STE2 Gene Research Articles

The Cryptococcus neoformans STE12alpha gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific.

Cryptococcus neoformans possesses two mating types, MATalpha and MATa. Alpha-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFalpha, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12alpha gene, however, has one striking difference from other fungal STE12 genes; it is found only in alpha-cells. The existence of STE12alpha in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

Functional interactions between synthetic alkyl phospholipids and the ABC transporters P-glycoprotein, Ste-6, MRP, and Pgh 1.

The ABC superfamily of transporters includes the mammalian P-glycoprotein family (Class I and Class II P-gps), the multidrug resistance-associated protein (MRP), the Pgh-1 product of Plasmodium falciparum gene pfmdr1, all of which are associated with cellular pleiotropic drug resistance phenomena. STE6, the yeast transporter for the farnesylated peptide pheromone a, is also a member of this family. Structural similarities in this family translate into functional homology as expression of mouse Mdr3S (P-gp), P. falciparum Pgh-1, and human MRP partially restore mating in a sterile yeast mutant lacking a functional STE6 gene. The demonstration that Class II P-gps function as phosphatidylcholine (PC) translocators raise the possibility that other ABC transporters may also interact with physiological lipids. We report the identification of the synthetic lipid and PC analog ET-18-OCH3 (edelfosine) as a substrate for not only Class II P-gp but also for Class I P-gps and surprisingly for the other ABC transporters MRP, Pgh-1, and STE6. Expression of these proteins in the yeast Saccharomyces cerevisiae JPY201 was found to confer cellular resistance to cytotoxic concentrations of this lipid by a factor of 4-20-fold in a growth inhibition assay. The noted activity of ABC transporters toward this synthetic lipid was specific as a mutant variant of Mdr3 (Mdr3F) with reduced activity could not convey cellular resistance to ET-18-OCH3. ET-18-OCH3 was also found capable of blocking a-peptide pheromone transport and STE6 complementation by these ABC proteins. The inhibitory effect of ET-18-OCH3 on cell growth and a-factor transport could be abrogated by incubation with the lipid acceptor protein BSA or by enzymatic cleavage by microsomal alkylglycerol mono-oxygenase (MAMO). MAMO and BSA reversal of the ether lipid effect was only seen in the presence of a functional transporter. These results suggest that the group of cytotoxic synthetic PC analogs studied reveal possible structural and functional aspects common to the ABC transporters tested. Furthermore, the studies with BSA and MAMO suggest that the mechanism of transport of ET-18-OCH3 by these ABC transporters may be related to the flippase mechanism of PC transport by Mdr2.

Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene.

The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the Whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fsw1 allele.

Expression and Purification of the Saccharomyces cerevisiae α-Factor Receptor (Ste2p), a 7-Transmembrane-segment G Protein-coupled Receptor

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor.

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone. M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe.

Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WHI3, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2Val19 and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development.

Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene

G protein coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a wide variety of extracellular signals. However, the structural basis for activation of this class of receptors by ligand binding is not well understood. We report here the use of a systematic genetic protocol for identifying interactions among the seven transmembrane helices of the GPCR responsible for cellular responses to the α-mating pheromone of the yeast Saccharomyces cerevisiae. Random mutations were introduced into the region of the STE2 gene encoding the third transmembrane segment of the α-factor receptor, followed by screening for loss of signaling. The limited spectrum of non-conservative mutations recovered, including removal of the only negatively charged side-chain in the transmembrane region, indicates that most substitutions in the third transmembrane segment do not affect receptor function. Three second-site intragenic suppressors of these initial mutations were isolated following mutagenesis of the remaining six transmembrane segments. One of these suppressors, Y266C in the sixth transmembrane segment, is allele specific and shows non-additivity of phenotypes indicative of a physical interaction between the third and sixth transmembrane regions of the receptor. A second suppressor, M218T in the fifth transmembrane segment, exhibits only partial allele specificity. A third suppressor, R58G, in the first transmembrane segment, suppresses a variety of starting alleles and appears to cause global stabilization of the receptor. Analysis of these suppressors and additional alleles can provide a database for modeling GPCR structure.

The Gβγ Complex of the Yeast Pheromone Response Pathway: SUBCELLULAR FRACTIONATION AND PROTEIN-PROTEIN INTERACTIONS

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction. A slower-migrating, presumably phosphorylated, form of Ste4p is enriched in the non-membrane fraction. The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to co-sediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum. Under the conditions of our assay, Gpa1p was not associated with either particle. The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G betagamma complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes. The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of the cell-surface receptors. These results indicate that at least 40% of Ste4p and Ste18p are part of a G betagamma complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G alpha.

