No abstract

A hormone-like substance is secreted by alpha mating-type cells of heterothallic yeast strains. It induces in cells of the opposite mating type, a, a morphological change characteristic of the mating process. Secretion of this substance and mating ability have some common genetic determinants. In partially purified preparations, the substance has properties of an oligopeptide.

Cells of Saccharomyces cerevisiae exhibiting the α mating type excrete into the culture medium a low‐molecular‐weight substance, termed α factor. This factor, which specifically inhibits DNA replication in cells of the opposite mating type a, has been purified more than 100000‐fold from culture filtrates of α cells. Purified α factor appears to be homogeneous as judged from thin‐layer chromatography and thin‐layer electrophoresis in different systems. It shows a positive‐ninhydrin reaction and, upon hydrolysis, gives rise to several ninhydrin‐positive substances of which the amino acids leucine, glycine, proline, glutamic acid, tyrosine, tryptophan and possibly histidine have been identified. The presence of tryptophan and tyrosine is also confirmed by the ultraviolet absorption spectrum of the factor. In addition, purified α factor contains cupric ions which can be separated from the ninhydrin‐positive material by thin‐layer electrophoresis at pH 3.6. Gel filtration on Sephadex G‐25 in 8 M urea indicates a molecular weight in the range of 1400. The properties of the purified α factor are consistent with those of a low‐molecular‐ weight peptide.

The amino acid sequences of the mating hormones produced by a-mating-type cells of the yeast Saccharomyces c*twvisiuu (a factor peptides a l , 012, a3 and a4) have been determined. By cleavage of the peptides with thermolysin and pepsin and structural analysis of the fragments the sequence H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH could be assigned to a 1. a2 lacks the N-terminal tryptophan residue. a3 and ct4 represent the methionine sulfoxides of a1 and a2 respectively.The control of sexual conjugation between haploid cells of the yeast Saccharomyces cerevisiae involves the function of mating-type specific conjugation factors which are excreted by the cells into the culture medium [l -31. These factors, which are now generally referred to as mating hormones [4], appear to mediate the interaction between cells of opposite mating-type by synchronizing the cells prior to fusion [5]. The hormonal activity produced by cells of the a mating-type, which was termed a factor [l], has recently been found to be associated with a group of structurally related oligopeptides consisting of 12 and 13 amino acids respectively [6]. The peptides, which were designated as a l , a2, ct3 and a4, were shown to possess the following common structure: H2N-His-Trp-Leu-(Gluz, Pro2, Gly,, Leul, Lys1)-Met-Tyr-COOH, the tridecapeptides a1 and a3 differing from the dodekapeptides a2 and a4by an additional N-terminal tryptophan residue. a3 and a4 were shown to represent oxidation products of a1 and a2 respectively, containing a methionine sulfoxide residue instead of a methionine residue in the penultimate position.The determination of the complete amino acid sequence of the a peptides was hampered by the fact that only very small amounts of the individual compounds were available and by the hydrophobicity of the peptides, which leads to extensive losses during Edman degradation. These difficulties have now been overcome by enzymic cleavage of preparations containing all four a peptides with endopeptidases and structural analysis of the fragments. By this approach the complete amino acid sequences of the a factor peptides have now been elucidated. MATERIALS AND METHODSThe a factor peptides were isolated from culture filtrates of the haploid wild-type strain X2180-1 B (mating-type a) as described previously [6]. Between 2 pmol and 4 pmol of a mixture of all four a peptides was digested overnight at 40 "C in 2.0 ml 0.01 M Tris/ HCI, pH 8.0, containing 0.05 M CaC12 with 50 pg/ml thermolysin. The digests were chromatographed on SP-Sephadex C-25 equilibrated with 0.17 M pyridinium acetate, pH 4.6. The column ( 2 x 8 0 cm) was eluted with 200 ml of the equilibration buffer followed by a sigmoidal gradient of pyridinium acetate prepared from 200 ml of 0.17 M buffer, pH 4.6, and 400 ml of 2.25 M buffer, pH 5.4, as described by Schroder [7]. Ninhydrin-positive material was determined in each fraction according to the method of Hirs [S] and the appropriate fractions were checked for homogeneity by thin-layer chromatography as descri...