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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.

The effect ofmata1,matα1 andmatα2 mutations in the mating type locus on the production of the sexual agglutination substances responsible for sexual agglutination was examined. Cells carrying themata1 mutation produceda agglutination substance as efficiently as cells ofMATa. Cells carryingmatα1 showed neither α nora agglutination ability. Cells carryingmatα2 behaved just likematα1 cells at 28°C, but at 36°C, or in glycerol or acetate medium, they produceda agglutination substance, showinga agglutination ability.matα2 cells showed α agglutination ability even at 28°C when treated with 2-mercaptoethanol which inactivates thea agglutination substance selectively, indicating that botha and α agglutination substances were produced simultaneously at 28°C, but no agglutination ability was expressed by mutual interaction of these two substances. This indication was confirmed by the fact that α agglutination substance was detected in the cell wall fraction ofmatα2 cells cultured at 28°C, by treatment with 2-mercaptoethanol followed by DEAE cellulose column chromatography. In the light of the above results and the α1-α2 hypothesis, the mechanism of regulation of production of agglutination substance by the mating type locus is discussed.

Sexual activity in homothallic strains of Saccharomyces cerevisiae was investigated. We succeeded in culturing homothallic haploid cells without conjugation, by lowering the pH value of the culture medium. In spore cultures of a homothallic strain both a and alpha pheromones were detected. Agglutination substance of a and alpha mating types were detected in homothallic haploid cells from spore cultures in early logarithmic phase regardless of mating type information at the HML and HMR loci, but either a or alpha agglutination substance was detected predominantly in homothallic haploid cells from spore culture in late logarithmic phase, depending on mating type information at the HML and HMR loci.

The substance responsible for the sexual agglutinability was successfully solubilized by a newly established autoclaving method from the surface of mating type a cells of Saccharomyces cerevisiae and purified by DEAE cellulose chromatography, gel filtration, affinity chromatography and electrophoresis. The substance was found to consist of at least two different glycoprotein subunits. The molecular weight of the substance was estimated to be about 23,000 daltons by gel filtration. The substance was univalent in its biological activity and specifically masked the sexual agglutinability of the mating type α cells. The substance formed a complementary complex with the agglutination substance from α cells in vitro.

We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. alpha Pheromone of S. exiguus (alpha se pheromone) induced formation of large pear-shaped cells (shmooing) in alpha strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible alpha strain of S. cerevisiae. alpha se Pheromone is a peptidyl substance a little different from alpha pheromone of S. cerevisiae. alpha Pheromone of S. exiguus acts only on alpha cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.

Two kinds of a-mating-type-specific proteins inactivating α pheromone (α factor) were purified from heat shock extract of MATa cells. Their molecular weights were estimated to be 400,000 and 200,000 by gel filtration. Both proteins were detected in MATa SST1 cells but not in MATα SST1, MATa sst1-1 and MATa/MATα SST1/SST1 cells. In addition, the proteins were detected in matα2-1 SST1 cells but not in matα1-2 SST1 cells. From these results, it is concluded that these proteins are synthesized under the control of the SST1 gene and responsible for the Barrier action of MATa cells. The relationship of these proteins to the secreted Barrier protein having a higher molecular weight is discussed.

Three peptides, αsk1, αsk2 and αsk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, αsk2 pheromone, having already been determined. The unknown N-terminus of αsk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from αsk1 pheromone, and the primary structure of αsk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of αsk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of αsk2 pheromone.

A peptidyl factor named α substance-IA, which induces sexual agglutinability in a type cells of the heterothallic Saccharomyces cerevisiae, was found to be composed of twelve amino acid residues. Further, the amino acid sequence was elucidated by the combined application of the Edman-dansyl procedure and the cleavage reaction with cyanogen bromide to be H- His-Trp-Leu-Gln-Leu-Lys-Pro-GIy-Gln-Pro-Met-Tyr-OH.

The relationship between the sporulation ability and the stage of the vegetative cell cycle was studied. The ability of mature cells to sporulate was the highest just before bud initiation and decreased rapidly as soon as detectable buds appeared. The mitotic DNA synthesis was competitive with the premeiotic one.

