Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation. Role of plastoquinol bound to the reduced cytochrome bf complex.

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.

Protein phosphorylation plays a major role in cellular signaling, developmental processes, and metabolism regulation of living cells (1)(2)(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)(14)(15)(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)(6)(7). In cytochrome bf-deficient mutants of algae (17) and higher plants (18 -20) the redox-controlled phosphorylation of the mobile subpopulation of LHCII enriched in the 25-kDa subunit (21) is abolished. Furthermore, a stable form of the LHCII kinase active in darkness in Acetabularia thylakoids and the light-activated kinase of pea thylakoids are rapidly deactivated by specific inhibitors of cytochrome bf reduction (22,23). These results suggest that the cytochrome bf complex is involved in LHCII kinase activation/deactivation. However, cytochrome bf deficiency does not abolish redox-dependent phosphorylation of photosystem II proteins as revealed from studies on Lemna (18) and maize mutants (19,20). These findings have been interpreted as evidence for the existence of more than one redox-controlled kinase or redox control mechanisms (11,12). As opposed to that, based on redox titration of the phosphorylation of 13 proteins in pea thylakoids it was suggested that the redox control involves a single endogenous agent (24). Differential phosphorylation of LHCII and proteins of photosystem II under different light intensities (16) and the possibility that the kinase activation process affects also the substrate specificity of * The project was financed by the Swedish Natural Science Research Council and the Swedish Research Council for Agriculture and Forestry. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  the enzyme were considered as well (23). Moreover, a dual control of protein phosphorylation in intact chloroplasts by redox and energy states, including dissipation of a trans-thylakoid pH gradient, was previously proposed (25)(26)(27).
Protein kinases isolated from thylakoid membranes have not shown redox-dependent activity (28). Attempts to obtain preparations enriched in kinase activity still exhibiting redox control resulted in a partial copurification of the LHCII kinase with the cytochrome bf (29). However, the redox-controlled kinase(s) and the corresponding phosphatase enzymes still remain unidentified.
Identification of the specific regulatory component(s) involved in the activation/deactivation of the thylakoid protein phosphorylation has been hampered by the fact that the activation process was so far achieved only under conditions in which all thylakoid electron carriers were reduced following either illumination or excess addition of reducing agents in darkness. In this work we introduce a new experimental system in which thylakoid kinase activation is induced in darkness by a short preincubation of the membranes at low pH in the absence of added reducing agents. Studies on the kinetics of the kinase activation and deactivation using this new experimental approach combined with low temperature EPR spectroscopy indicate that control of the thylakoid protein phosphorylation is related to the maintenance of a plastoquinol bound to a cytochrome bf complex in which the Rieske Fe-S center is in the reduced state.

Plant Growth and Preparation of Thylakoid Membranes-Spinach
(Spinacea oleracea L.) was grown hydroponically in the nutrient solution (30) at 25°C and light intensity of 475 mol of photons m Ϫ2 s Ϫ1 using a regime of 10/14-h light/dark period. Thylakoids were isolated from 6-week-old plants according to Andersson et al. (31) except that the last washing step and final suspension (3-4 mg of chlorophyll/ml) were made in 10 mM sodium phosphate, pH 7.5, 100 mM sorbitol, 5 mM MgCl 2 , and 20 mM NaCl.
Lemna perpusilla strain 6746 (wild type) and mutant strain 1073 were grown at 22°C under dim light in sterilized medium (32). Plants were washed with 25 mM Tris/HCl, pH 7.6, 0.4 M sorbitol, 10 mM NaCl, 0.5 mg/ml ascorbate, collected by filtration on a porcelain Buchner funnel, and homogenized in the same buffer using a Warring blender (3 ϫ 10 s, 15 ml of the buffer per 1 g of wet weight). The homogenate was filtered through eight layers of nylon mesh and centrifuged at 1,000 ϫ g for 2 min. The supernatant was collected and centrifuged at 6,000 ϫ g for 10 min. The pellet was resuspended in 50 mM Tricine/NaOH, pH 7.5, 5 mM MgCl 2 , and 10 mM NaCl, and recentrifuged at 12,000 ϫ g for 10 min. The thylakoid pellet was suspended in 10 mM sodium phosphate, pH 7.5, 100 mM sorbitol, 5 mM MgCl 2 , and 20 mM NaCl and stored on ice in darkness until use. Chlorophyll was determined as in Refs. 33 and 34).
