Abstract
Mitoproteases are emerging as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Using multi‐omic analyses and machine learning, we have predicted a distinct association between one mitoprotease in Saccharomyces cerevisiae, Oct1p, and coenzyme Q (CoQ) biosynthesis—a pathway essential for mitochondrial respiration. More specifically, we have discovered that Oct1p directly processes the N‐terminus of at least one CoQ‐related biosynthesis enzyme. Mutations to the N‐terminus of this substrate inhibit Oct1p processing in vivo, leading to CoQ deficiency and subsequent respiratory in competency. The objective of this study is to confirm in vitro that Oct1p and its disease‐related human ortholog MIPEP assist in the biosynthesis of CoQ via processing of one or more proteins in the CoQ pathway. Recombinant Oct1p and MIPEP were expressed in Escherichia coli cells and were purified via dual immobilized metal affinity chromatography (IMAC). Protease activity was quantified using Fluorescence Resonance Energy Transfer (FRET) peptides. Activity for both Oct1p and MIPEP was verified using generic control peptides, enabling us to test CoQ‐related proteins as direct substrates. This work reveals the importance of a post‐translational processing event for CoQ biosynthesis, and connects a mitoprotease to a new mitochondrial function.
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