Interaction between light harvesting chlorophyll-a/b protein (LHCII ...

THE JOURNAL OF BIOLOGICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 32, Issue of November 15, pp. 19742-19749, 1990 Printed in U.S. A.

Interaction between Light Harvesting Chlorophyll-u/b Protein (LHCII) Kinase and Cytochrome be/f Complex IN VITRO CONTROL OF KINASE ACTIVITY*

(Received for publication, July 12, 1990)

Alma Gal, Giinter Hauska$, Reinhold Herrmann& and Itzhak Ohad From the Department of Biological Chemistry, The Hebrew University, 91904 Jerusalem, Israel, the $Department of Botany, Regensburg University, D-8400 Regensburg, and the $Znstitute of Botany, Miinchen University, 8000 Miinchen, Federal Republic of Germany

We have previously reported that the cytochrome be/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome ha/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome be/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome be/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome be/f. Phosphorylation of LHCII by fractions containing both cytochrome be/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of @-D-O&y1 glucoside but not when 3-[(cholamidopropyl)dimethyl- ammoniol- I-propanesulfonate was used as the solu- bilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3- methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo- 5-t-butyl-p-benzoquinone) known to inhibit cytochrome ha/f activity. However, kinase activity was inhibited by these analogues in all preparations in- cluding those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome beIf while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.

The phosphorylation of the light harvesting chlorophyll- a/b protein complex associated with photosystem II (LHCII)’ is considered to be the major regulatory mechanism

* This work was supported by Grant SFB-184 (to I. 0. and R. H.) from the Deutsche Forschungsgemeinschaft and partially by a grant (to I. 0.) from the Israeli Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: LHCII, light harvesting chlorophyll a/b protein complex of photosystem II; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DBMIB, 2,5-dibromo-3- methyl-6-isopropyl-p-benzoquinone; DIBB, 2,3-diiodo-5-t-butyl-p- benzoquinone; OG, octyl-fl-D-glucopyranoside; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-l-propanesulfonate.

of energy distribution between the two photosystems in oxygen-evolving thylakoids (state transition) (l-3). The most important feature of the membrane-bound LHCII kinase is its reversible activation responding to the redox state of the photosynthetic electron transfer chain (2, 4). Based on experimental results obtained both in viuo and in vitro it was proposed that the activation of LHCII kinase is induced by reduced plastoquinone (l-5). A 64-kDa polypeptide isolated from spinach or pea thylakoids was identified as the light harvesting kinase by Coughlan and Hind (6, 7). The purified enzyme phosphorylated LHCII or histone added as substrates as well as membrane-bound LHCII (8). However, the purified enzyme did not show a redox controlled activation (6-8).

Recently, we have demonstrated that LHCII is not phosphorylated in a Lemna mutant lacking the cytochrome be/f complex (9). Similar results have been obtained independ- ently with other organisms by Wollmann et al. (lo), Bennett et al. (ll), and Coughlan (12).

The LHCII kinase activity is specifically inhibited by cytochrome bs/f inhibitors while the plastoquinone pool is still reduced (13). Furthermore, the activated kinase can be deactivated by cytochrome be/f inhibitors (13).* Thus it had been proposed that the activation of the LHCII kinase in vivo by plastoquinol is mediated by the cytochrome b,/f complex (9-13).

A putative association between the cytochrome be/f complex and the LHCII kinase in uivo could also be inferred from the fact that phosphorylation activity was detected in cytochrome-enriched preparations. Indeed, kinase activity measured using histone as a substrate was reported to be associated with the cytochrome bs/f complex during the initial kinase purification procedure (7, 14) and persisted even after extensive purification of the cytochrome be/f complex (15).

The aim of this work was to establish whether association of the kinase with the cytochrome complex might be neces- sary for its redox-controlled activation in vitro. For this purpose fractions containing kinase activity and cytochrome be/f complex were prepared by various procedures. The kinase activity of such fractions, using both isolated LHCII and histone as substrates, was enhanced by addition of plastoquinol-1 and was inhibited by inhibitors of cytochrome bs/f activity such as DBMIB (16).

EXPERIMENTAL PROCEDURES

Preparation of Thylakoids, Extraction of Cytochrome be/f Complex and LHCZZ-Spinach (Spinacea oleracea, cultivar Hybrid 424) and

2D. Frid, A. Gal., W. Oettemeir., and I. Ohad., manuscript in preparation.

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Redox Control of LHCII Kinase Activity by Cytochrome be/f Complex 19743

pea (Pisum satiuum, Dan variety) plants were grown at 25 “C under a 12-h light/l2-h dark regime for 2-3 weeks.

Spinach thylakoid preparation and extraction of cytochrome b6/f complex from 2 M NaBr-washed thylakoids (equivalent of 50- 200 mg of chlorophyll as starting material) were carried out according to Hauska (17). For solubilization the O-D-OCtyl glucoside deter- eent (30 mM) was used. The solubilized material was fractionated by ammonium sulfate precipitation (lo-40%, w/v). The pellet was removed by centrifugation and the supernatant was precipitated again (40-55%. w/v, ammonium sulfate). The resulting pellet (AMS) was resuspended in 30 mM OG, 0.5% (w/v) sodium cholate or 10 mM CHAPS both in 50 mM Tris-Cl. DH 8.0) (AMS-OG or AMS-CHAPS, respectively) as described under “Results.” The detergent-resuspended material was subjected to affinity column purification, immunoprecipitation, or sucrose density centrifugation as described below.

LHCII was extracted from pea thylakoids (equivalent of 30 mg of chlorophyll) using the procedure of Mullet and Arntzen (18).

Preparation of Cytochrome b,Jf Fractions Containing Kinase Ac- tiuity-Affinity columns were prepared by coupling of either histone III-S or the respective antibodies to cyanogen bromide-activated Sepharose, according to Cuatrecasas (19). Histone (5 mg) or lo-12 mg of the ammonium sulfate-concentrated IgG fraction were bound ner 1 g of swollen CNBr-activated Sepharose. The binding yield was 80-1010%.

