Regulation of thylakoid protein phosphorylation in intact chloroplasts by the activity of kinases and phosphatases

Abstract

Regulation of thylakoid protein phosphorylation from saturating up to photoinhibitory light conditions was studied in intact chloroplasts with high rates of CO 2 fixation and functioning protein biosynthesis. Comparing steady-state level phosphorylation with the phosphorylation in the presence of the phosphatase inhibitor NaF, evidence was found that phosphorylation of light harvesting chlorophyll-protein complex II (LHC II) and Photosystem II (PS II) polypeptides was catalysed by at least two different kinase / phosphatase systems. Whereas the steady-state level of LHC II phosphorylation declined with increasing light intensity, the phosphorylation level of PS II polypeptides remained stable even at photoinhibitory conditions. Decrease of steady-state level of LHC II phosphorylation at higher light intensities was partially due to an inhibition of the phosphorylation reaction. Its inactivation was observed before any significant loss of PS II electron transfer activity occurred. Quenching analysis of chlorophyll fluorescence revealed that the high light inhibition of the LHC II phosphorylation reaction was caused by an increased membrane energetisation, and not by an oxidation of the plastoquinone pool. In contrast, the kinase activity responsible for the phosphorylation of the PS II polypeptides seemed to be exclusively under the redox control of the plastoquinone pool and was not influenced by membrane energetisation. Evidence was found that also the phosphatase activities specific for LHC II and PS II proteins were different. As indicated by the high turnover of phosphate groups bound to LHC II, the LHC II specific phosphatase showed a high activity. Its activity was stimulated at higher light intensities and determined to a main extent the steady state level of LHC II phosphorylation. The phosphatase specific for the PS II phosphoproteins showed almost no activity in the light as indicated by the absence of a phosphate group turnover in the light. Dephosphorylation of PS II could only be observed in the dark and in contrast to LHC II dephosphorylation could not be inhibited by NaF. Even the increased turnover of the D1 protein at higher light intensities, which was followed by the light-dependent incorporation of [ 14C]leucine into the protein, did not accelerate the D1 protein phosphorylation. The steady-state level of PS II protein phosphorylation was therefore in principle determined by its kinase activity.