Sexual agglutination occurred only between cells of opposite mating types of the same species in all the Sacharomyces, Hansenula, Saccharomycodes, and Pichia yeasts tested. We succeeded in solubilizing the sex-specific glycoprotein, cell wall agglutination substance responsible for sexual agglutinability by briefly autoclaving these yeasts. The agglutination substances of all the above yeasts were univalent and sensitive to the enzyme pronase. The formation of complementary complexes was observed only between agglutination substances of opposite mating types of the same species. In general, the agglutination substance of one mating type was more resistant to heat treatment at 100°C in 3% acetic acid and more sensitive to 5% 2-mercaptoethanol treatment than the agglutination substance of the other mating type in these yeasts. On the basis of these results together with the pheromone response and production, we expect that almost all ascosporogenous yeasts can be classified into the two mating types corresponding to a and α mating types in Saccharomyces cerevisiae, respectively.

Spores of heterothallic diploid cells of Saccharomyces cerevisiae had neither a nor α agglutination substance in either cell wall or cytoplasmic fraction; they, however, showed selfagglutination not caused by sex-specific agglutination substances. Meanwhile, practically no sexual agglutination was detected during germination and outgrowth of the spores; it arose after emergence of the first buds and progressed with incubation time. Its ability increased gradualy until the first bud emergence and rapidly thereafter. a and α agglutination substances were detected in both cell wall and cytoplasmic fractions of cells from an 8h-old spore culture. Only germinated spores with buds had the ability to produce and to respond to the a pheromone.

We have isolated the mutants, T55s-41(a) and T562s-161 (α) which have no sexual agglutinability when cultured at 28°C, but become sexually agglutinable by the action of the sex pheromone produced by respective opposite mating type. The sex-specific glycoproteins responsible for sexual agglutination were detected in the mutants treated with the opposite mating type pheromone, but not in those treated with the same mating type pheromone.

Three peptides, αsk1, αsk2 and αsk3 pherornones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, αsk2 pheromone, having already been determined. The unknown N-terminus of αsk1 pheromone was elucidated to be l, 2, 3, 4-tetrahydro-β-carbolme-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from αsk1 pheromone, and the primary structure of αsk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of αsk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of αsk2 pheromone.

The yeast, Hansenula wingei has two mating types designated 5 and 21. Cells of each mating type were found to produce mating type-specific sex pheromone which induces sexual agglutinability of the opposite mating type. Crude fractions of these pheromones were prepared by using an Amberlite CG 50 (H+ type) column. The agglutinability-inducing action of the pheromones required glucose as carbon source, but no external nitrogen source. The action of the pheromones was inhibited by 5 μg/ml cycloheximide. The optimum pH for the pheromone action was 4.0. α Pheromones of Saccharomyces cerevisiae and Saccharomyces kluyveri induced sexual agglutinability of 5 mating type cells but did not that of 21 mating type cells. a Pheromones of the Saccharomyces yeasts had no effect on both 5 and 21 mating type cells. The sex pheromones of H. wingei had no effect on the sexual agglutinability of inducible a cells of S. cerevisiae. From the experimental results obtained so far, we propose to call 5 and 21 mating types in H. wingei a and α mating types, respectively.

Plasma membrane invagination structures were found to occur in all the sexually agglutinatable a strains, independently of physiological and genetic conditions. The structures were not observed in the a strains without sexual agglutination ability tested so far. The number of invagination structures was around 30 per cell. An increase in sexual agglutination ability was preceded by an increase in number of the invagination structures during induction of sexual agglutination ability of inducible a cells by α pheromone, indicating that the structures play an important role in the occurrence of sexual agglutination ability.

Effects of ethyl N-phenylcarbamate (EPC) on the mating reaction of Saccharomyces cerevisiae were studied, with special attention on the effect on the alpha pheromone action. EPC inhibited zygote formation at a concentration which promoted induction of sexual agglutinability. EPC enhanced agglutinability induction by alpha pheromone, but inhibited alpha-pheromone-induced formation of large pearshaped cells in a mating type. The enhancement of agglutinability induction was accompanied with increased production of a agglutination substance and inhibition of alpha pheromone inactivation. EPC arrested the cell cycle of a cells probably in the step controlled by CDC19, CDC35, cAMP etc., just before the step controlled by CDC28, alpha pheromone etc.

