A redox-active FKBP-type immunophilin functions in accumulation … · A redox-active FKBP-type immunophilin functions in accumulation of the photosystem II supercomplex in Arabidopsis

A redox-active FKBP-type immunophilin functions inaccumulation of the photosystem II supercomplexin Arabidopsis thalianaAmparo Lima*, Santiago Lima†, Joshua H. Wong*, Robert S. Phillips†‡, Bob B. Buchanan*§, and Sheng Luan*§

*Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720; and Departments of †Chemistry and ‡Biochemistry andMolecular Biology, University of Georgia, Athens, GA 30602

Contributed by Bob B. Buchanan, June 30, 2006

Photosystem II (PSII) catalyzes the first of two photosyntheticreactions that convert sunlight into chemical energy. Native PSII isa supercomplex consisting of core and light-harvesting chlorophyllproteins. Although the structure of PSII has been resolved by x-raycrystallography, the mechanism underlying its assembly is poorlyunderstood. Here, we report that an immunophilin of the chloro-plast thylakoid lumen is required for accumulation of the PSIIsupercomplex in Arabidopsis thaliana. The immunophilin,FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamilythat functions as peptidyl-prolyl isomerases (PPIases) in proteinfolding. FKBP20-2 has a unique pair of cysteines at the C terminusand was found to be reduced by thioredoxin (Trx) (itself reducedby NADPH by means of NADP-Trx reductase). The FKBP20-2 pro-tein, which contains only two of the five amino acids required forcatalysis, showed a low level of PPIase activity that was unaffectedon reduction by Trx. Genetic disruption of the FKBP20-2 generesulted in reduced plant growth, consistent with the observedlower rate of PSII activity determined by fluorescence (usingleaves) and oxygen evolution (using isolated chloroplasts). Anal-ysis of isolated thylakoid membranes with blue native gels andimmunoblots showed that accumulation of the PSII supercomplexwas compromised in mutant plants, whereas the levels of mono-mer and dimer building blocks were elevated compared with WT.The results provide evidence that FKBP20-2 participates specificallyin the accumulation of the PSII supercomplex in the chloroplastthylakoid lumen by means of a mechanism that has yet to bedetermined.

chloroplast thylakoid lumen � protein folding � photosynthetic electrontransport

Much of life on Earth is sustained by oxygenic photosyn-thesis, a process that utilizes sunlight to produce oxygen

and organic carbon from water and carbon dioxide. The absorp-tion of light and its conversion into chemical energy is broughtabout by two photosystems [photosystem II (PSII) and photo-system I (PSI)] acting sequentially. PSII catalyzes the light-dependent oxidation of water that results in the evolution ofoxygen. The electrons released in this reaction are transferredalong a photosynthetic electron transport chain that leads, bymeans of PSI, to the production of NADPH and ATP, thechemical energy currency used for carbon fixation.

The chloroplast PSII core complex consists of �17 proteinsubunits that include the D1 and D2 reaction centers forchlorophyll (Chl) P680 binding, cytochrome b559, CP43 andCP47 for building the Chl antennae, and other proteins whosefunction is less well characterized (1–4). The native form of PSIIresiding in the thylakoid membrane is believed to be a super-complex consisting of the core and peripheral light-harvestingcomplex II (LHCII) components. The light harvested by LHCIIis transferred to the core complex that brings about chargeseparation, thereby driving the transfer of electrons from waterto plastoquinone and initiating photosynthetic electron trans-port. Recent studies using x-ray and cryoelectron crystallography

have resolved the three-dimensional structure of the PSII com-plexes in both cyanobacteria and land plants (5–9), providing aplatform for further elucidation of PSII’s properties.

