-surface expression, ligand-binding affinity, and endocytosis of the receptor. However, these mutations confer partial signaling defects, as determined by cell cycle arrest and transcriptional induction assays, as well as in vitro assays of receptor-G protein interaction. Physiological tests suggested that receptors bearing third loop substitutions promote recovery from pheromone-induced growth arrest. Genetic evidence indicated that the third loop and the C-terminal domain of the receptor control independent recovery or adaptation processes. In contrast, receptor third loop substitutions caused rapid adaptation only if cells express a functional SST2 gene. Thus, disruption of pheromone receptor-G protein interaction concomitantly blunts signaling and specifically promotes the function of an SST2-dependent adaptation pathway. Possible functions for the Sst2 protein are discussed.

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.

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.

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.

Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.

Dominant genetics using a yeast genomic library under the control of a strong inducible promoter.

In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 x 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.

Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway.

The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.

Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction.

In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone signal. Cells carrying a mutation in the ste8 gene are unable to induce transcription of mat1-Pm in response to pheromone, suggesting that the ste8 gene product functions in the signal transduction pathway. The ste8+ gene encodes a 659 amino acid putative protein kinase, which is identical to the previously identified byr2 suppressor of the ras1 defect. Furthermore, ste8+ is highly homologous to the Saccharomyces cerevisiae STE11 gene, which functions in signal transduction in budding yeast. Expression of the S. cerevisiae STE11 gene in S. pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone. Also, such cells become capable of conjugation and sporulation. When mat1-Pm is artifically expressed from a heterologous promoter, ste8 mutant cells will enter meiosis. This demonstrates that the meiotic defect of ste8 mutants is due to the absence of the mat1-Pm gene product.

Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1.

The FUS3 and KSS1 kinases are components of the pheromone-dependent signal transduction pathway in yeast. We show that FUS3 and KSS1 become rapidly phosphorylated after pheromone treatment. Similar to mammalian MAP kinases, this modification occurs at two amino acids of FUS3, threonine-180 and tyrosine-182. A mutation introduced at either position results in complete loss of function in vivo. Amino acid substitutions that destroy catalytic activity of the kinase do not prevent phosphorylation of the mutant products, a result that excludes an autocatalytic activation pathway. The modification of FUS3 is dependent on kinases encoded by the STE11 and STE7 genes. Furthermore, a hyperactive allele of STE11 causes increased phosphorylation of FUS3 in the absence of pheromone stimulation. Thus, either STE7 or STE11 could be the kinase responsible for the phosphorylation of FUS3.

Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase.

The signal transduction pathway that mediates the response of haploid yeast cells to peptide mating pheromones involves several components including the protein kinases STE7 and STE11. We have isolated and characterized a dominant allele of the STE11 gene and have demonstrated that expression of an amino-terminally truncated form of STE11 protein causes constitutive activation of the mating pathway. Expression of this dominant STE11 allele also restored mating ability to certain sterile strains. In conjunction with the results of others, our epistasis results establish the following order of action of pathway components: STE2, GPA1(SCG1), STE4, STE5, STE11, STE7, STE12. Transduction of the signal from STE11 to STE7 may involve phosphorylation because STE7 displays several phosphorylation forms, and STE7 is multiply phosphorylated in response to either pheromone or coexpression of dominant STE11 protein. Further signal propagation appears to require STE7 protein kinase activity, because a catalytically impaired STE7 mutant is defective in the mating response.

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.

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.

New mechanisms of hormone secretion: MDR-like gene products as extrusion pumps for hormones?

P-glycoprotein, the product of the multidrug resistance (MDR1) gene, is an ATP-driven transmembrane pump that increases the resistance of cells by actively exporting toxic chemicals. In addition to transporting anticancer drugs, P-glycoprotein has been reported to extrude a variety of lipophilic drugs, such as calcium channel blockers, phenothiazines, cyclosporines etc. Interestingly, recent experiments suggest that steroid hormones may be physiologic substrates for P-glycoprotein. In addition, there exists a family of transporter genes with high structural homology to P-glycoprotein, the so-called ABC (ATP-binding casette) family. Although the physiological ligands for most of these transporters are unknown, there is increasing evidence that peptides may be transported by some of these proteins. Thus, the a-factor, a farnesylated pheromone with 13 amino acids, is exported from yeast cells by the product of the STE6 gene, a transporter protein with high homology to P-glycoprotein. Recently, we have cloned a novel member of the ABC-transporter gene family from neuroblastoma x glioma hybrid (NG-108-15) cells. This putative transporter gene ("NG-TRA") is expressed in the adrenal gland, kidney and in the brain. High amounts of NG-TRA mRNA are found in a variety of human brain tumors. Whether NG-TRA and/or other MDR-related transporters are involved in the transport of steroids, peptide hormones or growth factors remains to be established. If so, the cellular export of hormones by active pumps may represent a new mechanism of hormone secretion.

A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.