The interactomes of the mitochondrial cristae-shaping protein Opa1 vary depending on the oxidative metabolism of carbon sources

Mitochondrial cristae, the bioenergetic units of life, are dynamic compartments with distinct structures that contribute to respiratory efficiency, control of cytochrome c release and cellular proliferation. Optic Atrophy 1 (Opa1), a dynamin-related inner mitochondrial membrane (IMM) protein, is a central regulator of cristae shape that is retrieved in oligomeric complexes modulated by metabolic changes. Whether and how Opa1 connects fuel availability to cristae dynamics is unexplored. To address this question, we studied the Opa1 interactome upon changes in fuel availability. We generated an Opa1-TurboID (Opa1TID) chimera that correctly localizes to the IMM, faces the intermembrane space, displays biotinylation activity, and lies in high molecular weight complexes of similar stoichiometry to Opa1. Once expressed in Opa1-/- cells, Opa1TID restores mitochondrial ultrastructure and fusion, confirming it can vicariate untagged Opa1. We therefore used Opa1TID to unbiasedly identify by label free proteomics the changes in the Opa1 interactome when Opa1fl/fl Murine Embryonic Fibroblasts stably expressing Opa1TID were exposed to glucose, starvation, fatty acids, amino acids, or a complete cell culture medium mimicking human plasma. A pipeline of liquid chromatography-mass spectrometry detection of the biotinylated proteins identified 231 bona fide mitochondrial proteins as Opa1TID interactors. A bioinformatic analysis indicated that unique proteins were significantly enriched in distinct metabolic conditions. This discovery of the carbon source-dependent Opa1TID interactome indicates an axis between fuel availability and Opa1-mediated cristae dynamics and pinpoints metabolic enzymes that can relay individual fuel source to the cristae biogenesis machinery.