Chloroplast protein import. Chloroplast envelopes and thylakoids have different abilities to unfold proteins.
Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocation 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 fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.
Endo
T
T
Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Japan.
Kawakami
M
M
Goto
A
A
America
T
T
Weisbeek
P
P
Nakai
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism*; D007887:Fabaceae / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008024:Ligands; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010946:Plants, Medicinal; D010970:Plastocyanin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013820:Thermolysin
D008024:Ligands; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D010970:Plastocyanin; D013762:Tetrahydrofolate Dehydrogenase; D013820:Thermolysin; D008727:Methotrexate