MATa Cells Research Articles

Purification, Functional Reconstitution, and Characterization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p

Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and alpha-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-beta-d-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure.

TUP1 disruption reveals biological differences between MATa and MATα strains of Cryptococcus neoformans

Cryptococcus neoformans exists in two mating types MATa and MATalpha. Although the morphology, growth characteristics and genetic segregation patterns among MATa and MATalpha strains are indistinguishable in the laboratory, the predominance of MATalpha strains in nature suggests that MATalpha strains are better suited for survival in nature. We disrupted the TUP1 gene, a global repressor, to find the possible biological differences in congenic MATalpha and MATa cells of C. neoformans. Disruption of TUP1 affected neither the yeast nor the hyphal cell morphology but resulted in a similar reduction of mating frequencies in both MATalpha and MATa cells. Disruption of TUP1, however, functionally manifested itself in several mating type-dependent phenotypes: (i) MATalpha cells became more sensitive to 0.8 M KCl while MATa cells showed no change in sensitivity, (ii) a temperature-dependent growth reduction was exhibited at both 30 degrees C and 25 degrees C in MATa but a similar growth reduction was not observed in MATalpha cells until the temperature was lowered to 25 degrees C and (iii) the transcriptional level of genes in several different biological pathways was markedly altered in a mating type-dependent manner. This work is the first case in which non-mating-related biological differences are observed between two congenic mating partners in yeast.

The Saccharomyces cerevisiae TRT2 tRNAThr gene upstream of STE6 is a barrier to repression in MATalpha cells and exerts a potential tRNA position effect in MATa cells.

A growing body of evidence suggests that genes transcribed by RNA polymerase III exhibit multiple functions within a chromosome. While the predominant function of these genes is the synthesis of RNA molecules, certain RNA polymerase III genes also function as genomic landmarks. Transfer RNA genes are known to exhibit extra-transcriptional activities such as directing Ty element integration, pausing of replication forks, overriding nucleosome positioning sequences, repressing neighboring genes (tRNA position effect), and acting as a barrier to the spread of repressive chromatin. This study was designed to identify other tRNA loci that may act as barriers to chromatin-mediated repression, and focused on TRT2, a tRNA(Thr) adjacent to the STE6 alpha2 operator. We show that TRT2 acts as a barrier to repression, protecting the upstream CBT1 gene from the influence of the STE6 alpha2 operator in MATalpha cells. Interestingly, deletion of TRT2 results in an increase in CBT1 mRNA levels in MATa cells, indicating a potential tRNA position effect. The transcription of TRT2 itself is unaffected by the presence of the alpha2 operator, suggesting a hierarchy that favors assembly of the RNA polymerase III complex versus assembly of adjacent alpha2 operator-mediated repressed chromatin structures. This proposed hierarchy could explain how tRNA genes function as barriers to the propagation of repressive chromatin.

Uniqueness of the mating system in Cryptococcus neoformans

Although the mating system of Cryptococcus neoformans shares many physiological and genetic characteristics with other fungi, there are an increasing number of features that make it unique. The sexual state of C. neoformans is distinct from other basidiomycetes. The mating-type loci contain several mating-type-specific pheromone response pathway genes, including some that function differently in the two mating types. There are also observations of uniparental mitochondrial inheritance from the MATa mating type and unidirectional nuclear migration from MATα to MATa cells. These factors lead us to postulate that MATα and MATa cells play disparate roles during mating. Furthermore, we surmise that the inherent genetic differences between the two mating types render MATα mating-type strains more fit for survival. This might explain the predominance of MATα mating types both in nature and in clinical isolates.

Switching mating types with one arm tied

![Graphic][1] HML is less constrained in MATa cells (top) than in MATα cells (bottom).The mating type (MAT) locus on Saccharomyces cerevisiae chromosome III can recombine with the HMRa locus near the right telomere or the HMLα locus near the left telomere of the same chromosome. On

Mating type-dependent constraints on the mobility of the left arm of yeast chromosome III.