Despite our advanced understanding of its structure andfunction, relatively little is known about molecular mechanismsunderlying the biogenesis and maintenance of the PSII super-complex. It is established that biogenesis of the complex involvesthe translation of chloroplast as well as nuclear-encoded proteinsubunits, the latter imported from the cytosol. Although muchwork has investigated the pathways and mechanisms of importinto both the stroma and the lumen (10, 11), aside from a studythat identified a thylakoid-associated protein involved in PSIIbiogenesis (12), little is known about how the protein subunitsare assembled. It is certain that interaction among differentsubunits is required during assembly, because mutation of asingle subunit gene resulted in mutants lacking PSII (13–16).Further, a number of proteins that participate in assembly andmaintenance, such as molecular chaperones, most likely actspecifically on one side of the thylakoid membrane (e.g., at eitherthe stromal or luminal face).

Earlier studies on PSII assembly concentrated on the role ofstromal factors, such as the translation and import machinery,because only a limited number of proteins were considered toreside in the thylakoid lumen. However, the number of possibleluminal participants in assembly has increased dramaticallybecause of recent proteomic findings that suggest a populationof 80–100 proteins in that compartment (17–19). One of thepredominant groups identified is the immunophilin family madeup of FKBP (FK-506 binding protein) and cyclophilin members.Originally defined as receptors for immunosuppressive drugs(FK506 and cyclosporin A) (20), these proteins are now knownto occur widely and function as protein foldases and chaperones.The identification of at least 16 immunophilins in the thylakoidlumen suggests that these protein-folding catalysts play a criticalrole in the assembly and maintenance of protein complexes suchas the two photosystems that reside at least in part in thethylakoid lumen (21–23).

To pursue this possibility, we have applied a systematic geneticapproach to dissect the function of lumen immunophilins in thecontext of photosynthesis. We now report that an FKBP-typeimmunophilin localized in the thylakoid lumen, FKBP20-2,functions in the accumulation of the PSII supercomplex inArabidopsis.

Results and DiscussionIsolation of fkbp20-2 Mutants of Arabidopsis. All immunophilins ofthe thylakoid lumen are transcribed from the nuclear genome,

Conflict of interest statement: No conflicts declared.

Abbreviations:Chl,chlorophyll;Trx,thioredoxin;NTR,NADP-Trxreductase;PPIase,peptidyl-prolyl isomerase; PSI, photosystem I; PSII, photosystem II; T-DNA, transfer DNA.

§To whom correspondence may be addressed. E-mail: [email protected] [email protected].

© 2006 by The National Academy of Sciences of the USA

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translated in the cytosol, and targeted to the lumen as shown inseveral studies (17, 18, 24). To characterize the function of thisfamily of proteins, we isolated multiple transfer DNA (T-DNA)insertional mutants for each of the relevant immunophilin genes.We then screened the mutants for potential growth defectsunder short-day conditions (10-h light�14-h dark cycle with lightintensity of 180 �mol of photons�m�2�s�1). Two independentmutant alleles for the gene encoding a 20-kDa FKBP, FKBP20-2,showed stunted growth. The T-DNA insertional alleles(SALK�080069 and SALK�134696), referred to as fkbp20-2a andfkbp20-2b, contained T-DNA insertions located in introns 3 and5 (Fig. 1A). The mRNA of FKBP20-2 was undetectable byRT-PCR in both mutants (Fig. 1B), indicating that the T-DNAinsertions ( fkbp20-2a and 2b) had disrupted the expression ofthe FKBP20-2 gene. Under normal growth conditions, leaves ofboth mutants were much smaller and less green than the WT(Fig. 1C). Chl analysis revealed a reduction in Chl of �25% perleaf area for both mutants. (Chl a: fkbp20-2, 16.6 � 1.4 �g�cm2;WT, 22.2 � 1.7 �g�cm2. Chl b: fkbp20-2, 5.4 � 1.1 �g�cm2; WT,7.5 � 0.4 �g�cm2.) By contrast, the Chl a�b ratio was the samein mutant and WT. The fact that the two independent mutantsshowed a similar phenotype suggested that the defect in plantgrowth was caused by disruption of FKBP20-2 gene expression.The experiments described below were carried out with both ofthe fkbp20-2 mutants. Although data are presented for only onemutant ( fkbp20-2b) for simplicity, the results were consistentlythe same with the other mutant ( fkbp20-2a) within experimentalerror.

