The early stage of chloroplast protein import involves Com70

The chloroplast envelope protein Com70 is a hsp70 homolog identified recently as a component of the protein translocation apparatus. The stage of protein import involving Com70 was determined by examining the nature of the association of Com70 with the envelope and its interaction with translocating proteins. Com70 is accessible to thermolysin, but its association with the envelope could not be disrupted by stringent washes. In light of the external membrane-bound location, the involvement of Com70 at the early stage of protein translocation was investigated using a combination of in vitro binding assays, chemical cross-linking, and coimmunoprecipitation. The results provide evidence that Com70 is in close physical proximity to different types of chloroplast protein precursors under conditions supporting binding rather than complete translocation. The formation of cross-linked complexes is dependent on the presence of a typical plastid transit signal and protease-accessible outer envelope components. The close proximity of Com70 and the translocating protein occurs while the protein is still exposed to the cytosol.