Phosphorylation Assay of Spinach and Lemna Thylakoid Proteins-Dark-adapted spinach thylakoids were diluted with 50 mM NaH 2 PO 4 , pH 4.3, 100 mM sorbitol, and 5 mM MgCl 2 (acidic medium) to 0.1 mg of chlorophyll/ml. The preincubation at low pH was performed in darkness at room temperature for times (2-20 min) as indicated in the figure legends and was terminated by centrifugation in a microcentrifuge for 30 s. The supernatant was removed, and the thylakoid membranes were resuspended (0.4 mg of chlorophyll/ml) in the phosphorylation reaction medium containing 50 mM sodium phosphate, pH 7.4 or 8.0, 100 mM sorbitol, 5 mM MgCl 2 , 10 mM NaF, and 0.1 mM [␥-32 P]ATP (1 Ci/30 l). Incubation in darkness at room temperature was performed for the indicated times and was terminated by the addition of electrophoresis sample buffer (10% SDS, 40% sucrose, 1 mM EDTA, 20% ␤-mercaptoethanol, and 100 mM Tris/HCl, pH 6.8) in a proportion of 1:3 (v/v) to the sample. In control experiments, thylakoids were both preincubated and phosphorylated in the dark either at pH 7.4 or at pH 4.3. To activate the kinase by light, thylakoids were preincubated in 50 mM sodium phosphate, pH 7.4, 100 mM sorbitol, and 5 mM MgCl 2 . Illumination was provided by a light projector (white light, 100 mol of photons m Ϫ2 s Ϫ1 ).
Lemna thylakoids (12 g of chlorophyll; 0.6 mg of chlorophyll/ml) were diluted 25 times with the acidic medium and incubated in dark-ness at room temperature for 1 min. The suspension was centrifuged in a microcentrifuge, and the supernatant was removed. The thylakoid pellet was resuspended in 30 l of 50 mM sodium phosphate, pH 8.0, 100 mM sorbitol, 10 mM MgCl 2 , 10 mM NaF, and 0.1 mM [␥-32 P]ATP (6 Ci). The incubation was continued for 15 min in darkness and was stopped by the addition of 10 l of electrophoresis sample buffer. For light activation (white light, 100 mol of photons m Ϫ2 s Ϫ1 ) all procedures were similar, but the preincubation medium contained 50 mM sodium phosphate, pH 8.0.
If not otherwise indicated, different compounds were added both to the thylakoid suspension at the end of preincubation in the low pH medium and to the phosphorylation assay medium. Nigericin, FCCP, DCMU, DBMIB and HQNO were dissolved in ethanol. The final ethanol concentration in the reaction medium did not exceed 1%.
Kinetics of Thylakoid Protein Kinase Deactivation-For measuring the kinase deactivation of spinach thylakoids preincubated at low pH the following procedure was used. A thylakoid suspension was diluted with the acidic medium to 0.1 mg of chlorophyll/ml (final volume, 0.9 ml) and incubated in darkness at room temperature for 2 min. An aliquot of 0.1 ml was transferred to an Eppendorf tube (time 0), and both thylakoid suspensions were centrifuged for 30 s. The phosphorylation activity of the time 0 sample was assayed immediately. The main thylakoid pellet was resuspended in 0.2 ml of phosphorylation buffer, pH 7.4, without addition of [␥-32 P]ATP. The kinase activity was assayed in aliquots of 25 l after the addition of 5 l of 0.6 mM [␥-32 P]ATP (2 Ci) and 60 mM NaF at times as indicated.
Kinetics of kinase deactivation in darkness under anaerobic conditions was performed as described above but using a combination of argon flushing and enzymatic removal of oxygen. Glucose (5 mM) was added to the phosphorylation buffer, which was bubbled with argon for 2 min prior to addition of glucose oxidase (0.4 mg/ml, Sigma) and catalase (4,000 units/ml, Sigma).
For assay of deactivation of the light-activated protein kinase a thylakoid suspension (0.4 mg of chlorophyll/ml) in 50 mM sodium phosphate, pH 7.4, 100 mM sorbitol, and 5 mM MgCl 2 was illuminated (white light, 100 mol of photons m Ϫ2 s Ϫ1 ) for 2 min and transferred to darkness (time 0). Aliquots of 25 l were removed at times as indicated and mixed with 5 l of 0.6 mM [␥-32 P]ATP (2 Ci) and 60 mM NaF.