--

Immunoaffinity columns or histone-Sepharose (l-2 ml of swollen gel) were packed in 2-ml syringes and were washed with 10 ml of 5 &M OG, 0.1% sodium cholate-(w/v) in 20 mM Tris-Cl buffer, pH 8.0. Histone affinitv columns used with CHAPS were washed with 10 ml of 10 mM CHAPS in the same buffer. AMS-OG or AMS- CHAPS diluted to a final detergent concentration as that of the washing buffer was bound to the gel for 1 h in the cold with constant shaking. Nonbound material was eluted with 5 ml of the washing buffer (flow-through fractions). The bound material was eluted with 2.5 ml of 1 M NaCl in the same buffer. The eluate was desalted using prepacked PD-10, Sephadex G-25 columns (Pharmacia LKB Biotechnology Inc.) and concentrated by centrifugation on Amicon Centricon 30 filters to a final volume of 0.5-1.0 ml. The resulting fractions were analyzed for their polypeptide composition, cytochrome bs/f content, and kinase activity.

For immunoprecipitation, the AMS-OG fraction was diluted to a detergent concentration of 5 mM OG and 0.1% sodium cholate in 20 mM Tris-Cl, pH 8.0, and incubated with the respective antibodies at %o dilution for 2 h in the cold with constant shaking. Antibody- protein conjugates were precipitated by addition of 100 ~1 of pres- wollen protein A-Sepharose for an additional hour as above and centrifugation for 10 min at 13,000 rpm in a cooled Eppendorf centrifuge. The resulting supernatant was analyzed as described. The amount of bound fraction was estimated by substracting the measured values of the free fraction from the initial amount loaded on the column. As a control for nonspecific binding or adsorption to protein A-Sepharose, either preimmune serum was used or no antibodies were added.

Fractionation of the AMS-OG or AMS-CHAPS was carried out using sucrose density centrifugation (14-30% (w/v) sucrose in 50 mM Tris-Cl buffer, pH 8.0, containing either 30 mM OG and 0.5% sodium cholate, or 10 mM CHAPS). AMS-OG or AMS-CHAPS (equivalent of lo-20 nmol of cytochrome fl were loaded onto the gradient and spun for 16 h in a Beckman SW 41 rotor at 250,000 x g at 4 “C. Fractions (0.9-l ml) were collected and analyzed as described above.

Preparation of Polyclonal Antisera against the 64-kDa Kinase- Rabbit polyclonal antiserum against the putative LHCII kinase was prepared using as antigen the 64-kDa polypeptide band present in a highly purified preparation of cytochrome be/f (15). This preparation phosphorylated histone III-S and in absence of added substrate, the 64-kDa polypeptide was phosphorylated. The polypeptides of this fraction were resolved by SDS-PAGE and electrotrans- ferred to nitrocellulose paper (0.45 wm). The band containing the 64-kDa polypeptide was excised, homogenized in phosphate-buff- ered saline and used for rabbit immunizations.

Immunogold Labeling of Stacked Thylakoids-Stacked pea thylakoids (20) were fixed with 1% (w/v) glutaraldehyde in the cold for 2 h and were dehydrated and embedded in LR-White medium as described (21). Thin sections were cut with an LKB Ultrome III microtome and the sections were immunodecorated with anti-cytochrome f or anti-64-kDa kinase rabbit antibodies followed by deco- ration with goat anti-rabbit antibodies tagged with 15 nm colloidal

gold particles (21). The sections were then stained with uranyl acetate and lead citrate and micrographs were taken with a Philips EM 300 electron microscope.

Phosphorylation Assays-Histone III-S phosphorylation by NaBr-washed thylakoids-was carried out as described (9). Phos- nhorvlation of added histone III-S or nurified LHCII bv the deter- gentlsolubilized fractions was assayed in a final volume of 100 ~1 containing 30-50 pg of protein of the respective fraction, 50 mM Tris-Cl, pH 8.0, 10 mM MgC12, 0.2 mM [-r-32P]ATP (300-500 cpm/ pmol), and 25 rg of histone III-S or 20 pg of LHCII as substrates. Where indicated, plastoquinol-1 and DBMIB or DIBB (100 or 10 pM final concentrations, respectively) dissolved in methanol were added. The methanol concentration in the reaction mixture did not exceed 2% (v/v). The reaction was carried out at 25 “C for 30 min and was stopped by addition of unlabeled ATP (5 mM final concentration). For quantitative determination of kinase activity, aliquots were absorbed on 2-cm* strips of Whatman 3MM filter paper followed by washing with 5% (w/v) cold trichloroacetic acid (22).

Kinase activity units are defined as picomoles of 32P incorporated per 30 min ml-‘. For resolution of phosphorylated polypeptides by SDS-PAGE, cold trichloroacetic acid (20% (w/v) final concentration) was added to the reaction mixture. After incubation for 1 h on ice the samples were centrifuged (13,000 rpm, 15 min) in an Eppen- dorf centrifuge. The pellets were solubilized in equal volumes of 1 M Tris-Cl, pH 9.5, and lithium dodecyl sulfate sample buffer (6% lithium dodecyl sulfate, 30% glycerol, 150 mM Tris-Cl, pH 8.0, 1.5 mM dithiothreitol, and 0.15% bromphenol blue).

Electrophoretic Separation of Polypeptides and Western Blot- ting--SDS-PAGE was performed according to Laemmli (23) using the Hoeffer minigel system. Gels were stained by Coomassie Bril- liant Blue. Radioactive gels were dried and exposed to x-ray film using an intensifier screen at -70 “C.

Western immunoblotting of SDS-PAGE-resolved polypeptides and dot-blotting of nondenatured fractions were carried out as described (24) using lZ51-protein A as a second detector.

Other Measurements-Cytochromes f and b, concentrations were determined spectrophotometrically according to Hauska (17), using a Kontron model UVIKCON-860 snectronhotometer. The extinction coefficients used were c551m540 of 18 m&-l cm-’ for cytochrome f and t563.570 14 mM-’ cm-’ for cytochrome bs.

Protein determination in presence of detergent or high sucrose concentrations was carried out according to Peterson (25). Chloro- phyll concentration was determined as described by Arnon (26).