Amino Acid Sequence Analysis and Enzymatic Activity of FKBP20-2.The FKBP20-2 protein has a single FKBP domain with a clearthylakoid lumen targeting signal (designated in Fig. 2 by a boxshowing the arginine residues and a line marking the hydropho-bic component), consistent with proteomic analyses localizingFKBP20-2 to that compartment (17, 18). Sequence analysisrevealed homologs in cyanobacteria as well as algae, suggestingconservation among oxygenic photosynthetic organisms (Fig. 2).Further, the positioning of two conserved Cys residues locatedat the C terminus of the algal and land plant sequences suggeststhe presence of a disulfide bridge that would be absent in thecyanobacterial homologs (see arrows in Fig. 2).

Protein mutational analysis with the human FKBP12 proteinrevealed five amino acids that are required for PPIase activity(identified with an asterisk for the homologs shown in Fig. 2)

(25). All five are conserved in FKBP13, whereas FKBP20-2possesses only two of the essential amino acids (positions D155and F217 shown for the Arabidopsis protein in Fig. 2).

To determine whether FKBP20-2 could act as a foldase andwhether the conserved cysteines influence function, we assayedthe enzyme for PPIase activity by using a modification of theprotocol (26) adopted from the original procedure of Kofron etal. (27). As seen in Fig. 3A, WT FKPB20-2 was enzymaticallyactive (kcat�Km � 0.021 �M�1�s�1). However, its activity was�1�500 of that observed with FKBP13 (28), a possible reflectionof the above-noted deficiency in residues required for activity. Asimilar deficiency can explain the apparent lack of activity ofanother Arabidopsis immunophilin, FKBP42 (29). In this case,four of the five required amino acids described above weremissing from the active site of the enzyme.

Mutations in the cysteine residues reduced the PPIase activityby 50% (kcat�Km � 0.01 �M�1�s�1), suggesting that these resi-dues may be involved in catalysis or regulation (Fig. 3A).FKBP20-2 also resembled FKBP13 in being reduced by Esche-richia coli thioredoxin (Trx), itself reduced by NADPH andNADP-Trx reductase (NTR) (Fig. 3B). Negligible reduction wasobserved with reduced glutathione (data not shown). As ex-pected, the FKBP20-2 (C225S, C241S) mutant showed no re-sponse to reduced Trx or DTT (Fig. 3C). Although the redoxproperties of FKBP20-2 and FKBP13 were similar, the enzymesdiffered with respect to the effect of reduction. The activity ofFKBP20-2 was unaffected by Trx reduced either by NADPH andNTR (Fig. 3A) or DTT (not shown), whereas that of FKBP13was decreased (28). The low PPIase activity coupled with thedisconnect between Cys to Ser mutagenesis (50% inhibition ofactivity) and disulfide reduction (no effect on activity) raises the

Fig. 2. Comparison of FKBP20-2 and related protein sequences. The aminoacid sequence of A. thaliana (At3g60307) FKBP20-2 was aligned with ho-mologs in rice (O. sativa, Q7XHRO), a green alga (C. reinhardtii, gene modelC�2101108), and a cyanobacterium (S. elongatus, Q8DJW8). Sequences werealigned with ClustalW (38). The line indicates the hydrophobic stretch afterthe two arginine residues (boxed) that constitute the derived thylakoid tar-geting sequence (21). Arrows indicate conserved cysteine residues in landplants and the green alga that are absent in the cyanobacterial proteins.Asterisks indicate positions essential for PPIase activity (25).