All phosphorylation reactions were continued for 20 min in the dark after the addition of [␥-32 P]ATP and stopped by the addition of 10 l of electrophoresis sample buffer.
Analysis of Thylakoid Phosphoproteins-Electrophoretic separation of the thylakoid proteins by SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (36). Thylakoid membranes suspended in electrophoresis sample buffer were heated to 70°C for 3-4 min, and electrophoresis was performed using 15% acrylamide slab gels. The gels were stained with Coomassie Brilliant Blue or with silver, dried, and autoradiographed using x-ray film. Quantification of the protein phosphorylation level was done by scanning the autoradiograms by laser densitometry using the software package Image Quant from Molecular Dynamics.
Lipid Extraction and Determination of the Plastoquinol:Plastoquinone Ratio-Lipid extraction was performed according to Åberg et al. (35). All procedures were carried out in darkness. The thylakoid suspension (0.5 ml; 0.1 mg of chlorophyll/ml) was extracted with 4 ml of methanol and 4 ml of petroleum ether (boiling point 40 -60°C) by extensive vortexing for 30 s. After phase separation the upper phase was removed and evaporated under nitrogen in the dark. The residue was dissolved in 100 l of chloroform:methanol (2:1 v/v), and 20 -30-l samples were injected directly into the HPLC column. Reverse phase HPLC was performed on a Hewlett-Packard Hypersil ODS 3-mm column with a linear gradient of methanol:H 2 O (9:1, v/v) to methanol:2propanol (2:1 v/v) for 20 min at a flow rate of 1.5 ml/min. The absorbance of the eluate was monitored at 210 nm. The elution times of plastoquinone and plastoquinol were determined using reference compounds.
Low Temperature EPR Spectroscopy-Samples of dark-adapted spinach thylakoids containing 3.8 mg of chlorophyll/ml were frozen in liquid nitrogen at various stages of the activation and deactivation of the protein kinase. Directly afterwards, the X-band EPR spectra were measured at 15 K on a Bruker ESP 300 spectrometer equipped with a helium flow cryostat and temperature controller (Oxford Instruments). All procedures were performed keeping the samples in darkness. EPR conditions for measurement of signals from reduced Rieske Fe-S center, plastocyanin, and oxidized cytochrome f were microwave power 6.3 milliwatts and modulation 0.12 millitesla.

Activation of Thylakoid Protein Phosphorylation by Transient Low pH Treatment-Preincubation of spinach thylakoid
membranes in a solution of monosubstituted phosphate, pH 4.3, and subsequent transfer to the phosphorylation buffer, pH 7.4 or 8.0, results in a significant activation of the protein kinase in darkness (Fig. 1). The pattern of protein phosphorylation induced by the transient low pH preincubation is identical to that induced by light. However, the extent of the phosphorylation is lower, varying between 30 and 60% of that induced by continuous illumination. Notably, the level of phosphorylation induced in darkness by preincubation at acidic pH is 100-fold higher than that of the basal phosphorylation at pH 4.3 or that at pH 7.4 without prior preincubation at low pH as judged by densitometry of autoradiograms (Fig. 1). The activation of protein phosphorylation could not be elicited in the dark by preincubation of the thylakoids at pH between 5.8 and 8.0 (not shown). This suggests that the low pH (pH 4.3) treatment before transfer of the membranes to a higher pH is required for the kinase activation. Among the phosphorylated polypeptides the most conspicuous are those of 25 and 27 kDa of LHCII. Considering the molar ratio of these two LHCII subunits (1:4), the relative phosphorylation of the mobile component, the 25-kDa protein is significantly higher than that of the stationary LHCII 27-kDa protein subunit (see also Ref. 21).
Addition of nigericin (0.2 M or 2 M) either in the presence or absence of KCl (10 mM), NH 4 Cl (10 mM), or FCCP (10 M) has no effect on the extent or rate of the low pH-induced phosphorylation in the time range between 0.5 and 30 min (not shown). Thus, the activation of the kinase by the above procedure is not due to the formation or dissipation of a transmembrane pH gradient.
