Inhibition of membrane Ca2(+)-ATPase of Saccharomyces cerevisiae by mating pheromone alpha-factor in vitro.

Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells.     

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.     

Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").     

MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein,is required for Ca2+ influx and mating.

By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating.     

Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating.

During conjugation in Saccharomyces cerevisiae, two cells of opposite mating type (MATa and MAT alpha) fuse to form a diploid zygote. Conjugation requires that each cell locate an appropriate mating partner. To investigate how yeast cells select a mating partner, we developed a competition mating assay in which wild-type MAT alpha cells have a choice of two MATa cell mating partners. We first demonstrated that sterile MAT alpha 1 cells (expressing no a- or alpha-specific gene products) do not compete with fertile MATa cells in the assay; hence, wild-type MATa and MAT alpha cells can efficiently locate an appropriate mating partner. Second, we showed that a MATa strain need not be fertile to compete with a fertile MATa strain in the assay. This result defines an early step in conjugation, which we term courtship. We showed that the ability to agglutinate is not necessary in MATa cells for courtship but that production of a-pheromone and response to alpha-pheromone are necessary. Thus, MATa cells must not only transmit but must also receive and then respond to information for effective courtship; hence, there is a "conversation" between the courting cells. We showed that the only alpha-pheromone-induced response necessary in MATa cells for courtship is production of a-pheromone. In all cases tested, a strain producing a higher level of a-pheromone was more proficient in courtship than one producing a lower level. We propose that during courtship, a MAT alpha cell selects the adjacent MATa cell producing the highest level of a-pheromone.     

Characterization of the energy-dependent, mating factor-activated Ca influx in Saccharomyces cerevisiae

The yeast mating pheromones, a and alpha factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early G1 phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [1]. We have characterized the alpha factor induced increase in cellular Ca2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the G0-G1 border at the nonpermissive temperature. We observed a 2-4 fold increase in the initial velocity of Ca2+ influx in alpha factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited by membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca2+ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.     

Analysis of Y-Linked Mutations to Male Sterility in DROSOPHILA MELANOGASTER

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is determined by a pair of alleles MATa and MAT alpha. Under various conditions haploid mating types can be interconverted. It has been proposed that transpositions of silent cassettes of mating-type information from HML OR HMR to MAT are the source of mating type conversions. A mutation described in this work, designated AON1, has the following properties. (1) MAT alpha cells carring AON1 are defective in mating. (2) AON1 allows MAT alpha/MAT alpha but not MATa/MATa diploids to sporulate; thus, AON1 mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AON1 are MATa phenocopies, i.e., MAT alpha/mata-1 AON1 diploids behave as standard MAT alpha/MATa cells; therefore, AON1 suppresses the defect of mata-1. (4) AON1 maps at or near HMRa. (5) Same-site revertants from AON1 lose the ability to convert mating type to MATa, indicating that reversion is associated with the loss of a functional HMRa locus. In addition, AON1 is a dominant mutation. We conclude that AON1 is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.     

Comparison of dose-response curves for alpha factor-induced cell division arrest, agglutination, and projection formation of yeast cells. Implication for the mechanism of alpha factor action

MAT alpha cells of the yeast Saccharomyces cerevisiae produce a polypeptide mating pheromone, alpha factor. MATa cells respond to the pheromone by undergoing several inducible responses: the arrest of cell division, the production of a cell surface agglutinin, and the formation of one or more projections on the cell surface commonly termed the "shmoo" morphology. Dose-response curves were determined for each of these inducible responses as a function of alpha factor concentration. It is shown that under conditions commonly employed in previous studies, the dose-response for cell division arrest is determined by the rate at which cells inactivate the alpha factor. In order to achieve conditions where inactivation would not be the dominant parameter, the cell division response to alpha factor was monitored at low cell densities. Under conditions of essentially no alpha factor destruction, the dose of alpha factor at which cells exhibit a half-maximal response for cell division arrest (2.5 X 10(-10) M) is nearly the same as that at which cells exhibit a half-maximal response for agglutination induction (1.0 X 10(-10) M). On the contrary, the half-maximal response for projection formation was obtained at doses of alpha factor 2 orders of magnitude higher (1.4 X 10(-8) M). These results are consistent with the same high affinity alpha factor receptor mediating both cell division arrest and agglutination induction. A different system of lower affinity must mediate projection formation. Alternatively, if the same system and receptor are used, then a much higher occupancy is required for the induction of projections compared to division arrest and agglutination induction.     

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.     

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.     