Fig. 1. Genetic characterization and phenotype of FKBP20-2 mutant plants.(A) Localization of the T-DNA insertions in the two mutant alleles. (B) RT-PCRamplification of FKBP20-2 mRNA (lane 1) and ACTIN2 control (lane 2). The WTand mutant alleles are indicated above the gel. (C) Phenotype of the twofkbp20-2 mutants and WT plants 6 weeks after planting.

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question of the true enzymatic function of FKBP20-2. Althoughthe question remains open, it is noted that disulfide reductionand Cys-to-Ser mutagenesis were reported to give contrastingeffects with a stromal cyclophilin (30). Another unansweredquestion concerns the significance of reduction of FKPB20-2 byTrx. As discussed for another immunophilin, FKBP13, reductionby Trx, taking place in the stroma, could facilitate transport intothe lumen (28, 31). Once in the lumen, the protein would beoxidized to the disulfide form. It is possible that these proteinsare also reduced in the lumen by a disulfide protein that ispresent in that compartment.

Analysis of fkbp20-2 Mutants. The observation that the FKBP20-2protein is located in the thylakoid lumen and that mutantslacking the protein were stunted suggested that the mutantsmight have defects in photosynthesis. Photosynthetic parameterswere initially determined by measuring Chl fluorescence in theleaves of WT and mutant plants. Electron transport rates(ETRs) were found to be similar in the two plants for lightintensities up to 200 �mol of photons�m�2�s�1 (Fig. 4A). How-ever, when the light intensity reached 500 �mol ofphotons�m�2�s�1 or higher, the ETR of the mutant was signifi-cantly (approximately one-third) lower than WT, suggesting adefect in the electron transport chain. At higher intensities, thedifference became more pronounced. That the problem mightreside at the level of PSII was suggested by the finding that theefficiency of PSII electron transport (�PSII) estimated as afunction of incident photon flux density was also lower in mutantplants (Fig. 4B). By contrast, nonphotochemical quenching washigher in mutant plants than in WT plants (Fig. 4C), likely aresult of the need for increased dissipation of excess energybecause of lower PSII activity and photochemical quenching.The maximum efficiency of PSII photochemistry (Fv�Fm) wasalso decreased in mutant plants (data not shown). Taken to-gether, these results suggested that fkbp20-2 mutants had defectsspecifically related to PSII function.

PSII was further analyzed by measuring ferricyanide-dependent oxygen evolution with isolated chloroplast thylakoidmembranes. As observed in the fluorescence experiments, ac-tivity in the fkbp20-2 mutant was significantly reduced (approx-imately one-third) relative to WT (Fig. 5). This finding issignificant in that it confirms the fluorescence data (based onmutant leaves containing less Chl per leaf area than WT) withan independent oxygen evolution assays (based on equal Chlconcentrations). The results with both approaches were consis-tent with a defect in PSII.

Function of FKBP20-2. Although they suggested a defect in PSIIfunction, the above results gave no indication as to the nature ofthe problem. We, therefore, examined the composition of PSIIin mutants and WT plants by using a native gel procedure toseparate and quantify the photosynthetic protein complexes.

Fig. 3. Enzymatic and redox activity of FKBP20-2. (A) PPIase activity. The values here have been corrected for controls lacking the FKBP20-2 enzyme. The ratesshown represent first-order rate fits. (B and C) Reduction of FKBP20-2 by the NADP�Trx system from E. coli. Ctrl, control (FKBP20-2 alone); Cpl, complete Trx system(NADPH plus NTR plus Trx plus FKBP20-2); -Trx, complete Trx system omitting Trx; DTT, DTT alone. ‘‘Protein’’ refers to a Coomassie blue stain of the completeTrx system. (B) FKBP20-2. (C) FKBP20-2 (C225S, C241S).

Fig. 4. Light response curves for Chl fluorescence parameters. Data aremeans � SE, n � 3. (A) Electron transport rates (ETR). (B) Efficiency of PSIIelectron transport (�PSII). (C) Nonphotochemical quenching (NPQ).

