Reaction Order of Saccharomyces cerevisiae Alpha-Factor-Mediated Cell Cycle Arrest and Mating Inhibition

Alpha-factor-mediated cell cycle arrest and mating inhibition of a mating-type cells of Saccharomyces cerevisiae have been examined in liquid cultures. Cell cycle arrest may be monitored unambiguously by the appearance of morphologically abnormal cells after administration of alpha factor, whereas mating inhibition is determined by comparing the mating efficiency in the absence or presence of added alpha factor. For both cell cycle arrest and mating inhibition, a dose-dependent response may be observed at limiting concentrations of the pheromone. If cell cycle arrest and mating inhibition require a small number of alpha-factor molecules, one might expect that responsive/nonresponsive cells = K(alpha factor)(N) where N is the order of dependence of cell cycle arrest (or mating inhibition) on alpha-factor concentration. The value of N has been determined to be 0.98 +/- 0.18 (standard error of the mean) for cell cycle arrest and 1.08 +/- 0.32 for mating inhibition. These results support the notion that saturation of a single site by alpha factor is sufficient to cause cell cycle arrest or mating inhibition of a mating-type cells.     

Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone

Temperature-sensitive mutations that produce insensitivity to division arrest by alpha-factor, a mating pheromone, were isolated in an MATa strain of Saccharomyces cerevisiae and shown by complementation studies to difine eight genes. All of these mutations (designated ste) produce sterility at the restrictive temperature in MATa cells, and mutations in seven of the genes produce sterility in MAT alpha cells. In no case was the sterility associated with these mutations coorectible by including wild-type cells of the same mating type in the mating test nor did nay of the mutants inhibit mating of the wild-type cells; the defect appears to be intrinsic to the cell for mutations in each of the genes. Apparently, none of the mutants is defective exclusively in division arrest by alpha-factor, as the sterility of none is suppressed by a temperature-sensitive cdc 28 mutation (the latter imposes division arrest at the correct cell cycle stage for mating). The mutants were examined for features that are inducible in MATa cells by alpha-factor (agglutinin synthesis as well as division arrest) and for the characteristics that constitutively distinguish MATa from MAT alpha cells (a-factor production, alpha-factor destruction). ste2 Mutants are defective specifically in the two inducible properties, whereas ste4, 5, 7, 8, 9, 11, and 12 mutants are defective, to varying degrees, in constitutive as well as inducible aspects. Mutations in ste8 and 9 assume a polar budding pattern unlike either MATa or MAT alpha cells but characteristic of MATa/alpha cells. This study defines seven genes that function in two cell types (MATa and alpha) to control the differentiation of cell type and one gene, ste2, that functions exclusively in MATa cells to mediate responsiveness to polypeptide hormone.     

Bud formation by the yeast Saccharomyces cerevisiae is directly dependent on "start"

Cells of the yeast Saccharomyces cerevisiae, which bear a cdc4 gene mutation, arrest early in the cell cycle but continue to produce buds in a periodic fashion. We show here that this periodic bud formation by cells already arrested at the CDC4 step is inhibited if the cell cycle regulatory step "start" is also specifically blocked by mutation or by the presence of the yeast mating pheromone alpha-factor. Thus, the characteristic periodic bud formation by cdc4 mutant cells requires the continued ability to perform start. This finding raises questions concerning the nature of start; these issues are discussed.     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.     

Saccharomyces cerevisiae Ste5 is important for induction and substrate specificity of Fus3 MAP kinase in the pheromone signaling pathway

The pheromone pathway is one of the mitogen activated protein kinase (MAPK) signaling pathways identified in Saccharomyces cerevisiae and is involved in both G1 cell cycle arrest and mating of cells. Fus3 functions at a branching point for G1 cell cycle arrest and mating responses in the signaling cascade, and the Fus3 MAPK uses components of both G1 arrest and mating routes as substrates. The Ste5 is a scaffold protein of the MAPK module and is essential for the activation of Fus3. However, it is not known how Ste5 is involved in the specific activation of Fus3 in G1 arrest and mating. In this study, we characterized several G1 arrest defective Ste5 mutants to better understand the roles of Ste5 in the regulation of Fus3. The level of Fus3 increased by treatment with alpha-factor. However, the alpha-factor effects were not readily apparent in the observation of yeast cells containing G1 arrest defective ste5 mutant. This suggests that Ste5 plays an essential role in Fus3 induction. Fus3 immune kinase assay of G1 arrest defective ste5 transformants revealed that Ste5 is important for substrate specificity of Fus3 for G1 arrest and/or mating.     

Mating Pheromones of Saccharomyces kluyveri: Pheromone Interactions Between Saccharomyces kluyveri and Saccharomyces cerevisiae

