Evaluating the energy-dependent "binding" in the early stage of protein import into chloroplasts

During protein import into chloroplasts, precursor proteins are synthesized in the cytosol with an amino-terminal extension signal and irreversibly bind to chloroplasts under stringent energy conditions, such as low levels of GTP/ATP and low temperature, to form the early translocation intermediates. Whether the states of the early-intermediates that are formed under different energy conditions are similar has not been well studied. To evaluate the early intermediate states, we analyzed how precursor proteins within the early intermediates behave by employing two different approaches, limited proteolysis and site-specific cross-linking. Our results indicate that three different combinations of three different early intermediate stages are present and that the extent of precursor translocation differs between these stages based upon temperature as well as hydrolysis of GTP and ATP. Furthermore, the transition from the second to the third stage was only observed by increasing the temperature. This transition is also accompanied by the hydrolysis of ATP and the movement of the transit peptide. These results suggest the presence of temperature-sensitive and temperature-insensitive ATP-hydrolyzing steps during the early stages of protein import.