Switching of a Mating-Type a Mutant Allele in Budding Yeast SACCHAROMYCES CEREVISIAE

Aimed at investigating the recovery of a specific mutant allele of the mating type locus (MAT) by switching a defective MAT allele, these experiments provide information bearing on several models proposed for MAT interconversion in bakers yeast, Saccharomyces cerevisiae. Hybrids between heterothallic (ho) cells carrying a mutant MAT a allele, designated mata-2, and MAT alpha ho strains show a high capacity for mating with MATa strains. The MAT alpha/mata-2 diploids do not sporulate. However, zygotic clones obtained by mating MAT alpha homothallic (HO) cells with mata-2 ho cells are unable to mate and can sporulate. Tetrad analysis of such clones revealed two diploid (MAT alpha/MATa):two haploid segregants. Therefore, MAT switches occur in MAT alpha/mata-2 HO/ho cells to produce MAT alpha/Mata cells capable of sporulation. In heterothallic strains, the mata-2 allele can be switched to a functional MAT alpha and subsequently to a functional MATa. Among 32 MAT alpha to MATa switches tested, where the MAT alpha was previously derived from the mata-2 mutant, only one mata-2 like isolate was observed. However, the recovered allele, unlike the parental allele, complements the matalpha ste1-5 mutant, suggesting that these alleles are not identical and that the recovered allele presumably arose as a mutation of the Mat alpha locus. No mata-2 was recovered by HO-mediated switching of MAT alpha (previously obtained from mata-2 by HO) in 217 switches analyzed. We conclude that in homothallic and heterothallic strains, the mata-2 allele can be readily switched to a functional MAT alpha and subsequently to a functional MATa locus. Overall, the results are in accord with the cassette model (HICKS, STRATHERN and HERSKOWITZ )977b) proposed to explain MAT interconversions.     

Single alpha-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger

Spliced isoforms of the Na+/Ca2+ exchanger, NCLX, truncated at the alpha-repeat region have been identified. The activity and functional organization of such proteins are, however, poorly understood. In the present work, we have studied Na+/Ca2+ exchange mediated by single alpha-repeat constructs (alpha1 and alpha2) of NCLX. Sodium-dependent calcium transport was fluorescently detected in both the reversal and forward modes; calcium-dependent outward currents were also recorded using a whole cell patch configuration in HEK293 cells heterologously expressing either the alpha1 or alpha2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an alpha2 mutant (alpha2-S273T). Thus, our data indicate that the single alpha-domain constructs mediate electrogenic Na+/Ca2+ exchange. The alpha1 domain, but not the alpha2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li+-dependent Ca2+ efflux was detected in cells expressing either the alpha1 or alpha2 construct. The functional organization of the single alpha-domain constructs was assessed using a dominant-negative approach. Coexpression of the alpha1 or alpha2 constructs with the nonfunctional alpha2-S273T mutant had a synergistic inhibitory effect on Na+/Ca2+ transport. Dose-dependence analysis of the inhibition of alpha2 construct activity by the alpha2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the alpha2 construct indeed interacts with the alpha2-S273T mutant. Taken together, our data indicate that although single alpha1 or alpha2 domain constructs are independently capable of Na+/Ca2+ exchange, oligomerization is required for their activity. Such organization may give rise to transport activity with distinct kinetic parameters and physiological roles.     

Nimodipine inhibits calcium-independent nitric oxide synthase activity in transient focal cerebral ischemia rats and cultured mouse astroglial cells

The effect of nimodipine on nitric oxide synthase (NOS) activities in brains in transient focal cerebral ischemia rats, in cultured mouse neurons and astroglial cells and bovine brain capillary endothelial cells (BCECs) was investigated. The administration of nimodipine (3 mg.kg(-1), p.o., twice a day, for 3 days) before middle cerebral artery (MCA) occlusion significantly reduced infarct size, decreased nitrite/nitrate (NOx) content and inhibited Ca2+-independent NOS activity in the infarct area. Nimodipine inhibited the Ca2+-independent NOS activity induced by lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF alpha) in mouse astroglial cells with an IC50 value of 0.036+/-0.003 mM and Ca2+-dependent NOS activity in mouse neurons with an IC50 value of 0.047+/-0.003 mM, but did not affect Ca2+-dependent NOS activity in BCECs. The inhibition of Ca2+-independent NOS activity by nimodipine in astroglial cells was competitive with respect to L-arginine. Nimodipine also inhibited the induction of Ca2+-independent NOS activity in vitro. These results suggest that nimodipine in addition to its cerebral vasodilating effect may protect brain from ischemic neuronal damage through modifying NOS activity.