Searching for the Function of Ecm14, a Pseudopeptidase, Using a Synthetic Lethal Assay

Abstract

Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme‐homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. The human M14 metallocarboxypeptidase (MCP) family is one such enzyme family that contains several active and inactive members. The baker's yeast Saccharomyces cerevisiae contains only one M14 MCP member, Ecm14, which is produced by a non‐essential gene in the yeast genome. We show through a phylogenetic analysis that this gene is present throughout the ascomycete lineage of fungi. Although Ecm14 is very similar to human CPA1 (a catalytically active member of the M14 MCP family), multiple key modifications in Ecm14 seem to indicate a loss of traditional peptidase‐like activity. A key technique for elucidating the function of non‐essential genes, including those producing pseudoenzyme products, is the genome‐wide synthetic lethal screen. Biochemical pathways typically involve multiple gene components and many of these gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color‐reporting plasmid that complements the deletion of the endogenous gene of interest. Here we demonstrate the insertion of the wild‐type ECM14 gene into this integral pSLS1 plasmid and the determination of gene insert orientation using restriction endonucleases. Additionally, we demonstrate the preparation of knockout yeast strains using drug resistant deletion cassettes, the transformation of these yeast with the above plasmid, and finally, genome‐wide mutagenesis attempts using the chemical mutagen ethyl methanesulfonate. Identification of synthetic lethal genes will be presented, as a step toward a better understanding of the biological role of Ecm14.Support or Funding InformationGraduate Student Grants in Aid of Research to RCM and Faculty Research Grants to PJL from the Office of Research & Creative Scholarship, Andrews University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.