Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae

Author

Susan Kay Raths

Date of Award

12-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey M. Becker

Committee Members

Stuart Riggsby, John Koontz, Tom Chen

Abstract

Alpha-factor is a peptide of thirteen amino acids which is required for mating between the haploid mating types, a and α, in Saccharomyces cerevisiae. An analogue of alpha-factor, DHP⁸ DHP¹¹ Nle¹² tridecapeptide, was catalytically reduced in the presence of ³H gas for production of a radiolabelled pheromone suitable for use in binding studies. Incorporation of tritium resulted in ³H-alpha-factor with high specific activity, purity, biological activity and long shelf-life. Binding studies revealed that alpha-factor interacts with its receptor via a simple, reversible process which obeys the law of mass action. Association and dissociation kinetics indicate values of 2.92 X 10⁶ M^-1 min^-1 for k₁ and between 4 and 7 X 10^-2 min^-1 for k_-1. Saturation binding studies reveal an equilibrium dissociation constant equal to 2.32 X 10^-8 M which approximates the kinetically-derived K_D of 2.12 X 10^-8 M. Scatchard and Hill analyses as well as dissociation behavior in the presence of excess unlabelled ligand indicate alpha-factor interacts with a homogeneous population of binding sites (13000 sites/cell) which do not interact and exhibit one affinity for the alpha-factor pheromone. Studies using unlabelled competitors confirm the specificity expected for a receptor-mediated process, i.e., binding affinities of alpha-factor analogues parallel their efficiency at inducing biological activities. Preliminary evidence obtained from the thermodynamic analysis of the temperature dependence of binding of alpha-factor (an agonist) and desTrp¹ Ala³ dodecapeptide (an antagonist) suggests that agonist binding is entropy-driven and antagonist binding is enthalpy-driven. This may reflect differences observed when binding of the ligand involves interaction with, or traversing of, the cell wall.

Recommended Citation

Raths, Susan Kay, "Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae. " PhD diss., University of Tennessee, 1987.
https://trace.tennessee.edu/utk_graddiss/12157