Doctoral Dissertations

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey Becker

Committee Members

Stuart Riggsby, Bruce McKee, David Brian

Abstract

During the life cycle of the yeast Saccharomyces cerevisiae, haploid cells of opposite mating type can fuse during sexual conjugation to form a diploid cell. In preparation for conjugation, haploid cells secrete small diffusible peptide molecules [α-factor, a tridecapeptide pheromone and α-factor, a modified dodecapeptide pheromone] that bind specifically to cell surface receptors found on the opposite mating type cell.

The basic structure of the receptors (Ste2p for α-factor and Ste3p for α-factor) is evolutionarily conserved and places them among the 7-transmembrane, G-protein coupled super-family of hormone receptors. Part I of this dissertation is an overview of the life and times of the yeast Saccharomyces cerevisiae as it relates to this work.

Part 2 of this dissertation is a specific study of one residue of the a-factor pheromone. This section describes the possible function of the His² side chain of the a-factor and its contributions to binding to the a-factor receptor and propagation of the extracellular mating signal. While conclusions drawn from assays performed on the altered position 2 side chain analogs indicate that the His2 residue is not essential for binding or biological activity alone, this residue does contribute to the overall efficient transduction of the mating signal.

Part 3 of this dissertation takes a broad look at the entire a-factor pheromone by alanine-scanning the entire peptide. Both the L- and D-isomers of alanine were synthesized in place of each natural amino acid to produce an Ala-scanned "library" of a-factor analogs. Biological and binding assays performed with these peptides on cells expressing the wild-type α-factor receptor led us to propose functional domains of the peptide that play dominant roles in signal transduction, receptor interaction, and stabilization of the biologically relevant structure of the α-factor itself.

In Part 4, an analysis of the x-factor receptor is carried out using random and site-directed mutagenesis to try to pinpoint those residues critical for pheromone binding and signal transduction. Receptors with random mutations were generated and screened for biological response to antagonist peptides. Mutant receptors containing multiple mutations that respond to antagonist molecules were identified by sequencing as candidates with residues responsible for association with the ligand and/or propagation of the mating signal. These candidate residues were targeted for site-directed mutagenesis and tested for an antagonist responsive phenotype. Four mutations were independently introduced into the a-factor receptor. One mutant receptor (S259P) demonstrated ligand dependent biological response to all peptides tested. The S219P mutation demonstrated biological response to a-factor, some antagonist peptides and the synergist, but not to other antagonists. The F55V mutant receptor responded only to a-factor and the synergist peptide and not to any antagonist analogs. The last mutant receptor (1263V) only responded to α-factor.

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