CHLOROPLAST PROTEIN IMPORT - CHLOROPLAST ENVELOPES AND THYLAKOIDS HAVE DIFFERENT ABILITIES TO UNFOLD PROTEINS

Proteins have to be at least partially unfolded upon passage through t he biological membranes. Previous studies with a dihydrofolate reducta se fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by bind ing of a ligand, methotrexate, failed to block its translocation acros s the envelopes, suggesting that chloroplast envelopes have strong act ivity to unfold proteins [America, T., Hageman, J., Guera, A., Rook, E , Archer, K., Keegstra, K. and Weisbeek, P (1994) Plant Mel. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocati on of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusi on protein was imported into the stroma but its translocation across t he thylakoid membrane was blocked. The fusion protein that bound to th e envelope became susceptible to digestion by thermolysin. These resul ts suggest that, while the envelope membranes can unfold the methotrex ate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.