The Activation of Protein Phosphorylation by Low pH Preincubation Is Related to a Change in the Membrane Redox Potential-Low pH incubation may alter the redox potential by affecting the equilibrium [PQ][H ϩ ] 2 /[PQH 2 ] and activate the kinase via reduction of plastoquinone. To test this possibility the oxidizing effect of ferricyanide on both the low pH-and light-induced activation of protein phosphorylation was studied. The results show that the induction of phosphorylation by both low pH treatment and by light is completely inhibited by ferricyanide ( Fig. 2A). This indicates that plastoquinol and/or an additional reduced redox component are involved in the process of kinase activation by transient low pH in darkness.
The kinase activation induced by transient low pH treatment may involve a mechanism different from that of the lightinduced process. If this is the case, the light-dependent reduction of the plastoquinone pool could have an additive effect to the low pH induced kinase activation. Therefore, the extent of thylakoid protein phosphorylation was assayed in the light subsequent to the preincubation of the membranes at low pH in darkness. Moreover, the experiments were done either in the presence or absence of DCMU, an inhibitor of light-dependent plastoquinone reduction by photosystem II (Fig. 2B). Light was found to have no additive effect on the phosphorylation of thylakoid proteins after preincubation of the membranes at low pH in the dark. (Fig. 2, A and B). DCMU completely inhibited the phosphorylation in the light but had no effect on the kinase activation by low pH in darkness. However, DCMU inhibited the phosphorylation performed in the light subsequent to the dark preincubation of thylakoids at low pH (Fig. 2B). These results can be explained by photosystem I-mediated oxidation of plastoquinol formed during the transient low pH treatment under conditions when electron transfer from photosystem II to the plastoquinone pool is inhibited by DCMU.
Kinetics of Activation/Deactivation of the Thylakoid Kinase Induced by Low pH Treatment: Relation to the Redox State of the Plastoquinone Pool-The protein kinase activity in darkness in the presence of reducing agents has previously been shown to be optimal in the range between pH 6.0 and 9.5 (37). Thus, one could expect a dual effect of the transient low pH treatment, a possible irreversible inactivation due to the acidic treatment being superimposed on the activation process. The kinetics of protein kinase activation by transient low pH incubation is shown in Fig. 3. The activation is rather rapid and is maximal in less than 2 min. However, an initial limited loss of activity occurs during the first 10 min of incubation at low pH.
Notably, the kinase activated by transient low pH treatment phosphorylates the thylakoid proteins in the dark for up to 20 min. Fig. 4 shows the kinetics of LHCII phosphorylation in darkness after the low pH and light induction of the kinase activity. The kinase activated by light phosphorylates LHCII only for about 4 min after transfer of the thylakoids to darkness. This difference in the deactivation kinetics of the kinase induced by preincubation at low pH and by light was further analyzed. The thylakoids were incubated either in the light at pH 7.4 or in darkness at pH 4.3 for 2 min and then transferred in darkness and at pH 7.4 for different times prior to the addition of [␥-32 P]ATP. The phosphorylation was then continued for 20 min, allowing the reaction to proceed to completion (see Fig. 4). The kinase deactivation proceeds similarly with respect to all thylakoid substrate polypeptides in both the lightand low pH-activated enzyme(s) (Fig. 5, A and B). However, the kinase activity induced by the low pH treatment is deactivated considerably more slowly in the dark as compared to the deactivation of the light-activated enzyme, with a half-life time of 4 and 1 min, respectively (Fig. 5C). The deactivation of the low pH-induced phosphorylation was further retarded under anaerobic conditions, while the electron acceptor ferricyanide deactivated the kinase completely in 1 min (Fig. 5C).
To test directly whether the reduction state of the plastoquinone pool is involved in the low pH activation/deactivation process, we have determined the relative levels of plastoquinol and plastoquinone in thylakoids under the experimental conditions used for the low pH-induced kinase activation. The ratio of PQH 2 to PQ in control thylakoids incubated in the dark at pH 7.4 was in the range of 0.02-0.07 (Fig. 6). After 2 min of incubation at pH 4.3 this ratio increased to 0.20 -0.27. Transfer of thylakoids from pH 4.3 to 7.4 resulted in reoxidation of plastoquinol in only 1 min (Fig. 6). Therefore, it should be noted that at the time when the kinase activity is maximal (see Fig.  5) the plastoquinone pool is already oxidized to a level incompatible with the activation process. These results, therefore, strongly suggest that the active state of the kinase, once achieved, is only indirectly related to the redox state of the "free pool" of plastoquinone.