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Isolated thylakoid membranes were treated with dodecyl mal-toside to solubilize the photosynthetic supercomplexes that werethen loaded at equal protein and Chl concentration onto bluenative gels and separated under nondenaturing conditions (Fig.6). The results revealed specific differences between the mutantand WT in the abundance of the PSII supercomplex. Thehigher-molecular-weight green bands corresponding to compo-nents of the supercomplex appeared less intense in the mutant(Fig. 6, row 1). Bands for the PSII monomer and dimer, on theother hand, showed increased accumulation in the mutantrelative to WT. Immunoblot scans revealed the following ratiosof D1 for mutant relative to WT: PS supercomplex, 38%; PSI,PSII dimer, 585%; PSII monomer, 326%. These differencesprovide additional evidence that the mutant has a defect in PSII.

To confirm and quantify these differences, we applied immu-noblot analysis to examine key components of the two photo-

systems and the cytochrome b6 f complex. The individual bluenative gel lanes were subjected to SDS�PAGE in a seconddimension to separate individual protein components in thecomplexes. Using antibodies against D1, the PSII reaction centerprotein, immunoblots clearly showed a reduced accumulation ofthis protein associated with the PSII supercomplex in fkbp20-2mutants (Fig. 6, row 2). By contrast, the levels of the D1 proteinin the lower-molecular-weight components (the monomers anddimers not incorporated in the PSII supercomplex) were higherin the mutant. The distribution of LHCII (light-harvestingcomplex II) proteins was not altered (Fig. 6, row 3). Thus, theresults indicated a change in supercomplex stoichiometry in themutant: less D1 and the same amount of LHCII. The PsaDprotein of the PSI complex (Fig. 6, row 4) and the cytochromef of the Cytb6 f complex (data not shown) showed no significantdifferences in mutant and WT.

Concluding Remarks. As the catalyst of the first of two photore-actions in the chloroplast electron transport chain, PSII convertswater into oxygen and a source of electrons (for the ultimatereduction of ferredoxin and NADP) and protons (for thesynthesis of ATP). The composition of this impressive proteincomplex has been largely elucidated. Many of the componentsare well characterized, and recent structural analysis has re-vealed the three-dimensional topology of the complex. A keyquestion that remains is how this complex, as well as its com-panion catalyzing the PSI photoreaction, is assembled andfunctionally maintained. Both complexes contain proteins thatare synthesized in the cytosol and chloroplast stroma and areultimately transported to the thylakoid membrane or luminalspace, where they are assembled. The present study shows thatFKBP20-2, an FKBP-type immunophilin of the thylakoid lumen,is essential for the accumulation of the PSII supercomplex.Understanding the mechanism underlying this function will befacilitated by identifying the immediate target(s) of FKBP20-2.

FKBP20-2 contains a disulfide bridge at the C terminus andwas reduced by Trx. Interestingly, this disulfide bridge is highly

Fig. 5. Ferricyanide-dependent O2 evolution from WT and fkbp20-2 mutantplants. Chloroplasts isolated from 12-week-old plants were used in theseexperiments. (A) Rates of O2 evolution. Data are means � SE, n � 9. (B)Dependence of O2 evolution on light.1, light on;2, light off.

Fig. 6. Composition of chloroplast thylakoid protein complexes determined by blue native gel and immunoblot analysis. Row 1 shows resolution of the WTand fkbp20-2 mutant complexes under native (nondenaturing) conditions. Rows 2–4 show immunoblots of WT (Left) and fkbp20-2 (Right) proteins resolvedunder denaturing conditions. Antibodies were applied as indicated.

