Investigations of the Saccharomyces cerevisiae a-factor lipopeptide : structure-function relationships and target cell responses

The budding yeast Saccharomyces cerevisiae can exist in stable haploid and diploid forms. Mating between the two haplotypes, termed a and a, is mediated by the reciprocal secretion of two peptide pheromones, a and α-factor. The a-factor is a 12 amino-acid lipopeptide which is post-translationally modified by the addition of a carboxymethyl group and a 15-carbon farnesyl moiety prior to export from the a-cell by Ste6p, the yeast homolog of mammalian multi-drug resistance P-glycoprotein. Both pheromones bind to a specific receptor on the surface of the opposite mating type and initiate a transduction cascade resulting in physiological and morphological changes.

Previous studies had established a role for endopeptidase action in recovery of cells from pheromone-induced growth arrest. In order to examine the identified α-cell-specific activity capable of degrading a-factor, attempts were made to clone the gene encoding this enzyme utilizing a variety of biochemical and genetic techniques (Part II).

The role of Ste6p as a transporter of a-factor has been well established. However, observations made previously suggested that this protein could also be involved in another aspect of the mating event, possibly by maintaining a gradient of a-factor concentration outside the cell. To further investigate this possibility, strains were constructed that contained deletions of STE6 and then challenged for the ability to mate in the presence of exogenous pheromone. This revealed that with Ste6p absent, these cells were still able to mate, thus indicating that this transporter does not play an additional role in mating (Part III).

Although only a small peptide, the structure of a-factor has been shown to be important for bioactivity. Using synthetic peptides and peptidomimetic compounds, studies were undertaken to clarify the suggested requirement for a bend in the peptide. By incorporating stereoisomers of alanine at position 5, it was discovered that substitution with a D-amino acid enhanced biological activity (Part IV). Subsequent synthesis of constrained peptidomimetic analogs confirmed this observation, with activities increased by up to 32 fold over the wild-type compound (Part V). These data indicate the structural requirement which allows this ligand to optimally associate with the receptor involves a bend centered around the gly⁵ residue.

The farnesyl component of a-factor had been suggested to be primarily a device to localize the peptide to the membrane. Analyzing the biological activities of synthetic analogs with altered farnesyl groups that contained substitutions of the 3-methyl moiety with bulkier additions demonstrated that enhancement of response was possible (Part VI). This suggested that the farnesyl group may also play a role in presentation of the peptide portion to the receptor in addition to membrane localization.