Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, 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, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL 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 CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

中文翻译：

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线粒体 ADP/ATP 载体上心磷脂结合位点的功能多样性。


脂质-蛋白质相互作用在膜稳态中发挥着多种重要作用。线粒体膜具有独特的脂蛋白环境，可确保生物能效率。心磷脂 (CL) 是线粒体的标志性脂质，在促进氧化磷酸化 (OXPHOS) 中发挥多种作用。在线粒体内膜中，ADP/ATP 载体（酵母中的 AAC；腺嘌呤核苷酸转运蛋白，哺乳动物中的 ANT）交换 ADP 和 ATP，从而实现 OXPHOS。 AAC/ANT 包含三个紧密结合的 CL，这些相互作用在进化上是保守的。在这里，我们结合生化方法、天然质谱和分子动力学模拟研究了这些埋藏的 CL 在 AAC/ANT 中的作用。我们将带负电荷的突变引入酵母 Aac2 的每个 CL 结合位点，并通过实验证实这些突变破坏了 CL 相互作用。虽然所有突变都破坏了 Aac2 三级结构的稳定性，但转运活性以结合位点特异性方式受到损害。此外，我们确定人类 ANT1 的一个 CL 结合位点中与疾病相关的错义突变损害了其结构和转运活性，导致 OXPHOS 缺陷。我们的研究结果强调了 CL 在 AAC/ANT 结构和功能中的保守意义，与特定的脂质-蛋白质相互作用直接相关。