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

Hitoshi Inoue,Mitsuru Akita

doi:10.1074/jbc.m709571200

Abstract

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.

Highlights

Most chloroplastic proteins are encoded in the nuclear genome
To conduct the docking reaction under stringent conditions, purified recombinant precursors, free from any cytosolic factors, were used. These precursors were applied to the in vitro protein import assay system to estimate the extent of translocation in the early intermediate that was formed under various conditions, and the outer surface of the chloroplast was subjected to limited proteolysis
Development of the Assay System—Translocation of the amino-terminal end of the precursor in the early intermediate inside chloroplasts was evaluated by analyzing the proteolytic fragments generated by limited proteolysis of the precursor docked to chloroplasts

Summary

EXPERIMENTAL PROCEDURES

Growth of Plants and Isolation of Chloroplasts—Pea (Pisum sativum) plants were grown for 8 –10 days in a growth chamber under a cycle of 14 h of light at 24 °C and 10 h of dark at 20 °C. Chloroplasts were suspended in import buffer (I-buffer3: 50 mM HEPES-KOH, pH 8.0, 330 mM sorbitol) at a concentration of 2 mg of chlorophyll/ml. After several washes with H2O, inclusion bodies were solubilized in denaturing/wash buffer (8 M urea, 50 mM HEPES-KOH, pH 7.0, 300 mM NaCl, 20 mM imidazole). Solubilized inclusion bodies and resins pre-equilibrated with denaturing/wash buffer were mixed in a microtube and incubated for 20 min at room temperature with frequent mixing. Precursor protein was dissolved in solubilization buffer (S-buffer: 8 M urea-25 mM HEPES-KOH, pH 7.5–50 mM KCl-2 mM MgCl2). The reaction was quenched by the addition of 5 mM dithiothreitol, and the mixture was further incubated for 15 min on ice. Excess biotin-maleimide was removed by trichloroacetic acid precipitation, and the final pellet was solubilized in S-buffer and stored in the freezer until use.

The abbreviations used are

RESULTS

DISCUSSION