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conserved in land plants and eukaryotic algae but is absent incyanobacteria, indicating that potential redox regulation is re-stricted to chloroplasts. However, the low level of PPIase activityassociated with FKBP20-2 did not appear to be affected afterreduction by Trx, raising the question of whether PPIase activityis the key property responsible for the accumulation (assemblyor maintenance) of the PSII complex. This question assumesspecial interest in view of the recent finding that the PPIaseactivity of FKBP13, another immunophilin of the thylakoidlumen, is regulated by a redox system (28, 31).

Materials and MethodsPlant Materials. All genotypes used were of the Arabidopsisecotype Columbia-0. AtFKBP20-2 (At3g60307) insertional mu-tants (SALK�080069 and SALK�134696) were isolated from theT-DNA-transformed Arabidopsis collection at the ArabidopsisBiological Resource Center (Columbus, OH). (For simplicity,we refer to the AtFKBP20-2 mutants as fkbp20-2a and fkbp20-2b.) Primers used for genomic PCR verification of T-DNAinsertion in the mutants were 5�-GGG AGG ATC CAT GGTGAC GAT TCT ATC AAC TCC-3� and 5�-CAT TCT CGAGTT AAC TGC ATG TGA CAT CTG AGT-3�. The left borderprimer used was 5�-GCG TGG ACC GCT TGC TGC AAC TCTCT-3�. Confirmation of null mutants was carried out by RT-PCRusing the same primer set described above for FKBP20-2.Expression levels of ACTIN2 were monitored as a quantifyingcontrol (5�-GGA AGG ATC TGT ACG GTA AC-3� and5�-TGT GAA CGA TTC CTG GAC CT-3�). For use in theexperiments described below, plants were grown for 12 weeks ina 10-h light (22°C)�14-h dark (20°C) cycle with a photon fluxdensity of 180 �mol of photons�m�2�s�1.

Amino Acid Sequence Alignment. The amino acid sequence ofFKBP20-2 was aligned with homologs in rice (Oryza sativa,Q7XHRO), a green alga (Chlamydomonas reinhardtii, genemodel C�2101108), and a cyanobacterium (Synechococcus elon-gatus, Q8DJW8). The targeting peptide was determined by themethods of He et al. (21).

Isolation of Thylakoid Membranes. Thylakoid membranes wereisolated as described in ref. 32 with minor modifications. Forblue native gel analyses, leaves were homogenized in ice-coldhomogenization buffer (330 mM sorbitol�30 mM Mops-KOH,pH 7.8�2 mM EDTA�2 mM ascorbate) and filtered through twolayers of Miracloth (Calbiochem, San Diego, CA). The filtratewas centrifuged (at 2,000 � g for 2 min at 4°C). The pellet wasresuspended in homogenization buffer, overlaid on 40% Percollsolution [40% (wt/vol) Percoll�330 mM sorbitol�30 mM Mops-KOH, pH 7.8�2 mM EDTA�2 mM ascorbate) and centrifuged(at 2,500 � g for 15 min at 4°C). Isolated thylakoids wereresuspended in the same buffer to a final Chl concentration of0.4 mg�ml (33).

Blue Native Gel Electrophoresis. Blue native gel electrophoresis(34) was performed as described in ref. 35 with modification.Isolated thylakoids (0.4 mg�ml Chl) were solubilized with anequal volume of 50 mM Bis-Tris�HCl, pH 7.0�1.5 M �-aminoca-proic acid�15% (wt/vol) glycerol containing 1.5% (wt�vol)N-dodecyl-�-D-maltoside (Sigma-Aldrich, St. Louis, MO) andincubated for 10 min on ice. Samples were centrifuged (at10,000 � g for 10 min at 4°C). After centrifugation, loadingbuffer [5% (wt/vol) Serva Blue G (Serva, Heidelberg, Germa-ny)�100 mM Bis-Tris�HCl, pH 7.0�0.5 M �-aminocaproic acid�75% (wt/vol) glycerol] was added to the supernatant solution(1�10 volume). For both mutant and WT, 18 �l of sample(corresponding to 88 �g of protein and 7.0 �g of Chl) was loadedto a blue native gradient gel containing 5–13.5% polyacrylamide(30:0.8 acrylamide:Bis) (Bio-Rad, Hercules, CA). Electrophore-

sis was performed in a MiniPROTEAN 3 cell (Bio-Rad) at 200V at 4°C. The cathode buffer initially contained 0.01% ServaBlue G dye and was replaced by buffer lacking dye halfwaythrough the run.

