Intermembrane space proteome of yeast mitochondria.
Intermembrane space proteome of yeast mitochondria.
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DOI：
10.1074/mcp.m112.021105
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发表时间：
2012-12
期刊：
影响因子：
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通讯作者：
Meisinger C
中科院分区：
文献类型：
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作者：
Vögtle FN;Burkhart JM;Rao S;Gerbeth C;Hinrichs J;Martinou JC;Chacinska A;Sickmann A;Zahedi RP;Meisinger C
膜间隙（IMS）代表线粒体的最小亚隔室。然而，它在蛋白质、脂质和金属离子的转运和修饰以及呼吸链复合物的调节和组装中起着重要作用。此外，它参与许多氧化还原过程，并协调程序性细胞死亡的关键步骤。IMS蛋白质的全面分析迄今尚未进行。我们已经建立了一种方法，使用促凋亡蛋白Bax从分离的线粒体释放IMS蛋白，我们分析了该隔室的蛋白质组成。使用稳定的同位素标记的线粒体从酿酒酵母，我们能够测量特定的IMS依赖的蛋白质释放和定量释放IMS蛋白质和基质蛋白的背景流出之间的区别。从已知的31种可溶性IMS蛋白中，可重复地鉴定出29种蛋白，对应于> 90%的覆盖率。此外，我们还发现了20个新的膜间隙蛋白，其中10个以前没有定位于线粒体。许多这些新的IMS蛋白具有未知的功能或已被报道在氧化还原调节中发挥作用。我们确认IMS本地化的15个蛋白质使用的organello进口，蛋白酶的可及性渗透溶胀，并释放测定。此外，我们确定了两种新的线粒体蛋白，Ymr 244 c-a（Coa 6）和Ybl 107 c（Mic 23），作为MIA输入途径的底物，具有不寻常的半胱氨酸基序，并发现蛋白磷酸酶Ptc 5是内膜蛋白酶（IMP）的新底物。对于CoA 6，我们发现了作为细胞色素c氧化酶复合物的新组装因子的作用。我们在这里提出了第一个和全面的蛋白质组IMS蛋白的酵母线粒体共51个蛋白质。IMS蛋白质组将作为一个有价值的来源，为进一步研究IMS在细胞生命和死亡中的作用。
The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.