Abstract 1951 Conserved and specific cardiolipin-mitochondrial ADP/ATP carrier interactions assume structural and functional roles

Lipid-protein interactions are crucial for maintaining membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin, the signature phospholipid in mitochondria, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP to facilitate OXPHOS. AAC/ANT contains three tightly bound cardiolipins, and these interactions are evolutionarily conserved. In this study, we investigated the role of these buried cardiolipins in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each cardiolipin-binding site of yeast Aac2 and established experimentally that the mutations disrupted the cardiolipin interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one cardiolipin-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of cardiolipin in AAC/ANT structure and function, directly tied to specific lipid-protein interactions. This work was supported in part by NIH grants, and post-doctoral fellowships from the Uehara Memorial Foundation, and the American Heart Association and the Barth Syndrome Foundation.