Saccharomyces Cerevisiae Mating Research Articles

Identification of Residues that Contribute to Receptor Activation through the Analysis of Compensatory Mutations in the G Protein-Coupled α-Factor Receptor

The alpha-factor receptor (Ste2p) stimulates mating of the yeast Saccharomyces cerevisiae. Ste2p belongs to the large family of G protein-coupled receptors that are characterized by seven transmembrane alpha-helices. Receptor activation is thought to involve changes in the packing of the transmembrane helix bundle. To identify residues that contribute to Ste2p activation, second-site suppressor mutations were isolated that restored function to defective receptors carrying either an F204S or Y266C substitution which affect residues at the extracellular ends of transmembrane domains 5 and 6, respectively. Thirty-five different suppressor mutations were identified. On their own, these mutations caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function. The majority of the mutations affected residues in the transmembrane segments that are predicted to face the helix bundle. Many of the suppressor mutations caused constitutive receptor activity, suggesting they improved receptor function by partially restoring the balance between the active and inactive states. Analysis of mutations in transmembrane domain 7 implicated residues Ala281 and Thr282 in receptor activation. The A281T and T282A mutants were supersensitive to S. cerevisiae alpha-factor, but were defective in responding to a variant of alpha-factor produced by another species, Saccharomyces kluyveri. The A281T mutant also displayed 8.7-fold enhanced basal signaling. Interestingly, Ala281 and Thr282 are situated in approximately the same position as Lys296 in rhodopsin, which is covalently linked to retinal. These results suggest that transmembrane domain 7 plays a role in receptor activation in a wide range of G protein-coupled receptors from yeast to humans.

High level expression of a recombinant acid phytase gene in Pichia pastoris

To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.

Phenotypic switching and mating type switching of Candida glabrata at sites of colonization.

Candida glabrata switches spontaneously at high frequency among the following four graded phenotypes discriminated on agar containing 1 mM CuSO(4): white, light brown, dark brown (DB), and very dark brown. C. glabrata also contains three mating type loci with a configuration similar to that of the Saccharomyces cerevisiae mating type cassette system, suggesting it may also undergo cassette switching at the expression locus MTL1. To analyze both reversible, high-frequency phenotypic switching and mating type switching at sites of colonization, primary samples from the oral cavities and vaginal canals of three patients suffering from C. glabrata vaginitis were clonally plated on agar containing CuSO(4). It was demonstrated that (i) in each vaginitis patient, there was only one colonizing strain; (ii) an individual could have vaginal colonization without oral colonization; (iii) phenotypic switching occurred at sites of colonization; (iv) the DB phenotype predominated at the site of infection in all three patients; (v) genetically unrelated strains switched in similar, but not identical, fashions and caused vaginal infection; (vi) different switch phenotypes of the same strain could simultaneously dominate different body locations in the same host; (vii) pathogenesis could be caused by cells in different mating type classes; and (viii) mating type switching demonstrated at both the genetic and transcription levels occurred in one host.

Fig1p Facilitates Ca2+ Influx and Cell Fusion during Mating of Saccharomyces cerevisiae

During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.

Cell-type-dependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p-Itc1p chromatin remodelling complex.

In Saccharomyces cerevisiae MATa haploid cells, the a-specific genes are expressed, whereas in the MATalpha haploid and MATa/alpha diploid cell types their transcription is repressed. It is shown in this report that Itc1p, a component of the ATP-dependent Isw2p-Itc1p chromatin remodelling complex, is required for the repression of a-specific genes. It has previously been reported that disruption of the ITC1 gene leads, in MATalpha cells, to an aberrant cell morphology resembling the polarized mating projection of cells responding to pheromone. The activation of the pheromone signalling pathway in itc1 mutants of both mating types was examined and found to be constitutively active in MATalpha itc1 but not in MATa itc1 cells. Furthermore, unlike the wild-type, MATalpha itc1 and MATa/alpha itc1/itc1 cells secrete a-factor and express significant levels of other a-specific genes. The results indicate that the inappropriate a-factor production in a MATalpha context, due to the derepression of the a-specific genes, produces an autocrine signalling loop that leads to the aberrant morphology displayed by MATalpha itc1 cells. It is suggested that the Isw2p-Itc1p complex contributes to maintain the repressive chromatin structure described for the asg operator present in the promoters of a-specific genes.

Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor.

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae.