Cytochrome bf Complex Is Involved in the Low pH Activation of the Protein Kinase-The deactivation of the thylakoid protein kinase described above (Fig. 5) may reflect a slow oxidation of either plastoquinol bound in the vicinity of the redox sensor or oxidation of the reduced sensor itself. A possible redox sensor could reside within the cytochrome bf complex (17)(18)(19)(20)(22)(23)(24).
To explore this possibility, we have assayed the effect of DB-MIB, a cytochrome bf inhibitor, on the low pH activation of thylakoid phosphorylation. This inhibitor binds to the cytochrome complex at a site permitting interaction with the Rieske Fe-S protein and the low potential cytochrome b, thereby preventing reduction of the cytochrome bf complex (38,39). As shown in Table I, DBMIB inhibits the low pH-induced phosphorylation of the thylakoid proteins to about 50% at a concentration of 1 M, and complete inhibition is obtained at 5 M.
It was reported before that DBMIB inhibits preferentially the light-induced LHCII phosphorylation relative to that of the photosystem II proteins (20,40,41), and consequently it was postulated that there are at least two thylakoid protein kinases with different sensitivity to DBMIB inhibition (11). Thus, it was of interest to analyze the effect of DBMIB on the degree of phosphorylation of various thylakoid polypeptides by the low pH-activated kinase in darkness. The results show a similar degree of inhibition of the transient low pH-induced phosphorylation for all of the thylakoid phosphoproteins by DBMIB ( Table I).
Oxidation of reduced cytochrome bf via NADP and ferredoxin inhibits to a similar extent the radioactive labeling of all the phosphoproteins. Moreover, this inhibitory effect is completely reversed by the addition of 5 M HQNO (Table I). At this concentration HQNO is known to block electron transfer from cytochrome b to plastoquinone, preventing the oxidation of the cytochrome complex (22,(42)(43)(44). These results, therefore, provide evidence supporting the involvement of reduced cytochrome bf complex in the activation of the thylakoid protein phosphorylation by transient low pH treatment.
This conclusion was further supported by analysis performed on the thylakoids of the cytochrome bf-deficient mutant 1073 of Lemna perpusilla, in which the phosphorylation of the LHCII polypeptides is not induced by light or by the addition of reducing agents in the dark. In contrast, redox-controlled phosphorylation of other thylakoid proteins can be induced in this mutant (18). As shown in Fig. 7, in the wild type Lemna thylakoids low pH preincubation in darkness as well as the light activation induce the phosphorylation of LHCII polypeptides. However, the phosphorylation of LHCII subunits could not be induced in darkness by the transient low pH treatment of the cytochrome bf-deficient thylakoids. Moreover, the acidic pH treatment induces a lower level of phosphorylation of other polypeptides in the mutant as compared with the wild type thylakoids. The labeling of a polypeptide(s) in the molecular mass range of about 100 kDa appeared to be enhanced in the mutant thylakoids (Fig. 7).

Changes in the Redox State of High Potential Path Components of Cytochrome bf during Activation/Deactivation of the
Kinase as Determined by EPR Spectroscopy-The experimental system described in this work permits a transient reduction of plastoquinone and change in the membrane redox potential without light excitation of the two photosystems. Taking advantage of this new approach we used EPR spectroscopy to identify the reduced redox component of the cytochrome bf involved in the activation as well as in the slow deactivation of the protein phosphorylation.
The slow protein kinase deactivation occurring after the reoxidation of the plastoquinol pool, which is another merit of this system, implies that the component involved in maintaining the kinase in the active state could not be in the low potential path of the cytochrome bf complex, since following reduction, cytochrome b is promptly reoxidized on a millisecond time scale (45)(46)(47).
