Functional consequences of structural alterations to the isoprenylated yeast mating pheromone a-factor

In the course of its biosynthesis, secretion, and biological action, the isoprenylated mating pheromone of Saccharomyces cerevisiae, a-factor, interacts with representative proteins of several biologically significant protein families. The a-factor is posttranslationally modified by the enzyme famesyl transferase, exported by a mechanism requiring the action of a yeast homolog of the mammalian multidrug resistance Pglycoprotein, and induces mating via interaction with a protein that shares homology to the G protein-coupled family of receptors. The main objective of this research was to establish and employ a system for the analysis of the structure-function relationships involving a-factor as it serves as either a substrate or ligand for its cellular target proteins. An overview of the literature, as it is related to this work, is presented in Part I of this dissertation. Part II of this dissertation outlines experimental procedures developed in order to establish a system for structure-function relationship studies on a-factor. This entailed the use of oligonucleotide-directed site-specific mutagenesis to introduce precise genetic mutations within the MFa1 gene, encoding a-factor, coupled with a physiological assay for pheromone bioactivity to discern the consequences of such alterations. These experiments led to the characterization of two specific mutations which blocked the production of a-factor in vivo. Part III of this dissertation describes an analysis of the biological consequences of altering the isoprene targeting motif within a-factor to code for posttranslational modification with a geranyl, geranyl isoprene group instead of the native farnesyl lipid. This study demonstrated that differential isoprenylation of a-factor with either farnesyl or geranyl, geranyl results in a secreted and bioactive peptide hormone. Furthermore, biochemical analysis of the peptide pheromones produced by yeast overexpressing a-factor containing a specific geranyl, geranyl targeting sequence indicated that cross-prenylation of such an a-factor substrate peptide with either isoprene can occur in vivo. Parts IV and V of this dissertation summarize the results obtained from an investigation into the bioactivity of a-factor peptides that contain amino acid substitutions or truncations. Specifically, Part IV discusses the identification of an a-factor analog which exhibits a stereospecific superactivity. In two separate bioassays, the [D-Ala⁵] a-factor was found to have biological activity higher than both a-factor and a [L-Ala⁵] a-factor analog. Part V reports the results of activity analyses on synthetic a-factor analogs containing truncations at the N-terminus of the peptide. In addition, a physiological screen was employed to identify non-active a-factor peptides selected from yeast expressing a pool of a-factor genes that were randomly mutated within the mature portion of the peptide. DNA sequence analysis of the genes encoding the selected peptides revealed amino acid substitutions predominantly at specific positions within the peptide, implicating these residues in pheromone bioactivity. These combined studies represent an initial inquiry into the relative contribution of specific amino acids to a-factor activity. Finally, Part VI of this dissertation serves to summarize and relate the findings of this work to current studies in progress and acts as a forum for the proposal of future directions of a-factor related research.