(Received for publication, June 20, 1995)
From the
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 = 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
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, 5, 6, 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) ()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, 19, 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 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.
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 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
g for 2 min. The
supernatant was collected and centrifuged at 6,000
g for 10 min. The pellet was resuspended in 50 mM Tricine/NaOH, pH 7.5, 5 mM MgCl
, and 10
mM NaCl, and recentrifuged at 12,000
g for 10
min. The thylakoid pellet was suspended in 10 mM sodium
phosphate, pH 7.5, 100 mM sorbitol, 5 mM
MgCl
, and 20 mM NaCl and stored on ice in darkness
until use. Chlorophyll was determined as in (33) and (34) ).
Lemna thylakoids (12 µg of
chlorophyll; 0.6 mg of chlorophyll/ml) were diluted 25 times with the
acidic medium and incubated in darkness 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
µl of 50 mM sodium phosphate, pH 8.0, 100 mM sorbitol, 10 mM MgCl, 10 mM NaF, and
0.1 mM [
-
P]ATP (6 µCi). The
incubation was continued for 15 min in darkness and was stopped by the
addition of 10 µl of electrophoresis sample buffer. For light
activation (white light, 100 µmol of photons m
s
) 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 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 was illuminated (white light, 100 µmol
of photons m
s
) for 2 min and
transferred to darkness (time 0). Aliquots of 25 µl were removed at
times as indicated and mixed with 5 µl of 0.6 mM [
-
P]ATP (2 µCi) and 60 mM NaF.
All phosphorylation reactions were continued for 20 min in
the dark after the addition of [-
P]ATP and
stopped by the addition of 10 µl of electrophoresis sample buffer.
Figure 1:
Activation of thylakoid protein
phosphorylation in the dark induced by low pH preincubation.
Autoradiogram of thylakoid membrane phosphoproteins resolved by
SDS-polyacrylamide gel electrophoresis. Spinach thylakoids were
preincubated in darkness at pH 4.3 for 20 min and then transferred to
the reaction medium containing [-
P]ATP at
pH 7.4 (indicated as 4.3
7.4). The
phosphorylation reactions were terminated after the time indicated. As
controls, the phosphorylation patterns induced by incubation of the
membranes with [
-
P]ATP in the dark at pH
4.3 or 7.4, or in the light at pH 7.4 are presented. No difference in
the membrane protein pattern between thylakoids preincubated at pH 4.3
and control thylakoids incubated at pH 7.4 was detected as judged by
staining of the gels with Coomassie Brilliant
Blue.
Addition of
nigericin (0.2 µM or 2 µM) either in the
presence or absence of KCl (10 mM), NHCl (10
mM), or FCCP (10 µM) 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.
Figure 2:
Effect
of ferricyanide and DCMU on the activation of the thylakoid protein
phosphorylation induced by low pH preincubation or light.
Phosphorylation of spinach thylakoids was assayed either in the dark or
light after preincubation in the dark at pH 4.3 (20 min) and transfer
to the phosphorylation medium containing
[-
P]ATP at pH 7.4 (pH 4.3
7.4, +) or without preincubation at low pH(-).
Phosphorylation was carried out for 5 min either in the
absence(-) or presence (+) of 30 mM
K
Fe(CN)
(panel A) or 10 µM DCMU (panel B).
The kinase activation induced by transient low pH treatment may involve a mechanism different from that of the light-induced 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.
Figure 3:
Dependence of the kinase activation on the
time of thylakoid preincubation at low pH. Thylakoids were resuspended
in the preincubation medium (pH 4.3, time 0) in the dark. At times as
indicated, the thylakoids were centrifuged, resuspended in the reaction
medium, pH 7.4, containing [-
P]ATP and
finally incubated in the dark for 20 min. Samples were subjected to
SDS-polyacrylamide gel electrophoresis, and the level of LHCII
phosphorylation was determined by scanning of the
autoradiograms.
Notably, the kinase activated by
transient low pH treatment phosphorylates the thylakoid proteins in the
dark for up to 20 min. Fig. 4shows 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
[-
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 light- and
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).