In the dark-adapted control thylakoids, kept at pH 7.4, cytochrome f is oxidized as indicated by the characteristic EPR signal (48) at g ϭ 3.53 (Fig. 8A). This signal disappears after incubation of the thylakoids for 2 min at pH 4.3 in darkness, indicating reduction of cytochrome f (Fig. 8A). Moreover, the plastocyanin also becomes reduced at pH 4.3 as indicated by FIG. 5. Kinetics of thylakoid protein kinase deactivation in darkness. Protein kinase activity was induced in spinach thylakoids by illumination for 2 min at pH 7.4 or by preincubation for 2 min in the dark at pH 4.3. At time 0, the sample activated by illumination was transferred to darkness, and that activated by preincubation at pH 4.3 in the dark was transferred to the buffer at pH 7.4, either under air or under anaerobic conditions. All samples were further incubated in the dark. At times as indicated [␥-32 P]ATP was added to aliquots of each sample, and the phosphorylation was performed for 20 min. Autoradiograms show the phosphorylation pattern induced by light (A) or low pH treatment under aerobic conditions (B). C, relative phosphorylation of LHCII. Empty circles, light activation; filled circles, low pH activation; triangles, low pH activation followed by the addition of 1 mM ferricyanide; squares, low pH activation followed by incubation under anaerobic conditions. The level of LHCII phosphorylation was determined by the scanning of autoradiograms as in panels A and B and normalized to the phosphorylation level at time zero. Phosphorylation of LHCII in the low pH-activated sample at time zero stands for 100%.

TABLE I Effect of cytochrome bf complex inhibitors and electron acceptors on thylakoid protein phosphorylation induced by transient low pH treatment
Low pH-induced phosphorylation of spinach thylakoid proteins was performed for 20 min in darkness either in the presence or absence of various additions. The concentration of NADP was 5 mM, and that of ferredoxin and HQNO was 5 M. The level of phosphorylation of each polypeptide was determined by scanning of autoradiograms and normalized to that in the control experiment (no additions). Autoradiograms were exposed for various times to permit quantitative estimation of polypeptides phosphorylated to different extents. The values are the mean of four identical experiments, and the standard deviation is Յ8%. the EPR signal at g ϭ 2.05 (49) (Fig. 8B), which is equal to that of a control sample reduced by 10 mM ascorbate (not shown). Transfer of the membranes from pH 4.3 back to pH 7.4, when the kinase is activated, coincides with the reduction of the Rieske Fe-S center as evidenced by the appearance of the characteristic EPR signal at g ϭ 1.90 (50, 51), (Fig. 9, compare with Fig. 8B). Unexpectedly, following subsequent incubation for up to 10 min, during which the kinase becomes slowly deactivated (see Fig. 5C), cytochrome f (not shown), plastocyanin (g ϭ 2.05), and the Rieske Fe-S center (g ϭ 1.90) remain reduced (Fig. 9). However, the spectra in Fig. 9 show a decrease of the EPR signal around g ϭ 2.03 occurring as a function of incubation time from 1 to 10 min, which reflects a decrease in the g z signal of the reduced Rieske Fe-S center (50,51). The g z signal is known to be affected by displacement of bound plastoquinol from the reduced Rieske Fe-S center when DBMIB or other quinone analogues compete for this binding site (39). As shown in Fig. 9 (lower trace), addition of DBMIB induces the appearance of a signal at g ϭ 1.94 arising from the interaction of the reduced Rieske Fe-S center with the quinone analogue (39) as well as a shift in the g z signal at g ϭ 2.03. Notably, binding of DBMIB inhibits the activation of the kinase induced by low pH treatment (Table I).
Thus, during deactivation of the protein kinase in the dark all redox components of the high potential path of the cytochrome bf complex remain in their reduced state. However, the correlation between the changes in the EPR g z signal and the kinase deactivation in darkness as well as the change of this signal in presence of DBMIB indicate a connection between the kinase active state and occupancy of a quinol binding site in the vicinity of the reduced Rieske Fe-S center. DISCUSSION In this work we demonstrate that thylakoid protein phosphorylation ascribed to activation of the redox-controlled thylakoid kinase(s) can be induced by transient exposure of the thylakoid membranes to low pH in the absence of light and without the addition of reducing agents. The kinase activation by the transient low pH treatment can be explained in terms of a pH-dependent shift of the membrane redox potential, causing the reduction of plastoquinone, cytochrome f, plastocyanin, and the Rieske Fe-S center. Transfer of dark-adapted thylakoids from pH 7.4 to pH 4.3 leads to the increase in the ratio of PQH 2 to PQ, from an average of 0.05 to 0.25, as measured by the total quinone extraction and HPLC quantification. One can therefore consider that basically all the plastoquinone pool is oxidized at pH 7.4 in darkness. The level of reduction of the plastoquinone pool after incubation of the thylakoids at pH 4.3 is comparable with the level that was determined to be sufficient for activation of thylakoid protein phosphorylation in experiments with single-turnover flashes (6). Furthermore, one should take into account that part of the plastoquinone present in the thylakoids is localized in plastoglobuli and therefore not accessible to the redox-mediated reactions (52). The fast reoxidation of the plastoquinol after the transfer of the thylakoid membranes from pH 4.3 back to pH 7.4, while the kinase is still in its active state, demonstrate that the free plastoquinol pool could not be responsible for maintaining its activity.
