Abstract
Most of chloroplast proteins are encoded in the nuclear genome and synthesized in the cytosol as precursors, and then imported into chloroplast through translocon at the outer and the inner envelopes. During protein import under certain conditions, precursors and translocon are irreversibly bound, forming the early-translocation intermediate. By examining the early translocation intermediate, we have discovered that there are three forms of the early-translocation intermediate depending on the GTP/ATP requirement and temperature.
We now start to investigate the interaction between precursors and translocon in the early-translocation intermediate by cross-linking. In brief, the series of a mutant precursor which has single cysteine residues at various positions was constructed and overexpressed in E. coli. These precursors were modified with SH-specific cross-linkers. After forming the different forms of early-translocation intermediate, cross-linking reaction was initiated. The cross-linked products were analyzed by SDS-PAGE. Our current efforts of cross-linking study will be presented at the meeting.