Materials-The detergents P-D-octyl glucopyranoside and CHAPS. cvanogen bromide-activated SeDharose. histone III-S. and .” - ultrapure ammonium sulfate were purchased from Sigma. Sodium cholate was obtained from Serva. Plastoquinol-1 was provided by G. Hauska (University of Regensburg). Protein A-Sepharose was from Pharmacia. [y-32P]ATP (30-70 Ci/mmol) was obtained from Du Pont-New England Nuclear. Y-Protein A (100 &i/ml, 32 mCi/ mg), was purchased from Amersham Corp. The antiserum against cytochrome f was kindly given by Dr. F. A. Wollman (Institute of Physico-chemical Biology, Paris). The other antibodies against spinach cytochrome 66/f complex components were prepared by G. Hauska. All the antibodies used in this work were in the purified IgG form (27). Molecular weight markers were obtained from Bio- Rad. All other chemicals and reagents used were of analytical grade.

RESULTS

LHCII Kinase Activity Copurifies with the Cytochrome bs/f Complex-Previous observations indicated presence of kinase activity in cytochrome bs/f preparations (14,15). Such preparations could also contain the components involved in redox-controlled kinase activation. To check this possibility the cytochrome complex was isolated from spinach thylakoids and the kinase activity was measured using histone III-S as a substrate at various steps of the purification procedure. The results of such an experiment are shown in Table I. The ratio of kinase/cytochrome during the purification steps increased slightly, and a 6-fold enrichment in the cytochrome complex and a 5-fold enrichment in the kinase specific activity were found in the AMS fraction.

The following step in the purification procedure, i.e. the sucrose density gradient centrifugation (17), partially sepa-


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19744 Redox Control of LHCII Kinase Activity by Cytochrome bG/f Complex

TABLE I Kinase activity copurifies with cytochrome bG/f complex

Cytochrome (cyt.) f concentration is expressed as nanomoles/mg protein using an extinction coefficient of 0.018 at 554 nm (17). Specific activity (Sp. act.) of kinase assayed with histone III-S as a substrate is given as units X 10~“/mg protein. The ratio of kinase/cytochrome f is given as kinase units x lo-“/nmol cytochrome f.

Fraction Total Kinase sp. act. Ratio of kinase/cvt.

w? nmol % cone units X IO-’ % unrts/nnol NaBr washed membranes 297 132 100 0.4 500 100 1.8 3.8

OG extract 80 80 60 1.0 256 51 3.2 3.2

Ammonium sulfate (40-55%) 16 38 29 2.4 183 36 8.6 4.8

,-LHCII I 2 3 4 5 6 +LHc11 ,,-LHCII I 2 3 4 5 6 +LHcII, 0 b

FIG. 1. LHCII phosphorylation by fractions separated by sucrose density gradient centrifugation. An AMS-OG preparation was centrifuged for 16 h on a sucrose density gradient as described under “Experimental Procedures”; 13 fractions (1 ml) were collected and assayed for cytochrome (cyt) content and kinase activity. Fractions (from bottom) 9-10 (a) and 11-12 (b) were pooled and concentrated to 0.2 ml; phosphorylation was assayed without or with added LHCII (lanes 1 and 4), and with further addition of plastoquinol-1 (lanes 2 and 5) and plastoquinol-1 and DBMIB (lanes 3 and 6). Aliquots were resolved by SDS-PAGE and autoradiographed. Cytochrome f concentration was 22 pM (a) and 4 pM (b); kinase activity with histone III-S as a substrate was 10,000 and 31,800 units in a and b, respectively. G, stained gel; AR, autoradi- ogram.

rates the cytochrome complex from the bulk of the kinase activity as also observed previously (10, 15). Fractions enriched either in the cytochrome b6/f complex or kinase activity collected from such gradients were assayed for autophosphorylation and LHCII phosphorylation activities (Fig. 1). In absence of added substrate, both fractions exhibited autophosphorylation activity. However, different polypeptides present in the preparations were phosphorylated. In the cytochrome b,Jf-enriched fraction the 64-kDa polypeptide was heavily phosphorylated. The kinase-enriched fraction phosphorylates mostly a low molecular mass peptide (lo-12 kDa) (Fig. 1, a and b, lanes 1-3). The phosphorylation activity of both fractions in absence of added substrate was enhanced by addition of plastoquinol-1 and inhibited by addition of DBMIB (Fig. 1).

However, only the cytochrome be/f-enriched fraction phosphorylated LHCII. This activity was enhanced by addition of plastoquinol-1 but only slightly inhibited by DBMIB (Fig. 1, a and 6, lanes 4-6).

Association of the Cytochrome b6/f Complex with LHCII Kinase-The data presented above indicate that at least a fraction of the total 64-kDa polypeptides present in the extract co-migrates with the cytochrome complex. The question arises whether the cytochrome complex and the kinase present in the AMS-OG preparation share common or distinct detergent micelles having the same specific density. To answer this question the 40-55% ammonium sulfate-precipitated fraction (see Table I) was solubilized in @-octyl D-

67

43

31

21

14

- -

-

-.“a

. . -

o- Kin ._YxL.

FIG. 2. LHCII kinase activity of cytochrome be/f-enriched fraction obtained by histone-Sepharose column. A, an AMS- OG preparation (lane I) was bound to a histone III-S-Sepharose column; the unbound material was washed through (lane 2), and the retained fraction was eluted with high salt, desalted, and concentrated 5-fold (lane 3). Aliquots were assayed for cytochrome (cyt) and kinase (Kin) activity with added histone III-S and LHCII as substrates. The polypeptide pattern of aliquots of equal volumes was resolved by SDS-PAGE; the amount of cytochrome f and kinase activity (histone III-S) present in lane 3 were 10 and 22%, respectively, of that loaded on the column. B, aliquots from lane 3 were phosphorylated in absence of added substrate, with addition of LHCII with or without plastoquinol-1. Equal samples from the phosphorylation assay were resolved by SDS-PAGE and autoradiographed as described. The specific kinase activity in absence of added substrate (auto phosphorylation) or using LHCII or histone III-S as a substrate was 5,900, 7,766, and 17,520 units mg protein-‘, respectively. PQH,, reduced plastoquinone-1.

glucoside and passed through a histone (a kinase substrate) affinity column which was reported before to bind the kinase specifically (6-8).