Two-Dimensional Immunoblot Analysis. After electrophoresis, lanesof WT and mutant samples on blue native gel were excised witha razor blade and incubated for 10 min at 25°C in NuPAGEsample buffer (Invitrogen, Carlsbad, CA) supplemented with2.5% 2-mercaptoethanol. Each lane was then placed on top of a1-mm NuPAGE Bis-Tris 4–12% gel (Invitrogen) and subjectedto second-dimension separation at constant voltage (110 V) at25°C for 60 min. After separation in the second dimension,proteins were transferred to a nitrocellulose membrane (Bio-Rad). Membranes were preincubated in PBS containing 0.1%Tween 20 and 5% skimmed powdered milk. Blots were incubatedovernight with primary antibody against D1, PsaD, and Cytf at4°C. Chicken (D1) and rabbit (PsaD and Cytf ) immunoglobulinscoupled to horseradish peroxidase were used as secondaryantibodies. Immunoblots were visualized by using the ECL PlusWestern Blotting Detection System (Amersham Pharmacia Bio-sciences, Buckinghamshire, U.K.). The blots were scanned witha desktop scanner and analyzed by using Bio-Rad Quantity One.

Chl Fluorescence Assays. Chl fluorescence measurements wereperformed with dark-adapted (overnight) plants by using anFMS2 instrument (Hansatech, King’s Lynn, U.K.). Six samples(three independently grown sets of WT and fkbp20-2 plants)were measured. Plants were subjected to a saturating light pulseand then illuminated for a programmed series of 5-min periodsof increasing light intensities. Between steps in actinic photonflux density, the minimum fluorescence in the light-adaptedstate (Fo�) was determined during a 1-s period of far-redillumination. Nonphotochemical quenching is defined as (Fm �Fm�)�Fm�, efficiency of PSII electron transport (�PSII) is definedas (Fm� � Fs)�Fm�, and the maximum efficiency of PSII photo-chemistry (Fv�Fm) is (Fm � Fo)�F, where Fm is the maximumfluorescence in the dark-adapted state, Fm� is the maximumfluorescence in any light-adapted state, and Fs is the steady-statevalue of fluorescence immediately before the light flash.

Oxygen Evolution. Thylakoid membranes were isolated as abovewith the following modifications. All procedures were carriedout at 4°C under dim light. Leaves were homogenized in ice-coldhomogenization buffer (10 mM NaCl�5 mM MgCl2�330 mMsorbitol�30 mM Mops-KOH, pH 7.8�2 mM EDTA). Chloro-plasts were used directly after filtration and centrifugation.Thylakoids equivalent to 20 �g�ml Chl were resuspended inhomogenization buffer. Potassium ferricyanide (500 �M) wasadded as an electron acceptor. O2 evolution was carried out at25°C in a DW1 Clark Electrode (Hansatech) in response toincreasing photon flux densities. Nine samples were measured(three independently grown sets of plants with three sampleseach).

