Biosynthesis and target cell interactions of the Saccharomyces cerevisiae a-factor mating pheromone

Author

Stevan Marcus

Date of Award

5-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Jeffrey M. Becker

Committee Members

Mary Ann Handel, W. Stuart Riggsby, Robert Villafane

Abstract

The a-factor mating pheromone of Saccharomyces cerevisiae is an isoprenylated, carboxy methyl esterified dodecapeptide (NH₂-YIIKGVFWDPAC[farnesyl]-COOCH₃] which is post-translationally modified in a manner analogous to that of the ras protooncogene product, Ras. The objectives of this research were to 1) detail steps involved in a-factor maturation and 2) gain an understanding of the interactions of a-factor and its MATα target cell. A comprehensive overview of a-factor-related research leading to this work is presented in Part I of this dissertation. Part II of this dissertation summarizes efforts made toward the characterization of the a-factor post-translational processing pathway. An in vitro maturation assay was developed which demonstrated that the order of a-factor -CAAX post-translational processing steps is as follows: 1) isoprenylation of the cysteine sulfur, 2) proteolysis of -AAX, and 3) carboxy terminal methyl esterification. Additional experiments demonstrated that the RAM gene product is required for farnesyl transferase activity and that the activity is dependent on a divalent cation. Part III of this dissertation discusses a detailed analysis of the role of the C-terminal cysteine modifications in the biological activity of a-factor. Both the farnesyl and carboxy methyl ester modifications of a-factor were found not to be absolutely essential for pheromone activity, and could be replaced by other moieties to produce analogs with varying degrees of biological activity. However, replacement of both the farnesyl and carboxy methyl ester groups by hydrogen atoms resulted in a pheromone with negligible activity. Mating restoration assays demonstrated that MATa cells do not have to actively produce a-factor in order to mate. Part IV of this dissertation outlines studies on an a cell specific a-factor degrading activity, which was found to be cell-associated, endoproteolytic, and not required for response to pheromone. a-Factor degradation was not energy-dependent nor did it require pheromone internalization or interaction with its receptor. Evidence is also presented which demonstrates that a-factor degradation most likely plays a role in the recovery of α cells from the pheromone response. Finally, Part V of this dissertation summarizes and interconnects Parts II - IV and suggests directions for future studies on a-factor.

Recommended Citation

Marcus, Stevan, "Biosynthesis and target cell interactions of the Saccharomyces cerevisiae a-factor mating pheromone. " PhD diss., University of Tennessee, 1991.
https://trace.tennessee.edu/utk_graddiss/11177