Characterization of atpA and atpB deletion mutants produced in Chlamydomonas reinhardtii cw15: Electron transport and photophosphorylation activities of isolated thylakoids

We produced atpA and atpB deletion mutants of C. reinhardtii cw15 in order to investigate the role of certain domains of the α and β subunits of the chloroplast ATP synthase (CF₀CF₁) by site-directed mutagenesis. The deletion mutants were obtained by transformation with constructs containing the aadA cassette inserted into the atpA or atpB genes in place of the corresponding open reading frames. Homoplasmic strains were obtained by continued selection under heterotrophic growth conditions in the presence of spectinomycin. The deletion mutants could be complemented by transformation with the wild-type atpA or atpB genes, respectively. In both deletion mutants none of the subunits of CF₁ and of CF₀ were detected by electrophoretic analysis. Since the mother 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 deletion mutants. Thylakoids from the deletion mutants were able to maintain a significantly higher light-induced proton gradient than thylakoids from cw15, confirming the absence of a functional proton channel in these deletion mutants. When substrates of photophosphorylation were added, the transmembrane proton gradient in wild-type thylakoids decreased and ATP was formed, while in the deletion mutant thylakoids the proton gradient was not affected and no ATP was formed. The phosphorylation/ΔpH relationship in C. reinhardtii thylakoids was shifted to lower ΔpH values by reduction with dithiothreitol, indicating that the C. reinhardtii enzyme is also similar to the higher plant chloroplast ATP synthase in this respect.