Division of Molecular Plant Biology, Hilgard Hall, University of California, Berkeley, California 94720. Received July 25,1988, and in revised form December 21, ...
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 271, No. 1, May 15, pp. 223-239,1989
Evidence for Function of the Ferredoxin/Thioredoxin System in the Reductive Activation of Target Enzymes of Isolated Intact Chloroplasts N. A. CRAWFORD, Division
of Molecular
M. DROUX,l
Plant Biology, Hilgard
N. S. KOSOWER,’ Hall, University
AND
of California,
B. B. BUCHANAN3 Berkeley,
California
94720
Received July 25,1988, and in revised form December 21,1988
Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodeeyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a Z-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering y globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes. o 1989Arademic P~.S lnc
The ferredoxin/thioredoxin system, composed of ferredoxin, ferredoxinithioredoxin reductase (FTR),4 and thioredoxin,
has been shown to link light to the regulation of enzyme activity in numerous reconstituted assays (l-5). In this system, thio-
’ Permanent address: UM 41 CNRS, Rh&e Poulenc Agrochimie, Centre de la Reeherche la Dargoire, B.P. 9163,69263 Lyon Cedex 09, France. ’ Permanent address: Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 69978. 3 To whom correspondence should be addressed. ’ Abbreviations used: mBBr, monobromobimane; FTR, ferredoxin/thioredoxin reductase; NADPMDH, NADP-linked malate dehydrogenase; FBPase,
fructose-1,6-bisphosphatase; DTT, dithiothreitol; NEM, N-ethylmaleimide; rub&o, ribulose-1,5-bisphosphatase carboxylase/oxygenase; PRK, phosphoribulokinase; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetracetic acid; DTNB, 5,5’dithiobis-(2-nitrobenzoic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Hepes, 4-(Z-hydroxyethyl)-l-piperazineethanesulfonic acid; ELISA, enzyme-linked immunosorbent assay. 223
0003-9861/89 $3.00 Copyright All rights
8 1989 by Academic Press, Inc. of reproduction in any form reserved
224
CRAWFORD
redoxins, small proteins with an active site consisting of two half-cystine residues which undergo reversible reduction and oxidation, are reduced in the light by ferredoxin via a catalytically active thiol group on FTR. In the case of biosynthetic enzymes, e.g., fructose-1,6-bisphosphatase (FBPase) and NADP-malate dehydrogenase (NADP-MDH), each subunit contains a regulatory disulfide group that is reduced to the sulfhydryl level by reduced thioredoxin, thereby leading to activation of the enzyme (6,7). Recent results with a reconstituted thylakoid assay containing homogeneous protein components provide evidence that the ferredoxin/thioredoxin system serves as a regulatory thiol chain that links light to target enzymes via the noncyclic electron transport chain (8,9). Specific inhibition of light activation of NADPMDH by an antibody against FTR suggests that the ferredoxin/thioredoxin system is also active in lysed chloroplasts (10). Aside from findings with lysed chloroplasts and reconstituted assays, the only physiological evidence for the ferredoxin/ thioredoxin systems rests on experiments m is more reshowing that thioredoxin duced in the light than in the dark in isolated intact chloroplasts (11) and cyanobacterial cells (12). If the current model of the ferredoxin/thioredoxin system is correct, one should see in addition to the thioredoxin m change in such systems (1) a light-dependent reduction of the catalytically active FTR disulfide (8, 9), and (2) in the interacting thiol groups of thioredoxin f(13) and the target enzymes (6-9). To test this point, we have developed a technique to monitor thiol changes in components of the ferredoxin/thioredoxin system in isolated intact chloroplasts maintained under aerobic conditions. The method is based on use of monobromobimane (mBBr), a membrane-permeable thiol labeling reagent that reacts rapidly with available sulfhydryl groups to yield stable fluorescent products (14). Proteins of interest are immunoprecipitated and thiols are quantified by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence techniques. The results provide evidence
ET AL
that the ferredoxin/thioredoxin system functions in enzyme regulation in intact chloroplasts by way of the light-induced thiol changes identified in earlier reconstitution experiments. EXPERIMENTAL
PROCEDURES
Materials Spinach (Sp&acea olerucea) was obtained from a local market. mBBr, also called “Thiolyte,” was purchased from Calbiochem-Behring and protein A-AffiGel beads from Bio-Rad. Other reagents were from commercial sources and were of the highest quality available. Stock solutions of 250 mM mBBr were prepared in acetonitrile and stored up to several months at room temperature protected from light. N-Ethylmaleimide (NEM), dithiothreitol (DTT), and Z-mercaptoethanol stock solutions were prepared fresh daily. Na-ascorbate was added to solutions just before use.
