Inoue Hitoshi, Akita Mitsuru
United Graduate School of Agricultural Science, Ehime University, Matsuyama, Ehime 790-8566, Japan.
J Biol Chem. 2008 Mar 21;283(12):7491-502. doi: 10.1074/jbc.M709571200. Epub 2008 Jan 16.
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.
在蛋白质导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与叶绿体结合，形成早期转运中间体。通过分析早期中间体，人们为鉴定参与蛋白质导入的成分付出了诸多努力。然而，到目前为止，中间体中前体的状态尚未得到充分研究。在本研究中，我们试图通过确定在各种条件下早期中间体中前体的状态，并分析与叶绿体对接的前体经有限蛋白酶解产生的片段，来评估前体的转运程度。我们的结果表明，根据温度以及GTP/ATP的水解情况，会形成三组不同的早期中间体。这些中间体已根据前体蛋白水解片段的大小被鉴定为“能量依赖性结合”、“插入”和“穿透”状态。这些发现表明在蛋白质导入的早期阶段存在两个独立的ATP水解步骤，其中一个对温度敏感。我们的结果还表明，通过外膜的转运主要依赖于内部ATP。
