The transition of early translocation intermediates in chloroplasts is accompanied by the movement of the targeting signal on the precursor protein

During protein import into chloroplasts, precursor proteins are docked to these organelles under stringent energy conditions to form early translocation intermediates. Depending on the temperature and the requirement for ATP, different types of early-intermediates are present, for which the extent of precursor protein translocation differs [H. Inoue, M. Akita, J. Biol. Chem. 283 (2008) 7491-7502]. However, it has not been determined whether the environment surrounding the precursor differs for each intermediate. We therefore employed a site-specific photo-crosslinking strategy in our current study to capture any components in close proximity to the targeting signal of the precursors within the early-intermediates. Various crosslinked products, one of which contains Toc75, were identified. The appearance of these products was found to be dependent on the position of the precursor upon modification by the crosslinker and also the intermediate state. This indicated that the transition of early translocation intermediates is accompanied with the movement of the targeting signal within the early-intermediates.