Dynamic protein phosphorylation shapes mitochondrial catalysis, import, and architecture

Abstract

Phosphorylation is a pervasive mitochondrial protein post-translational modification, yet the extent of its influence over mitochondrial functions remains unclear. To identify dynamic mitochondrial phosphoisoforms, we profiled the phosphoproteome of AML12 cells following individual genetic perturbations of 10 resident mitochondrial phosphatases. Guided by these results, we investigated the functional implications of phosphorylation on mitochondrial processes related to catalysis, import, and morphology. We find that phosphorylation of adenylate kinase 2 suppresses its nucleotide binding and catalytic activity. Elevated phosphorylation proximal to the mitochondrial targeting sequence of branched chain ketoacid dehydrogenase kinase disrupts its import and processing, resulting in enhanced catabolism of branched chain amino acids. Most notably, we discover extensive phosphorylation on proteins involved in mitochondrial cristae architecture, including multiple members of the MICOS complex and ATP synthase, upon silencing the phosphatase PGAM5. Collectively, our work connects reversible phosphorylation and resident phosphatases to the regulation of diverse mitochondrial processes. HIGHLIGHTS Perturbation of 10 mitochondrial resident phosphatases maps dynamic phosphosites. AK2 S151 phosphorylation tunes catalysis by augmenting adenine nucleotide binding. MTS-proximal BCKDK phosphorylation impairs protein import and gates BCAA catabolism. MIC19 and ATP5I phosphorylation remodels cristae and modulates bioenergetics.