The results (Fig. 2) show that a fraction of kinase activity is retained by the column and can be eluted only by high salt washing. Examination of the absorption spectrum of this fraction and its polypeptide pattern (average of five experiments) demonstrate that about 10% of the cytochrome bs/f complex present in the loaded material was retained as well, as compared with 25% retention of the initial kinase activity measured with histone III-S as a substrate. More- over, this fraction phosphorylated LHCII and the activity was enhanced by addition of plastoquinol-1 (Fig. 2B) indi- cating that part of the kinase and the cytochrome bs/f share common detergent micelles.

The polypeptide pattern of the retained fraction showed enrichment in the 64-kDa polypeptide as well as in the cytochrome bs/f components relative to the other few con- taminating polypeptides. A large number of polypeptides present in the AMS fraction loaded on the column were not


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Redox Control of LHCII Kinase Activity by Cytochrome be/f Complex 19745

1 anti anti Kinase cyt-f e*

B Mr anti anti x10.3 Kinase cyt.f

e-lelr-i

94 67 -- -

43

31

21

14 .

FIG. 3. Specificity of antibodies against the native and denatured 64-kDa kinase and cytochrome (cyt) f. A, dot-blot showing antibodies interaction with the AMS-OG nondenatured fraction; B, Western blot using AMS-OG resolved by denaturing SDS-PAGE and detected with ““I-protein A. The amount of protein loaded on the dots was 40, 20, 10, and 5 pg, respectively, and the antibodies dilution was %SOO for cytochrome f and %OO for the kinase antibodies. For the Western blots, 32 pg of protein was loaded on each slot. The exposure time was 48 h for the anti-64-kDa kinase and 18 h for the anti-cytochrome f antibodies.

TABLE II Ratio of cytochrome f/kinase activity in fractions obtained by

immunoprecipitation or affinity columns The 100% value for cytochrome (cyt.) f (nanomoles) and kinase

activity (units) was 6.8 and 3,870 and 7.0 and 19,000 for the immunoprecipitated and affinity column experiments, respectively. Kf = kinase and cytochrome f sharing the same micellar domains precipitated or bound by anti-cytochrome f or anti-kinase antibodies; f = total cytochrome f precipitated or bound by anti-cytochrome f antibodies; k = total kinase precipitated or bound by anti-kinase antibodies.

Measured Cross-Pre- Method ~ cipitation

cyt. f Kinase Kf/f Kf/k nmol % units % %

Immunoprecipitation ct cyt. f 2.6 38 232 6 50 (Y kinase 1.3 19 805 21 29

Immunoaffinity column acyt.f 5.5 79 4,085 21 40 (Y kinase 2.2 32 17,100 90 23

bound and were eluted in the flow-through wash (Fig. 2A). Additional support for the possibility that part of the

kinase and the cytochrome share the same micellar domains was found in results of experiments in which immunoaffinity columns using antibodies against the kinase or cytochrome bs/f components were used. The specificity of the anti-64- kDa kinase and anti-cytochrome f antibodies toward their respective antigens as well as the antibodies ability to react with both the denatured and the native protein is demonstrated in Fig. 3. The antibodies against the 64-kDa kinase reacted also with Chlamydomonas (data not shown) and pea thylakoids (see below).

Immunoaffinity columns using both anti-kinase or anti- cytochrome f antibodies retained kinase activity and cytochromes as demonstrated by the data shown in Table II and Fig. 4. As in the case of the substrate affinity purification, the fractions retained by the column and eluted only with high salt concentration were enriched in the 64-kDa kinase polypeptide and the cytochrome components relative to other polypeptides. The flow-through fraction of the column contained most of the other polypeptides present in the

Mr AhWOG x10-3

g4- -wm 67--e

43 - u

31 - u-

21 .._

14 .

anti cyt f

00

c e

- a-

FIG. 4. Polypeptide pattern of fractions retained by immunoaffinity columns using antibodies against cytochrome f and 64-kDa kinase. An AMS-OG fraction was incubated with anti-cytochrome f or anti-kinase antibodies covalently attached to CNBr-Sepharose. The nonbound material was removed by washing (lane I) and the bound fraction was eluted with high salt, desalted, and concentrated as described (lane 2). Aliquots of equal volume were resolved by SDS-PAGE and stained as described. Closed arrow, the 64-kDa polypeptide (kinase); open arrow, cytochrome f.

loaded material (Fig. 4) as observed also with the substrate affinity column (compare with Fig. 2A). Similar results were obtained when an anti-cytochrome bs immunoaffinity column was used (data not shown).

Immunoprecipitation using anti-kinase and anti-cytochrome f antibodies also showed similar results. Both antibodies precipitated cytochrome bs/f components and kinase activity (Table II). The ratio of kinase activity to the cytochrome bs/f content bound by the respective antibodies differed in various AMS preparations. However, if one con- siders that AMS fractions consist of a mixture of micelles containing either free kinase (k), free cytochrome complex, (f), or cytochrome bs/f sharing the same domains with the kinase (kf), one can calculate the ratio of the components sharing the same micelles relative to the total amount of cytochrome or kinase immunoprecipitated or retained by the affinity columns (Table II). Such a calculation indicates that the ratio of the kinase and cytochrome complex sharing the same domains to the bound kinase (kf/k), or to the bound cytochrome (kf/f), is quite similar irrespective of the method used for enrichment, immunoprecipitation, or immunoaffinity columns.

It has been demonstrated before that the cytochrome bs/f complex is located in both the grana partitions as well as in the stroma lamellae (28, 29). LHCII kinase was reported to fractionate with the grana thylakoids (30). To ascertain that both cytochrome b6/f and the 64-kDa polypeptide share similar membrane domains, thin sections of isolated pea thylakoids were immunodecorated with anti-cytochrome f and anti-kinase antibodies by the immunogold technique.

The results (Fig. 5) show that cytochrome f (Fig. 5A) is localized in the grana partitions and partially in the stroma lamellae while the kinase seems to be located more specifically in the grana domains, particularly at the edges of the grana stacks (Fig. 5B).

CHAPS Treatment Abolishes the Activation of LHCII Ki- nase by Plastoquinol-1 in Preparations Enriched in Cyto- chrome b,Jf-The affinity enrichment using substrate or specific antibodies against cytochrome be/f components or kinase, demonstrate the existence of a kinase/cytochrome fraction (kf, Table II) present in the AMS preparations. The presence of kinase and cytochrome be/f in the same domains may imply that their association or interaction is a required condition for the activation of the kinase by addition of plastoquinol-1 and inhibition of this activation by DBMIB. To test this possibility another detergent, CHAPS, which


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19746 Redox Control of LHCII Kinase Activity by Cytochrome be/f Complex

. - . . : -J

1

c

. : * ,_ .