When α pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.

We reviewed our investigations on sexual behaviors and interactions including sexual cell agglutination and pheromone action mainly in non-conventional yeasts, Hansenula anomala, H. wingei, Pichia amethionina, P. heedi, P. opuntiae, Saccharomyces kluyveri, S. globsus, S. exiguus, Saccharomycodes ludwigii. The techniques and genetic models including the cassette model and alpha 1-alpha 2 hypothesis which had been developed largely in S. cerevisiae were applicable to these yeasts in principle. The sexual agglutination was distinctly species-specific while sex pheromones were cross-reactive beyond species' barriers. The successful induction of heterothallic strains from homothallic strains in S. exiguus by mutagenesis enabled to the subsequent biochemical and genetical analysis of sexual behavior in the yeast. The phylogenetic consideration on sex differentiation is also included.

The agglutinability-masking factors (AMFs) of a and alpha mating types of Issatchenkia scutulata var. scutulata were prepared from culture fluids. AMFs masked the agglutinability of opposite mating-type cells sex-specifically, just like agglutination substances responsible for sexual cell agglutination. a AMF adsorbed to alpha cells was eluted by incubating the cells at 60 degrees C for 10 min. alpha AMF was prepared directly from culture fluids of alpha cells by DEAE-cellulose ion exchange chromatography. The active part of the AMFs is thought to be a peptidyl moiety because of the sensitivity to subtilisin. The pretreatment of cells with AMF of the opposite mating-type was shown to promote zygote formation. alpha AMF slightly inhibited growth in a cells but not in alpha cells, while a AMF did not show any growth-inhibitory effect on either a or x cells.

Three analogues of the peptidyl pheromone, α pheromone of Saccharomyces kluyveri, synthesized based on the amino acid sequence proposed by Sato et al. (Agric Biol Chem 45:1531–1533, 1981) were tested for both shmoo-inducing and agglutinability-inducing actions. Purified natural α pheromone of the yeast showed the highest activity among the peptides tested. When methionine in the peptides was oxidized, the activity decreased significatly. α Pheromone of S. kluyveri induced sexual agglutinability in a cells of Saccharomyces cerevisiae, and shmoo in a cells of S. cerevisiae and S. kluyveri. a Pheromone of S. kluyveri had no agglutinability-inducing action on α cells of S. cerevisiae. a Cells of S. kluyveri inactivated only α pheromone of the same species, but a cells of S. cerevisiae inactivated α pheromones of both S. cerevisiae and S. kluyveri.

A Saccharomyces cerevisiae a strain carrying the secretory mutation sec1, sec7 or sec18 showed no sexual agglutination ability when treated with α pheromone at the restrictive temperature 36° C, although the a agglutination substance had accumulated in the cytoplasm. These cells became sexually agglutinable, with a concomitant decrease in the agglutination substance in the cytoplasm, when the temperature was shifted from 36° C down to the permissive temperature 24° C after the addition of, cycloheximide. The a agglutination substance was barely detectable in sec53 cells (a) treated with α pheromone at 36° C, indicating that the active a agglutination substance was formed after the export of its precursor into the endoplasmic reticulum. These results indicate that the a agglutination substance is exported through the yeast secretory pathway and that α pheromone acts at the level of synthesis of the precursor molecule of the substance. An α strain carrying sec1, sec7 or sec18 behaved just like an a strain carrying the sec gene in the induction of agglutination ability by the opposite mating type sex pheromone.

In Saccharomyces cerevisiae α mating-type strains carrying the sec1, sec7 and sec18 genes, pheromone a induces α agglutination ability at 24 °C, but not at 36 °C. Even when cells were treated at 36 °C, a biologically active α agglutination substance was found in cytoplasm although this activity was not detected in the cell surface fraction. These 36 °C-treated cells became agglutinable with a concomitant appearance of α agglutination substance activity in the cell surface fraction. The cells remained agglutinable even when the treatment temperature was dropped to 24 °C under conditions where no de novo synthesis of the agglutination substance occurred. These results indicate that the α agglutination substance is transported through the yeast secretory pathway and that pheromone a acts at the step of synthesis of the precursor molecule of the a agglutination substance, similar to the case ofthe α-pheromone-induction of the a agglutination substance. Differences in the action of the sex pheromones between agglutination ability induction and induction of G1 arrest or shmooing is discussed.

