Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks (original) (raw)
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Journal of Biological Chemistry, 2013
Background: Chloroplast ATP synthase activity is regulated by both light and metabolic factors, but the relationship between these regulatory modes is not established. Results: Mutating three highly conserved acidic amino acid residues in the ␥ subunit alters light-but not metabolism-induced regulation. Conclusion: Metabolism and light regulation operates via distinct mechanisms. Significance: The chloroplast ATP synthase is a key control point for the light and dark reactions of photosynthesis.
European Journal of Biochemistry, 1989
FoFl ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion Fo. The smaller subunits of F1, especially subunit 6, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast 6, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CFo. In view of controversies in the literature, we reinvestigated two further aspects related to subunit 6, namely (a) its stoichiometry in CFoCFl and (b) whether or not 6 is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified 6, we confirmed a stoichiometry of one 6 per CFoCF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit 6 but, surprisingly, also by CFl(~ 6). We found that the latter was attributable to a contamination of CF1(-6) preparations with integral CF1. To lesser extent CF1(-6) acted by complementary rebinding to CFo channels that were closed because they contained 6 [CFo(+6)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit 6 to control proton flow through CFo and the absolute requirement for 6 in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: 6 may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme. ATP synthesis in thylakoid membranes, in the inner mitochondria1 membrane and in the plasma membrane of microorganisms is performed by FoFl ATP synthases which couple ATP synthesis to proton translocation. ADP and P, bind to the water-soluble F1 part of the enzyme. According to current concepts, ATP is formed spontaneously but remains firmly bound to F1 [l]. Its energy-requiring release is driven by proton flow through the membrane-embedded Fo portion which causes conformational changes in F1 that promote the release of bound ATP [l, 21. For recent reviews see [l-71. The smaller subunits of C F l , namely y and 6, probably link F1 to Fo. As possible transducing elements between electrochemical, conformational and chemical events they are of particular interest. We focussed on the role of subunit 6. CFI consists of five different subunits (a to c) with a stoichiometry of C I~[~~~~S~E~ [S-101 (and this paper). Only one group has reported a stoichiometry of 6/CFoCF1 greater than one [I I]. For the analogous subunit to chloroplast 6 in Correspondence to S. Engelbrecht, Universitat Osnabruck, Biophysik, Barbarastrak 11, D-4500 Osnabriick, Federal Republic of Gcrmany Abbreviutions and definitions. CFo = chloroplast coupling factor 0 (proton channel); opcn CFo = CF,-depleted CFo, active in proton conduction upon membrane encrgization; exposed CF,, = CF,, without CF1 counterpart, not necessarily active in proton conduction; CF, = chloroplast coupling factor 1 (integral ATPase); CF1(-6) .= CFI lacking the 6 subunit; CFl(-c) = CF1 lacking the E subunit; Mega 9 = N-(~-gluco-2,3,4,5,6-pentahydroxyl)-~-methylnonanamide. Enzyme. Chloroplast ATP synthase (EC 3.6.1.34).
FEBS Letters, 1984
Protein phosphorylation in isolated, intact pea chloroplasts was measured during the onset of CO2‐dependent O2 evolution. Total incorporation of 32P (from 32Pi) into the light‐harvesting chlorophyll a/b—protein was found to be less sensitive than O2 evolution to inhibition by the uncouplers FCCP and NH4C1 It is concluded that changes in the rate of ATP synthesis cannot affect protein phosphorylation without also affecting the rate of CO2‐fixation in this system. The ATP/ADP ratio is therefore unlikely to regulate photosynthetic protein phosphorylation under normal physiology conditions.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2012
Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b 6 f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studie...
A Survey of Chloroplast Protein Kinases and Phosphatases in Arabidopsis thaliana
Current Genomics, 2008
Protein phosphorylation is a major mode of regulation of metabolism, gene expression and cell architecture. In chloroplasts, reversible phosphorylation of proteins is known to regulate a number of prominent processes, for instance photosynthesis, gene expression and starch metabolism. The complements of the involved chloroplast protein kinases (cpPKs) and phosphatases (cpPPs) are largely unknown, except 6 proteins (4 cpPKs and 2 cpPPs) which have been experimentally identified so far. We employed combinations of programs predicting N-terminal chloroplast transit peptides (cTPs) to identify 45 tentative cpPKs and 21 tentative cpPPs. However, test sets of 9 tentative cpPKs and 13 tentative cpPPs contain only 2 and 7 genuine cpPKs and cpPPs, respectively, based on experimental subcellular localization of their N-termini fused to the reporter protein RFP. Taken together, the set of enzymes known to be involved in the reversible phosphorylation of chloroplast proteins in A. thaliana comprises altogether now 6 cpPKs and 9 cpPPs, the function of which needs to be determined in future by functional genomics approaches. This includes the calcium-regulated PK CIPK13 which we found to be located in the chloroplast, indicating that calcium-dependent signal transduction pathways also operate in this organelle.
Proceedings of the National Academy of Sciences, 1968
The autonomous, biosynthetic capacity of chloroplasts is determined by their ability to generate adenosine triphosphate (ATP) and reducing power at the expense of radiant energy. The most common biosynthetic activity of chloroplasts, and the one usually associated with photosynthesis, is the assimilation of carbon dioxide to the level of carbohydrate. This process requires both ATP and reducing power.1 Other biosynthetic activities of chloroplasts, such as the synthesis of protein from free amino acids, require only ATP. It should be possible, therefore, to demonstrate that, unlike CO2 assimilation, protein synthesis by isolated chloroplasts depends solely on their photochemical capacity to form ATP and is independent of their photochemical capacity to generate reducing power.
A Potential Function for the Ȗ2 Subunit (atpC2) of the Chloroplast ATP Synthase
Higher plants possess two, distinct genes for the ATP synthase Ȗ subunit, atpC1 and atpC2. In Arabidopsis, atpC1 is the predominant form, and atpC2 is only weakly expressed in photosynthetic tissues. There is no evidence that it plays any role in energy transduction. Indeed, mutants lacking atpC1 are incapable of photoautotrophic growth, while those lacking atpC2 have no noticeable phenotype. To elucidate the possible function of these orthologs, we analyzed mutants expressing exclusively atpC1 or atpC2 in Arabidopsis thaliana. In vivo chlorophyll fluorescence and electrochromic shift (ECS) analyses demonstrated that both atpC1 and atpC2 can function in ATP synthesis, though even under a strong promoter, the activity of atpC2-containing ATP synthase was low. However, we observed a striking difference in the regulation of ATP synthase containing the two orthologs. With atpC1, the ATP synthase was inactivated in the dark, likely via oxidation of the regulatory Ȗ subunit thiols. ATP sy...