In the experimental system we have introduced here, the low pH treatment activates the phosphorylation of all the thylakoid phosphoproteins as in the case of light activation. Moreover, experiments using inhibitors and the cytochrome bf-deficient mutant demonstrate that the cytochrome bf complex is involved in the control of the phosphorylation induced by the transient low pH treatment.
While it is generally accepted that cytochrome bf complex is involved in the process of the kinase activation (for review, see Refs. 11 and 12) the molecular mechanism of this process has so far not been elucidated. It was previously proposed that binding of a reduced quinone to the cytochrome bf complex may be responsible for the kinase activation (22). This hypothesis was based on an extensive study of the Acetabularia thylakoid LHCII kinase, which retains its activity in the dark for very long periods of time but could be rapidly inactivated by the binding of quinone analogues to the cytochrome bf quinol oxidizing site (23). The data of the present work further resolve the process of plastoquinol/cytochrome bf involvement in the kinase activation. Thus the kinase active state is induced and maintained as long as the high potential path of the cytochrome bf complex is reduced and plastoquinol is bound to the quinol-oxidizing site. Halogenated quinone analogues including DBMIB displace plastoquinol from the quinol binding site and induce a shift in the g z EPR signal (g ϭ 2.03) (39) as also shown in this work. Replacement of the plastoquinol by DBMIB prevents the kinase activation. We interpret the decrease in the EPR signal at g ϭ 2.03 paralleling the kinase deactivation as a change in the interaction between the reduced Rieske Fe-S center and a plastoquinol bound to a site in its vicinity. A slow dissociation of the bound plastoquinol from this site may allow its oxidation by ambient oxygen and/or exchange with the oxidized plastoquinone pool. This interpretation is supported by the slower rate of the kinase deactivation under anaerobic conditions.
The observed persistence in the reduction state of the cytochrome bf high potential path in darkness and the related active state of the kinase after reduction of part of the plastoquinone pool by the low pH treatment can be explained by the fact that the reduced plastoquinol, the high potential path cytochrome bf components, and plastocyanin cannot be oxidized by photosystem I in the absence of light excitation.
The observation that the deactivation of the light-activated kinase in darkness is significantly faster than that of the kinase activated by the transient low pH exposure can be explained by the rapid re-reduction of P700 ϩ in the dark after illumination. Consequently, one electron from the high potential path of the cytochrome bf complex is consumed. This corresponds to withdrawal of the first electron from plastoquinol bound to the quinol oxidizing site of cytochrome bf complex (47). The second electron of semiquinone reduces cytochrome b, which in turn is very rapidly reoxidized (45)(46)(47). Thus, the bound plastoquinol is rapidly oxidized when the thylakoids are transferred from light to dark, causing the observed rapid kinase deactivation.
The molecular identity of the redox-controlled thylakoid kinase(s) is not yet established. Involvement of the cytochrome bf complex in the kinase(s) activation would suggest a physical interaction between the two entities. Indeed, protein kinase activity was found to be associated with the purified cytochrome bf complexes (29,53). On the other hand, phosphorylation of some of the thylakoid polypeptides can be induced by light in cytochrome bf-less mutants (Refs. 18 -20 and this work) but only partially by the transient low pH treatment. This could be explained if the kinase involved has a putative plastoquinol binding site with a low affinity for plastoquinol or readily susceptible to oxidation in the absence of cytochrome bf complex.
Despite the close interaction between the plastoquinol and the Rieske Fe-S center, there is no evidence for quinol binding directly to the Rieske protein (petC). Possible involvement of subunit IV (petD) of the cytochrome bf complex in close connection with the Rieske protein should also be considered (for review see Ref. 54).
We conclude that the activation of thylakoid protein phosphorylation requires reduction of the cytochrome bf complex only inasmuch as to maintain a plastoquinol bound in the quinol oxidizing site of the complex. The deactivation of the kinase is consequently due to the release or oxidation of this bound plastoquinol. The binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center could correspond to a ligand-receptor interaction in a signal transduction system of the photosynthetic membrane.