Methods Analysis of Thiols in Immunoprecipitated Proteins from Light- or Dark-Treated Chloroplasts Step 1: Preparation of intact chloroplasts. All of the following steps were carried out in subdued light. Chloroplasts were isolated by a modified procedure of Reeves and Hall (15). Spinach leaves (3 kg) were washed in distilled water, deribbed, and added in ZOOg lots to a chilled quart size Waring blender in which razor blades replaced the original knife. Four hundred milliliters of ice-cold blending buffer (20 mM Na pyrophosphate buffer, pH 6.5, 330 mM sorbitol, 5 mM MgCIP, and 2 mM sodium ascorbate) was added so that the ratio of buffer to leaves was 2:l (v:w). Following four to six short pulses, the homogenate was filtered through four layers of filtering nylon (30 threads/cm) and immediately centrifuged for 30 s timed from the moment the rotor was started. Final speed was 4OOOg. Total elapsed time from start to finish of centrifugation was 90 s. The green supernatant fraction was decanted and the soft top layer of the pellet, which contained a high proportion of broken chloroplasts was removed by gently swirling with a few milliliters of a solution containing 50 mM Hepes, pH 7.5, 330 mM sorbitol, 2 mM EDTA, 2 mM Na-ascorbate (buffer A). The remaining pellet was resuspended in buffer A to a chlorophyll concentration of 1 mg/ml and was stored on ice in the dark until use. Chloroplasts prepared in this manner were greater than 70% intact as determined by a ferricyanide test for intactness (16) with a yield of 50 mg chlorophyll/kg leaves.
FERREDOXIN/THIOREDOXIN Step 2: Illumination and labeling of chloroplasts with mBBr. Chloroplasts were poured into a glass beaker and placed on a plexiglass shelf suspended over a light source. The total volume of chloroplast suspension in each beaker was determined by the number of assays to be performed using 7 mg of chlorophyll per assay (1 mg chlorophyll/ml). The size of the beaker was selected so that the chloroplast suspension formed a layer of sufficient thinness to receive uniform illumination. For example, a 2-liter beaker was used for four assays (28 ml suspension). Dark controls were wrapped in foil. Chloroplasts were illuminated (100 PLE.mm’. s ’ of 400 to 700 nm light transmitted through 20 cm of water) at room temperature for 15 min, unless otherwise specified, and were frequently mixed by swirling. mBBr was added from the stock solution to give a fisusnal concentration of 1 mM, and the chloroplast pension was kept in the light (or dark) for another 5 min. The reaction was stopped by diluting the chloroplast suspension with 1 vol of buffer A and adding 2mercaptoethanol to a final concentration of 1 mM to bind excess mBBr. The suspension was then kept in the dark until further treatment. DTT controls were prepared in the dark and treated in the same manner except that 1 mM DTT was added to either the intact or lysed chloroplasts, the final mBBr concentration concentrawas 3 mM, and the final 2-mercaptoethanol tion was 3 mM. Step 3: Preparation of chloroplast extract. Chloroplast suspensions were centrifuged at 3000~ for 1 min taking care to keep samples in the dark. The supernatant fraction was discarded, and the chloroplasts were lysed (in darkness) by resuspending to a concentration of 1 mg chlorophyll/ml with a solution containing 50 mM Tris, pH 7.5, and 200 mM NaCI. [Breakage was complete under these conditions based on the finding that similar amounts of soluble protein (ca. 2 mg/mg chlorophyll) were recovered with 0 and 50 mM NaCl.] The solution was centrifuged at 40,OOOgfor 20 min, and the supernatant fraction containing soluble chloroplast proteins was concentrated 12.fold or more using an Amicon Diaflow apparatus fitted with a YM5 membrane. The precipitate, containing thylakoid membranes, was saved and analyzed in certain experiments as described below. Samples containing the supernatant fraction were clarified by centrifugation at 14,000 rpm for 10 min in an Eppendorf bench top microfuge and the protein concentration was determined. Typical final protein content was 25-30 mg per ml. Step 4: Immunoprecipitation of labeled proteins. During this work we found that our antibody against corn thioredoxin m immunoprecipitated reduced thioredoxin m (from either corn or spinach) more effectively than oxidized thioredoxin m. To overcome this problem, oxidized thioredoxin was reduced with DTT and derivatized with NEM, a nonfluorescent
SYSTEM
225
probe. The NEM- and mBBr-derivatized thioredoxin m samples showed no difference in immunoprecipitation. Accordingly, chloroplast extracts from step 3 were reduced by a 15.min incubation with 1 mM DTT at room temperature and then treated for 10 min with 3 mM NEM. Excess NEM was blocked by addition of 2-mercaptoethanol to 3 mM prior to application of the antibody against thioredoxin m. This reduction step was not necessary for proteins other than thioredoxin m and in fact it interfered with the mBBr labeling of denatured proteins (see below). Immunoprecipitation was carried out according to a modified procedure of Anderson and Blobel(17). To all samples was added a solution containing 1% Triton X-100,0.2% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and the following other protease inhibitors (1 KM each): leupeptin, antipain, and