Mating-Type Regulation of Methyl Methanesulfonate Sensitivity in SACCHAROMYCES CEREVISIAE

Heterozygosity at the mating-type locus (MAT) in Saccharomyces cerevisiae has been shown previously to enhance X-ray survival in diploid cells. We now show that a/α diploids are also more resistant to the radiomimetic agent methyl methanesulfonate (MMS) than are diploids that are homozygous at MAT (i.e., either a/a or α/α). Log-phase a/α cultures exhibit biphasic MMS survival curves, in which the more resistant fraction consists of budded cells (those cells in the S and G2 phases of the cell cycle). Survival curves for log-phase cultures of a/a or α/α diploids have little if any biphasic nature, suggesting that the enhanced S- and G2-phase repair capacity of a/α cells may be associated with heterozygosity at MAT. The survival of cells arrested at the beginning of the S phase with hydroxyurea indicates that MAT-dependent MMS repair is limited to S and G2, whereas MAT-independent repair can occur in G1.     

Dependence on Mating Type for the Overproduction of Iso-2-Cytochrome c in the Yeast Mutant CYC7–H2

The CYC7–H2 mutation causes an approximately 20-fold overproduction of iso–2–cytochromo c in a and α haploid strains of the yeast Saccharomyces cerevisiae due to an alteration in the nontranslated regulatory region that is presumably contiguous with the structural region. In this investigation, we demonstrated that heterozygosity at the mating type locus, a/α or a/a/α/α, prevents expression of the overproduction, while homozygosity, a/a and α/α, and hemizygosity, a/0 and α/0, allow full expression of the CYC7–H2 mutation, equivalent to the expression observed in a and α haploid strains. There is no decrease in the overproduction of iso-2-cytochrome c in a/α diploid strains containing either of the other two similar mutations, CYC7–H1 and CYC7–H3. It appears as if active expression of one or another of the mating-type alleles is required for the overproduction of iso-2-cytochrome c in CYC7–H2 mutants.     

A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Interconversion of Yeast Cell Types by Transposable Genes

The a and α cell types of budding yeast Saccharomyces cerevisiae are controlled by alternate alleles of the mating-type locus (MAT), MATa and MATα. The cell types can be interconverted by switching alleles of MAT. The loci HMRa and HMLα, which are loosely linked to MAT, are involved in mating-type switching. Experimental evidence for their role in MAT interconversion is presented. As a result of switching, the homothallic and heterothallic strains containing the amber and ochre mutations within the HMRa locus yield corresponding amber and ochre mutant mata loci. Similarly, the hmlα mutant strain generates matα mutant alleles. That is, specific mutations from HMRa and HMLα are transmitted to MAT. A replica of the mating-type coding information originating from these loci is transposed to MAT, where it replaces the existing information. Furthermore, "Hawthorne deletions" in strains containing hmra-amber/ochre result in production of mata-amber/ochre alleles. Therefore, genetic information for MATa resides at HMRa. The switches occur in a defined set of clonally related cells. Thus, the efficient interconversion of yeast cell types is mediated by an unidirectional transfer of genetic information between nonallelic sites in a nonrandom and programmed fashion. The results are inconsistent with the "flip-flop" models, but satisfy a key prediction of the general controlling element and the specific cassette models proposed for mating-type interchange.     

Interconversion of Yeast Mating Types III. Action of the Homothallism (HO) Gene in Cells Homozygous for the Mating Type Locus

Mating type interconversion in homothallic Saccharomyces cerevisiae has been studied in diploids homozygous for the mating type locus produced by sporulation of a/a/a/α and a/a/α/α tetraploid strains. Mating type switches have been analyzed by techniques including direct observation of cells for changes in α-factor sensitivity. Another method of following mating type switching exploits the observation that a/α cells exhibit polar budding and a/a and α/α cells exhibit medial budding.—These studies indicate the following: (1) The allele conferring the homothallic life cycle (HO) is dominant to the allele conferring the heterothallic life cycle (ho). (2) The action of the HO gene is controlled by the mating type locus—active in a/a and α/α cells but not in a/α cells. (3) The HO (or HO-controlled) gene product can act independently on two mating type alleles located on separate chromosomes in the same nucleus. (4) A switch in mating type is observed in pairs of cells, each of which has the same change.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and α-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MATa-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH₂-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH₂-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases.     

