Disentangling signaling gradients generated by equivalent sources

Yeast cells approach a mating partner by polarizing along a gradient of mating pheromones that are secreted by cells of the opposite mating type. The Bar1 protease is secreted by a-cells and, paradoxically, degrades the α-factor pheromones which are produced by cells of the opposite mating type and trigger mating in a-cells. This degradation may assist in the recovery from pheromone signaling but has also been shown to play a positive role in mating. Previous studies suggested that widely diffusing protease can bias the pheromone gradient towards the closest secreting cell. Here, we show that restricting the Bar1 protease to the secreting cell itself, preventing its wide diffusion, facilitates discrimination between equivalent mating partners. This may be mostly relevant during spore germination, where most mating events occur in nature.     

Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells

Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiae a-cells to sense and respond to spatial gradients of the mating pheromone α-factor produced in a microfluidics chamber; the focus was on bar1Δ strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient (∼80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average α-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the K_d of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Δ and ste2^300Δ) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of α-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.     

Role of α-Factor and the Mfα1 α-Factor Precursor in Mating in Yeast

The peptide pheromones secreted by a and α cells (called a-factor and α-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major α-factor structural gene (MFα1) that should result in an altered product inhibits α mating. These results suggested that the pheromone precursors (the MFα1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of α-factor and the MFα1 precursor in α mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MFα1 pro region peptide but no α-factor could not be alleviated by addition of exogenous α-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce α-factor, but with a variety of alterations in MFα1 precursor structure, mated at levels proportional to the levels of α-factor produced, suggesting that the only role of the α-factor precursor in mating is to produce α-factor. Both of these results argue against a role for the MFα1 pro region separate from its role in α-factor production. We also describe results that show that in vivo production of α-factor'' (the form of α-factor encoded by one of the two α-factor repeats of MFα2) is equivalent to the major form of α-factor for induction of all responses necessary for mating. We discuss the implications of these results on the role of the pheromones in mating.     

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.     

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.     

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

The α-factor mating pheromone induces a transient intracellular alkalinizatin 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-dinotrophenol 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 α-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25^ts mutation causes MATa cells to become nonresponsive to α-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.     

Discovery and Functional Study of a Novel Genomic Locus Homologous to Bα-Mating-Type Sublocus of Lentinula edodes

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.     

Synthetic Morphology Using Alternative Inputs

Designing the shape and size of a cell is an interesting challenge for synthetic biology. Prolonged exposure to the mating pheromone α-factor induces an unusual morphology in yeast cells: multiple mating projections. The goal of this work was to reproduce the multiple projections phenotype in the absence of α-factor using a gain-of-function approach termed “Alternative Inputs (AIs)”. An alternative input is defined as any genetic manipulation that can activate the signaling pathway instead of the natural input. Interestingly, none of the alternative inputs were sufficient to produce multiple projections although some produced a single projection. Then, we extended our search by creating all combinations of alternative inputs and deletions that were summarized in an AIs-Deletions matrix. We found a genetic manipulation (AI-Ste5p ste2Δ) that enhanced the formation of multiple projections. Following up this lead, we demonstrated that AI-Ste4p and AI-Ste5p were sufficient to produce multiple projections when combined. Further, we showed that overexpression of a membrane-targeted form of Ste5p alone could also induce multiple projections. Thus, we successfully re-engineered the multiple projections mating morphology using alternative inputs without α-factor.     

Localized secretion of acid phosphatase reflects the pattern of cell surface growth in saccharomyces cerevisiae

Secretion of cell wall-bound acid phosphatase by Saccharomyces cerevisiae occurs along a restricted portion of the cell surface. Acid phosphatase activity produced during derepressed synthesis on a phosphate-limited growth medium is detected with an enzyme-specific stain and is localized initially to the bud portion of a dividing cell. After two to three generations of phosphate-limited growth, most of the cells can be stained; if further phosphatase synthesis is repressed by growth in excess phosphate, dividing cells are produced in which the parent but not the bud can be stained. Budding growth is interrupted in α-mating-type cells by a pheromone (α-factor) secreted by the opposite mating type; cell surface growth continues in the presence of α-factor and produces a characteristic cell tip. When acid phosphatase synthesis is initiated during α-factor treatment, only the cell tip can br stained; when phosphate synthesis is repressed during α-factor treatment, the cell body but not the tip can be stained. A mixture of derepressed α cells and phosphatase-negative α cells form zygotes in which mainly one parent cell surface can be stained. The cell cycle mutant, cdc 24 (Hartwell, L.H. 1971. Exp. Cell Res. 69:265-276), fails to bud and, instead, expands symmetrically as a sphere at a nonpermissive temperature (37 degrees C). This mutant does not form a cell tip during α-factor treatment at 37 degrees C, and although acid phosphatade secretion occurs at this temperature, it is not localized. These results suggest that secretion reflects a polar mode of yeast cell- surface growth, and that this organization requires the cdc 24 gene product.     

Pheromone-regulated Genes Required for Yeast Mating Differentiation

Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for β-galactosidase (β-gal) expression in the presence and absence of α factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: β-gal and Fig2::β-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.     

Multiple Genes Encoding Pheromones and a Pheromone Receptor Define the Bβ1 Mating-Type Specificity in Schizophyllum Commune

The genes defining multiple B mating types in the wood-rotting mushroom Schizophyllum commune are predicted to encode multiple pheromones and pheromone receptors. These genes are clustered in each of two recombinable and independently functioning loci, Bα and Bβ. A difference in specificity at either locus between a mated pair of individuals initiates an identical series of events in sexual morphogenesis. The Bα1 locus was recently found to contain genes predicted to encode three lipopeptide pheromones and a pheromone receptor with a seven-transmembrane domain. These gene products interact in hetero-specific pairs, the pheromone of one Bα specificity with the receptor of any one of the other eight Bα specificities, and are likely to activate a signaling cascade similar to that known for mating in Saccharomyces cerevisiae. We report here that the Bβ1 locus also contains at least three pheromone genes and one pheromone receptor gene, which function similarly to the genes in the Bα1 locus, but only within the series of Bβ specificities. A comparison of the DNA sequences of the Bα1 and Bβ1 loci suggest that each arose from a common ancestral sequence, allowing us to speculate about the evolution of this unique series of regulatory genes.     

The White Cell Response to Pheromone Is a General Characteristic of Candida albicans Strains

For Candida albicans, evidence has suggested that the mating pheromones activate not only the mating response in mating-competent opaque cells but also a unique response in mating-incompetent white cells that includes increased cohesion and adhesion, enhanced biofilm formation, and expression of select mating-related and white cell-specific genes. On the basis of a recent microarray analysis comparing changes in the global expression patterns of white cells in two strains in response to α-pheromone, however, skepticism concerning the validity and generality of the white cell response has been voiced. Here, we present evidence that the response occurs in all tested media (Lee's, RPMI, SpiderM, yeast extract-peptone-dextrose, and a synthetic medium) and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone is therefore a general characteristic of MTL-homozygous strains of C. albicans.     

Genetically Controlled Self-Aggregation of Cell-Surface-Engineered Yeast Responding to Glucose Concentration

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and α-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agα1p (α-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and α-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.     

Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction

Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180^o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change.     

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.     

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone, M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe. Received: 20 December 1996 / Accepted: 29 January 1997