pepstatin. Samples were divided into aliquots containing 17 mg of protein and 50 pg of affinity purified antibody or purified preimmune IgG was added. Following incubation for 12 h at 4”C, tubes were clarified by centrifugation for 10 min at 14,000 rpm in an Eppendorf bench top microfuge. Protein A-Affi-Gel beads (Bio-Rad, Richmond, CA), which had been previously washed with a solution containing50 mM Tris, pH 7.5,200 mM NaCl, 0.15% (v/v) Triton X-100,0.03% (w/v) SDS, and 5 mM EDTA (immunoprecipitation wash buffer), were added to each sample (30 ~1 of a 50% suspension), and the samples were mixed mechanically by turning them end over end at room temperature for 1 to 2 h. The beads were pelleted by centrifugation in an Eppendorf bench top centrifuge (1 min, 4000 rpm) and the supernatant fraction was decanted and discarded. The beads were washed four times with 1.5 ml of immunoprecipitation wash buffer and then once with 1.5 ml of 50 mM Tris, pH 7.5, each time being centrifuged as above. The pellet was analyzed as described below. Step 5: Analysis qf mBBr labeling by SDS-PAGE. Excess wash buffer was drawn off the bead pellet with a Hamilton syringe, 20 ~1 of solubilizing buffer [125 mM Tris pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.01% (w/v) bromphenol blue] was added and samples, containing 1 to 4 fig of the protein in question, were heated 4 min in a boiling water bath. The liquid was drawn off using a Hamilton syringe and was applied to a 1.5mm-thick lo-20% SDS-polyacrylamide gradient gel. To increase recovery, another 20 ~1 of half-strength solubilizing buffer was added to the beads, removed as before, and applied to gel wells containing the first sample. After overnight electrophoresis at 8 mA, gels were fixed by soaking them for 24 h in a solution containing 40% methanol and 10% acetic acid (fixing solution). Gels were examined for labeled proteins that appeared as fluorescent bands with an ultraviolet light box fitted with a 365nm source (Spectroline transilluminator Model TS365) and photographed with Polaroid Type 55 posi-
226
CRAWFORD
tive-negative Land film using a yellow Wratten gelatin filter No. 8 (cutoff = 460 nm) and an exposure time of 2 min. Gels were stained for protein for 2 h in fixing solution containing 0.25% (w/v) Coomassie blue R2.50, and then destained 24 h in a solution of 10% methanol and 10% acetic acid. A Polaroid positivenegative film photograph was taken of the stained gel (18). Fluorescence was quantified by scanning the Polaroid negatives of both the fluorescent and stained gels with a Hoefer gel scanner and relating the reading to a standard curve prepared for each pure mBBrlabeled protein as described below. Care was taken that photographic and staining/destaining conditions were consistent throughout.
Related mBBr Labeling Procedures Monobromobimane Labeling of SDSDenatured Proteins Chloroplasts were prepared and treated in the light or dark with mBBr, and extracts immunoprecipitated as described above. Solubilizing buffer minus 2-mercaptoethanol was added to the Afh-Gel beads containing immunoprecipitate and the samples were heated 4 min in a boiling water bath. The samples were cooled to room temperature and mBBr was added to 1 mM. After 15 min, 2 ~1 of 14 M 2-mercaptoethanol was added and the samples were electrophoresed as described above.
“Reverse labeling ” of Chloroplast Proteins by mBBr In the reverse labeling procedure described below, proteins of intact illuminated or dark-adapted chloroplasts were first derivatized with NEM to block sulfhydryl (SH) groups and then following reduction with DTT were derivatized with mBBr. In this way, the oxidized (S-S) groups of a given protein were rendered fluorescent following its isolation and analysis on SDS-PAGE. It is noted that while mBBr and NEM would compete for sulfhydryl (SH) groups, this problem did not arise in the below procedure because the free (underivatized) probes were not present together at any one time. Chloroplasts were prepared, treated in the light or dark with NEM, and immunoprecipitated as described in steps 1-4 above except that NEM replaced mBBr. The Affi-Gel beads containing immunoprecipitated proteins were heated 4 min with 20 el of a solution containing 125 mM Tris, pH 6.8, 4% SDS, and 20% glycerol in a boiling water bath. Samples were brought to room temperature and NEM to 2 rtIM was added to block newly exposed thiols that were unavailable in the first NEM treatment. After 30 min excess DTT was added (4 mM) and samples were flushed with argon for 10 min and then incubated for
ET AL. 90 min. mBBr was added to a final concentration of 10 and samples were incubated for another 15 min. The reaction was stopped with 2-mercaptoethanol added to 700 mM and samples were analyzed on SDSPAGE as described above. mM
Analysis of Total Soluble Protein and Thylakoid Fractions Chloroplasts were prepared, illuminated, labeled with mBBr and the soluble protein fraction (chloroplast extract) was prepared as described in steps l-3 above. Time of illumination was 15 min. Aliquots of the concentrated protein solution were analyzed by SDS-PAGE as described above for the immunoprecipitated proteins. The thylakoid fraction prepared as given in step 3 was analyzed by the same SDS-PAGE procedure.
