Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro.

Abstract

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of C. reinhardi was studied in vivo and in vitro. Intact cells or purified membranes were labeled with 32Pi or [.gamma.-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis]. The 32P was esterified to serine and threonine residues. At least 6 polypeptides were phosphorylated in vitro and in vivo and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU [3-(3,4-dichlorophenyl-1,1-dimethyl) urea] binding site (32-35 kdaltons) and the reaction center (26 kdaltons). The membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without DCMU, or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44-54 kdaltons were missing. All the phosphoproteins appear to be components of the photosystem II comlex and are only partially dephosphorylated in vivo, which suggest strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The LHC is not phosphorylated in the absence of the reaction center, which lends support to this idea.