Sexual Agglutination Research Articles

Postzygotic reproductive isolation among three Saccharomyces yeast species.

We have previously isolated heterothallic haploid strains from original homothallic diploids of two yeast species in the family Saccharomycetaceae. In this study, heterothallic haploid strains were isolated from an original homothallic diploid of Saccharomyces kudriavzevii type strain, followed by investigation of sexual interactions among these yeast strains, in addition to S. cerevisiae laboratory strains. It has been shown that prezygotic reproductive isolation was observed between Kazachstania naganishii and S. cerevisiae with α-factor mating pheromones representing crossaction with each other beyond the genus boundary. Using heterothallic strains, postzygotic reproductive isolation system was shown to reside in the genus Saccharomyces by mass mating and cell-cell contact experiments. In mass mating experiments, crossaction of α-factor and a-factor mating pheromones and sexual agglutination effectively occurred beyond species boundaries among S. kudriavzevii, S. paradoxus, and S. cerevisiae. When the fates of cell-cell pairs from these Saccharomyces yeast species were systematically chased one by one, interspecific F1 hybrids were effectively produced, while sporulations were partially prohibited, with spore germination perfectly blocked in the hybrids. These results indicated that postzygotic reproductive isolation definitively resides among these Saccharomyces yeast species and that disorder of chromosome organization had to some extent occurred in interspecific F1 hybrids.

High-throughput characterization of protein–protein interactions by reprogramming yeast mating

High-throughput methods for screening protein-protein interactions enable the rapid characterization of engineered binding proteins and interaction networks. While existing approaches are powerful, none allow quantitative library-on-library characterization of protein interactions in a modifiable extracellular environment. Here, we show that sexual agglutination of Saccharomyces cerevisiae can be reprogrammed to link interaction strength with mating efficiency using synthetic agglutination (SynAg). Validation of SynAg with 89 previously characterized interactions shows a log-linear relationship between mating efficiency and protein binding strength for interactions with Kds ranging from below 500 pM to above 300 μM. Using induced chromosomal translocation to pair barcodes representing binding proteins, thousands of distinct interactions can be screened in a single pot. We demonstrate the ability to characterize protein interaction networks in a modifiable environment by introducing a soluble peptide that selectively disrupts a subset of interactions in a representative network by up to 800-fold. SynAg enables the high-throughput, quantitative characterization of protein-protein interaction networks in a fully defined extracellular environment at a library-on-library scale.

Distal and Proximal Actions of Peptide Pheromone M-Factor Control Different Conjugation Steps in Fission Yeast

Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of “distal pheromone action”, but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of “proximal pheromone action”.

Inhibitory effects of nucleoside 5'-alkylphosphates on sexual agglutination in Saccharomyces cerevisiae.

Among various nucleoside 5'-alkylphosphates, uridine 5'-hexadecylphosphate (UMPC16) and adenosine 5'-hexadecylphosphate (AMPC16) inhibited the sexual agglutination between a and alpha haploid cells of Saccharomyces cerevisiae. The effect of AMPC16 accompanied severe growth inhibition of the yeast cells but it was not observed with UMPC16. Sexual agglutination was not inhibited by the presence of UMPC16 or AMPC16 when the yeast cells had been pretreated with the mating pheromone. UMPC16 was characterized as a specific inhibitor of sexual agglutination without direct influence on the agglutinin function, being distinguishable from any of those ever known.

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.

The RGS domain‐containing fission yeast protein, Rgs1p, regulates pheromone signalling and is required for mating

