Three sets of translocation intermediates are formed during the early stage of protein import into chloroplasts
Three sets of translocation intermediates are formed during the early stage of protein import into chloroplasts
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DOI：
10.1074/jbc.m709571200
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发表时间：
2008-03-21
影响因子：
4.8
通讯作者：
Akita, Mitsuru
中科院分区：
文献类型：
--
作者：
Inoue, Hitoshi;Akita, Mitsuru
在蛋白质输入叶绿体的早期阶段，在严格的能量条件下，在细胞质中合成的前体蛋白不可逆地结合到叶绿体形成早期易位中间体。通过分析早期中间体，已经做出了许多努力来鉴定参与蛋白质输入的组分。然而，到目前为止，中间体中前体的状态还没有得到很好的研究。在这项研究中，试图通过确定在各种条件下的早期中间体中的前体的状态和分析由停靠到叶绿体的前体的有限蛋白水解产生的片段来评估前体的易位的程度。我们的研究结果表明，三组不同的早期中间体形成的温度和水解的GTP/ ATP。这些已被确定为“能量依赖性协会”，“插入”和“渗透”状态的前体的蛋白水解片段的大小的基础上。这些发现表明，在蛋白质输入的早期阶段，有两个单独的ATP水解步骤，其中之一是温度敏感的。我们的研究结果还表明，通过外膜转运主要依赖于内部ATP。
During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far. In this study, an attempt was made to evaluate the extent of translocation of the precursor by determining the state of the precursor in the early intermediate under various conditions and analyzing the fragments generated by limited proteolysis of the precursors docked to chloroplasts. Our results indicate that three different sets of early intermediate are formed based on temperature and the hydrolysis of GTP/ ATP. These have been identified based on the size of proteolytic fragments of the precursor as "energy-dependent association," "insertion," and "penetration" states. These findings suggest two individual ATP-hydrolyzing steps during the early stage of protein import, one of which is temperature-sensitive. Our results also demonstrate that translocation through the outer envelope membrane is mainly dependent on internal ATP.