title: Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation. Role of plastoquinol bound to the reduced cytochrome bf complex.
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 (t 1/2 = 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, 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 gz 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.
authors.affiliation: Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
authors.forename: A V
authors.initials: AV
authors.lastname: Vener
authors.forename: P J
authors.initials: PJ
authors.lastname: Van Kan
authors.forename: A
authors.initials: A
authors.lastname: Gal
authors.forename: B
authors.initials: B
authors.lastname: Andersson
authors.forename: I
authors.initials: I
authors.lastname: Ohad
mesh_terms: D003573:Cytochrome b Group / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D010940:Plant Proteins; D010971:Plastoquinone / Q000031:analogs & derivatives* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D008565:Membrane Proteins; D010940:Plant Proteins; C042085:thylakoid membrane phosphoprotein, plant; C003165:plastoquinol; D045346:Cytochrome b6f Complex; D011494:Protein Kinases; D010971:Plastoquinone