When nutritionally starved, the fission yeast Schizosaccharomyces pombe enters a cell differentiation process which leads to mating and meiosis. The Ste11 protein is a key regulator of this differentiation pathway, activating the transcription of mating and meiotic genes upon starvation. Here, we describe rgs1, a member of the Regulator of G-protein Signalling (RGS) family, as a novel Ste11 target gene. rgs1 expression requires both an Ste11-mediated nitrogen starvation signal and the pheromone-induced activation of the Byr2/Byr1/Spk1 MAPK pathway. We show that rgs1 deletion results in a sensitivity to pheromone and in a mating defect. Deltargs1 cells initiate the mating pathway normally, undergoing sexual agglutination and G1 arrest, while inducing pheromone-dependent transcription, but then fail to fuse with a mating partner while elongating abnormal conjugation tubes. Endogenous Rgs1 tagged with GFP localizes to the nucleus and cytoplasm, and this localization pattern is not altered during pheromone treatment. Importantly, Rgs1 function requires its C-terminal RGS domain, as well as a central DEP domain and a novel homology domain present in its N-terminal region (Fungal-DR domain). These results demonstrate that rgs1 expression requires nutritional starvation and pheromone signalling. Rgs1 negatively regulates pheromone signalling during mating, acting in a negative feedback loop that is essential for the mating process.

Cell-cell recognition and pheromone response of the yeast Saccharomyces globosus.

Sexual agglutination and pheromone interaction between cells of two mating types, a and alpha, in the yeast Saccharomyces globosus were studied. S. globosus was shown to produce mating-type-specific factors analogs to a- and alpha-mating pheromones of Saccharomyces cerevisiae and to undergo the sexual agglutination reaction between cells of two mating types. While the sexual agglutination of cells of different species was not observed, mating type a cells of each species were shown to respond to alpha-factors produced by the other species. Thus, the mating response of S. globosus was shown to be identical to what has been observed in two other species of the same genera: S. cerevisiae and Saccharomyces kluyveri.

Cell–cell recognition and pheromone response of the yeast Saccharomyces globosus

Sexual agglutination and pheromone interaction between cells of two mating types, a and α, in the yeast Saccharomyces globosus were studied. S. globosus was shown to produce mating-type-specific factors analogs to a- and α-mating pheromones of Saccharomyces cerevisiae and to undergo the sexual agglutination reaction between cells of two mating types. While the sexual agglutination of cells of different species was not observed, mating type a cells of each species were shown to respond to α-factors produced by the other species. Thus, the mating response of S. globosus was shown to be identical to what has been observed in two other species of the same genera: S. cerevisiae and Saccharomyces kluyveri.

Inhibitory effects of nucleoside 5′-alkylphosphates on sexual agglutination in Saccharomyces cerevisiae

Among various nucleoside 5′-alkylphosphates, uridine 5′-hexadecylphosphate (UMPC16) and adenosine 5′-hexadecylphosphate (AMPC16) inhibited the sexual agglutination between a and α haploid cells of Saccharomyces cerevisiae. The effect of AMPC16 accompanied severe growth inhibition of the yeast cells but it was not observed with UMPC16. Sexual agglutination was not inhibited by the presence of UMPC16 or AMPC16 when the yeast cells had been pretreated with the mating pheromone. UMPC16 was characterized as a specific inhibitor of sexual agglutination without direct influence on the agglutinin function, being distinguishable from any of those ever known.

Mutations Affecting O-Glycosylation in Chlamydomonas reinhardtii Cause Delayed Cell Wall Degradation and Sex-Limited Sterility.

We describe a mutation, gag-1, that affects in a temperature-dependent manner a specific type of O-glycosylation in the green alga Chlamydomonas reinhardtii. In the mutant, all the major glycoproteins, in particular cell wall proteins, show a decreased apparent molecular weight in polyacrylamide gels, and their antigenicity is affected. The mutant forms multicellular aggregates (palmelloid colonies) at the restrictive temperature due to the delayed release of the daughter cells from the mother cell wall after mitosis. In addition, the mutation causes sterility by preventing sexual agglutination. In contrast to the other phenotypes, the sterility phenotype is temperature independent, and it is expressed only by cells of the plus mating type. We show that imp-8, a previously described nonagglutinating sex-limited mutation, causes the same glycosylation defect and is allelic to gag-1. Thus, expression of mt+ agglutinability appears to require the specific type of O-glycosylation that is defective in these mutants. More generally, these observations show that a sex-limited phenotype can be caused by a mutation in a gene that is not itself sex limited in its expression.