Mating-type gene (MAT) switching in budding yeast exhibits donor preference. MAT a preferentially recombines with HML near the left telomere of chromosome III, whereas MATα prefers HMR near the right telomere. Donor preference is controlled by the recombination enhancer (RE) located proximal to HML. To test if HML is constrained in pairing with MATα, we examined live-cell mobility of LacI-GFP–bound lactose operator (lacO) arrays inserted at different chromosomal sites. Without induction of recombination, lacO sequences adjacent to HML are strongly constrained in both MATα and RE-deleted MAT a strains, compared with MAT a. In contrast, chromosome movement at HMR or near a telomere of chromosome V is mating-type independent. HML is more constrained in MAT a Δre and less constrained in MAT a RE+ compared with other sites. Although HML and MAT a are not prealigned before inducing recombination, the three-dimensional configuration of MAT, HML, and HMR is mating-type dependent. These data suggest there is constitutive tethering of HML, which is relieved in MAT a cells through the action of RE.

Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p.

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall beta-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the alpha-agglutinin Agalpha1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATalpha cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

Haploid fruiting in Cryptococcus neoformans is not mating type α-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATα strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATα-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.

Roles of sexual cell agglutination in yeast mass mating.

The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia.

Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer.

Saccharomyces mating-type switching results from replacement by gene conversion of the MAT locus with sequences copied from one of two unexpressed donor loci, HML or HMR. MATa cells recombine with HMLalpha approximately 90% of the time, whereas MATalpha cells choose HMRa 80%-90% of the time. HML preference in MATa is controlled by the cis-acting recombination enhancer (RE) that regulates recombination along the entire left arm of chromosome III. Comparison of RE sequences between S. cerevisiae, S. carlsbergensis, and S. bayanus defines four highly conserved regions (A, B, C, and D) within a 270-bp minimum RE. An adjacent E region enhances RE activity. Multimers of region A, D, or E are sufficient to promote selective use of HML. Regions A, D, and E each bind in vivo the transcription activator forkhead proteins Fkh1p and Fkh2p and their associated Ndd1p, although there are no adjacent open reading frames (ORFs). Deletion of FKH1 significantly reduces MATa's use of HML, as does mutation of the Fkh1/Fkh2-binding sites in a multimer of region A. We conclude that Fkh1p regulates MATa donor preference through direct interaction with RE.

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.

The role of mating type and morphology in Cryptococcus neoformans pathogenesis

AbstractCryptococcus neoformans is a major fungal pathogen of both humans and animals. The fungus can be divided into two varieties, with each variety being composed of two serotypes. A sexual phase has been identified, which classifies C. neoformans as a bipolar heterothallic fungus with two mating types, MATa and MATα. The analysis of mating and mating type in this organism is important for a number of reasons. Both clinical and environmental isolates display a severe bias of the MATα mating type over MATa. MATα cells are also more virulent than MATα cells. Molecular and genetic analyses of the genes that make up the mating pathway have revealed that some of these genes are required for virulence. Finally, although it is well known that infection begins in the lungs after inhalation of infectious particles, it still remains unclear what constitutes the infectious particle. This review will discuss current information about what is known about the role that mating type and morphology play in virulence.

Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving mating between haploid MATa and MATalpha cells. Here we describe the isolation of part of the MATa mating-type locus encoding a Ste20 kinase homolog, Ste20a. We show that the STE20a gene cosegregates with the MATa mating type in genetic crosses, maps within the mating-type locus on a 1.8-Mb chromosome, and is allelic with the MATalpha locus. We identify the first MATa isolate of the most common pathogenic variety of C. neoformans (serotype A, variety grubii) which had been thought to be extinct. This serotype A MATa strain is sterile, fails to produce mating pheromone, and is less virulent than a serotype A MATalpha strain in an animal model. Our studies illustrate an association of mating type with virulence and suggest that, like Candida albicans, pathogenic isolates of C. neoformans may be largely asexual.

