Mitochondrial ADP/ATP carriers (Aac) mediate the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix, an activity that is required for oxidative phosphorylation. Previously, we made the exciting discovery that the major yeast ADP/ATP carrier, Aac2, associates with the respiratory supercomplex (RSC; higher order assemblies of individual respiratory complexes) but only in the context of mitochondrial membranes that contain the unique phospholipid cardiolipin. Subsequently, we established that there is substantial overlap between the interactomes of yeast Aac2 and two human Aac isoforms. When combined, our results demonstrate that cardiolipin is of general importance to the extended and clinically relevant Aac family which participate in numerous evolutionarily conserved and cardiolipin-dependent protein-protein interactions that are therefore presumed to be functionally important. These collective findings strongly support our central hypothesis that the cardiolipin-dependent Aac interactome represents the mitochondrion’s “Achilles’ heel” in the multiple disease states that result from altered cardiolipin metabolism. In our ongoing efforts to drill into the cardiolipin- dependency of Aac2 we determined that cardiolipin promotes both the tertiary and quaternary assembly of Aac2, and excitingly, it does so via distinct mechanisms. We hypothesize that these two separable structural roles of cardiolipin with respect to Aac2 assembly reflect specific Aac2-cardiolipin interactions occurring within the folded carrier or on its periphery. From within, we speculate that three conserved cardiolipin-binding sites support the carriers folded structure and potentially enable its transport-related conformational dynamics. Armed with a series of rationally designed cardiolipin-binding Aac2 mutants, we will test our hypothesis using a suite of structural, biochemical, biophysical, and functional analyses. On the periphery, we hypothesize that the defining role of cardiolipin for the association of Aac2 with respiratory supercomplexes, composed in yeast of a complex III dimer and 1-2 copies of complex IV, involves individually weak interactions between Aac2-cardiolipin, Aac2- cardiolipin-RSC, and Aac2-RSC that when combined stabilize these multi-protein complexes. In Aim 2, mutations will be engineered into both Aac2 and specific complex III and IV subunits to disrupt this conserved interaction and then test our hypothesis that the cardiolipin-dependent association between Aac2 and the respiratory supercomplex is functionally and reciprocally beneficial. In testing a novel sixth model as to the functional relevance of RSCs, in this case those RSCs physically associated with Aac, results from this aim may help provide a contextual framework for the other proposed RSC-related models which are currently debated. Overall, results from this proposal will significantly impact our understanding of the consequences of alterations in the Aac interactome that may occur due to mutations in Aac and/or perturbations in cardiolipin metabolism. In turn, a greater understanding of basic mechanisms contributing to cardiovascular disease, the number one cause of death in the United States, will be obtained.

Project Narrative: As most cellular energy is generated by mitochondria, perhaps it is not surprising that about 1 in 5000 children will develop mitochondrial disease. This proposal focuses on a protein component of mitochondrial membranes, the ADP/ATP carrier, that is indispensable for energy production by mitochondria and which participates in a number of evolutionarily conserved physical interactions that are dependent on a single phospholipid, cardiolipin, only found in mitochondrial membranes. Several mutations in the ADP/ATP carrier cause heart and muscle disease and alterations in the normal metabolism of the phospholipid cardiolipin, which may result in defective ADP/ATP carrier assembly and/or function, have been documented in numerous distinct cardiomyopathies.