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.