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 whose structure contributes to cellular function. Optic Atrophy 1 (Opa1), a dynamin-related inner mitochondrial membrane (IMM) protein, is a central regulator of cristae shape. Opa1 containing oligomeric complexes are modulated by metabolic changes. To study the specific metabolically dependent Opa1 interactome we generated an Opa1-TurboID (Opa1TID) chimera that localizes to the IMM, displays biotinylation activity, and lies in native complexes of similar stoichiometry to Opa1. Once expressed in Opa1-/- cells, Opa1TID restores mitochondrial ultrastructure and fusion, confirming it vicariates untagged Opa1. We therefore used Opa1TID to unbiasedly identify by label free proteomics 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 media mimicking human plasma. A liquid chromatography-mass spectrometry pipeline identified 231 bona fide mitochondrial proteins as Opa1TID interactors and bioinformatic analysis indicated 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 pinpointing metabolic enzymes relay individual fuel sources to cristae biogenesis machinery. A novel Opa1TID gain-of-function mouse will next uncover the cardiac specific Opa1TID interactome.