Preparation
of Standard Curves
1. Thioredoxins. Homogeneous thioredoxin for m (12 fig in 20 ~1) was treated with or without 1 mM DTT for 15 min. Monobromobimane was added to a concentration of 3 mM. After 10 min incubation the reaction was stopped by adding a slight excess of 2-merof thiorecaptoethanol (2 mM). Complete inhibition doxin activity was observed in standards labeled by mBBr in the presence of DTT, whereas only a lo-40% inhibition was observed in standards labeled in the absence of DTT, as measured in standard DTT-linked FBPase or NADP-MDH assays. Samples were prepared for standard curves by solubilizing and analyzing amounts ranging from 0.1 to 3 pg by the SDS-PAGE method described in step 5 of the thiol analysis procedure above. Standard curves were obtained by plotting height of the fluorescent band scans versus height of the protein band scans. 2, Ferredoxin/thioredoxin reductase. Purified FTR was reduced in the light in the presence of spinach thylakoids (60 pg chlorophyll) in a clear microfuge tube of 1.5 ml capacity, containing 25 mM Tris-HCI, pH 8.0, 10 mM sodium ascorbate, 0.1 mM 2,6-dichlorophenol-indeophenol, 29 Fg spinach ferredoxin, and 55 fig spinach FTR (vol = 400 ~1). The tubes were fitted with serum stoppers, flushed with argon for 10 min, and either illuminated or kept in the dark for 15 min. Percentage reduction was determined with 5,5’-dithiobis-(2.nitrobenzoic acid) (DTNB) as described previously (8). A standard curve with mBBr was prepared with 0.1 to 3 pg protein as given above for thioredoxin. 3. NADP-malate dehydrwenase. Homogeneous corn NADP-MDH (70 Fg in 20 ~1) and spinach thioredoxin m (60 pg in 20 ~1) were incubated with 2 mM DTT for 30 min and mBBr, was added to 5 mM. After 10 min, 20 ~1 of 14 M 2-mercaptoethanol was added and a standard curve for NADP-MDH was generated with 0.1 to
FERREDOXIN/THIOREDOXIN 3 fig protein as given above for thioredoxin. A reverselabeled NADP-MDH curve was generated by reducing the enzyme as described above except that 1 mM DTT was used. NEM was added to 4 mM, and after 15 min, the sample was denatured through addition of 125 mM Tris, pH 6.8, 4% SDS, and 20% glycerol (in equal volume) by heating 4 min. Additional NEM was added to give a final concentration of 3 mM, and after 30 min, DTT was added to 5 mM. The samples were then flushed with argon for 10 min and treated with mBBr as described above for reverse labeling of chloroplast proteins.
Immunological Antibody
Procedures
Preparation
Previous preparations of antibodies against spinach thioredoxin f and corn thioredoxin m (18), were used in the current studies. Anti-spinach FTR was prepared using a homogeneous FTR sample which contained predominantly the similar subunit (the subunits of FTR are identified in Ref. (19) and are also described below). The innoculation protocol followed that previously described for corn thioredoxin m (18). Anti-corn NADP-MDH antibody against a mixture of reduced and oxidized enzyme was custom-prepared by Manzanita Immunological Co., Oracle, Arizona. Reduced enzyme, 1.3 mg purified as in Ref. (20), was prepared under argon by reaction with DTT for 16 h at room temperature. Thiols were prevented from reforming a disulfide bridge by reaction for 15 min with 35 mM NEM. The reaction was stopped by the addithe preparation tion of 14 mM 2-mercaptoethanol; was then dialyzed overnight against a l-liter solution containing 20 mM Tris, pH 7.5, and 150 mM NaCl (TBS). Oxidized corn NADP-MDH (0.7 mg) was added to give the final antigenic mixture. The injection, booster, and bleeding treatments were carried out as described previously (18).
Preparation
of Immunoafinity
Columns
Affinity columns were prepared from purified antigens and Affi-Gel 10 matrix using a solution of 0.1 M Hepes, pH 7.5, and 80 mM CaCI, as coupling buffer (21). To ensure its maximal binding to antibody, spinach thioredoxin m (2 mg) was reduced and derivatized with NEM prior to coupling to the matrix. The protein was reduced with 1 mM DTT for 30 min. The thi01s were blocked with 3 mM NEM for 30 min, 2-mercaptoethanol was added to 3 mM, and the protein was then coupled to 1 ml of Affi-Gel 10 matrix. For antibody isolation, corn NADP-MDH (20 mg) was reduced and blocked as described above to ensure maximal antibody interaction, and was coupled to 5 ml of Affi-Gel matrix. Oxidized thioredoxinf(3 mg) and oxidized spinach FTR (2 mg) were coupled to 1 ml each
227
SYSTEM
of Affi-Gel 10 matrix. A spinach FBPase Sepharose 4B column was prepared as previously (18).
Immunoafinity
PuriJication
of Antibodies
Antibodies were purified on affinity columns by mixing the column matrix with 2-5 ml of the corresponding antiserum end over end for 1 h at room temperature. The mixture was poured into a column (1 cm diameter) and the eluting serum was collected to be mixed again with the column once specifically bound antibodies were eluted. This process was repeated three to five times until the eluting serum was depleted of the specific antibody. After the column was washed with TBS, nonspecifically bound y globulins were eluted with 5 column vol of a solution containing 1 M Tris, pH 7.9, and 500 mM NaCl. Antibodies were eluted by sequential washes with 5 column vol of each of the following solutions: 25% (v/v) ethylene glycol in 50 mM glycine (pH 11.8); 20% (v/v) ethylene glycol in 100 mM acetic-formic acid (pH 2.3); and 0.5 M NaCl in 1 M Tris-HCI (pH 7.9). Antibodies were recovered in both the glycine and acetic-formic acid washes as well as in the final 1 M Tris wash as determined by ELISA (18). The column was reequilibrated with TBS and then mixed again with serum. Affinity purified fractions were pooled, and the pH was measured and, if necessary, adjusted to neutrality with 1 M Tris, pH 7.5. The solution was then concentrated to about 1 mg/ml in an Amicon Diaflow apparatus fitted with a YMlO membrane. Preimmune IgG was purified on a protein A-Afh-Gel column according to Ref. (22). Affinity purified antibodies were stored up to 6 months at 4°C in the presence of 0.02% sodium azide.
