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...

The molecular structure of a factor, a mating hormone produced by a mating type cells of Saccharomyces cerevisiae has been investigated. From culture filtrates of a cells four oligopeptides exhibiting a factor activity have been isolated. These peptides, designated as a 1, a 2 , a3, and a 4 are structurally closely related, being composed of thirteen (a1 and a3) and twelve ( a 2 and a4) amino acids, respectively. The peptides were found to be composed of the following amino acids: 2 glutamic acid or glutamine, 2 proline, 1 glycine, 1 methionine or methionine sulfoxide, 2 leucine, 1 tyrosine, 1 lysine, 1 histidine, 1 or 2 tryptophan. The tridekapeptides differ from the dodekapeptides by an additional NH2-terminal tryptophan residue. Tyrosine was identified as the C-terminal amino acid in all four peptides. a3 and a4 are oxidation products containing an internal methionine sulfoxide instead of methionine. The mechanisms which could introduce the observed heterogeneity of the peptides are discussed.In the life cycle of the yeast Saccharomyces cerevisiae the transition from the haploid to the diploid growth phase is achieved by the conjugation of two haploid cells of different mating type. This process appears to be controlled by specific regulatory factors which are now generally referred to as mating hormones or sexual hormones (for review see [l]). Haploid yeast cells belong to either one of two alternative mating types designated as a and a, each of which produces one or, probably, several hormones which are excreted into the culture medium and act specifically on cells of the opposite mating type [2-51. Thus, culture filtrates of a cells contain an activity designated as a factor which specifically inhibits cell division in cells of mating type a [2]. A corresponding a factor which is produced by a cells and acts on a cells has been recently described by several groups [4,5]. It has been shown that a factor interferes with an essential cell cycle function of the a cells arresting them at an early stage of the cycle during the G1 period [6]. As a consequence initiation of DNA synthesis and bud formation are inhibited [6,7]. Since the mating ability of haploid yeast cells is restricted to the early part of the cell cycle it has been proposed that under physiological conditions the hormones could serve to increase the fraction of competent cells in a mating mixture, thus enhancing the efficiency of conjugation [5,8].

The biological activities of two synthetic oligopeptides (His-Trp-Leu-Gln-Leu and Trp-Leu-Gln-Leu), which represent part of the primary structure of the mating hormone alpha factor from Saccharomyces cerevisiae, were studied. The peptides did not exhibit hormonal activity by themselves. However, both intensified the mating-type-specific inhibitory effect of native alpha factor on the division of haploid cells of mating type a. Random peptides or mixtures of the corresponding amino acids did not stimulate alpha factor activity. Likewise, a synthetic peptide representing another part of the alpha factor sequence was ineffective. In addition, the activity of a factor, the mating hormone produced by a cells, was not influenced by the synthetic peptides, indicating that the compounds specifically affect the interaction between alpha factor and its target cells. The analysis of the utilization of the tetrapeptide as a source of amino acids for auxotrophic a strains suggested an extracellular site of action for the observed enhancement of alpha factor activity.