Arabidopsis thaliana cDNA isolated by functional complementation shows homology to serine/threonine protein kinases

The yeast Saccharomyces cerevisiae ste6 mutant is defective in transport of a-mating factor, resulting in an inability of ste6 a cells to mate with α cells. The gene encodes an ATP-binding cassette, ABC transporter. We used functional complementation of a yeast ste6 mutant with an Arabidopsis thaliana expression library in an attempt to clone an Arabidopsis homolog. Sequence analysis of the isolated Arabidopsis complementing cDNA however showed no homology to the STE6 gene. High sequence similarity was detected to members of the mitogen-activated serine/threonine protein (MAP) kinase family involved in signal transduction: STE20, STE11, BCK1, Byr2 and p65PAK. The Arabidopsis clone failed to complement a fus3/ kss1 mutant of S. cerevisiae, but did complement a defect in ste11, ste20, as well as ste6. The isolated clone encodes a protein that is truncated at its amino-terminal, and might function in a similar way as a dominant STE11 truncation allele. These results suggest that the Arabidopsis cDNA encodes a putative serine/threonine kinase that can function in the mating response pathway upstream of FUS3/KSS1 in S. cerevisiae, at the level of STE11 gene. Interestingly, this clone is able to restore the ability of the ste6 yeast mutant to export a-factor.

Schizosaccharomyces pombe pcr1+ Encodes a CREB/ATF Protein Involved in Regulation of Gene Expression for Sexual Development

The Schizosaccharomyces pombe pcr1 gene encodes a bZIP protein that apparently belongs to the cyclic AMP response element (CRE)-binding protein/activating transcription factor family. The deduced pcr1 gene product consists of 171 amino acid residues and is most similar to the mammalian CRE-BP1. A glutathione S-transferase-Pcr1 fusion protein produced in Escherichia coli was able to bind specifically to the CRE motif in vitro. Analysis with anti-Pcr1 serum suggested that Pcr1 is included in the major CRE-binding factors present in the S. pombe cell extract. Disruption of the pcr1 gene was not lethal, but the disruptant showed cold-sensitive growth on rich medium. The disruptant was also inefficient in mating and sporulation, though it was not completely sterile. Expression of the ste11 gene, which encodes a key transcription factor for sexual development, was greatly reduced in the disruptant, and overexpression of ste11+ suppressed the deficiency of the pcr1 disruptant in sexual development. It has been shown that expression of ste11 is negatively regulated by cyclic AMP-dependent protein kinase (PKA) and that the loss of PKA activity results in ectopic sexual development. Disruption of pcr1 blocked ectopic sexual development. Furthermore, disruption of pcr1 reduced expression of fbp1, a glucose-repressible gene negatively regulated by PKA. These results suggest that Pcr1 is a putative transcriptional regulator whose activity may be controlled by PKA. Alternatively, its activity may be independent of PKA, and full induction of ste11 and fbp1 expression requires the function of Pcr1 in addition to elimination of the repression by PKA.

Regulation of Meiosis in Fission Yeast.

The fission yeast Schizosaccharomyces pombe initiates sexual development under starved conditions. Nutritional starvation decreases the level of intracellular cAMP. This decrease induces expression of the ste11 gene, which encodes a key transcription factor for genes required for mating and meiosis. Mutational analyses of S. pombe genes encoding components of the cAMP cascade have shown that S. pombe cells stay in the mitotic cell cycle as long as the level of cAMP-dependent protein kinase activity is high, but are committed to mating and meiosis if this activity is lowered. To initiate meiosis in S. pombe, a protein kinase encoded by pat1 (also called ran1) should be inactivated. This inactivation results from deprivation of nutrients via a cascade of expression of genes including ste11. The mei2 gene encodes a factor indispensable for the initiation of meiosis, and its expression is regulated directly by Ste11. If Pat1 kinase is intact, it blocks Mei2 function. Mei2 is required at two distinct stages of meiosis, once prior to premeiotic DNA synthesis and then prior to the first meiotic division (meiosis I). Mei2 is an RNA-binding protein, and forms a complex with a specific RNA species to promote meiosis I. This RNA species, named meiRNA, is polyadenylated but is unlikely to encode a protein product. It is essential for meiosis I, but not for either cell growth or premeiotic DNA synthesis. These observations unequivocally demonstrate that RNA plays a critical role in the control of meiosis.

