Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase
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(2020) 21:86
BMC Molecular and Cell Biology
RESEARCH ARTICLE
Open Access
Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase R. Christian McDonald, Matthew J. Schottˆ, Temitope A. Idowu and Peter J. Lyons*
Abstract Background: Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results: A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions: We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest 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. Keywords: Saccharomyces cerevisiae, Fungi, Protease, Carboxypeptidase, Prodomain, Pseudoenzyme, Synthetic lethal assay, Cell wall, Extracellular matrix
Background Most major enzyme families contain inactive enzyme homologs, or pseudoenzymes. Pseudoenzymes are characterized by highly conserved domains that are structurally similar to their catalytically active homologs, but with substitutions within key motifs suggesting a loss of catalytic function [1, 2]. Research is just now beginning to show the large part that these proteins play in biology; * Correspondence: [email protected] ˆMatthew J. Schott is deceased. Department of Biology, Andrews University, Berrien Springs, MI, USA
they are much more than just innocent bystanders [3]. Pseudoenzymes have been shown to play key roles as allosteric modulators and non-catalytic competitors in a variety of signa
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