The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase
The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase
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DOI：
10.1111/j.1742-4658.2006.05480.x
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发表时间：
2006-11-01
期刊：
影响因子：
5.4
通讯作者：
Anderka, Oliver
中科院分区：
文献类型：
--
作者：
Bachmann, Julie;Bauer, Brigitte;Anderka, Oliver
Rieske [2Fe-2S]蛋白（ISP）是线粒体和细菌呼吸链中细胞色素bc(1)复合物的重要亚基。基于两个连续的精氨酸的存在，我们认为革兰氏阴性土壤细菌反硝化副球菌的ISP是通过双精氨酸易位（Tat）途径插入细胞质膜的。在这里，我们提供了实验证据，证明细菌ISP的膜整合确实依赖于Tat易位。我们发现ISP的靶向性取决于双精氨酸基序。特别对第二精氨酸残留物（R16）建立了严格的要求；第一个精氨酸（R15K）的保守替换仍然允许大量的ISP传输。比较序列分析揭示了几种细菌isp中Tat信号肽的共同特征；然而，与假定的ISP Tat信号同时充当膜锚相关的事实有明显的特征。这些差异包括与一般Tat信号相比h区疏水性提高，以及缺乏保守的“+5”一致基序赖氨酸残基。+5赖氨酸（Y20K）的取代在一定程度上损害了ISP输出和/或细胞色素bc(1)的稳定性，并指出了这种偏离标准Tat基序的特定作用。EPR光谱证实了[2Fe-2S]辅因子的细胞质插入。必需辅因子结合残基（C152S）的突变降低了ISP膜水平，可能表明辅因子插入是沿Tat途径有效易位的先决条件。
The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.