Cloning, Expression, and Purification of Recombinant FKBP20-2. ThecDNA region encoding the FKBP20-2 mature protein wasamplified from total mRNA isolated from leaf tissue of WTArabidopsis thaliana. Primers used were 5�-GGG AGG ATCCAT GGT GAC GAT TCT ATC AAC TCC-3� and 5�-CATTCT CGA GTT AAC TGC ATG TGA CAT CTG AGT-3�. Themutant form of the protein lacking both cysteines at the C-terminal region was constructed by site-directed mutagenesiswith the following primer sets: 5�-TCA GAT GTC ACA TCCAGT TAA CTC GAG-3� and 5�-CTC GAG TTA ACT GGATGT GAC ATC TGA-3�; 5�-CTC AGT ATC CAG AAT TCTGAG AGG AGG ACT ATA-3� and 5�TAT AGT CCT CCTAGA ATT CTG GAT ACT GAG-3�. Amplification products

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were cloned into the expression vector pGEX-4T (AmershamPharmacia Biosciences). Ultracompetent Rosetta E. coli cellswere transformed according to the manufacturer’s instructions(Novagen, Madison, WI). Cultures were grown for 18 h at 25°Cin ZYP-5052 (36). Cells were collected by centrifugation (at10,000 � g for 15 min at 4°C), resuspended in PBS solution, andlysed by sonication (Fisher 300, Dynatech Labs, Chantilly, VA).Recombinant proteins were purified by affinity chromatographyby using glutathione Sepharose beads (Amersham PharmaciaBiosciences). Fusion proteins were cleaved by thrombin andfurther purified by using 5,000 and 50,000 molecular-weightcutoff Vivaspin columns (Amersham Pharmacia Biosciences).Purified proteins were analyzed by 4–12% SDS�PAGE.

Enzyme Assay. PPIase was assayed (27) at 20°C as in ref. 26.Activity was followed by the increase on absorbance at 390 nmdue to release of p-nitroanilide by the proteolytic cleavage of thechromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroani-lide (N- succinyl-AAPF-p-nitroanilide) (Sigma-Aldrich). Thereaction mixture contained 30 mM Tris (pH 7.0), 71 �M N-succinyl-AAPF-p-nitroanilide, 0.68 �M �-chymotrypsin (Sigma-Aldrich), and 0.15% 2,2,2 trif luorethanol. N-Succinyl-AAPF-p-nitroanilide was dissolved in 2,2,2 trif luorethanol containing 250mM LiCl. Progress curves were fit to first-order rate constants(Kobs, s�1), and the second-order rate constant (kcat�Km) wasdetermined from the slope of a plot of FKBP20-2 concentrationvs. Kobs. FKBP20-2 and FKBP20-2 were reduced by Trx, which

was itself reduced by the NADP�Trx system of E. coli (added inthe same ratios as described below) or by DTT (added at2.5 mM).

Redox Analysis of FKBP20-2 Protein. Reduction of FKBP20-2 by theNADP�Trx system of E. coli was performed as described byWong et al. (37). Recombinant FKBP20-2 and FKBP20-2(C225S, C241S) (4.5 �g) proteins were incubated in reactionbuffer (50 mM Tris�HCl, pH 7.5) at 37°C for 60 min. Thecomplete sample contained 2.5 mM NADPH, 5 �g NTR, and 5�g Trx. Newly exposed cysteines resulting from disulfide reduc-tion were labeled by addition of the thiol-specific f luorescentprobe monobromobimane to 2 mM. After labeling, the proteinsample was incubated with 10 mM 2-mercaptoethanol for 2 minand was then subjected to electrophoresis on NuPAGE Bis-Tris(Invitrogen) using 4–12% gels. Fluorescence was recorded byusing a Gel Doc-1000 fitted with a UV 365-nm Transilluminatorand the Quantity One data analysis program (Bio-Rad). Subse-quently, proteins in the gels were visualized by staining withcolloidal Coomassie blue G-250.

We thank K. Niyogi, L. Curatti, Y. Balmer, Y. H. Cheong, A. Melis, andJ. R. Dominguez for helpful discussions and R. Malkin (University ofCalifornia, Berkeley, CA) for providing antibodies. This work wassupported by grants from the U.S. Department of Agriculture NationalResearch Initiative (to B.B.B. and S. Luan) and the U.S. Department ofEnergy (to S. Luan).

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