Characterization of the MFalpha pheromone of the human fungal pathogen cryptococcus neoformans.

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well-developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATalpha. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATalpha locus was cloned, and a presumptive pheromone gene, MFalpha1, was identified by its ability to induce conjugation tube-like filaments when introduced by transformation into MATa cells. Here, the ability of the MFalpha1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure-function analysis of the gene. We show that the MFalpha1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C-terminal CAAX motif is required for activity of the MFalpha1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFalpha1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFalpha1 expression. We identify a second pheromone gene, MFalpha2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFalpha1 pheromone and show that only the predicted mature modified form of the alpha-factor peptide triggers morphological responses in MATa cells.

A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor.

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)ălpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.

A STE12 homolog is required for mating but dispensable for filamentation in candida lusitaniae.

Candida lusitaniae is a dimorphic yeast that is emerging as an opportunistic fungal pathogen. In contrast to Candida albicans, which is diploid and asexual, C. lusitaniae has been reported to have a sexual cycle. We have employed genetic approaches to demonstrate that C. lusitaniae is haploid and has a sexual cycle involving mating between MATa and MATalpha cells under nutrient deprivation conditions. By degenerate PCR, we identified a C. lusitaniae homolog (Cls12) of the Ste12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae, C. albicans, and Cryptococcus neoformans. Comparison of the CLS12 DNA and protein sequences to other STE12 homologs and transformation experiments with selectable markers from S. cerevisiae (URA3, KanMX, HphMX) and C. albicans (CaURA3) provide evidence that the CUG codon encodes serine instead of leucine in C. lusitaniae, as is also the case in C. albicans. The C. lusitaniae CLS12 gene was disrupted by biolistic transformation and homologous recombination. C. lusitaniae cls12 mutant strains were sterile but had no defect in filamentous growth. Our findings reveal both conserved and divergent roles for the C. lusitaniae STE12 homolog in regulating differentiation of this emerging fungal pathogen.

Ca2+ signal is generated only once in the mating pheromone response pathway in Saccharomyces cerevisiae.

The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating. In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling. Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response. Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration. In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone. These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway. Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway [Iida et al., (1990) J. Biol. Chem., 265: 13391-13399] Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.

Characterization of Novel Peptide Agonists of the α Mating Factor of Saccharomyces cerevisiae

α-Factor [WHWLQLKPGQPMY], a secreted tridecapeptide pheromone, is required for mating between the a- and α-haploid mating types of Saccharomyces cerevisiae (MATa, MATα). New analogues of α-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies. The Y0Nle12F13 analogue [YWHWLQLKPGQPNleF] (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone. Binding of 125I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native α-mating factor. Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (Kd = 6.4 × 10−8). Specific binding of 125I-labeled MFN5 was significantly reduced by guanosine nucleotides. Affinity cross-linking of 125I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein. The novel synthetic α-factor analogue MFN5 can be easily iodinated and used as a probe for the α-factor receptor.

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.

Physical monitoring of HO-induced homologous recombination.

The repair of chromosomal double-strand breaks (DSBs) in Saccharomyces cerevisiae occurs most efficiently by homologous recombination. Homothallic mating-type (MAT) switching provides the most well-characterized system to study DSB repair by recombination in mitotic cells (1,2,3). MAT switching is a genetically programmed event in yeast haploid cells, initiated by the site-specific HO endonuclease (Fig. 1). This creates a DSB at MAT that can be repaired by homologous donor sequences, HMLα or HMR a, located near the ends of the same chromosome. These donor loci are maintained in a silent chromatin structure that prevents both their transcription and cleavage by HO, though they can still serve as donors in recombination. Most of the time MAT a cells use HMLα and thus switch to MATα, whereas MATα cells use HMR a to switch to MAT a. This change of mating type can be scored genetically and molecularly, since Y a and Yα sequences are different and have restriction endonuclease polymorphisms (Fig. 1).