Primary Structure of α‐Factor Peptides from Saccharomyces cerevisiae

Abstract

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...

Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone.

“…Many fungal sex hormones have been described (for a review see ref. 21), perhaps the best studied of these being the yeast a factor (22), a peptide believed to be involved in mating (for a review see ref. 23).…”

Nuclear‐magnetic‐resonance studies on the conformations of tridecapeptide α‐mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution

“…For simplicity, we use the abbreviated notations: the natural a-mating factor is denoted as (1 -13)peptide, and the des-Trp' dodecapeptide as (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)peptide. Thus, truncated peptides are denoted with the numbering of the N-terminal and C-terminal residues.…”

“…It may readily be noted that these peptides are classified into three groups, A,, A, and B, from the spectral patterns of amide proton resonances, especially in the region 8.5 -8.0 ppm. The group B includes the inactive analogs, (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), (3)(4)(5)(6)(7)(8)(9)(10) and his^] (1 -13)peptide, and a number of N-H proton resonances of this group are localized around 8.4-8.3 ppm. The group A, includes active (1-n)peptides (n = 13, 12 and 10) and inactive (1 -9)peptide, and the group A, includes active (2-n)peptides ( n = 13 and 12) and inactive (2-9)peptide.…”

“…Masui et al have reported that the activity of a-mating factor is not affected if the N-terminal Trp' residue is deleted [7], although Shenbagamurthi et al have recently reported that the dodecapeptide is less active than a-mating factor [lo]. However, if the two residues (Trp' and His') are eliminated, the truncated peptide, (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)peptide, shows no activity. On the other hand, the successive truncation of the C-terminal residues lowers the biological activity step by step.…”

Secretion of human insulin by a transformed yeast cell

“…One pheromone, the a-factor, is a peptide of 13 amino acid residues [2] which is synthesized in the yeast cells as a high molecular mass precursor [3]. The sequencing of the mating factor cyl precursor gene (MFal) revealed that the a-factor is present in four repeats separated by spacer peptides with the sequences of Lys-Arg-(Glu-Ala)3 or Lys-Arg-GluAla-Asp-Ala-Glu-Ala [3].…”