CHARACTERIZATION OF ATPA AND ATPB DELETION MUTANTS PRODUCED IN CHLAMYDOMONAS-REINHARDTII CW15 - ELECTRON-TRANSPORT AND PHOTOPHOSPHORYLATIONACTIVITIES OF ISOLATED THYLAKOIDS

We produced atpA and atpB deletion mutants of C. reinhardtii cw15 in o rder to investigate the role of certain domains of the alpha and beta subunits of the chloroplast ATP synthase (CF0CF1) by site-directed mut agenesis. The deletion mutants were obtained by transformation with co nstructs containing the aadA cassette inserted into the atpA or atpB g enes in place of the corresponding open reading frames. Homoplasmic st rains were obtained by continued selection under heterotrophic growth conditions in the presence of spectinomycin. The deletion mutants coul d be complemented by transformation with the wild-type atpA or atpB ge nes, respectively. In both deletion mutants none of the subunits of CF 1 and of CF0 were detected by electrophoretic analysis. Since the moth er strain cw15 as well as the deletion mutants are cell wall-deficient , we were able to prepare photosynthetically active thylakoids and to study the photosynthetic characteristics of cw15 thylakoids and mutant thylakoids lacking the ATP synthase. Electron transport measurements showed that the PSII and PSI activities were not affected in the delet ion mutants. Thylakoids from the deletion mutants were able to maintai n a significantly higher light-induced proton gradient than thylakoids from cw15, confirming the absence of a functional proton channel in t hese deletion mutants. When substrates of photophosphorylation were ad ded, the transmembrane proton gradient in wild-type thylakoids decreas ed and ATP was formed, while in the deletion mutant thylakoids the pro ton gradient was not affected and no ATP was formed. The phosphorylati on/Delta pH relationship in C. reinhardtii thylakoids was shifted to l ower Delta pH values by reduction with dithiothreitol, indicating that the C. reinhardtii enzyme is also similar to the higher plant chlorop last ATP synthase in this respect.