Performing discovery-based proteomics using a novel Opa1-TurboID stable cell line to determine the metabolically dependent Opa1 proteome

Cristae are dynamic subcompartments of mitochondria 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. However, 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, facing the intermembrane space and displays biotinylation activity. Once expressed in Opa1-/- cells, OPA1-TurboID restores mitochondrial ultrastructure and fusion, confirming that OPA1-TurboID can vicariate OPA1 and that it can therefore be used in our proteomics workflow to study the molecular determinants of the connection between metabolism and mitochondria ultrastructure. We next immortalized Opa1 fl-fl Murine Embyronic Fibroblasts and used a lentivirus to stably express Opa1-TurboID in our cells. We next cultured our Opa1-TurboID expressing cells in different metabolic conditions (DMEM high glucose, HBSS, Fatty Acids alone, Amino Acids alone, or in a physiologic like cell culture medium called Metablomax) while inducing biotinylation. We next collected protein lysates, trypsin digested the peptides, and performed streptavidin magnetic bead pulldowns to identify biotinylated peptides using liquid chromatography mass spectrometry. Samples are now being run through the LC-MS and we will soon uncover and report the metabolically dependent Opa1 protein interactome.