,- . ,

FIG. 5. Localization of cytochrome f and 64-kDa kinase by immunogold labeling of stacked pea thylakoids. Electron micrographs of pea stacked thylakoids immunodecorated with anti- cytochrome f (A) or anti-64-kDa kinase (B) antibodies as detected by protein A gold 20 nm particles. A total of 409 and 359 immunogold particles were counted in five different micrographs using anti- cytochrome f and anti-64-kDa kinase antibodies, respectively, and the relative distribution of the particles over grana and stroma lamellae was analyzed. About 60 and 83% of the immunogold particles were localized in the grana regions for cytochrome f and the kinase antigens, respectively. Within the grana thylakoids, 80% of the anti-kinase protein A gold particles were localized at the edges of the stacks. g and s, particles localized over grana or stroma lamellae regions. e, particles localized at the edges of grana stacks. (bar, 1 pm). Magnification x 23,400.

was shown before to separate the kinase from the cytochrome bs/f complex (6) was used.

The fraction precipitated by 40-55% ammonium sulfate was solubilized with either OG or CHAPS. Assay of kinase activity using histone III-S as a substrate and the effect of plastoquinol-1 and DBMIB is shown in Fig. 6. Both AMS- OG and AMS-CHAPS had a low autophosphorylation activity, phosphorylated well histone as an added substrate, and their phosphorylation activity was inhibited by DBMIB. However, only the AMS-OG preparation exhibited an enhanced kinase activity toward histone in the presence of plastoquinol-1.

To test whether the lack of kinase activation by plastoquinol-1 in the AMS-CHAPS preparation is due to a sepa-

12341234

FIG. 6. Effect of plastoquinol and DBMIB on histone phosphorylation by AMS-OG and AMS-CHAPS fractions. AMS- OG and AMS-CHAPS fractions were assayed for histone III-S phosphorylation. Lane 1, no additions; lane 2, with addition of histone III-S; lanes 3 and 4, same as lane 2 with addition of plastoquinol-1 or plastoquinol-’ and DBMIB, respectively. Aliquots (30 pg) were resolved by SDS-PAGE for their polypeptide composition and the gels were autoradiographed as described under “Ex- perimental Procedures.”

TABLE III Effect of detergents on LHCII phosphorytation by cytochrome bG/f

kinase-enriched fractions prepared by histone III-S affinity columns AMS-OG and AMS-CHAPS preparations were passed through

histone affinity columns and the retained fractions were eluted with high salt. After desalting, the fractions were assayed for their polypeptide composition and kinase activity as described under “Experi- mental Procedures.” The polypeptide pattern was similar to that shown in Fig. 2. The specific kinase activity in absence of added substrate was 1920 and 1100 units mg protein-’ for the OG- and CHAPS-treated samples, respectively. For added histone III-S phosphorylation, the specific kinase activity was 9900 and 7700 for the OG- and CHAPS-treated samples, respectively. 6, net change in phosphorylation. PQH2, reduced plastoquinone-1.

Detergents

Additions OG CHAPS

pm01 RPP incorpo- 6 pm01 ‘*P incorporated/assay rated/assay

d

None 34 18 LHCII 52 18 20 2 LHCII, PQH2 71 37 23 5 LHCII, PQH,, DBMIB 21 -13 10 -8 LHCII, PQH,, DIBB 22 -12 7 -11

ration between the kinase and the cytochrome complex, AMS-CHAPS and AMS-OG were fractionated by a substrate affinity column and tested for kinase activity and cytochrome bs/fcontent. After loading, the columns were washed with low salt buffer and then eluted with high salt buffer as described under “Experimental Procedures.” The desalted eluates were assayed for cytochrome be/f content and kinase activity on histone III-S or LHCII as added substrates. The results of such experiments demonstrated that the fractions retained by both columns contained cytochrome be/f and kinase. The ratio of kinase (units. 10-3)/cytochrome f (nanomoles) was 6 for the AMS-CHAPS and 3.2 for the AMS- OG eluents, respectively.

The kinase activity of these fractions was measured without or with addition of isolated LHCII, in the presence or absence of plastoquinol-1 and inhibitors of cytochrome b,Jf complex activity (DBMIB and DIBB (16, 31)) as shown in Table III. Neither significant increase in the phosphorylation activity by addition of LHCII nor stimulation by plastoquinol-1 could be demonstrated in the CHAPS-treated fraction. In contrast, the OG-treated fraction exhibited a higher phosphorylation activity in the presence of added LHCII, and this activity was doubled by addition of plasto-


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Redor Control of LHCII Kinase Activity by Cytochrome be/f Complex

Mr AMS- xIO-~ OG

94 -a _^

CHAPS

.

OCTYL-GLUCOSIDE

I )L - - - Kin - __- - _ . r 7 -. -

5 6 7 8 9 IO II 12 5 6 7 8 9 IO II I2 fraction no. froct1on “0

FIG. 7. Different polypeptide pattern of fractions obtained by sucrose density centrifugation of AMS-OG or AMS-CHAPS. AMS-OG and AMS-CHAPS preparations were loaded on sucrose density gradients and centrifuged as described. Fractions were collected and equal aliquots of fractions 5-12 were analyzed for their polypeptide pattern by SDS-PAGE. Analysis of the cytochrome (cyt) be/f content and kinase (Kin) activity of such gradient fractions were carried out as well (data not shown; see also Ref. 15) and were in agreement with the presence of the polypeptides identified as cytochrome b6/f components and 64-kDa kinase.

TABLE IV

LHCII phosphorykztion activity of OG- or CHAPS-treated fractions obtained by sucrose density centrifugation

AMS-OG and AMS-CHAPS fractions were prepared and centrifuged on sucrose density gradients as described under “Experimental Procedures.” Fractions containing the highest kinase activity toward histone III-S in both preparations (equivalent to fraction 10 in Fig. 7), were analyzed for their LHCII phosphorylation activity as described under “Experimental Procedures.” The kinase specific activity in absence of added substrate was 1300 and 750 and with histone III-S as a substrate was 5,200 and 6,000 units.mg protein-’ for the OG- and CHAPS-treated fractions, respectively. 8, net change in phosphorylation. PQHZ, reduced plastoquinone-1.

