An intimate and multifaceted partnership: cardiolipin and the mitochondrial ADP/ATP carrier

线粒体ADP/ATP载体(AAC)介导ADP进出线粒体的1：1交换 基质，氧化磷酸化所需的一种活性。此前，我们有一个令人兴奋的发现 主要的酵母ADP/ATP载体Aac2与呼吸超复合体(RSC；更高级)相关 单个呼吸复合体的组装)，但仅在包含以下内容的线粒体膜的背景下 独一无二的磷脂心磷脂。随后，我们发现，在这两个 酵母Aac2与两种人Aac亚型的相互作用。综合起来，我们的结果表明 心磷脂对扩大的和临床相关的AAC家族具有普遍重要性，这些家族参与 许多进化上保守的和心磷脂依赖的蛋白质-蛋白质相互作用，因此 被认为在功能上很重要。这些集体发现有力地支持了我们的中心假设 心磷脂依赖的AAC相互作用组代表了线粒体在多发性疾病中的“阿喀琉斯之踵” 由于心磷脂代谢改变而导致的状态。我们正在努力钻探心磷脂- Aac2的依赖性我们确定心磷脂促进Aac2的三级和四级组装， 令人兴奋的是，它是通过不同的机制做到这一点的。我们假设这两个可分离的结构角色 与Aac2组装相关的心磷脂反映了折叠内发生的Aac2-心磷脂的特异性相互作用 载体或在其外围。从内部来看，我们推测有三个保守的心磷脂结合位点支持 载体折叠结构，并可能使其运输相关的构象动力学。装备着一把 一系列设计合理的心磷脂结合Aac2突变体，我们将使用一套 结构、生化、生物物理和功能分析。在外围，我们假设定义 心磷脂在Aac2与呼吸超复合体结合中的作用 III二聚体和1-2个拷贝的复合体IV，涉及Aac2-心磷脂、Aac2-心磷脂和Aac2- 心磷脂-RSC和Aac2-RSC结合时可以稳定这些多蛋白复合体。在目标2中，突变 将被工程化为Aac2以及特定的复合体III和IV亚基来破坏这种保守的相互作用 然后测试我们的假设，即Aac2和呼吸之间的心磷脂依赖关系 超复合体在功能和互惠方面都是有益的。在测试新的第六种型号时，关于功能 RSC的相关性，在这种情况下，那些物理上与AAC相关的RSC，这一目标的结果可能会有所帮助 为目前正在辩论的其他拟议的RSC相关模型提供一个背景框架。总的来说， 这项提案的结果将极大地影响我们对 由于AAC突变和/或心磷脂代谢紊乱而可能发生的AAC相互作用体。反过来, 更好地了解心血管疾病的基本机制，心血管疾病的头号原因 在美国的死亡，将被获得。

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.