Origin of mitochondrial proton leak: comparative investigation of Adenine Nucleotide, Translocase, Phosphate and Aspartat/Glutamate Carriers

在氧化磷酸化过程中，质子通过F1/F0-ATP合成酶被驱回基质或通过替代途径返回(泄漏)，从而降低了ATP合成的效率。质子泄漏的起源和意义尚不清楚。如果由解偶联蛋白亚家族的成员催化，它会导致棕色脂肪组织的非颤抖产热(UCP1)，或者可能参与其他组织中反应性氧合酶物种的调节(UCP2-UCP5)。对分离的线粒体的实验表明，其他线粒体载体也可能对H+泄漏起到重要作用。在本项目中，将使用由高度纯化的蛋白质：腺核苷酸转位酶(ANT)、磷酸(PIC)或(III)天冬氨酸/谷氨酸载体(AGC)重组的模型膜来研究后者。将研究膜脂组成、跨膜电位和偶极电位的调节作用。脂肪酸与蛋白质结合的必要性将通过定点突变得到证明。比较ANT、PIC、AGC和解偶联蛋白的单分子质子电导，评估它们对基础质子泄漏和对抑制剂敏感的膜质子泄漏的相对贡献。

During oxidative phosphorylation protons are driven back into the matrix through the F1/F0-ATP synthase or return through alternative pathways (leak), lowering the efficiency of ATP synthesis. Origin and significance of the proton leak is not well understood. If catalyzed by members of uncoupling protein subfamily it results in nonshivering thermogenesis in brown adipose tissue (UCP1) or it may participate in the regulation of reactive oxygene species in other tissues (UCP2-UCP5). Experiments on isolated mitochondria indicate that also other mitochondrial carriers may significantly contribute to H+ leak. The latter will be investigated in the present project using model membranes reconstituted with highly purified proteins: adenine nucleotide translocase (ANT), phosphate (PIC) or (iii) aspartate/glutamate carriers (AGC). The modulating role of membrane lipid composition, transmembrane and dipole potentials will be studied. The necessity of the fatty acid binding to proteins will be proved by site-directed mutagenesis. Comparing the single molecule proton conductances of ANT, PIC, AGC and uncoupling proteins, their relative contributions to basal and inhibitor-sensitive membrane proton leaks will be evaluated.