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.     

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.     

Saccharomyces cerevisiae alpha-factor prevents formation of glycoproteins in a cells

alpha-Factor inhibits incorporation of [14C]glucosamine into water-soluble and into SDS-extractable glycoproteins to about 90%. The incorporation into chitin is not affected and the same is true for [14C]phenylalanine incorporation into protein. The inhibition of glycoprotein synthesis is specific for a cells.     

Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist.

Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.     

Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells.

Saccharomyces cerevisiae mating type a cells enlarged and elongated when exposed to alpha-factor, a sex pheromone produced by mating-type alpha cells. This morphogensis required exogenous-D-glucose, nitrogen, and phosphate, and cells in exponential phase responded better than stationary-phase cells. Morphogenesis was blocked by cycloheximide and by inhibitors of cell wall biosynthesis such as 2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-deoxy-2-fluoro-D-mannose, but not by polyoxin D. One to two hours after addition of pheromone, a cells became more susceptible to lysis by glucanases, a change that was dependendent on the dose of alpha-factor and was blocked by drugs that block morphogenesis. On the other hand, treatment with alpha-factor did not increase susceptibility to attack by trypsin, subtilisin, or exo-alpha-mannanase. Radioactive label, incorporated into cell wall polysaccharides during treatment with alpha-factor, was not secreted into the medium during morphogenesis. Analysis of the labeled wall polymers showed that alpha-factor-treated cells contain more glucan and less mannan than control cells, and that the mannan of treated cells contains an increased proportion of shorter side chains and unsubstituted backbone mannose units. Thin-section electron microscopy of treated cells revealed that the cell wall possesses a diffuse outer layer in the extension and is thinner at the tip.     

Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.     

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.     

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae

alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.     

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.     

Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.     

The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae

The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for alpha-factor in MATa haploids. Methods were devised to prepare membrane fractions from MATa cells that retained high levels of alpha-factor binding activity, consistent with the view that the alpha-factor receptor resides in the plasma membrane. To demonstrate that the membrane constituent responsible for alpha-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis. Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000. Affinity labeling of yeast membrane preparations by chemical cross-linking to 35S-alpha-factor indicated that a molecule of 49,000 molecular weight was the major alpha-factor-binding species. This alpha-factor-binding species was shown to be the product of the STE2 gene in three ways. First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced alpha-factor binding activity about 15-fold. Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of alpha-factor (equivalent to the level displayed by MAT alpha haploids) and possessed no species that could be cross-linked to 35S-alpha-factor. Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to 35S-alpha-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide. These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast alpha-factor receptor.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

Agglutination and mating activity of the MF alpha 2-encoded alpha-factor analog in Saccharomyces cerevisiae.

The MF alpha 2-encoded Asn-5,Arg-7 alpha-factor-like peptide has been shown shown to have similar activity to Gln-5,Lys-7 alpha-factor in morphogenesis and growth arrest studies (S. Raths, P. Shenbagamurthi, F. Naider, and J. M. Becker, J. Bacteriol. 168:1468-1471, 1986). We tested the Asn-5,Arg-7 peptide in agglutination and mating assays and found that its activity was similar to or slightly less than that of the Gln-5,Lys-7 alpha-factor. The Asn-5,Arg-7 alpha-factor-like peptide is thus the most active analog of the Gln-5,Lys-7 alpha-factor known.     

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.     

Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating.

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.     

The alpha-factor receptor C-terminus is important for mating projection formation and orientation in Saccharomyces cerevisiae

Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p, the alpha-factor receptor. Besides receiving the pheromone signal and transducing it through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishment and orientation of the mating projection. We investigated the role of the carboxyl terminus of the receptor in mating projection formation and orientation using a spatial gradient assay. Cells carrying the ste2-T326 mutation, truncating 105 of the 135 amino acids in the receptor tail including a motif necessary for its ligand-mediated internalization, display slow onset of projection formation, abnormal shmoo morphology, and reduced ability to orient the mating projection toward a pheromone source. This reduction was due to the increased loss of mating projection orientation in a pheromone gradient. Cells with a mutated endocytosis motif were defective in reorientation in a pheromone gradient. ste2-Delta296 cells, which carry a complete truncation of the Ste2p tail, exhibit a severe defect in projection formation, and those projections that do form are unable to orient in a pheromone gradient. These results suggest a complex role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional adjustment of the mating projection, and that endocytosis of the receptor is important for this process. In addition, mutations in RSR1/BUD1 and SPA2, genes necessary for budding polarity, exhibited little or no defect in formation or orientation of mating projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in budding.     

Induction of sexual cell agglutinability of A mating type cells by alpha-factor in Saccharomyces cerevisiae.

Mating hormone, α-factor, which inhibits DNA synthesis and causes characteristic changes in cell morphology in $\text{a}$ mating type cells, was also responsible for induction of sexual cell agglutinability of $\text{a}$ mating type cells.