Saccharomyces kluyveri is a heterothallic yeast with two allelic mating types denoted as a-k and alpha-k by analogy with Saccharomyces cerevisiae and from the work described here. S. kluyveri produces mating pheromones analogous to those of S. cerevisiae, but which appear to have different specificity. S. kluyveri thus differs from S. cerevisiae, Hansenula wingei, and Schizosaccharomyces pombe in that it exhibits both strong constitutive agglutination and mating pheromones. alpha-k cells produce a pheromone ("alpha-k-factor") which causes a-k cells to arrest in the G1 phase of the cell cycle and to undergo a morphological change. After a period of time dependent on the concentration of alpha-k-factor, cells exposed to the factor resume cell division. alpha-k-factor has no effect on a-k/alpha-k diploids or on alpha-k cells, but at high concentration does induce G1 arrest of S. cerevisiaea cells (a-c). a-k cells produce a pheromone ("a-k-factor") which causes alpha-k cells to exhibit a morphological change. In addition, a-k cells exhibit the Bar phenotype with respect to alpha-k-factor. Partially purified preparations of S. cerevisiae alpha-factor are more active in inducing G1 arrest of a-k cells than of a-c cells. A more purified preparation of alpha-c-factor is less active against a-k cells than a-c cells, suggesting that an additional factor (KRE, kluyveri response enhancer) may be lost during purification. Attempts to mate S. kluyveri and S. cerevisiae cells by prototroph selection and by cell-to-cell mating have been unsuccessful with all combinations of mating types. Thus, S. cerevisiae and S. kluyveri are incompatible for mating even though their pheromones exhibit some physiological cross-reaction.     

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.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and alpha-factor. Partially purified fractions exhibiting a-factor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both G1 arrest and morphological alterations in cells of alpha-mating type, whereas a cells, a/alpha diploids, and nonmating alpha mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of alpha or a/alpha cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight greater than 600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for alpha-factor, the chemical properties of these two hormones appear to be quite different.     

Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the alpha mating pheromone, alpha-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BAR1, mat alpha 2 BAR1, and mat alpha 1 mat alpha 2 BAR1 strains, but not by MAT alpha BAR1, MATa/MAT alpha BAR1, mat alpha 1 BAR1, or MATa barl strains, demonstrating that it is under the control of both the MAT alpha 2 and the BAR1 genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to alpha-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for alpha-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The alpha-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for alpha-factor was found to be relatively insensitive to pH over a range of several units, the alpha-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BAR1 gene product in recovery of mating-type a cells from cell division arrest by alpha-factor.     

Yeast alpha-factor and somatostatin enhance binding of [3H]estradiol to proteins in rat pancreas and Saccharomyces cerevisiae

Pancreatic tissue contains an [3H]estradiol-binding protein that requires a coligand in the steroid-binding reaction. The endogenous coligand appears to be the tetradecapeptide somatostatin. Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of [3H]estradiol to partially purified pancreatic protein. Supernatant fractions from yeast cells also contain an [3H]estradiol-binding protein. alpha-Factor can enhance specific binding of [3H]estradiol to such yeast fractions. Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of [3H]estradiol but did not inhibit cell growth or induce morphological changes in S. cerevisiae. Thus, it appears that coligand-requiring [3H]estradiol-binding activity and mating in yeast are not directly related.     

The immunosuppressive drug leflunomide affects mating-pheromone response and sporulation by different mechanisms in Saccharomyces cerevisiae

Leflunomide (LFM) is a novel anti-inflammatory and immunosuppressive drug, and inhibits the growth of cytokine-stimulated lymphoid cells in vitro. The effect of LFM on haploid and diploid cells of Saccharomyces cerevisiae was investigated to elucidate the molecular mechanism of action of the drug. Using a halo assay, LFM was shown to enhance the cell cycle arrest of haploid cells induced by mating pheromone alpha-factor. LFM also inhibited sporulation of diploid cells completely. S. cerevisiae genes which were cloned to suppress the anti-proliferative effect when present in increased copy number were introduced and examined for their activity to suppress the effect of LFM. Out of them, MLF4/SSH4, was found to suppress the sporulation-inhibitory effect of LFM. However, MLF4 failed to suppress the enhancing effect of LFM on pheromone response. Thus, LFM is suggested to act on haploid and diploid cells by different mechanisms.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

Characterization of novel peptide agonists of the alpha mating factor of Saccharomyces cerevisiae.

Alpha-factor [WHWLQLKPGQPMY], a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae (MATa, MATalpha). New analogues of alpha-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies. The Y(0)Nle(12)F(13) analogue [YWHWLQLKPGQPNleF] (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone. Binding of (125)I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native alpha-mating factor. Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (K(d) = 6.4 x 10(-8)). Specific binding of (125)I-labeled MFN5 was significantly reduced by guanosine nucleotides. Affinity cross-linking of (125)I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein. The novel synthetic alpha-factor analogue MFN5 can be easily iodinated and used as a probe for the alpha-factor receptor.     

Induction of yeast histone genes by stimulation of stationary-phase cells.

In the cell cycle of the budding yeast Saccharomyces cerevisiae, expression of the histone genes H2A and H2B of the TRT1 and TRT2 loci is regulated by the performance of "start," the step that also regulates the cell cycle. Here we show that histone production is also subject to an additional form of regulation that is unrelated to the mitotic cell cycle. Expression of histone genes, as assessed by Northern (RNA) analysis, was shown to increase promptly after the stimulation, brought about by fresh medium, that activates stationary-phase cells to reenter the mitotic cell cycle. The use of a yeast mutant that is conditionally blocked in the resumption of proliferation at a step that is not part of the mitotic cell cycle (M.A. Drebot, G.C. Johnston, and R.A. Singer, Proc. Natl. Acad. Sci. 84:7948, 1987) showed that this increased gene expression that occurs upon stimulation of stationary-phase cells took place in the absence of DNA synthesis and without the performance of start. This stimulation-specific gene expression was blocked by the mating pheromone alpha-factor, indicating that alpha-factor directly inhibits expression of these histone genes, independently of start.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product

The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.