CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

Abstract

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK͞ CP12͞GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two Nterminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamideadenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP͞NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin͞thioredoxin system, but in addition, by reversible dissociation of the PRK͞CP12͞

The integration of activity in saline environments: problems and perspectives

“…In addition, there are membrane complexes such as clathrin (Batchelder and Yarar 2010) and multifunctional enzymes in primary metabolism (e.g. pyruvate dehydrogenase, Behal et al 1993;Perham 2000), the Calvin cycle (Wedel et al 1997), the phenylpropanoid pathway (Winkel 2004), glycolysis (Graham et al 2007), polyamine biosynthesis (Alcázar et al 2011) and starch synthesis (Hennen-Bierwagen et al 2009;Liu et al 2009). Although some of the complexes 'make sense' based on well-known biochemistry, others, such as the association of aldolase with the vacuolar ATPase, are less obvious (Lu et al 2004).…”

Thioredoxin-mediated reversible dissociation of a stromal multiprotein complex in response to changes in light availability

“…Incubation of the isolated, partially purified complex with DTT or NADPH induced dissociation of the complex, which was accompanied by activation of PRK and NADP-GAPDH (12). Evidence has now accumulated indicating that the PRK/GAPDH complex also includes the chloroplast protein, CP12 (13)(14)(15)(16). CP12 is a small protein (Ϸ8.5 kDa) containing four highly conserved cysteine residues, which, in the oxidized state, have been shown to form two intramolecular disulfide bridges (15,17,18).…”

“…CP12 is a small protein (Ϸ8.5 kDa) containing four highly conserved cysteine residues, which, in the oxidized state, have been shown to form two intramolecular disulfide bridges (15,17,18). It has been hypothesized that the function of the PRK/GAPDH/CP12 complex is to provide an additional mode of light/dark regulation of the Calvin cycle mediated by changes in the levels of NADP(H) (16). Much of our more recent knowledge about this complex has been gained from studies on in vitro coexpression of PRK, CP12 and the GapA subunit of GAPDH (13,17,(19)(20)(21), which has led to the proposal that the function of the PRK/GAPDH/CP12 even in higher plants, is to provide redox control of the A 4 GAPDH isoform (21).…”

Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein

“…In vitro analysis of Arabidopsis CP12 mutants has shown that both pairs of redox-regulated Cys residues are required for ternary complex formation: an N-terminal pair for PRK binding and a C-terminal pair for GAPDH binding (Wedel et al, 1997). The red alga Galdieria sulphuraria CP12 does not have an N-terminal Cys pair, and although the GAPDH-PRK-CP12 complex forms, PRK is not completely inactivated (Oesterhelt et al, 2007).…”

“…In eukaryotic organisms, CP12 is located in the chloroplast, where the only function thus far identified is in the regulation of the Calvin cycle in response to changes in light availability by reversibly binding glyceraldehyde-3-P dehydrogenase (GAPDH) and, subsequently, phosphoribulokinase (PRK; Wedel et al, 1997;Graciet et al, 2003aGraciet et al, , 2003bMarri et al, 2005aMarri et al, , 2008Erales et al, 2008a;Howard et al, 2008;Carmo-Silva et al, 2011). The formation of the GAPDH-PRK-CP12 complex inhibits GAPDH and PRK activity leading to down-regulation of the Calvin cycle; this has been demonstrated in plants, Chlamydomonas reinhardtii, and cyanobacteria (Wedel et al, 1997;Graciet et al, 2003a;Marri et al, 2005b;Tamoi et al, 2005). The GAPDH-PRK-CP12 complex dissociates under reducing conditions mediated by thioredoxin, thereby restoring GAPDH and PRK activity (Lebreton et al, 2003;Marri et al, 2005bMarri et al, , 2009Howard et al, 2008).…”