Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts.
Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts.
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DOI：
10.1083/jcb.139.7.1677
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发表时间：
1997-12-29
期刊：
影响因子：
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通讯作者：
Schnell DJ
中科院分区：
文献类型：
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作者：
Kouranov A;Schnell DJ
我们使用标记转移交联法研究了两个核编码的前蛋白与叶绿体蛋白输入机制在输入的三个阶段的相互作用。在外膜能量无关的结合过程中，前蛋白与外膜转位复合体的三个已知成分Toc34、Toc75和Toc86相互作用。虽然已知Toc75和Toc86在进口过程中与前蛋白有关，但以前还没有观察到Toc34在前蛋白结合中的作用。Toc34与前蛋白的相互作用受GTP的结合调节，但不受GTP的水解调节。这些数据为Toc34在进口中的直接作用提供了第一个证据，并为GTP作为前蛋白识别调节因子的功能提供了洞察力。Toc75和Toc86是前蛋白跨外膜插入时的主要交联靶，支持这两种蛋白在外膜转位和前蛋白识别中的作用。内膜蛋白Tic(21)和Tic22，以及一个以前未知的14kD蛋白是进口后期交联剂的主要目标。这些数据为这些成分在蛋白质跨内膜转运过程中的作用提供了额外的支持。我们的结果表明，一个明确的分子相互作用序列，导致核编码的前蛋白从细胞质运输到叶绿体基质中。
We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.