Detergents

Additions Octyl glucoside CHAPS

pm01 32P incorpo- 6 pm01 “P incorporated/assay rated/assay

d

None 5.5 5.0 LHCII 8.8 3.3 7.3 2.3 LHCII, PQHZ 11.5 6.0 7.5 2.5 LHCII, PQHZ, DBMIB 7.9 2.4 6.8 1.8

quinol-1. Accordingly, autoradiograms of the SDS-PAGE- resolved polypeptides of the reaction mixture of these experiments indicated phosphorylation of the LHCII polypeptide in the OG-treated fraction (data not shown). The kinase activity of both fractions was significantly inhibited by DBMIB and DIBB (Table III). These results show that although cytochrome bs/f is present in both preparations, phosphorylation of LHCII and activation of the kinase by plastoquinol-1 occurred only in the fraction solubilized in OG.

To ascertain further that this is the case, ammonium sulfate-precipitated fractions solubilized either in CHAPS or OG were separated on sucrose density gradients as described under “Experimental Procedures.” Fractions were collected and analyzed for their polypeptide pattern, cytochrome be/f content, and kinase activity as above (Fig. 7, Table IV). The kinase activity and the corresponding 64- kDa polypeptide was found in the same positions in both gradients (fractions 10-11). However, the cytochrome distribution was significantly different. Most of the cytochrome complex was found in fractions 9-10 in the OG-solubilized

material while in the CHAPS-treated sample the cytochrome be/f progressively dissociated from the kinase and appeared in fractions 5-9 (Fig. 7). Several heavily stained polypeptide bands (18-19 kDa, 33-50 kDa) were found to be located in both preparations in fractions 11-12. The presence of these polypeptides did not correspond to the kinase activity.

Assay of the kinase activity in the absence or presence of added LHCII as a substrate and its stimulation or inhibition by plastoquinol-1 or DBMIB respectively, are shown in Table IV. As in the previous experiment, the activity of LHCII phosphorylation, its enhancement by plastoquinol-1, and inhibition by DBMIB are significantly higher in the OG-solubilized fractions as compared with that of the CHAPS-solubilized fractions.

DISCUSSION

The aim of this work was to test whether preparations of LHCII kinase can retain the ability to be activated by reduced plastoquinone and inhibited by cytochrome beIf inhibitors as exhibited by the LHCII kinase in situ (2, 4, 5, 9, 11-13). Availability of such a preparation could serve as an additional proof for the involvement of the cytochrome b6/f complex in the activation of LHCII kinase and facilitate the investigation of its mechanism. The strategy used was to start from crude preparations containing all the components possibly involved in the process and gradually remove those which may not be required for, or interfere with, the activity of the redox-controlled LHCII kinase. The results of this work demonstrate that kinase activity partially copurifies with the cytochrome be/f complex. The detergent extraction procedure results in the removal of about 60% of the cytochrome complex from the membranes and about 50% of the kinase activity assayed with histone as a substrate as reported before (6). The association of cytochrome be/f and kinase activity persisted throughout the purification procedure. Similar results were obtained using spinach (14) and Chlamydomonas (10) thylakoids. Preliminary results indicated that kinase activity is retained even in cytochrome b6/f complex preparations following additional purification steps (15).

The presence of both cytochrome complex and kinase in these preparations could be fortuitous. Their copurification


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19748 Redox Control of LHCII Kinase Activity by Cytochrome bs/f Complex

could thus be due to their similar properties relative to the purification procedures. Indeed, data obtained by sucrose density centrifugation show that the fraction mostly enriched in the cytochrome bs/f complex does not exactly coincide with that enriched in the kinase activity (10, 15, and this work).

The membrane extraction procedure might have largely disrupted the association between the kinase and cytochromes. The extract appears to consist of a mixture of micelles (kf), containing various amounts of cytochrome be/f and kinase as well as a population of micelles containing free kinase (k) or cytochrome bs/f (f). Thus fractionation by sucrose density gradient centrifugation can partially separate the micelles enriched in cytochromes from those enriched in kinase, while immunoprecipitation or affinity columns may precipitate or bind all the micelles containing the respective components.

Indeed, the immunoprecipitation and binding to immunoaffinity columns of both the cytochrome b,/f and kinase using antibodies to either one of them (cytochrome f, cytochrome bs, or to the kinase), as well as the affinity columns using the substrate (histone III-S), indicate that at least part of the cytochrome complex and kinase share common detergent micelles. The relative amounts of cytochromes and kinase sharing such micelles seem to be quite similar irrespective of the technique used for their separation from the mixture (Table III, Figs. 2 and 4).

The presence of the cytochrome complex and kinase in the same micelles per se could be the basis for their functional association. So far such an association was inferred only from experimental results obtained in uiuo using mu- tants lacking cytochrome be/f (9-12) or inhibitors specifically binding to the cytochrome be/f plastoquinol oxidizing site (Q,) (13). The possibility that such a functional association is retained in vitro in fractions obtained by use of the detergent OG is indicated by the fact that these fractions retain the ability to phosphorylate LHCII and to be activated by reduced plastoquinone-1 (Tables III and IV, Figs. 1, 2, and 6). Whether the functional association inferred from the data presented here results from a reversible interaction between the cytochrome be/f and kinase or from a stable association between these components cannot be decided yet.

Nevertheless, fractions prepared by use of CHAPS as a detergent, despite the fact that they contain both kinase and cytochromes as those prepared by OG, have only a residual LHCII phosphorylation activity which is not enhanced by plastoquinol-1. This indicates that the functional association of the kinase and cytochrome complex is disrupted by CHAPS. This explanation is supported by previous reports showing that CHAPS facilitated complete dissociation of the cytochrome complex from the kinase (6-8). In addition, CHAPS might interfere directly with the ability of the enzyme to use LHCII as a substrate. LHCII kinase purified by use of this detergent has a lo-fold higher activity toward histone as compared with LHCII as a substrate (6). In our fractionation procedure, the kinase was not completely separated from the cytochrome bs/f complex by CHAPS treatment as reported (6-8) possibly due to the fact that the residual ammonium sulfate in the AMS preparation was not removed prior to the next purification step.