Evidence for Co-Dominance of the Homothallic Genes, HMalpha/hmalpha and HMa/hma, IN SACCHAROMYCES YEASTS

To investigate the dominance and recessiveness of the homothallism genes, HMα/hmα and HMa/hma, for mating-type conversion, we constructed hybrids with various configurations of the homothallic genes by fusion of protoplasts prepared from haploid strains having identical mating types. Eight different combinations of the homothallic genes were tested for their function by observing the mating and sporulation abilities of the fusion products. With few exceptions, nonmating and sporogenous fusion products were obtained from the following combinations: α HO hmα HMa + α ho hmα hma, α HO hmα HMa + α ho HMα hma, α HO hmα HMa + α ho HMα HMa, a HO HMα hma + a ho hmα hma, a HO HMα hma + a ho hmα HMa and a HO HMα hma + a ho HMα HMa. All the fusion products from the α HO hmα HMa + α ho hmα HMa and a HO HMα hma + a ho HMα hma combinations showed mating types identical to those of the respective haploid strains. These results clearly support the co-dominance of the HMα/hmα and HMa/hma alleles and indicate that the hmα allele has the same function as the HMa allele and that the hma allele has the same function as the HMα allele.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Pathways of ultraviolet mutability in Saccharomyces cerevisiae. III. Genetic analysis and properties of mutants resitant to ultraviolet-induced forward mutation.

Non-allelic mutants of Saccharomyces cerevisiae with reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were assigned to seven distinct genetic loci, each with allele designations umr1-1, umr2-1, …, umr7-1 to indicate UV mutation resistance. Each allele complemented rev1-1, rev2-1, and rev3-1. None conferred a great deal of UV sensitivity. When assayed on yeast extract-peptone-dextrose complex growth agar, umr1, umr3, and umr7 (a mating type) were the most UV-sensitive, with a dose-reduction factor of approximately 1.2 at 10% survival. When assayed on synthetic agar lacking arginine, however, umr3 was the most UV-sensitive (dose-reduction factor of 1.5 at 10% survival). UV revertability of his5-2, lys1-1, and ura4-1 was normal in strains carrying the single genes umr4, umr5, umr6 and umr7; umr1 reduced revertibility of his5-2 and ura4-1 but not lys1-1; umr2 reduced only ura4-1 revertibility; umr3 reduced UV reversion of all three test alleles. Five a/α homozygous umr diploids (except umr1 and umr4) failed to sporulate. One of these, umr7, blocked normal secretion of alpha hormone in α segregants and could not conjugate with a strains. The phenotypes of umr mutants are consistent with the existence of branched UV mutation pathways of different specificity, some of which may function in the single RAD6-dependent error-prone pathway for repair of UV damage. Other possible pathways of action are discussed. It is also suggested that regulatory functions interacting with the mating-type locus or its gene products may play some role in UV mutagenesis or error-prone repair.     

Receptor Inhibition of Pheromone Signaling Is Mediated by the Ste4p Gβ Subunit

The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of α-factor to the α-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the β-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its G_α binding surface. A STE4 allele containing several of these mutations, called STE4^SD13, reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4^SD13 were indistinguishable from those of STE4 in wild-type MATa and MATα cells. These results demonstrate that the effect of the STE4^SD13 allele is specific to the receptor inhibition function of STE4. STE4^SD13 suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein α-subunit gene; however, STE4^SD13 had no effect in a MATα strain containing a GPA1 deletion. Suppression of receptor inhibition by STE4^SD13 in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the α-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.     

Evidence of the Insensitivity of the alpha-inc Allele to the Function of the Homothallic Genes in Saccharomyces Yeasts

The possible function of the α-inc allele (an α mating-type allele that is insensitive to the function of the homothallic gene system) was investigated by means of protoplast fusion. The fusion of protoplasts prepared from haploid strains of α-inc HO HMα HMa and α ho hmα HMa gave rise mainly to nonmating clones (58 of 64 isolates) and a few clones (six of 64 isolates) showing α mating type. Thirty of the 58 nonmating clones showed the diploid cell size and 28 clones had a larger cell size. Tetrad analysis of the nonmating clones with diploid cell size indicated that they were a/α-inc diploid; the normal α allele in α/α-inc cells was preferentially switched to an a allele. This observation further indicated that the HO/ho HMα/hmα HMa/HMa genotype is effective for the conversion of the α to a and that the inconvertibility of the α-inc allele is due to the insensitivity of the mating-type allele to the functional combination of the homothallic genes. It was suspected that fusion products larger than diploid cells might have been caused by multiple fusion of protoplasts.     

Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm

Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate.