Institute of Molecular Biology and Biotechnology, Crete, Greece.
Antioxid Redox Signal. 2010 Nov 1;13(9):1327-39. doi: 10.1089/ars.2010.3200.
Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.
Erv1 和 Mia40 构成了介导线粒体膜间隙氧化蛋白折叠的二硫键中继系统的两个重要组成部分。Mia40 是识别引入二硫键的底物的输入受体，而它处于还原状态。Erv1 的一个关键功能是将 Mia40 循环回其活性氧化状态。我们的目的是剖析介导 Erv1 与 Mia40 之间蛋白-蛋白相互作用的 Erv1 结构域，并研究 Erv1 的穿梭结构域与其催化核心之间的相互作用及其与 Mia40 的相关性。我们分别纯化了这些结构域以及穿梭和活性核心结构域中的半胱氨酸突变体。通过等温滴定量热法测量 Mia40 与 Erv1 的非共价相互作用，而在体外和在体重建实验中分析它们的穿梭结构域和催化核心之间的共价混合二硫键中间体。我们确定 Erv1 的 N 端穿梭结构域是相互作用所必需和充分的。此外，我们为从 Erv1 的穿梭半胱氨酸对到核心结构域的分子内电子转移提供了直接证据。最后，我们通过添加 Erv1 的 N 和 C 末端结构域以及其底物 Mia40 来重建该系统。