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae MATa cells, export of the a-factor mating pheromone is mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of transporters and a close homologue of mammalian multidrug transporter P-glycoproteins (Pgps). We have used functional complementation of a ste6delta mutation to isolate a gene encoding an ABC transporter capable of a-factor export from the pathogenic yeast, Candida albicans. This gene codes for a 1323-amino acid protein with an intramolecular duplicated structure, each repeated half containing six potential hydrophobic transmembrane segments and a hydrophilic domain with consensus sequences for an ATP-binding fold. The predicted protein displays significant sequence similarity to S. cerevisiae Ste6p and mammalian Pgps. The gene has been named HST6, for homologue of STE6. A high degree of structural conservation between the STE6 and the HST6 loci with respect to DNA sequence, physical linkage and transcriptional arrangement indicates that HST6 is the C. albicans orthologue of the S. cerevisiae STE6 gene. We show that the HST6 gene is transcribed in a haploid-specific manner in S. cerevisiae, consistent with the presence in its promoter of a consensus sequence for Mata1p-Matalpha2p binding known to mediate the repression of haploid-specific genes in S. cerevisiae diploid cells. In C. albicans, HST6 is expressed constitutively at high levels in the different cell types analysed (yeast, hyphae, white and opaque), demonstrating that HST6 transcription is not repressed in this diploid yeast, unlike in diploid S. cerevisiae, and suggesting a basic biological function for the Hst6p transporter in C. albicans. The strong similarity between Hst6p and the multidrug transporter Pgps also raises the possibility that Hst6p could be involved in resistance to antifungal drugs in C. albicans.

Radicicol Leads to Selective Depletion of Raf Kinase and Disrupts K-Ras-activated Aberrant Signaling Pathway

Activation of Ras leads to the constitutive activation of a downstream phosphorylation cascade comprised of Raf-1, mitogen-activated protein kinase (MAPK) kinase, and MAPK. We have developed a yeast-based assay in which the Saccharomyces cerevisiae mating pheromone-induced MAPK pathway relied on co-expression of K-Ras and Raf-1. Radicicol, an antifungal antibiotic, was found to inhibit the K-ras signaling pathway reconstituted in yeast. In K-ras-transformed, rat epithelial, and K-ras-activated, human pancreatic carcinoma cell lines, radicicol inhibited K-Ras-induced hyperphosphorylation of Erk2. In addition, the level of Raf kinase was significantly decreased in radicicol-treated cells, whereas the levels of K-Ras and MAPK remained unchanged. These results suggest that radicicol disrupts the K-Ras-activated signaling pathway by selectively depleting Raf kinase and raises the possibility that pharmacological destabilization of Raf kinase could be a new and powerful approach for the treatment of K-ras-activated human cancers.

Periplasmic Bar1 protease of Saccharomyces cerevisiae is active before reaching its extracellular destination.

Saccharomyces cerevisiae MATa and MAT alpha cells secrete a-factor and alpha-factor pheromones. These peptides act on cells of the opposite mating type. They induce physiological changes which allow the formation of diploid cells. MATa strains produce an extracellular protease which cleaves, and thus inactivates the MAT alpha cell-specific alpha-factor pheromone. This pepsin-like enzyme is encoded by the BAR1(SST1) gene and is secreted into the periplasmic space of MATa cells. We found that the Bar1p protease is already active in early compartments of the secretory pathway. Our results indicate that Bar1 protease tolerates large N-terminal extensions of its substrate and does not require Golgi-specific modifications such as outer-chain glycosylation for activity.

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

Differential regulation of FUS3 MAP kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase MSG5 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae mating pheromone response is mediated by activation of a MAP kinase (Fus3p and Kss1p) signaling pathway. Pheromone stimulation causes cell cycle arrest. Therefore, inactivation of the Fus3p and Kss1p MAP kinases is required during recovery phase for the resumption of cell growth. We have isolated a novel protein tyrosine phosphatase gene, PTP3, as a negative regulator of this pathway. Ptp3p directly dephosphorylates and inactivates Fus3p MAP kinase in vitro. Multicopy PTP3 represses pheromone-induced transcription and promotes recovery. In contrast, disruption of PTP3 in combination with its homolog PTP2 results in constitutive tyrosine phosphorylation, enhanced kinase activity of Fus3p MAP kinase on stimulation, and delayed recovery from the cell cycle arrest. Both tyrosine phosphorylation and kinase activity of Fus3p are further increased by disruption of PTP3 and PTP2 in combination with MSG5, which encodes a dual-specificity phosphatase. Cells deleted for all three of the phosphatases (ptp2delta ptp3delta msg5delta) are hypersensitive to pheromone and exhibit a severe defect in recovery from pheromone-induced growth arrest. Our data indicate that Ptp3p is the major phosphatase responsible for tyrosine dephosphorylation of Fus3p to maintain a low basal activity; it also has important roles, along with Msg5p, in inactivation of Fus3p following pheromone stimulation. These data present the first evidence for a coordinated regulation of MAP kinase function through differential actions of protein tyrosine phosphatases and a dual-specificity phosphatase.

Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis.

Saccharomyces cerevisiae mating pheromones induce production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required for normal formation of the projection of cell growth that becomes the site of cell fusion during conjugation. Afr1p interacts with Cdc12p, which belongs to a family of filament-forming proteins termed septins that have been studied primarily for their role in bud morphogenesis and cytokinesis. The significance of the interaction between Afr1p and Cdc12p was tested in this study by examining the effects of AFR1 mutations that destroy the Cdc12p-binding domain. The results demonstrate that sequences in the C-terminal half of Afr1p are required for interaction with Cdc12p and for proper localization of Afr1p to the base of the mating projection. However, the Cdc12p-binding domain was not required for regulation of receptor signaling or for mating projection formation. This result was surprising because cells carrying a temperature-sensitive cdc12-6 mutation were defective in projection formation, indicating a role for Cdc12p in this process. Although the Cdc12p-binding domain was no essential for Afr1p function, this domain did improve the ability of Afr1p to promote morphogenesis, suggesting that the proper localization of Afr1p is important for its function.

Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification. NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDS-PAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.

Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p.

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycle-dependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.

Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1.

The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate during the cell cycle. In the absence of a functional Ste12 transcription factor, both the levels and the cell cycle pattern of expression of these genes were affected. FUS1 and AGA1 levels, which are maximally expressed only in G1-phase cells, were strongly reduced in ste12- cells. The cell cycle transcription pattern for FAR1 was changed in ste12- cells: the gene was still significantly expressed in G2/M, but transcript levels were strongly reduced in G1 phase, resulting in a lack of Far1 protein accumulation. G2/M transcription of FAR1 was dependent on the transcription factor Mcm1, and expression of a gene with Mcm1 fused to a strong transcriptional activation domain resulted in increased levels of FAR1 transcription. The pattern of cell cycle-regulated transcription of FAR1 could involve combinatorial control of Ste12 and Mcm1. Forced G1 expression of FAR1 from the GAL1 promoter resorted the ability to arrest in response to pheromone in ste12-cells. This indicates that transcription of FAR1 in the G1 phase is essential for accumulation of the protein and for pheromone-induced cell cycle arrest.

Polarization of yeast cells in spatial gradients of alpha mating factor.

The process of cell fusion during mating of the yeast Saccharomyces cerevisiae is mediated by factors secreted by the mating partners. Spatial gradients of one of these mating factors, alpha-factor, polarized the growth of projections by MATa cells. The site of previous budding did not affect the direction of polarization, and subsequent budding was also polarized if mating factor was removed. Orientation occurred in the presence of nocodazole, suggesting that microtubules were not critical. At extremely low concentrations of alpha-factor, sst2-mutants (which in genetic studies do not discriminate between partners producing different amounts of alpha-factor) were able to polarize their projections. The sensitivity of this spatial sensing mechanism in wild-type cells is such that differences in receptor occupancy estimated to be about 1% are sufficient for orientation.

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.

STE6, the yeast a-factor transporter

In eukaryotic cells, most extracellular proteins exit the cell via the classical secretory pathway (ER→Golgi→secretory vesicles). A notable exception to this pattern is the Saccharomyces cerevisiae mating pheromone a-factor, an isoprenylated, methylated, oligopeptide signaling molecule which uses a distinctly non-classical mechanism for secretion. Export of a-factor from the yeast cell is mediated by STE6, a member of the ABC protein superfamily. STE6 is one of the few eukaryotic ABC proteins for which a true physiological substrate is known. The ability to carry out molecular manipulations with ease in yeast, together with the possibility of probing substrate-transporter interactions via genetic analysis, affords an excellent opportunity to rigorously dissect the workings of this ABC family member.

A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae.

This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways. STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa. Its expression pattern is similar to that of several other protein kinases in the mating response pathway. Deletion of the kinase domain of STE20 causes sterility in both haploid mating types. This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele. A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone. This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12. The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12.