Discovery-based proteomics using a novel Opa1-TurboID stable cell line reveals the metabolically dependent Opa1 protein interactome

Mitochondrial cristae, the cellular bioenergtic units of life, are dynamic subcompartments involved in respiratory efficiency, control of cytochrome c release and cellular proliferation. Optic Atrophy 1 (OPA1), a dynamin-related IMM protein, is a central regulator of mammalian cristae shape that is retrieved in oligomeric complexes modulated by changes in nutrients and metabolism. Whether and how OPA1 connects fuel availability to cristae dynamics is unexplored. To address this question we generated an OPA1-TurboID chimera that allows us to study the OPA1 interactome upon metabolic changes. The OPA1-TurboID fusion protein correctly localizes to the inner mitochondrial membrane, faces the intermembrane space and displays biotinylation activity. Once expressed in Opa1-/- cells, OPA1-TurboID restores mitochondrial ultrastructure and fusion, confirming OPA1-TurboID can vicariate OPA1. After immortalizing Opa1 fl-fl Murine Embyronic Fibroblasts we stably expressed Opa1-TurboID with a lentivirus and cultured Opa1-TurboID expressing cells in different metabolic conditions (glucose, starvation, ketones and fatty acids, amino acids, or in a physiologic like cell culture medium) while inducing biotinylation. Protein lysates were collected, trypsin digested, biotinylated peptides were enriched with streptavidin beads, and peptides were identified with liquid chromatography mass spectrometry. Bioinformatic analysis identified ~1900 peptides in total and 371 peptides were significantly enriched under specific metabolic conditions revealing the metabolically dependent Opa1 interactome.