The human β2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

We have developed a rapid and efficient expression system to study the human β 2 adrenergic receptor (hu β 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu β 2AR gene, modified by replacement of the 5′ untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (α-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu β 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [ 125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu β 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.

Truncated Forms of a Novel Yeast Protein Suppress the Lethality of a G Protein α Subunit Deficiency by Interacting with the β Subunit

In Saccharomyces cerevisiae, the mating pheromone-initiated signal is transduced by a heterotrimeric G protein and normally results in transient cell cycle arrest and differentiation. A null allele of the G alpha (GPA1/SCG1) subunit results in cell death due to unchecked signaling from the G beta gamma (STE4, STE18, respectively) heterodimer. We have identified three high copy suppressors of gpa1 lethality. Two of these genes encode known transcription factors. Mat alpha 2p and Mcm1p. The third is a truncated form of a novel gene, SYG1. Overexpressed wild type SYG1 is a weak suppressor of gpa1. In contrast, the isolated mutant allele SYG1-1 is a strong suppressor that completely blocks the cell cycle arrest and differentiation phenotypes of gpa1 cells of both mating types. One deletion mutant (SYG1 delta 340) can suppress the cell cycle arrest associated with gpa1, but the cells retain a differentiated morphology. SYG1-1 can suppress the effects of overexpressed wild type G beta but is not able to suppress the lethality of an activated G beta mutant (STE4Hpl). Consistent with these genetic observations, the suppressing form of Syg1p can interact with the STE4 gene product, as determined by a two-hybrid assay. SYG1-1 is also capable of promoting pheromone recovery in wild type cells, as judged by halo assay. The sequence of SYG1 predicts eight membrane-spanning domains. Deletion mutants of SYG1 indicate that complete gpa1 suppression requires removal of all of these hydrophobic regions. Interestingly, this truncated protein localizes to the same plasma membrane-enriched subcellular fraction as does full-length Syg1p. Three hypothetical yeast proteins, identified by their similarity to the SYG1 primary sequence within the gpa1 suppression domain, also appear to have related structures. The properties of Syg1p are consistent with those of a transmembrane signaling component that can respond to, or transduce signals through, G beta or G beta gamma.

Regulation of Calcineurin Gene Expression in Schizosaccharomyces pombe: DEPENDENCE ON THE ste11 TRANSCRIPTION FACTOR

Calmodulin and its target enzymes are important regulators of numerous cellular processes, including reversible protein phosphorylation. The calmodulin-dependent protein phosphatase (calcineurin) has been suggested to play roles in activation of T cells and in the mating response of yeast. Recently, studies have shown it to be the target of immunosuppressant drugs such as cyclosporin and FK-506. In this study, we have cloned the gene for the catalytic subunit of calcineurin, CnA, from the yeast Schizosaccharomyces pombe. The gene (named ppb1+) has been mapped to chromosome II by analysis of the hybridization of a genomic DNA probe to an ordered library. The gene produces a single mRNA species of 2.5 kilobases, which varies during the cell cycle in exponentially growing cells. In addition, expression of ppb1+ mRA is induced by nitrogen starvation, a condition that favors mating in S. pombe. The ppb1+ gene promoter contains a cis-acting element for the ste11 transcription factor, and we have shown that induction of the ppb1+ mRNA during nitrogen starvation is dependent on the ste11 gene product. Together with earlier studies showing that disruption of the ppb1+ gene in S. pombe results in sterility (Yoshida, T., Toda, T., and Yanagida, M. (1994) J. Cell Sci., 107, 1725-1735), our studies suggest that the ppb1+ gene plays a role in the gene expression cascade that is essential for mating and sporulation in S. pombe.

Two novel genes involved in the sexual development of Schizosaccharomyces pombe.

We isolated two sterile mutants of Schizosaccharomyces pombe. One of them was mapped close to ste13 (8 cM). Since it turned out to be allelic with the hitherto unmapped ral2, its linkage with ste13 localizes ral2 on the right arm of chromosome II. The other mutant defines a novel class-I ste gene, ste15, closely linked to ste7 (4 cM) on chromosome I. ste15 is conjugation-specific and acts upstream of pat1 and ras1. During its genetic analysis, a phenotypic suppression of ste12-N9 was observed which was caused by mutations in the unlinked gene ssw1.

Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes

The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.