Presence of kinase activity in the various fractions coin- cided with that of a 64-kDa polypeptide band putatively identified as LHCII kinase (6) and used in this work for the preparation of antibodies. The phosphorylation of this polypeptide as shown here could be thus considered as autophos-

phorylation in agreement with previously published data (30). The phosphorylated lo-12-kDa polypeptide in fractions obtained by sucrose density centrifugation could be identical to the polypeptide of the same apparent molecular mass which was reported to be phosphorylated in isolated thylakoids and subject to redox control (9, 32). The 10-12- kDa polypeptide was present in similar amounts in both fractions as judged from its staining intensity (Fig. 1). The enhanced phosphorylation of this minor polypeptide band in the kinase-enriched fraction as compared to that found in the cytochrome b,Jf-enriched fraction could be due to a modulation of the 64-kDa kinase substrate specificity in this fraction containing a reduced amount of the cytochrome b,Jf complex. Modulation of the kinase substrate specificity is also indicated by the fact that only the cytochrome- enriched fraction phosphorylated LHCII (see below). The phosphorylation of the lo-12-kDa polypeptide was increased by addition of plastoquinol-1 and inhibited by DBMIB, as shown also to be the case for the lo-12-kDa polypeptide in isolated pea (32) and Lemna thylakoids (9), and thus is similar to phosphorylation of the LHCII polypeptides (32).

The weak phosphorylation of other polypeptides present in the sucrose gradient fractions could be due to the co- migration of another protein kinase of the thylakoid membrane, previously defined as CPKl by Lin et al. (33). This enzyme, a 25-kDa polypeptide, was purified to homogeneity by Coughlan et al. (7) and found to be a weak kinase relative to the 64-kDa kinase and could not phosphorylate LHCII polypeptides as added substrate (7).

Using the immunogold labeling technique, the 64-kDa polypeptide was localized almost exclusively in the grana region of the thylakoids, whereas the cytochrome bs/f appeared also in the stroma lamellae as reported before (28, 29). This finding is in agreement with the expected localization of LHCII kinase, which should be close to the photosystem II-bound LHCII, acting as the kinase substrate in situ (34), as well as with previous data showing its co- migration with photosystem II (30). The observed higher relative concentration of the immunogold-labeled kinase at the edges of grana stacks could be also in agreement with the observation that only a fraction of the LHCII polypeptides serve as the kinase substrate and, following phosphorylation, migrates to the stroma domains (35). This LHCII polypeptide fraction was found to be located at the edges of the grana stacks as well (36). Thus it is possible that the LHCII kinase, whose redox-dependent activation is mediated via cytochrome b6/f, is mostly located at the edges of the grana near its substrate and mediator. Other redox- controlled kinase(s) present in the thylakoids (9, 32) may be involved in the phosphorylation of other photosystem II polypeptides (37-39). It is intersting to consider the possibility that the distribution of the cytochrome b,Jf complex between the grana and stroma domains reflect the presence of two populations, one associated with the kinase in the grana and one in the stroma free from such association.

So far it has been reported that the purified LHCII kinase was not activated by redox reagents (6,8). It is possible that following extraction and complete dissociation from the cytochrome complex the kinase has lost both its ability to phosphorylate LHCII and its redox-negative control. The process of LHCII kinase deactivation is not yet well under- stood. A persisting activated state of the kinase in situ in absence of reduced plastoquinol was reported to exist in isolated thylakoids of Acetabularia (13).

The novelty of the data presented here is that LHCII phosphorylation by kinase preparations associated with cy-


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Redox Control of LHCII Kinase Activity by Cytochrome b6/f Complex

tochrome be/f can be activated in vitro by addition of plastoquinol-1 and inhibited by DBMIB or DIBB both known to interact specifically with the cytochrome complex (16, 31). The maximal redox activation obtained in uitro when using LHCII as a substrate in the various preparations described here was -2-fold as compared with a IO-20-fold activation in situ (1, 2). The residual 2-fold activation of the kinase by plastoquinol-1 in the micelles containing cytochrome b,=,/f and kinase may thus indicate that at least lo- 20% of the total kinase present in these fractions retains the potential to functionally interact with the cytochromes. The possibility that some additional minor polypeptides required for the activation process are lost during the extraction and fractionation procedure cannot be excluded.

Addition of DBMIB or DIBB completely abolished the stimulation of LHCII phosphorylation by plastoquinol-1 and also reduced the amount of incorporated [32P]phosphate even below the basal activity in absence of added substrate. This indicates that the autophosphorylation activity was also partially inhibited by the quinone analogue (Table III). Inhibition of autophosphorylation activity by DBMIB could be demonstrated also in fractions obtained by use of CHAPS. This activity in fractions obtained by sucrose gradient centrifugation in presence of OG could also be stimulated by plastoquinol-1 and inhibited by DBMIB irrespective of their LHCII phosphorylation capability and cytochrome b6/f content (Fig. 1).

It is possible that while the activation of the LHCII kinase is mediated by the cytochrome bs/f complex the deactivation may not be related to the kinase interaction with the cytochrome complex. The question arises whether deactivation may be achieved via binding of plastoquinone or its analogues to a quinone binding site within the kinase itself. This mechanism may account for the observed deactivation of the kinase when plastoquinone/plastoquinol ratio in- creases (4, 5). The putative binding site may not be affected by the detergent treatment in the same way as the kinase interaction with the cytochrome complex. This is indicated by the results presented in Figs. 1 and 6 and Tables III and IV. DBMIB may inhibit the kinase either by preventing the interaction of the cytochrome be/f complex with plastoquinol, or by binding to the putative quinone binding site of the enzyme. This site may also be involved in the activation of the kinase by plastoquinone toward substrates other than LHCII.

Thus, based on the data presented in this work as well as on previously reported results (9-13, 15), we propose as a working hypothesis that the redox-controlled activation of the kinase toward LHCII as a substrate is mediated by a cytochrome bs/f complex following its interaction with a plastoquinol molecule at the QZ site (40). The deactivation may be induced by binding of plastoquinone or an apropriate quinone analogue (DBMIB, DIBB) to a distinct quinone binding site of the LHCII kinase. The specificity of interaction of plastoquinone, plastoquinol, and analogues with the putative quinone binding site of the enzyme and the resulting effects on the enzyme activity and substate speci-

ficity could now be probed in vitro using the kinase preparations described in this work.

