Abstract Mitochondria are complex organelles found in virtually all eukaryotic cells. These organelles orchestrate diverse functions such as energy expenditure, nutrient selection, and ion homeostasis, and do so through the coordination of over 1,000 mitochondria-resident proteins. Most of these mitochondrial proteins are phosphorylated under select physiological conditions, but surprisingly little has been done to characterize these organellar modifications. Motivated by the observation that mitochondria house numerous protein phosphatases, we predicted that regulated protein phosphorylation may play a larger role in organellar homeostasis than is currently appreciated. Indeed, our studies show that the knockout of one mitochondrial phosphatase, Pptc7, leads to stark metabolic dysfunction culminating in fully penetrant perinatal lethality in mice. This surprisingly severe pathophysiology indicates that proper management of protein phosphorylation is requisite for mitochondrial homeostasis. Despite these data, it remains unclear how mitochondrial proteins become phosphorylated and the extent to which individual phosphorylation events contribute to mitochondrial function. We will begin to address these gaps in knowledge by mapping the full breadth of substrates of matrix-localized kinases and by testing the cellular compartment in which mitochondrial-destined proteins become phosphorylated. These studies will begin to address longstanding questions as to the mechanisms enabling mitochondrial protein phosphorylation as well as and the genetic identities of its regulators. To complement this work, we will utilize mechanistic, hypothesis-driven approaches to test the effects of phosphorylation on two proteins, Timm50 and Idh2. These two proteins are reproducibly hyperphosphorylated in Pptc7 KO conditions, suggesting they drive at least a subset of the stark phenotypes associated with the knockout of this phosphatase. Furthermore, these two proteins play key roles in mitochondrial protein import and TCA cycle-mediated metabolism and their regulation would likely have broad influence on mitochondrial function. We will test the effects of Timm50 and Idh2 phosphorylation at the biochemical level (determining how this modification affects protein functions), at the cellular level (determining how modulation of these phosphorylation events affect organellar processes such as protein import and metabolic flux), and at the organismal level (testing how phosphorylation of these proteins may mediate pathophysiology – particularly of phenotypes manifested in Pptc7 KO mice). Collectively, this work will link kinases to mitochondrial function and will establish a workflow to delineate the functions of individual phosphorylation events from the biochemical to the physiological level. As kinases are druggable and have had positive clinical impact in human disease, these studies may uncover novel therapeutic targets through which we can resolve mitochondrial dysfunc...