Reddehase Silvia, Grumbt Barbara, Neupert Walter, Hell Kai
Adolf-Butenandt-Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, Butenandtstrasse 5, 81377 München, Germany.
J Mol Biol. 2009 Jan 16;385(2):331-8. doi: 10.1016/j.jmb.2008.10.088. Epub 2008 Nov 7.
Cells protect themselves against oxygen stress and reactive oxygen species. An important enzyme in this process is superoxide dismutase, Sod1, which converts superoxide radicals into water and hydrogen peroxide. The biogenesis of functional Sod1 is dependent on its copper chaperone, Ccs1, which introduces a disulfide bond and a copper ion into Sod1. Ccs1 and Sod1 are present in the cytosol but are also found in the mitochondrial intermembrane space (IMS), the compartment between the outer and the inner membrane of mitochondria. Ccs1 mediates mitochondrial localization of Sod1. Here, we report on the biogenesis of the fractions of Ccs1 and Sod1 present in mitochondria of Saccharomyces cerevisiae. The IMS of mitochondria harbors a disulfide relay system consisting of the import receptor Mia40 and the thiol oxidase Erv1, which drives the import of substrates with conserved cysteine residues arranged in typical twin Cx(3)C and twin Cx(9)C motifs. We show that depletion of Mia40 results in decreased levels of Ccs1 and Sod1. On the other hand, overexpression of Mia40 increased the mitochondrial fraction of both proteins. In addition, the import rates of Ccs1 were enhanced by increased levels of Mia40 and reduced upon depletion of Mia40. Mia40 forms mixed disulfides with Ccs1, suggesting a role of Mia40 for the generation of disulfide bonds in Ccs1. We suggest that the disulfide relay system transfers disulfide bonds via Mia40 to Ccs1, which then shuttles disulfide bonds to Sod1. In conclusion, the disulfide relay system is crucial for the import of Ccs1, thereby affecting the transport of Sod1, and it can control the distribution of Ccs1 and Sod1 between the IMS of mitochondria and the cytosol.
细胞保护自身免受氧化应激和活性氧的影响。此过程中的一种重要酶是超氧化物歧化酶Sod1，它将超氧自由基转化为水和过氧化氢。功能性Sod1的生物合成依赖于其铜伴侣Ccs1，Ccs1将一个二硫键和一个铜离子引入Sod1。Ccs1和Sod1存在于细胞质中，但也存在于线粒体膜间隙（IMS），即线粒体外膜和内膜之间的间隔区。Ccs1介导Sod1的线粒体定位。在此，我们报告酿酒酵母线粒体中Ccs1和Sod1各部分的生物合成情况。线粒体的IMS含有一个由导入受体Mia40和硫醇氧化酶Erv1组成的二硫键中继系统，该系统驱动具有保守半胱氨酸残基的底物导入，这些残基以典型的双Cx(3)C和双Cx(9)C基序排列。我们发现，Mia40的缺失导致Ccs1和Sod1水平降低。另一方面，Mia40的过表达增加了这两种蛋白质的线粒体部分。此外，Mia40水平的增加提高了Ccs1的导入速率，而Mia40缺失时则降低。Mia40与Ccs1形成混合二硫键，表明Mia40在Ccs1中二硫键的形成中起作用。我们认为，二硫键中继系统通过Mia40将二硫键转移到Ccs1，然后Ccs1将二硫键穿梭到Sod1。总之，二硫键中继系统对于Ccs1的导入至关重要，从而影响Sod1的转运，并且它可以控制Ccs1和Sod1在线粒体IMS和细胞质之间的分布。