AcknowEedgments-We wish to thank Rina Timberg for her skill- ful help and performance of the immunogold labeling experiments. The isolated chloroplasts used for the electron microscope experiments were prepared by Dr. Ioana Akoyonoglou, N. R. DEMOK- RITOS, Athens, as part of a larger work project for localization of various thylakoid components in the grana stacks before and after unstacking. We are grateful to Dr. F.-A. Wollman for his kind gift of anti-cytochrome f antibodies.

REFERENCES 1.

2.

::

i:

ii: 9.

10.

11.

12. 13.

14.

15.

16. 17. 18.

2 21.

22.

23. 24. 25. 26. 27.

28. 29.

30. 31.

ii:

34.

35.

36.

37.

38.

39.

40.

Bennett, J., Steinback, K. E., and Arntzen, C. J. (1980) Proc. Notl. Acad. Sci. U. S. A. 77,5253-5257

Allen, J. F., Bennett, J., Steinback, K. E., and Amtzen, C. J. (1981) Nature 29 1,25-29

Staehelin, L. A., and Amtzen, C. J. (1983) J. Cell Biol. 97, 1327-1337 Allen, J. F., and Horton, P. (1981) Biochim. Biopkys. Acta 636, 290-295 Wollman, F.-A., and Delepelaire, P. (1984) J. Cell Biol. 95, l-7 Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385 Coughlan, S. J., and Hind, G. (1986) J. Biol. Ckem. 261,14062-14068 Coughlan, S. J., and Hind, G. (1987) J. Biol. Chem. 262,8402-8408 Ga$$lSghahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221,

W;hnan, F.-A., and Lemaire, C. (1988) Eiochim. Biopkys. Acta 933,85-

By;-;tttJ., Shaw, E. K., and Michel, H. (1988) Eur. J. Biochem. 171,

Coughlan, S. 3. (1988) B&him. Biopkys. Acta 933,413-422 Gal, A., Schuster, G., Frid, D., Canaani, O., Schweiger, H. G., and Ohad,

1. (1988) J. Biol. Ckem. 263,7785-7791 Clark, R. D., Hind, G., and Bennett, J. (1985) in Molecular Biology o the

Photosynthetic Apparatus (Steinback, K. E., Bon&, S., Arntzen, tt J., and Bogorad, L., eds) pp. 259-267, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Gal, A., Mor, T. S., Hauska, G., Herrmann, R., and Ohad, I. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., ed) Vol. II, pp. 783-786, Kluwer Academic Publishers, Dordrecht, Netherlands

Malkin, R. (1982) Biochemistry 21,2945-2950 Hauska, G. (1986) Methods Enzymol. 126, 271-285 M$t, J. E., and Amtzen, C. J. (1980) Btochrm. BLophys. Acta 559, lOO-

Cuatrecasas, P. (1970) J. Biol. Chem. 246,3059-3065 Tzinas, R., Argyroudi-Akayunoglou, J., and Akayunoglou, G. (1987) Pho-

tosyntk. Res. 14, 241-245 Schuster, G., Timberg, R., and Ohad, I. (1988) Eur. J. Biockem. 177,

403-410 Bollum, F. (1965) in Procedures in Nucleic Acid Research (Cantoni, G.

L., and Davis, D. R., eds) pp. 296-300, Harper and Row, New York Laemmli, U. K. (1970) Nature 227,680-685 Gershom, J. M., and Palade, G. E. (1983) Anal. Biochem. 131, 1-15 Peterson. G. L. (1977) Anal. Biochem. 83.346-356 Arnon, fi. (1948) Plant Pkys ,iol. 24, 1-15’ Chua, N.-H., Bartlet, S. G., and Weiss. M. (19821 in Methods in Chlora- ,

plast Molecular Biolog (Edelman, M., H.&ck,k. B., and Chua, N.-H., eds) pp. 1063-1080, E 8evler Scienti r

Albertson, P.-A. (1985) Ph fit Publishing Co., Amsterdam

siol. Anderson, J. M., Goodchd ,x

Veg. 23, 731-739 , D. J., and Thomson, W. W. (1990) in Current

Research in Photos nthesis (Baltscheffsky, M., ed) Vol. II, pp. 803-808, Kluwer Academic 8 ubhshers, Dordrecht, Netherlands

Coughlan, S. J., and Hind, G. (1987) Biochemistry 26, 6515-6521 Oettkmei r, W., Masson, K., and Dostatni, R. (1987) .Biochim. Biophys.

Acta 890, 260-269 Bennett, J., Shaw, E. K., and Bakr, S. (1987) FEBS Lett. 210, 22-26 Lucero, H. A., Lm, Z.-F., and Racker, E. (1982) J. Biol. Chem. 257,

12157-12160 Kyle, D. J., Kuang, T.-Y., Watson, J., and Amtzen, C. J. (1984) Biochim.

Biophys. Acta 765,89-96 Larsson, U. K., Sundby, C., and Andersson, B. (1987) Biochim. Biophys.

Acta 694. 59-W Bassi, R., Hoyre-Hansen, G., Barbato, R., Giacometti, G. M., and Simp-

son, D. J. (1987) J. Biol. Chem. 262, 13333-13341 Millner, P. A.. Marder. J. B.. Gounaris. K.. and Barber. J. (1986) Biochim.

Biophys. A&a f362,>0-37 ,.

Wollman, F.-A., and Lemaire, C. (1988) in Photocatalytyic Production of Energy-rich Compounds (Hall, D., and Grass, I., eds) pp. 210-214, Elsevier Scientific Publishing, Amsterdam

Michel, H., Hunt, D. F., Shabanowitz, J., and Bennett, J. (1988) J. Biol. Chem. 263, 1123-1130

Cramer, W. A., Black, M. T., Widger, W. R., and Girvin, M. F. (1987) in The Lizkt Reactions (Barber. J.. ed) DD. 447-493. Elsevier Scientific Publi&ingCo., New York *’


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A Gal, G Hauska, R Herrmann and I Ohadcytochrome b6/f complex. In vitro control of kinase activity.

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and

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