Date of Award

2020

Document Type

Thesis

Degree Name

Master of Science

College

College of Arts and Sciences

Program

Biology, MS

First Advisor

Peter J. Lyons

Second Advisor

Denise L. Smith

Third Advisor

H. Thomas Goodwin

Abstract

Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains several pseudoenzymes predicted to lack enzyme activity and with unknown molecular function. The genome of the yeast Saccharomyces cerevisiae encodes only one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. Ecm14 is found throughout ascomycete fungi, with a group of related pseudoenzymes found in basidiomycetes. Although the prodomain of Ecm14 can be cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism, no activity has been detected using standard carboxypeptidase substrates.

In order to examine the function of Ecm14 using an unbiased screen, a series of synthetic lethal assays were performed. Because biochemical pathways typically involve multiple key gene components, many of these key gene players provide redundancy if other genes experience deleterious mutations. The synthetic lethal screen identifies novel mutants whose survival depends on a particular gene of interest. Approximately 27,000 yeast colonies were screened; however, only twenty-two putative ecm14 synthetic lethal mutants were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker (plasmid reporting and selection) genes and synthetic lethality due to multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14. Recent research by others suggests a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae. Thus, Ecm14 is likely an important, conserved, and “activatable” component of extracellular processes that are not critical to most modern yeast strains.

Subject Area

Saccharomyces cerevisiae; Enzymes; MCPs (Metallocarboxypeptidases)

DOI

https://dx.doi.org/10.32597/theses/188/

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