THE IN-VITRO ASSEMBLY OF THE NADPH-PROTOCHLOROPHYLLIDE OXIDOREDUCTASEIN PEA-CHLOROPLASTS

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1. 6.99.1) is the major protein in the prolamellar bodies (PLBs) of etiop lasts, where it catalyzes the light-dependent reduction of protochloro phyllide to chlorophyllide during chlorophyll synthesis in higher plan ts. The suborganellar location in chloroplasts of light-grown plants i s less clear. In vitro assays were performed to characterize the assem bly process of the pchlide reductase protein in pea chloroplasts. Impo rt reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an i mport reaction revealed that imported protein was targeted to the thyl akoid membranes. No radiolabelled protein could be detected in the str omal or envelope compartments upon import. Assembly reactions performe d in chloroplast lysates showed that maximum amount of radiolabelled p rotein was associated to the thylakoid membranes in a thermolysin-resi stant conformation when the assays were performed in the presence of h ydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermor e, membrane assembly was optimal at pH 7.5 and at 25 degrees C. Howeve r, further treatment of the thylakoids with NaOH after an assembly rea ction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for mem brane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and t hat both the precursor and the mature form of the protein can act as s ubstrates for membrane assembly.