Characterization offus1ofSchizosaccharomyces pombe: a Developmentally Controlled Function Needed for Conjugation

In Schizosaccharomyces pombe, the fus1 mutation blocks conjugation at a point after cell contact and agglutination. The cell walls separating the mating partners are not degraded, which prevents cytoplasmic fusion. In order to investigate the molecular mechanism of conjugation, we cloned the fus1 gene and found that it is capable of encoding a 1,372-amino-acid protein with no significant similarities to other known proteins. Expression of the fus1 gene is regulated by the developmental state of the cells. Transcription is induced by nitrogen starvation and requires a pheromone signal in both P and M cell types. Consequently, mutants defective in the pheromone response pathway fail to induce fus1 expression. The ste11 gene, which encodes a transcription factor controlling expression of many genes involved in sexual differentiation, is also required for transcription of fus1. Furthermore, deletion of two potential Ste11 recognition sites in the fus1 promoter region abolished transcription, and expression could be restored when we inserted a different Ste11 site from the mat1-P promoter. Since this element was inverted relative to the fus1 element, we conclude that activation of transcription by Ste11 is independent of orientation. Although the fus1 mutant has a phenotype very similar to that of Saccharomyces cerevisiae fus1 mutants, the two proteins appear to have different roles in the process of cell fusion. Budding yeast Fus1 is a typical membrane protein and contains an SH3 domain. Fission yeast Fus1 has no features of a membrane protein, yet it appears to localize to the projection tip. A characteristic proline-rich potential SH3 binding site may mediate interaction with other proteins.

Lithium in the Mating Response and Cell Cycle of Saccharomyces cerevisiae

Studies with Li+, an inhibitor of phosphoinositide metabolism, demonstrated that an early response of Saccharomyces cerevisiae to a-factor pheromone was negatively affected by this cation. This was monitored by the production of β-galactosidase from a reporter construct containing the promoter region of the yeast STE3 gene and the coding region of the E. coliLacZ gene. Growth and progression through the cell cycle were also affected by Li+ and analysis of budded/unbudded ratios revealed that Li+ caused yeast cells to arrest in G1. These data provide support for the role of inositol phosphates in the mating response and cell cycle of Saccharomyces cerevisiae.

Transcriptional regulation of a Ras nucleotide-exchange factor gene by extracellular signals in fission yeast.

The ste6 gene of Schizosaccharomyces pombe encodes a putative GDP-GTP exchange factor for the ras1 gene product. Genetic analysis of the ste6 and ras1 genes has shown that they are required for mating and for the response to mating pheromones. In this study we show that expression of the ste6-encoded mRNA is induced by nitrogen starvation, the physiological signal that triggers mating and sexual differentiation. Exposure to mating pheromones enhances the induction of ste6 expression upon nitrogen starvation. Pheromone-induced expression requires not only the function of components of the pheromone-signalling pathway, but also ras1 function. Furthermore, mutants in which the Ras1 protein is activated have higher basal and induced levels of ste6 gene expression than wild-type cells. These observations indicate the existence of a positive-feedback loop through which Ras1 stimulates the expression of its own activator. Since Ste6 is likely to promote the exchange of guanine nucleotides on Ras1 protein, our results suggest an important role for GDP-GTP exchange in the regulation of Ras1 activity during the mating process in S. pombe.

Heterologous Expression of the Human D2S Dopamine Receptor in Protease‐Deficient Saccharomyces cerevisiae Strains

The cDNA for the human D2S dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The original D2S gene and an elongated D2S gene with an N-terminal fusion to the first 24 amino acids of the STE2 gene from S. cerevisiae were introduced into the episomal yeast expression vector YEp51 under the control of the GAL10 promoter. Expression studies performed in a wild-type strain and in two protease-deficient strains of S. cerevisiae revealed that the receptor was functionally expressed with respect to its ligand-binding properties. The KD values for the binding of the dopamine antagonist [3H]spiperone were calculated to be 1.6 nM for the D2S receptor alone and 1.9 nM for the STE2-D2S chimaera. Both membrane proteins could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists. D2S dopamine-receptor-specific polyclonal antibodies were used to monitor the heterologous expression of the receptor. Western-blot analysis of membranes prepared from transformed yeast cells producing either the receptor protein alone or the receptor fusion protein revealed apparent molecular masses of 40 kDa (D2S receptor alone) and 42 kDa (STE2/D2S receptor fusion protein). It could be shown that, in comparison to the expression in a wild-type S. cerevisiae strain, the amount of receptor degradation was drastically reduced in the protease-deficient strains. The localizations of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microscopy and were found to be restricted mainly to the vacuole of the cells.