Activation/Deactivation Cycle of Redox-controlled Thylakoid Protein Phosphorylation

Abstract

Signal transduction via light-dependent redox control of reversible thylakoid protein phosphorylation has evolved in plants as a unique mechanism for controlling events related to light energy utilization. Here we report for the first time that protein phosphorylation can be activated without light or the addition of reducing agents by a transient exposure of isolated thylakoid membranes to low pH in darkness. The activation of the kinase after incubation of dark-adapted thylakoids at pH 4.3 coincides with an increase in the plastoquinol: plastoquinone ratio up to 0.25. However, rapid plastoquinol reoxidation (<1 min) at pH 7.4 contrasts with the slow kinase deactivation (t1 ⁄2 ‫؍‬ 4 min), which indicates that the redox control is not directly dependent on the plastoquinone pool. Use of inhibitors and a cytochrome bf-deficient mutant of Lemna demonstrate the involvement of the cytochrome bf complex in the low-pH induced protein phosphorylation. EPR spectroscopy shows that subsequent to the transient low pH treatment and transfer of the thylakoids to pH 7.4, cytochrome f, the Rieske Fe-S center, and plastocyanin become reduced and are not reoxidized while the kinase is slowly deactivated. However, the deactivation correlates with a decrease of the EPR g z signal of the reduced Rieske Fe-S center, which is also affected by quinone analogues that inhibit the kinase. Our data point to an activation mechanism of thylakoid protein phosphorylation that involves the binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center.Protein phosphorylation plays a major role in cellular signaling, developmental processes, and metabolism regulation of living cells (1-3). In chloroplasts a unique light mediated redox-controlled phosphorylation (4 -7) of a number of proteins associated with the thylakoid membrane has evolved. Phosphorylation of the major light-harvesting chlorophyll a/b protein complex (LHCII) 1 regulates the balance of excitation energy between the two photosystems (5, 6, 8), protects oxygen-evolving organisms against photoinhibition by excessive light excitation (9) and may affect the process of LHCII degradation related to the long term acclimation of the light-harvesting antenna size to the prevailing ambient light intensity (10). Other identified phosphoproteins belong to photosystem II and include the D1 and D2 reaction center protein subunits as well as the chlorophyll a binding protein CP43 and the 9-kDa psbH protein (11,12). Phosphorylation of the D1 polypeptide in higher plants is implicated in the regulation of its degradation during the light induced turnover and repair of photoinhibitory damage to the photosystem II reaction center (13-16).The specific mechanism involved in the redox-mediated activation of the thylakoid kinase(s) is not yet understood. Activation of thylakoid protein phosphorylation is dependent on the redox state of the plastoquinone pool (5-7). In cytochrome bf-deficient mutants of algae (17) and higher plants (18 -20) the redox-controlle...

Redox Control of psbA Gene Expression in the Cyanobacterium Synechocystis PCC 6803. Involvement of the Cytochrome b6/fComplex

“…It has been recently demonstrated that occupancy of the Q 0 site in the Cyt b 6 /f by a plastoquinol molecule is the signal for the activation of a light-dependent kinase of light-harvesting complex II involved in the balance of excitation energy between the two photosystems (Vener et al, 1995(Vener et al, , 1997Zito et al, 1999). DBMIB, which prevents the PQ reoxidation by its binding to the Q 0 site of the Cyt b 6 /f, largely inhibits the accumulation of psbA mRNA upon cell illumination or in darkness in the presence of Glc.…”

Chimeric Fusions of Subunit IV and PetL in the b6 f Complex of Chlamydomonas reinhardtii

“…One unsolved issue in understanding LHCII kinase activation is the mechanism by which PQH 2 binding to Q o , on the luminal site of the membrane, activates an enzyme that operates in the stroma. One model involves conformational changes of the Rieske subunit (23,27,28), whose flexibility has been demonstrated in both the bc 1 (65,66) and b 6 f (67, 68) complexes. Recently, we have proposed (69) that the activating signal is transduced to the active site of the kinase via conformational changes occurring in the transmembrane region of the cytochrome b 6 f. Recent electron microscopy data indeed suggest that such changes, which are peculiar of the b 6 f complex, accompany the movements of the Rieske protein catalytic domain (13).…”

“…In Arabidopsis thaliana, the consequences of expressing antisense RNAs (25) suggest the involvement in state transitions of a family of thylakoid-associated, presumably transmembrane, kinases (thylakoid-associated kinases) (26). Although the molecular mechanism by which the redox state of the PQ pool controls the kinase is not known, it has been shown in vitro with thylakoid preparations from spinach (27,28) and in vivo in C. reinhardtii cells (23) that it depends on plastoquinol (PQH 2 ) binding to the oxidizing (Q o ) site of the cytochrome b 6 f complex.…”

Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes ofArabidopsis thaliana

“…In vitro studies using 32 P labeling have suggested that a number of thylakoid proteins are extensively phosphorylated in the light (22) by a kinase controlled by the photosynthetic electron transport chain (12,23,24), and dephosphorylated in the dark by phosphatase(s) that are not light sensitive (12,25). More recent studies using phosphothreonine antibodies questioned the magnitude of this phosphorylation by showing that some thylakoid phosphoproteins remain phosphorylated even in dark-adapted plants (26 -28).…”