Figure 4:
Kinetics of LHCII phosphorylation in the
dark after activation of the kinase by low pH pretreatment or
illumination. Spinach thylakoid kinase was activated by light at pH 7.4 (empty circles) or by preincubation at pH 4.3 in the dark (filled circles) for 2 min in both cases. Then thylakoids were
transferred to the reaction medium containing
[-
P]ATP at pH 7.4 (time 0), and incubation
was continued in the dark. The reaction was stopped at the indicated
times, and aliquots of the thylakoid suspensions were processed for
SDS-polyacrylamide gel electrophoresis and autoradiography. The
relative level of LHCII phosphorylation was determined by scanning of
the autoradiograms.
Figure 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
[-
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%.
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 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.
Figure 6:
Reversible reduction of plastoquinone
induced by low pH treatment of the dark-adapted thylakoids.
Plastoquinone and plastoquinol were extracted from spinach thylakoids
after incubation in the dark at pH 7.4 or 4.3 (2 min) or 1 min after
transfer from pH 4.3 back to pH 7.4 (4.3 7.4). The content of the both compounds was determined by
HPLC. Left panel, example of HPLC elution profile for the
sample preincubated at pH 4.3; right panel, PQH
:PQ
ratios calculated from HPLC elution
profiles.
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 DBMIB, 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 1, DBMIB inhibits the low pH-induced phosphorylation of the thylakoid proteins to about 50% at a concentration of 1 µM, and complete inhibition is obtained at 5 µM.
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 1).
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 µM HQNO (Table 1). 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).
Figure 7:
Transient low pH treatment activates
phosphorylation of LHCII in thylakoids of Lemna wild type but
not in those of cytochrome bf-deficient mutant. Thylakoids of Lemna wild type (Wt) or mutant (Mt) were
phosphorylated by [-
P]ATP at pH 8.0 for 15
min either in the light (+) or in the dark(-). Thylakoids
were either preincubated in the dark at pH 4.3 and then transferred to
the phosphorylation medium, pH 8.0 (pH 4.3
8.0, +), or preincubated and phosphorylated at pH 8.0
only(-).
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 = 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 the EPR signal at g = 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 = 1.90 (50, 51) , (Fig. 9, compare with Fig. 8B).
Figure 8: Low temperature EPR spectra of dark-adapted spinach thylakoids at pH 7.4 and 4.3. The thylakoids (3.8 mg of chlorophyll/ml) were incubated in the dark at pH 7.4 for 1 h and frozen (pH 7.4) or transferred to pH 4.3 for 2 min and frozen afterwards (pH 4.3). The EPR spectra in the region of cytochrome f (A) and plastocyanin and Rieske Fe-S center (B) were measured. EPR conditions were as follows: 15 K; microwave frequency 9.24 GHz; microwave power 6.3 milliwatts; magnetic field modulation 0.12 millitesla. The g = 3.53 corresponds to the signal of the oxidized cytochrome f(48) ; the g values 2.05 and 1.90 correspond to the signals of plastocyanin and Rieske Fe-S center, respectively(49, 50, 51) .
Figure 9:
Changes in the low temperature EPR
spectra of the Rieske Fe-S center during kinase deactivation.
Dark-adapted spinach thylakoids were preincubated at pH 4.3 for 2 min,
transferred back to pH 7.4 (time 0), and frozen at times as indicated (upper traces). DBMIB (5 µM) was added to the
thylakoid suspension after 2 min of preincubation at pH 4.3, and
thylakoids were transferred to the buffer, pH 7.4, containing DBMIB in
the same concentration and frozen after 1 min (lower trace).
All procedures were performed in the dark. The samples contained 3.8 mg
of chlorophyll/ml. The EPR conditions were as in Fig. 8. The
g = 1.90 and g
= 2.03 signals of
the reduced Rieske Fe-S center (46, 47) are indicated.
The g = 1.94 signal is attributed to the shift in the g
signal by DBMIB(39) . DBMIB did not saturate all centers
at the chlorophyll concentration used.
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 = 2.05), and the Rieske Fe-S center (g
= 1.90) remain reduced (Fig. 9). However, the spectra in Fig. 9show a decrease of the EPR signal around g = 2.03
occurring as a function of incubation time from 1 to 10 min, which
reflects a decrease in the g signal of the reduced Rieske
Fe-S center (50, 51) . The g
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 = 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
signal at g
= 2.03. Notably, binding of DBMIB inhibits the activation of the
kinase induced by low pH treatment (Table 1).
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 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.
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 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 EPR signal (g = 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 = 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 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 ( (18, 19, 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 (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.