Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells.
Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells.
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DOI：
10.1091/mbc.e12-12-0862
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发表时间：
2013-07
影响因子：
3.3
通讯作者：
Riemer J
中科院分区：
文献类型：
--
作者：
Fischer M;Horn S;Belkacemi A;Kojer K;Petrungaro C;Habich M;Ali M;Küttner V;Bien M;Kauff F;Dengjel J;Herrmann JM;Riemer J
氧化折叠促进蛋白质进入线粒体膜间间隙。对完整哺乳动物细胞中这一过程的分析揭示了Mia40、ALR、谷胱甘肽和膜电位的贡献。依赖氧化折叠的蛋白质保持稳定，并在胞浆中还原几分钟。半胱氨酸残基氧化成二硫化物促使许多蛋白质进入线粒体膜间隙。最近在酵母中的研究揭示了线粒体蛋白氧化的基本原理，但在生理条件下的动力学尚不清楚。我们开发了在活的哺乳动物细胞中跟踪蛋白质氧化的方法，这表明蛋白质的输入和氧化折叠是动力学和功能耦合的，并且依赖于氧化还原酶Mia40、肝再生的巯基氧化酶增强因子(ALR)和细胞内的谷胱甘肽池。底物氧化的动力学取决于Mia40的量，并且需要严格平衡的ALR量。依赖Mia40的Cox19在人类细胞中的输入依赖于内膜电位。我们的观察显示，线粒体导入途径的速度有相当大的差异：带有两部分靶向序列的前蛋白在几秒钟内导入，而Mia40的底物在细胞质中停留几分钟，显然避免了过早降解和氧化。
Oxidative folding facilitates protein import into the mitochondrial intermembrane space. An analysis of the process in intact mammalian cells reveals the contributions of Mia40, ALR, glutathione, and the membrane potential. Proteins that rely on oxidative folding remain stable and reduced in the cytosol for several minutes. Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.