ANALYSIS OF THE CHLOROPLAST PROTEIN IMPORT MACHINERY

Chloroplast destined precursor proteins bind to distinct areas on the organellar surface as visualized by immunogold decoration. Binding see ms to occur predominantly in regions where the space between outer and inner envelope membranes appears dense in electron microscopic pictur es. When complete protein translocation is blocked by antibodies, whic h bind specifically to the precursor protein, this precursor can still be partially translocated into the organell as deduced from its matur ation by the stromal processing peptidase. It remains, however, sensit ive to exogenous protease, thus indicating that the preprotein spans b oth membranes while in transit through the plastid envelopes. Chloropl ast envelope polypeptides that are involved and in close proximity to the precursor protein in the translocation event are identified by che mical crosslinking. Crosslinking experiments, using different transloc ation systems, i.e. intact organelles, outer envelope vesicles and an isolated import complex, gave identical labelled products. These cross linked polypeptides are likely candidates for active participants in t he import process.