In vivo mitochondrial matrix proteome profiling reveals RTN4IP1/OPA10 as an antioxidant NADPH oxidoreductase
Targeting proximity labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice expressing a mitochondrial matrix-targeted ascorbate peroxidase (MAX-Tg) to analyze tissue-specific matrix proteomes. Desthiobiotin-phenol labeling of muscle tissues from MAX-Tg mice allowed for efficient profiling of mitochondrial-localized proteins in these tissues. Comparative analysis of matrix proteomes from MAX-Tg muscle tissues revealed differential enrichment of mitochondrial proteins related to energy production in between different muscle groups. Reticulon 4 interacting protein 1 (RTN4IP1), also known as Optic Atrophy-10 (OPA10), was highly enriched in the cardiac and soleus muscles and was found to localize to the mitochondrial matrix via a strong mitochondrial targeting sequence at its N-terminus. Protein structure analysis revealed that RTN4IP1 is an NADPH oxidoreductase with structural homology to bacterial quinone oxidoreductase. Enzymatic activity assays, interactome analysis, and metabolite profiling confirmed a function for RTN4IP1 in coenzyme Q (CoQ) biosynthesis. Rtn4ip1-knockout C2C12 cells had reduced CoQ9 levels, were vulnerable to oxidative stress, and had decreased oxygen consumption rates and ATP production. Collectively, RTN4IP1 is a mitochondrial antioxidant NADPH oxidoreductase supporting oxidative phosphorylation activity in muscle tissue.