Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis

Abstract

20functional relevance of these modifications are largely unclear. Nonetheless, mitochondria 21 possess multiple resident phosphatases, suggesting that protein dephosphorylation may be 22 broadly important for mitochondrial activities. To explore this, we deleted the poorly characterized 23 matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7 -/mice 24 exhibited marked hypoketotic hypoglycemia, elevated acylcarnitines, and lactic acidosis, and died 25 soon after birth. Pptc7 -/tissues had significantly diminished mitochondrial size and protein content 26 despite normal transcript levels, but consistently elevated phosphorylation on select mitochondrial 27 proteins. These putative Pptc7 substrates include the protein translocase complex subunit 28 Timm50, whose phosphorylation reduced import activity. We further find that phosphorylation in 29 or near the mitochondrial targeting sequences of multiple proteins can disrupt their import rates 30 and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for 31 proper mitochondrial function and biogenesis during the extrauterine transition. 33 34Mitochondria are multifaceted organelles required for metabolic and signaling processes within 35 almost every eukaryotic cell type 1 . Beyond their production of ATP through oxidative 36 phosphorylation, mitochondria play key roles in macromolecular biosynthesis, ion homeostasis, 37 redox signaling, and apoptotic cell deathactivities that must be calibrated to changing cellular 38 needs. Recent investigations suggest that these and other mitochondrial functions may be 39 affected by post-translational modifications (PTMs), such as phosphorylation 2,3 and acylation 4,5 . 40These PTMs are found on hundreds of mitochondrial proteins 6,7 , and can alter enzyme function 6 , 41 complex assembly 8 , and metabolic flux 9,10 . However, other studies suggest that these 42 mitochondrial PTMs can arise non-enzymatically 11,12 and are often found at low stoichiometry 2,13-43 15 , calling into question whether and to what extent these modifications exert regulatory functions. 44Although much remains to be established about the overall nature and importance of 45 reversible phosphorylation in mitochondria, it is clear that these organelles possess a number of 46 resident phosphatases [16][17][18] . For example, it has long been known that the pyruvate 47 dehydrogenase 9 and branched chain ketoacid dehydrogenase complexes 19 include bound 48 phosphatases (and kinases) that regulate their activities. However, beyond these, there exist 49 other poorly characterized mitochondrial proteins that possess known or predicted protein 50 phosphatase domains [16][17][18] , suggesting that protein dephosphorylation may be of more widespread 51 importance in mitochondria than is currently appreciated. To begin exploring this, we recently 52 analyzed three S. cerevisiae mitochondrial phosphatase deletion strains (∆ptc5, ∆ptc6, and 53 ∆ptc7) 18 and identified phenotypes and putative substrates uni...