In the MitoFORMSinHF project we first solely feed cells single carbon sources and characterized how specific carbon sources contributed to Opa1-dependent mitochondrial morphology using super resolution microscopy. Next, we set out to precisely determine how metabolic sources regulate Opa1-macromolecular assembly by fusing Opa1 to a proximity dependent biotin ligase named TurboID generating an Opa1-TurboID chimeric protein. 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 endogenous Opa1 and that Opa1-TurboID is appropriate to use to evaluate how changes in the Opa1 protein interactome. We therefore used Opa1-TurboID to unbiasedly identify by label free proteomics changes in the Opa1 protein interactome by culturing cells stably expressing Opa1-TurboID 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 231 bona fide mitochondrial proteins as candidate Opa1-TurboID interactors and that interactors were uniquely and significantly enriched in distinct metabolic conditions. We also generated a Slc25a12 knockout cell line and subsequent investigation into this cell line unveiled the mitochondrial aspartate-glutamate carrier is a metabolic sensor that orchestrates organelle morphology and cristae maintenance through interactions with Opa1. Last, we also have generated a first-of-its-kind conditional Opa1-TurboID gain-of-function murine model which we have breed to obtain a cardiac specific, tamoxifen inducible Opa1-TurboID gain-of-function mice (Opa1iHTID). In future studies we will subject the Opa1iHTID mouse model to ischemia-reperfusion injury to identify specific protein candidates implicated in Opa1-mediated cristae remodeling in I/R injury and during heart failure.
Overall our results indicate axes exists between fuel availability and Opa1-mediated cristae dynamics pinpointing metabolic enzymes relay individual fuel sources to cristae biogenesis machinery.
The Opa1-TurboID plasmid is being deposited into the addgene plasmid depository. The large-scale proteomics dataset of the results of the discovery based proteomics screen is being deposited onto public repositories of mass spectrometry datasets. Novel genetically modified cell and mouse models will made available to researchers upon request.