Sexual agglutination substances require a 'carrier' glycoprotein for integration into the cell wall of Saccharomyces cerevisiae

Sexual agglutination, caused by agglutination substance (AS) on a and alpha cell walls, is the first indispensable step of the mating reaction in ascosporogenous yeasts including Saccharomyces cerevisiae. The AS biosynthetic process in S. cerevisiae was investigated by pulse label-chase experiments with analysis by polyacrylamide gel electrophoresis (PAGE) for 16 h in the presence of urea. Because of its low mobility, AS can be separated from other proteins by prolonged PAGE. Nascent AS was integrated into cell walls after it linked covalently to a 'carrier' glycoprotein. The results suggest that the 'carrier' is synthesized stepwise through three distinct precursors (III-->II-->I). The 'carrier' glycoprotein (I) and its precursors (II, III) were synthesized in both a, alpha haploid and a/alpha diploid cells. The N-glycosylation linkage inhibitor, tunicamycin, and protein synthesis inhibitor, puromycin, inhibited the III to I maturation. The results indicated that both the 'carrier' and the nascent active site of AS linked to the 'carrier' are integrated into the wall in a haploid cell while the 'carrier' alone is integrated in a diploid cell.

Mating in the heterothallic haploid yeast Clavispora opuntiae, with special reference to mating type imbalances in local populations

Mating was studied in the haploid, heterothallic yeast Clavispora opuntiae to assess the importance of nutritional, genetic, and other factors that may favour mating and recombination. Local populations of this yeast generally exhibit dramatic inequalities in mating type distributions, suggesting that mating is rare in nature even though most isolates mate freely in the laboratory. The absence of assimilable nitrogen is prerequisite to mating competence, presumably by causing G1 arrest. Maximum mating competence is found in cells entering stationary phase in nitrogen-limited media. Unlike the vast majority of mating yeasts, C. opuntiae does not appear to produce diffusible mating factors (sex pheromones), and mating-competent cells do not undergo sexual agglutination. Pairwise cell contact appears to be the only signal that triggers the sexual process in this case. In order to determine if mating type imbalances in nature are caused by reduced fertility of 'consanguine' crosses, meiotic recombination was measured in pairs of strains that varied in their genetic distances as indicated by restriction mapping. That hypothesis was rejected, as recombination efficiency decreased with increasing genetic distance. We conclude that the rarity of mating in local populations is exacerbated by the stringent physical (pairwise cell contact) and nutritional (nitrogen depletion) conditions that will allow mating to proceed. Parallels are drawn with mating patterns observed in Clavispora lusitaniae.

Sexual agglutination in Chlamydomonas eugametos is mediated by a single pair of hydroxyproline-rich glycoproteins

The sexual mating reaction between gametes of the green alga Chlamydomonas eugametos starts by cell-cell contacts involving sex-specific cell-adhesion molecules (agglutinins) at the flagellar membrane. An in vitro adhesion assay is described using glutaraldehyde-fixed gametes. In vitro adhesion was fully comparable to in vivo adhesion, making it a reliable assay to study the initial recognition step of sexual adhesion in vivo. It was shown that both agglutinins are capable of inhibiting sexual adhesion at similar concentrations (1−2×10 −10 M), indicating that mt + and mt − agglutinins interact with each other during binding. This was confirmed by demonstrating that charcoal particles adsorbed with purified agglutinins of the opposite mating type aggregate with each other.

Sexual agglutination inChlamydomonas eugametosis mediated by a single pair of hydroxyproline-rich glycoproteins

The sexual mating reaction between gametes of the green alga Chlamydomonas eugametos starts by cell-cell contacts involving sex-specific cell-adhesion molecules (agglutinins) at the flagellar membrane. An in vitro adhesion assay is described using glutaraldehyde-fixed gametes. In vitro adhesion was fully comparable to in vivo adhesion, making it a reliable assay to study the initial recognition step of sexual adhesion in vivo. It was shown that both agglutinins are capable of inhibiting sexual adhesion at similar concentrations (1−2×10−10 M), indicating that mt+ and mt− agglutinins interact with each other during binding. This was confirmed by demonstrating that charcoal particles adsorbed with purified agglutinins of the opposite mating type aggregate with each other.

Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins.

