Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.