Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE
Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE
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DOI：
10.1093/pcp/pcj002
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发表时间：
2006-03-01
影响因子：
4.9
通讯作者：
Nakai, M
中科院分区：
文献类型：
--
作者：
Kikuchi, S;Hirohashi, T;Nakai, M
叶绿体外包膜上的前蛋白转座子介导核编码前蛋白的识别和输入到叶绿体中。两个受体组分Toc159和Toc34以及通道Toc75形成了Toe复合物。在这项研究中，我们用蓝色原生PAGE （BN-PAGE）分析了Toe复合物的分子结构和组织，这是一种在非变性条件下分离膜蛋白复合物的高分辨率方法。在蛋白酶抑制剂混合物存在下分离的豌豆叶绿体直接溶解在洗涤剂溶液中，用BN-PAGE和粒径隔离色谱分析。随后的免疫印迹分析表明，由Toc75、Toc159和Toc34组成的复合物分子量为800-1,000 kDa。有限的蛋白水解揭示了Toc复合物的核心，它对蛋白酶和洗涤剂处理具有抗性。在Toc159: Toc75: Toc34之间，计算出三种Toe蛋白的化学计量量约为1:3:3。我们还分析了病质体和根质体的Toc复合物。这些质体基本上与叶绿体具有相同大小的趾复合体。
The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toe complex. In this study, we have analyzed the molecular architecture and organization of the Toe complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toe proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toe complex as that of the chloroplast.