CP12 PROVIDES A NEW MODE OF LIGHT REGULATION OF CALVIN CYCLE ACTIVITYIN HIGHER-PLANTS

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-pho sphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of t he reductive pentosephosphate cycle (Calvin cycle), Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interac t with CP12, led to the identification of a second member of the Calvi n cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12, The exchange of cysteines for serines in CP 12 demonstrate that interaction with PRK occurs at the N-terminal pept ide loop of CP12, Size exclusion chromatography and immunoprecipitatio n assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH compl ex in the stroma of higher plant chloroplasts, Its stoichiometry is pr oposed to be of two N-terminally dimerized CP12 molecules, each carryi ng one PRK dimer on its N terminus and one A2B2 complex of GAPDH subun its on the C-terminal peptide loop, Incubation of the complex with NAD P or NADPH, in contrast to NAD or NADH, causes its dissociation, Assay s with the stromal 600-kDa fractions in the presence of the four diffe rent nicotinamideadenine dinucleotides indicate that PRK activity depe nds on complex dissociation and might be further regulated by the acce ssible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reduc tive activation of different proteins by the well-established ferredox in/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).