Other Methods Light Activation of Target Enzymes in Intact Chloroplasts Chloroplasts were illuminated as described above (step 2 of thiol analysis section) and were assayed for FBPase, NADP-MDH, and phosphoribulokinase (PRK) activity by removing an aliquot containing 20 fig chlorophyll and injecting it into a l-ml cuvette containing hypotonic reaction mixture. The activity of each target enzyme was measured by following the change in absorption at 340 nm with a Cary 19 spectrophotometer (18). Final volume for each reaction mixture was 1 ml. The FBPase reaction mix (23) contained 100 mM Tris-HC1 buffer (pH 8,2):phosphoglucase isomerase (4 units); glucose-6-phosphate dehydrogenase (2 units); Triton X-100 (0.02% v/v); and the following (in mM): fructose 1,6-bisphosphate, 1; EDTA 1; NADP, 0.4; MgCI,, 5; and S-mercaptoethanol, 14. The NADP-MDH reaction mix (modified from (20)) contained 100 mM Tris-HCl buffer, pH 7.9, Triton X-100, 0.02%, v/v) and the following (in mM):
228
CRAWFORD
EDTA, 1 mM; NADPH, 0.2; oxaloacetate, 2.5; and 2mercaptoethanol, 14. The PRK (24) mix contained 50 mM Tris-HCI buffer (pH 7.9), lactate dehydrogenase (0.4 unit), pyruvate kinase (0.5 unit), phosphoriboisomerase (0.3 unit), Triton X-100, 0.02% (v/v), and the following (in mM): MgCl,, 10; KCl, 40; NADH, 0.2; phosphoenolpyruvate, 2.5; ATP, 1; ribose 5-phosphate, 2; and 2-mercaptoethanol, 5. For PRK assays a control for ATPase activity was tested in a mixture in which ribose 5-phosphate and phosphoriboisomerase were omitted. This control activity was subtracted from the final activity.
Analytical
Methods
Protein concentrations were determined by the Bradford method using human y globulin as a standard (25). The Laemmli system was used for SDSPAGE, all gels being lo-20% acrylamide gradients of 1.5 mm thickness (19). Chlorophyll was estimated according to Arnon (26).
Protein PurQication Methods for purification of proteins are described elsewhere: spinach or corn thioredoxin in and thioredoxinf(lS), spinach FTR (19), corn NADP-MDH (20), spinach FBPase (27), spinach ferredoxin (28). Thylakoid membranes for assaying FTR were prepared as described by Jacquot et al. (29) and stored at -80°C in aliquots of 100 ~1. RESULTS
AND
DISCUSSION
Our first attempts to identify light-induced thiol changes of isolated intact chloroplasts were made with the total soluble protein fraction in which mBBr was used as a probe to detect the individual labeled proteins separated by SDS-PAGE (Fig. 1). The total incorporation of mBBr was 20% higher in the light than in the dark as determined by weighing scans of the photograph negatives ((30) and data not shown). This finding is consistent with the earlier work of Slovacek et al. (31, 32) who reported a 50% increase in sulfhydryl groups under similar conditions with DTNB as probe. With mBBr under our conditions, light-dependent increase in fluorescence was observed with some protein bands, particularly in the low-molecular-weight region (Fig. l), but changes were very difficult to quantitate and, furthermore, the individual protein bands could not be identified. When subjected to electropho-
ET AL.
DL Reference
FBPase NADP- MDH/
FTR--L Thioredoxin D Thioredoxln f 7
Fluorescence 3
‘6.5
IL Protein
FIG. 1. Fluorescence of the soluble protein fraction of chloroplasts labeled with mBBr in the light or dark and resolved on SDS-PAGE. Each lane contained 120 pg of soluble chlorolast protein. The names of the proteins on the left are given only as reference and do not represent either fluorescent or Coomassie-stained bands shown in the gel.
