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J. Biol. Chem., Vol. 283, Issue 12, 7491-7502, March 21, 2008

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Three Sets of Translocation Intermediates Are Formed during the Early Stage of Protein Import into Chloroplasts^*

Hitoshi Inoue¹ and Mitsuru Akita^¶2

From the United Graduate School of Agricultural Science and Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan and ^¶Satellite Business Venture, Ehime University, Matsuyama, Ehime 790-8577, Japan

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.

Received for publication, November 26, 2007 , and in revised form, January 16, 2008.

¹ A Research Fellow of the Japan Society for the Promotion of Science.

² To whom correspondence should be addressed. Tel.: 81-89-946-9825; Fax: 81-89-946-9825; E-mail: akita{at}agr.ehime-u.ac.jp.

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