The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.

Fusion-defective mutants ofChlamydomonas eugametos

Mutant strains of the unicellular green algaChlamydomonas eugametos are described which are defective in sexual fusion. All mutants are mating type plus (mt+). They are unable to fuse because none of them is capable of protruding a mating structure through the cell wall, neither during sexual agglutination nor after adding dibutyryl-cAMP or compounds that raise the intracellular calcium level, treatments that are effective in wild type cells. Evidence is presented that these mutants lack the lytic enzyme activity which is normally involved in the local hydrolysis of the cell wall to allow the protrusion of the mating structure. Furthermore, a simple light microscopic method is presented to determine the presence of activated mating structures.

Purification and structure determination of the bacterial mating inhibitor for Chlamydomonas reinhardtii and Alexandrium catenella.

A mating inhibitor which inhibits sexual agglutination and gametic fusion of Chlamydomonas reinhardtii and hypnozygote formation of Alexandrium catenella was detected from bacterium strain NT4. The inhibitor (MI) has been successfully purified from a culture supernatant by a combination of Amberlite IRC-50, CM-cellulose 52, and high preformance liquid chromatographies. MI was separated into four derivatives (MI-1, MI-2, MI-3, MI-4), of which MI-1 was identified by spectral analyses (HR-MS, NMR) and chemical degradations to be 3-(4-hydroxyphenyl)-β-alanyl-2-hydroxy-β-alanyl-3-aminoalanyl-ζ-hydroxy-α, e-diamino-η-azelayl-N-(3-((4-aminobutyl)amino) propyl)glycinamide. This is identical with antibiotic edeine Al, a known inhibitor of DNA synthesis. Bacterial strain NT4 producing the inhibitor was identified as Bacillus brevis.

Thiamin regulates agglutination and zygote formation in Schizosaccharomyces pombe.

Nutritional conditions regulate mating of the fission yeast S. pombe. To investigate how nutritional signals are monitored by the cell and translated into appropriate mating behaviour, effects of unique and specific growth factors would be desirable. We show that thiamin can inhibit sexual agglutination and zygote formation in S. pombe. A concentration of 50 nM thiamin in the culture medium is required for full growth of a thiamin auxotrophic strain. At this concentration thiamin starts to inhibit mating of wild-type cells of opposite heterothallic mating type and at a 1 microM concentration zygote formation is inhibited by more than 95%. Growth conditions modulate the inhibitory effect of thiamin. Thiamin acts only for a restricted period of time and seems to inhibit commitment to zygote formation rather than the cell aggregation and fusion process itself. Pyrithiamin, a thiamin antagonist, inhibits growth as well as mating.

Induction of sexual agglutinability by unsaturated fatty acids in Saccharomyces cerevisiae

Agglutinins in S. cerevisiae are necessary for mating, for recognition between cells of opposite mating type. The mode of agglutinin synthesis is altered by the growth temperature and by the carbon source, from constitutive to inducible synthesis and vice versa. Some of the unsaturated fatty acids tested induced synthesis of agglutinins in cells grown at an elevated temperature, even in the absence of the pheromone. However, synthesis of agglutinins in glycerol-grown cells, that are inducible by the pheromone, was not induced by linolenic acid. Hence, the change in the mechanism of regulation of agglutinability produced by differences in temperature probably differs from that produced by differences in carbon source.

Bacterial inhibitor for the mating reaction in Chlamydomonas reinhardtii and Alexandrium catenella.

Sexual agglutination and zygospore formation of Chlamydomonas reinhardtii, a heterothallic organism, were used for screening of mating inhibitors in culture supernatants and cell extracts of 150 bacterial strains. Both fractions of a gram-negative bacterium, strain NT4, inhibited the mating reaction. When algal vegetative cells were treated with the mating inhibitor, both sexual agglutination and zygospore formation were inhibited. However, when mature gametes were treated with the inhibitor, zygospore formation was inhibited but sexual agglutination was not. The mating inhibitor also blocked hypnozygote formation of a toxic dinoflagellate, Alexandrium catenella, at a concentration 128 times as high as that of C. reinhardtii.