resis and analyzed with mBBr, the thylakoid membrane fraction showed a 5% increase in fluorescence in the light ((30) and data not shown). Since the bulk of the labeling was observed in the stroma, we developed an immunoprecipitation techand monitor these nique to identify changes in individual components, especially those of the ferredoxin/thioredoxin system. Comment on recovery of the individual proteins is in order. To confirm equivalence of recovery of the oxidized and reduced forms of the proteins studied, we included in all experiments two gel lanes, later stained with Coomassie blue, containing equal volumes of each protein precipitated from light- or dark-treated chloroplasts. We observed over numerous repetitions of these experiments that, while the oxidized (“dark”) and reduced (“light”) forms of FTR and thioredoxin f precipitated equally, thioredoxin m precipitated more effectively when reduced. Introduction of the NEM derivatization step described above overcame this problem and ensured
FERREDOXIN/THIOREDOXIN
equal precipitation of the oxidized and reduced forms of thioredoxin m. The point of equal precipitation was independently confirmed by immunoprecipitating reduced and oxidized forms of pure samples of each of the three proteins. In all cases, recoveries of about 30% were observed. Thus, in short, while there were significant unavoidable losses of FTR and thioredoxins f and m during isolation, the loss was uniform between the oxidized and reduced forms using above techniques. In the case of NADP-MDH, the reduced form precipitated about 50% as effectively as the oxidized form. This problem is minimized by fitting the data to a standard curve relating fluorescence to concentration of mBBr derivatized protein. As pointed out below, we were unable to quantitate data with the FBPase due to the fact that the antibody (y globulin) added for precipitation interfered with protein analysis by SDSPAGE. Immunoprecipitation
of Specific Proteins
According to the procedure developed, each protein is isolated from broken chloroplasts by precipitation with a polyclonal antibody (Fig. 2). Important to the success of the procedure is the use of relatively large amounts of affinity purified antibody (50 pg for each experiment) and the inclusion of Triton X-100 and SDS to eliminate nonspecific contamination by ribulose-1,5bisphosphate carboxylase/oxygenase (rubisco) and other proteins during precipitation. It was also found that chloroplast extract could not be frozen prior to immunoprecipitation and that the samples had to be clarified by centrifugation after the 12-h incubation and before addition of the protein A-Affi-Gel beads. Some specific antigen-antibody precipitate may be lost by this step, but the centrifugation is nevertheless necessary for removing nonspecific precipitates which form during the incubation period. Rubisco can be especially troublesome as a contaminant because of its abundance and because its small subunit migrates to a position near that of FTR and thioredoxin m. Careful observation of the necessary precautions
SYSTEM
229
given above eliminated interfering proteins as determined by a Western blot of the immunoprecipitates (data not shown) and by individual controls shown below in which preimmune IgG was added instead of a specific antibody. Further confirmation of the identity of the precipitated band was made by including additional controls in which purified unlabeled protein was added in large excess to the chloroplast extract containing its mBBr-labeled counterpart. As seen below, the fluorescence of the resolved immunoprecipitated proteins was greatly if not completely diminished under these conditions.
Th,ioredoxin j After immunoprecipitation and analysis on SDS-PAGE, thioredoxin f was found to be more highly fluorescent from lightthan dark-treated chloroplasts (Fig. 3, lanes D and L of treatments 1 and 2). That this increase in labeling was due to net reduction rather than conformational change (the most recent evidence shows that thioredoxin f contains three cyst(e)ine groups; P. Schiirmann personal communication) is supported by the retention of the light effect in these two treatments, i.e., fluorescence treatment 1 (without SDS) and treatment 2 (with SDS). In the case of treatment 2, the immunoprecipitated thioredoxin f sample, which had previously been labeled with mBBr in the light or dark as in treatment 1, was denatured with SDS in the absence of reductant (2-mercaptoethanol) to expose potential sulfhydry1 groups not available without denaturation and was then again treated with mBBr. This modification of the procedure resulted in only a slight increase in the fluorescence of the thioredoxin f band from chloroplasts kept in the dark and the band from illuminated chloroplasts was still more highly labeled (Fig. 3, lanes D and L of treatment 2). That the protein precipitated from chloroplasts in Fig. 3 was reduced thioredoxin f was confirmed by (i) its reduction by DTT in lysed chloroplasts (treatment 3); (ii) comparison to pure authentic thioredoxin f labeled with mBBr in presence and absence of DTT (treatment
230
CRAWFORD Intact
ET AL.
Chlaraplasts
mBBr BMET
LIGHT for DARK)
4
centrifuge lyse
chloroplo!sts
4
osmoticolly
centrifuqe
\
Soluble
Thylakaids -mBBr
Labeled
centrifuge
Proteins
(Measure UV fluorescence relate to protein content Triton
Affinity against
10.x to clarify
EDTA )
Iihibitors purified specific
IgG protein
4
incubate
overnighl
centrifuge A Affigel
incubate wash
solubilizing
to
clarify
beods 4
SDS
I
and of gel) SDS
Proteose
Protein
Proteins
eoncel/trote
SDS-IPAGE 4
buffer
l-2hr beads
-4
Solubillzed
\L mBBr Labeled
Protein
I SDS-‘PAGE
Resolved
mBBr Labeled Protein
(Measure UV fluorescence relate to protein content
FIG. 2. Flow chart of the protocol for labeling and analyzing selected proteins on SDS-PAGE.
