THE ASSEMBLY OF CYTOCHROME B(6)/F COMPLEXES - AN APPROACH USING GENETIC-TRANSFORMATION OF THE GREEN-ALGA CHLAMYDOMONAS-REINHARDTII

As an approach to the study of the biogenesis of the cytochrome b(6)/f complex, we characterized the behaviour of its constitutive subunits in mutant strains of Chlamydomonas reinhardtii bearing well-defined mutations. To this end, we have constructed three deletion mutant strains, each lacking one of the major chloroplast pet genes: the Delta petA, Delta petB and Delta petD strains were unable to synthesize cyt f, cyt b(6) and subunit IV (suIV) respectively. Western blotting analysis, pulse-labelling and pulse - chase experiments allowed us to compare the cellular accumulation, the rates of synthesis and the turnover of the cyt b(6)/f subunits remaining in the various strains. We show that the rates of synthesis of cyt b(6) and suIV are independent of the presence of the other subunits of the complex but that their stabilization in the thylakoid membranes is a concerted process, with a marked dependence of suIV stability on the presence of cyt bg In contrast, mature cyt f was stable in the absence of either suIV or cyt b(6) but its rate of synthesis was severely decreased in these conditions. We conclude that the stoichiometric accumulation of the chloroplast-encoded subunits of the cyt b(6)/f complex results from two regulation processes: a posttranslational regulation leading to the proteolytic disposal of unassembled cyt bb and suIV and a co-translational (or early post-translational) regulation which ensures the production of cyt f next to its site of assembly.