Large cell mutants were produced with high frequency when a haploid strain of Saccharomyces cerevisiae was cultured on a nutrient agar containing 500mg/l α-naphthaleneacetic acid (NAA). These mutants had no selective advantage in the presence of NAA. Biochemical and genetic analyses showed that most of these variants involve changes in genome structure.

Effect of a homothallism-controlling gene on sporulation was studied, using isogenic yeast strains carrying and not carrying D gene. In all cases tested, D gene showed distinct promotive effect on sporulation, irrespective of the period of incubation in presporulation or sporulation medium. However, D gene has no significant effect on vegetative growth in presporulation culture.

Gibberellic acid (GA) promoted sporulation in yeast when added to the sporulation medium. When added together with GA, metabolic inhibitors of RNA synthesis such as 8-azaguanine, 2-thiouracil, and actinomycin D suppressed selectively the promoting effect of GA on sporulation. The effect of 8-azaguanine and 2-thiouracil was alleviated by simultaneous addition of guanine and uracil, respectively. The promoting effect of GA on sporulation was also suppressed by inhibitors of protein synthesis such as ethionine, chloramphenicol, and puromycin. Methionine eliminated the inhibitory effect of ethionine on the GA action.

Effect of auxin and gibberellic acid on sporulation of a yeast, Saccharomyces ellipsoideus, was studied. When added to the sporulation media, gibberellic acid promoted sporulation. The sporulation rate was higher in the medium SGV with vitamins than in the vitamin-free SG, but the effect of gibberellic acid was more pronounced in the latter. Auxin (IAA, 2,4-D, and NAA) inhibited sporulation in SGV, but promoted it in SG. This sporulation-promoting effect of IAA was reversed by an antiauxin, 2,4,6-T. Preculturing in the presence of added IAA increased sporulation. Added to the preculture medium, gibberellic acid alone showed little effect on sporulation, but in combination with IAA it enhanced sporulation conspicuously. IAA and gibberellic acid were effective in sporulation promotion only when added before the nuclear enlargement occurred in sporulation culture.

Hormone-like substances were isolated from culture media of haploid strains of Saccharomyces cerevisiae. The one excreted by cells of mating type a made cells of the α type expand; the other, excreted by α type cells, made cells of the a type expand. Tentatively we call the former a hormone and the latter α hormone. The cell-expanding action of the a hormone was inhibited by actinomycin D, chloramphenicol and cycloheximide. The a hormone was shown to be heat-stable and dialyzable. Both hormones could be extracted with methylene chloride. The abilities of cells to produce these hormones and to respond to them are under control by the mating-type genes.

The effect of β-1, 3-glucanase on the cell expansion was studied with diploid strains of Saccharomyces ellipsoideus and S. cerevisiae, and their mutants differing in the response to auxin. The following results were obtained. Cell expansion was induced by β-1, 3-glucanase only in auxin-responsive or potentially auxin-responsive strains. β-1, 3-Glucanase induced cell expansion more rapidly than auxin. The cell wall of the auxin-responsive strain was more susceptible to digestion by β-l, 3-glucanase than that of the auxin-unresponsive one. The sensitivity of yeast cells to auxin action is discussed in relation to the nature of cell wall.

Using an auxin-responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts. 1 Indole-3-acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred. 2 The auxin-induced expansion growth was inhibited by an antiauxin, trans-cinnamic acid. 3 Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin-induced cell expansion growth. 4 Protoplasts did not expand in response to auxin under the condition where intact cells did. 5 The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion. 6 High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

Two kinds of proteinacious substances which bind to α pheromone were isolated from heat-shock extract of a mating type cells of Saccharomyces cerevisiae. One has Mr ∼300 000 and the other Mr 58 000. We purified the smaller binding protein by successive application of DEAE and CM-cellulose chromatography, gel filtration, and isoelectrofocusing. The purified binding protein showed a single band by polyacrylamide gel electrophoresis. This binding protein was detected in a cells but not in α cells.