4); (iii) dilution by added unlabeled purified thioredoxin f (treatment 5); and (iv) absence from a control in which preimmune IgG replaced thioredoxin f antibody (treatment 6). On the basis of the intensity of fluorescence determined by scanning photograph negatives, thioredoxin f was fully oxidized in the dark and almost fully reduced in the light. The low level of incorporation of mBBr in oxidized thioredoxin f, either in pure form or in dark-adapted chloroplasts (see Fig. 3), is attributed to the above-mentioned “third” cysteine residue that is not a part of the active site (Cys-Gly-ProCys). This conclusion is supported by the small but consistent differences in electrophoretic mobility of the oxidized and re-
chloroplasts
of
and gel)
with mBBr and immunoprecipitating
duced forms of thioredoxin$ When labeled with mBBr or NEM the reduced form of pure thioredoxin f migrated detectably slower than its oxidized counterpart (compare lanes D and L of protein treatment 1, Fig. 3). Hence, if the fluorescence of thiorechloroplasts doxin f from dark-treated were due to a partial reduction, one would expect to see the protein band split into a doublet. To confirm that the incorporation of mBBr into oxidized thioredoxin f was due to the third cysteine, we performed a reverse labeling experiment in which the oxidized protein was first treated with NEM to block available thiols and then immunoprecipitated in the usual manner up to the point of solubilization. Then the sample
FERREDOXIN/THIOREDOXIN
231
SYSTEM
Protein
Fluorescence
-
FIG:. 3. Demonstration of the photoreduction of thioredoxinf’in intact chloroplasts. Thioredoxinf was labeled in light(L)- or dark @-treated chloroplasts, immunoprecipitated, and resolved on SDSPAGE with the following variations. 1, Treatment as described in Fig. 2. 2, mBBr labeling of SDS denatured immunoprecipitate (see Experimental Procedures). 3, Thioredoxinfimmunoprecipitated from chloroplast extract following reduction with DTT and labeling with mBBr. 4, Pure thioredoxin fstandard labeled in the presence (+) or absence (p) of DTT. 5, Inhibition of the precipitation of mBBr-labeled thioredoxinfbg the addition of 100 kg of unlabeled purified thioredoxinfto the chloroplast extract. 6, Preimmune IgG control. Each lane contained ca. 1 pg thioredoxinf:
was denatured with SDS (in the absence of 2-mercaptoethanol) to expose any free sulfhydryl groups that previously were not accessible for reaction with NEM. NEM was again added and following 30 min incubation, the samples were reduced with DTT and then labeled with mBBr as before. In this way, the oxidized (disulfide) groups of the isolated protein appear as fluorescence bands on the gel, i.e., the reverse of the usual procedure. In the current case with thioredoxin f, reverse labeling of the nonreduced protein of intact chloroplasts resulted in a 27% decrease in mBBr incorporation compared to the control not previously blocked with NEM (data not shown). This value approximates the 33% one would expect to see in a protein with one sulfhydryl group and one disulfide bond. Thus the data confirm that the weak mBBr labeling of thioredoxinf’in intact chloroplasts kept in the dark is due
to reaction with the third cysteine rather than to partial reduction of the active site of the protein. When corrected for values obtained for samples in which mBBr label was incorporated in the dark, thioredoxin f was found to be 95% reduced in the light (Table I). Thioredoxin
m
When intact chloroplasts were incubated and treated with mBBr in the light, the fluorescence of thioredoxin m, like that of thioredoxin f, greatly increased relative to dark controls (Fig. 4, D and L lanes of treatment 1). Thioredoxin m was recovered as its two isoforms described by Maeda et al. (33). The authenticity of the recovered product as thioredoxin m was confirmed by other treatments of Fig. 3viz., (i) reduction by DTT in lysed chloroplasts (treatment 3); (ii) similarity to au-
232
CRAWFORD
ET AL.
in the measurements because of its ability to activate NADP-malate dehydrogenase EXTENTOFREDUCTIONOFCOMPONENTSOFTHE under certain assay conditions (18, 34). FERREDOXIN/THIOREDOXINSYSTEMINLIGHTThat point aside, Scheibe (11) found in the ORDARK-TREATEDINTACTCHLOROPLASTS first light/dark transition that thiore% Reduction doxin m was 62-77% reduced in the light and 8-30% reduced in the dark. Our values Protein Dark Light DTT control with mBBr support this conclusion but suggest a somewhat lower level of reduced Thioredoxinf 0 95 f 9.4 100 thioredoxin m in the dark (4%) and a Thioredoxin m higher level in the light (75 and 87%), de13 kDa 87 f 4 101 f 1 4&3 pending on the isoform measured (Table I). 11 kDa 4-c5 75 f 6 97f 5 These levels of photoreduction of thiore31t 2 FTR 3 f 0.2 38 f 5 doxin m are sufficient to activate NADPNADP-MDH 74 f 22 2fl 33 f 5 MDH according to the studies of Rebeille and Hatch who have reported that very Note. Intensity of fluorescence and protein content high reduced to oxidized thioredoxin ratios were measured as described under Experimental Proare required for activation of the enzyme cedures to determine percentage reduction; varia(35,36). tions in protein content of each band were compenIt should be noted that thioredoxin m sated for by fitting the data onto a standard curve. was consistently recovered from isolated Variations are given as standard deviations. DTT was chloroplasts as two bands with molecular added to lysed chloroplasts prior to labeling with mass values of 13 and 11 kDa, irrespective mBBr to obtain DTT controls. The dark control for of whether the reduced protein was unlathioredoxinfwas subtracted from the value obtained in the light and from the DTT control for reasons exbeled or labeled with mBBr or NEM. The plained under Results and Discussion. The value obprecipitation of both mBBr-labeled forms tained in the light was then related to the DTT conwas inhibited