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.

Sexual behavior and its pheromonal regulation in ascosporogenous yeasts.

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.

Interspecific actions of alpha mating pheromones on the a mating-type cells of three Saccharomyces yeasts.

The alpha mating pheromones synthesized in three Saccharomyces yeasts (S. cerevisiae, S. kluyveri, and S. exiguus) displayed interspecific actions on the a cells of all three species despite the fact that the amino acid sequences of all three alpha pheromones are different. Mating between species, however, did not occur. The interspecific alpha pheromone--a cell reaction was not necessarily more effective than the interspecific one.

Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae.

It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner. MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature. When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis reinitiation in a dose-dependent manner and even in the presence of cycloheximide. Yeast MPF exerted its activity in starfish oocytes as well. MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle. Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6 S. In contrast to the current assumption that MPF is a Ca-sensitive phosphoprotein stabilized by phosphorylated small molecules, such as ATP and Na-beta-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase. Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost undetectable when either of them was present alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Sexual differentiation and interactions in yeasts.

Results of analytical studies on sex-specific substances, together with behavioural analysis of mating yeast cells, allow some comprehension of the mechanisms regulating the mating reaction of Saccharomyces cerevisiae. Interspecific and intergeneric sexual interactions are studied with the hope of elucidating the ecological and phylogenetic significance of sexual differentiation and interaction in yeasts.

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus.

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.

Secreted agglutinability-masking factors in Issatchenkia scutulata var. scutulata.

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.

Genetic characterization of an alpha-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect.

A recessive ag alpha 1 mutation leads to specific defect in sexual agglutinability specifically in alpha cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cryR 1, closely linked to the mating type locus, was used to select alpha/alpha strains which emerged from alpha/alpha strains by mitotic nonreciprocal recombination, to genetically analyse ag alpha 1, since ag alpha 1 is expressed only in alpha mating type. The ag alpha 1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv 3 at 12 cM on chromosome X. Sexual agglutinability of alpha cells was shown to be dependent on the dose of the AG alpha 1 gene, using alpha/alpha isogenic strains carrying AG alpha 1/AG alpha 1, AG alpha 1/ag alpha 1 or ag alpha 1/ag alpha 1. The sst2-1 mutation did not suppress the ag alpha 1 mutation. Based on these results, function of the AG alpha 1 gene is discussed.

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae.

We have identified a recessive α-mating-type-specific gene agαl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agαl showed sexual agglutination with α cells but α cells carrying agαl showed sexual agglutination with neither α cells nor a cells. α cells carrying agαl produced α pheromone and responded to a pheromone just like wild α cells. α cells carrying agαl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agαl mutant is different from any α-specific ste mutants found so far in many sexual activities. The agαl gene is recessive, and unlinked to the mating type locus. Biological significance of the α mating type agglutinability is discussed based on the results obtained with the mutant.

Alpha-mating-type-specific suppression and a-mating-type-specific enhancement by tunicamycin of sexual agglutinability in the yeast Saccharomyces cerevisiae.

Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in alpha mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in alpha mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in alpha mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of alpha pheromone which induces or enhances the sexual agglutinability of a cells. Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.

Selective inhibition of transition from sexual agglutination to zygote formation by ethyl N-phenylcarbamate in Saccharomyces cerevisiae.

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.

Purification and genetic control of α-pheromone-inactivating glycoproteins in the yeast Saccharomyces cerevisiae.

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.

Changes in production of the mating-type-specific glycoproteins, agglutination substances in association with mating type interconversion in homothallic strains of the yeast, Saccharomyces cerevisiae.

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.

Recessive and dominant genes controlling inducible sexual agglutinability in Saccharomyces cerevisiae.

We have characterized the two dominant genes, IND 1 and IND 2, responsible for inducible sexual agglutinability. The strains carrying these genes differ from the inducible strains carrying the recessive gene, saa 1 in the following points. The former strains produce agglutination substance at 22 degree, 28 degree, and 37 degree C only response to sex pheromone of the opposite mating type, but the latter strains produce the substance constitutively without the pheromone at 22 degree C, only in response to the pheromone at 28 degree C, and do not produce the substance, even in the presence of the pheromone, at 37 degree C. We suggest that strains carrying one of the dominant, inducible genes are wild type and have a pheromone-controlled regulatory system of sexual agglutinability.

Promotion of sporulation by caffeine pretreatment in Saccharomyces cerevisiae. II. Changes in ribonuclease activity during sporulation.

Changes in RNase activity during sporulation of a homothallic diploid strain of Saccharomyces cerevisiae were measured in caffeine-treated and non-treated cells. 1. In caffeine-treated cells soon after the transfer to the sporulation medium a significant increase in RNase activity was observed; in control cells the rise of RNase activity was less and started after a lag period of 5 h. The final activity of RNase activity was about twice as high in caffeine-treated cells as in control cells. 2. Increase in RNase activity during sporulation was sensitive to cycloheximide in control cells, but insensitive in caffeine-treated cells. 3. RNases from vegetative cells and from sporulating ones are different in their Km values. Relation of the changes in RNase activity to premeiotic DNA synthesis is discussed.

Mating reaction in Saccharomyces cerevisiae. IX. Regulation of sexual cell agglutinability of a type cells by a sex factor produced by alpha type cells.

A diffusible sex-specific substance called alpha substance-I (alphaS-I) was isolated from culture filtrate of alpha type strains of the yeast Saccharomyces cerevisiae. The isolated alphaS-I, an oligopeptide, induced sexual cell agglutinability in inducible alpha type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 mug/ml of alphaS-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by alphaS-I. The aAF produced in response to alphaS-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of alphaS-I.