by the addition of excess trol which was assumed to be 100%. NEM-labeled thioredoxin m (Fig. 4, treatment 5). The 13-kDa form, the form used for the controls of Fig. 3, is the major species recovered by our preparative purificathentic DTT reduced thioredoxin m (13kDa form) (treatment 4); (iii) dilution by tion procedure (18). Two points pertinent to the thioredoxin NEM-labeled authentic thioredoxin m (treatment 5); and (iv) by its absence in a m analyses warrant comment. The first is that reduced thioredoxin m precipitated control in which preimmune IgG replaced more effectively from either crude or purithioredoxin m antibody in the isolation fied preparations than did oxidized thioreprocedure (treatment 6). doxin m. This finding suggests that the Because thioredoxin m has cysteine resiprotein undergoes a significant conformadues only in its active site (33), the lightdependent increase in mBBr labeling seen tional change on reduction. Furthermore, since oxidized thioredoxin m was the form in Fig. 4 could be attributed to photoreducused for antibody production, the protein tion of the protein. In significant earlier may have become reduced in the rabbits work, Scheibe (11) reported similar findafter injection via indigenous thiols such ings with thioredoxin m of isolated pea system (37). chloroplasts. She used NEM to bind re- as the NADP/thioredoxin The problem of unequal immunoprecipiduced thioredoxin m and measured the retation of the thioredoxin m was readily cirmaining thioredoxin m (i.e., the oxidized cumvented by reducing samples with 1 mM portion) enzymatically in an NADP-MDH DTT and labeling with NEM prior to the assay with DTT. By this method, reduced step. This precauthioredoxin was measured indirectly by antibody precipitation tion also avoided the presence of small mithe difference between total and oxidized grational differences between the unlathioredoxin m. This method has a complibeled and mBBr-labeled proteins as obcation in that thioredoxin f could interfere TABLE
I
FERREDOXIN/THIOREDOXIN
Fluorescence 1
2
345
233
SYSTEM
Protein 6
1
6
FIG. 4. Demonstration of the photoreduction of thioredoxin m in intact chloroplasts. Thioredoxin m was labeled with mBBr in light (L)- or dark (D)-treated chloroplasts, immunoprecipitated, and resolved on SDS-PAGE with the following variations. 1, Treatment as described in Fig. 2.2, Addition of DTT and mBBr to darkened intact chloroplasts prior to lysis and immunoprecipitation. 3, Same as 2 except DTT and mBBr were added after chloroplast lysis. 4, Pure thioredoxin 11~(la-kDa form) reduced with DTT and labeled with mBBr. 5, Inhibition of the immunoprecipitation of mBBr-labeled thioredoxin m from chloroplast extract by the addition of 70 fig of purified NEM-labeled thioredoxin 1~. 6, Preimmune IgG control. Each lane contained ca. 3 pg (total) thioredoxin m.
served for thioredoxin$ However, because of the DTT and NEM treatments, it was not possible to perform the mBBr labeling of SDS-denatured controls as described above for thioredoxinj The other pertinent noteworthy point concerns the entry of DTT into chloroplasts. When DTT was added to darkadapted chloroplasts that were labeled with mBBr prior to lysis, the incorporation of fluorescent label was only about 10% that observed for controls lysed prior to DTT addition (Fig. 4, treatment 2 vs treatment 3). Thus, according to our results, DTT enters plastids, but thioredoxin reduction under these conditions is clearly limited. Similar findings were made with thioredoxin f (data not shown) and FTR (see below). Such low levels of reduced thioredoxins generated by DTT under these conditions are seemingly sufficient to enhance carbon dioxide assimilation as
was observed in model studies carried out in the dark with isolated intact chloroplasts (38).
Ferredoxin/Thioredoxin
Reductase
Like the f and m thioredoxins, FTR was found to be photoreduced in intact chloroplasts (Fig. 5). That the observed fluorescence increase was due to net reduction, rather than to conformational change, was confirmed by retention of the light effect in samples treated with SDS as described above for thioredoxin f (Fig. 5, lanes D and L of treatment 1 vs D and L of treatment 2). That the recovered component was FTR was confirmed by controls of Fig. 5 similar to those described above for thioredoxins f and m-i.e., (i) reduction by DTT in lysed chloroplasts (treatment 4); (ii) comparison to authentic FTR, either oxidized or photoreduced (treatment 5, lanes 0 and R), (iii)
234
CRAWFORD
Fluorescence
ET AL.
Protein
W
FTR -
uuuuuuuuu FIG. 5. Demonstration of the photoreduction of FTR in intact chloroplasts. FTR was labeled with mBBr in (L)- or dark (D)-treated chloroplasts, immunoprecipitated, and resolved on SDS-PAGE with the following variations. 1, Treatment as described in Fig. 2.2, mBBr labeling of SDS-denatured immunoprecipitate (see Experimental Procedures). 3, Addition of DTT and mBBr to darkened intact chloroplasts prior to lysis and immunoprecipitation. 4, Same as 3 except DTT and mBBr were added after chloroplasts were lysed. 5, Pure FTR standard protein, oxidized (0) or DTT reduced (R) and labeled with mBBr. 6, Inhibition of the immunoprecipitation of mBBr-labeled FTR by the addition of 100 fig of pure unlabeled FTR to chloroplast extract. 7, Preimmune IgG control. Each lane contained ca. 2 pg (total) FTR.
dilution by unlabeled authentic FTR (treatment 6); and (iv) by its absence from a control in which preimmune IgG replaced FTR antibody (treatment 7). As also seen in Fig. 5, several higher molecular weight chloroplast proteins were photoreduced and recovered with FTR under these conditions (see below). Reduction of FTR occurred on the catalytically active disulfide group that was identified earlier on the 13-kDa (similar) subunit of the enzyme (8). The extent of reduction of FTR was 38% in the light and 3% in the dark (Table I). The finding that the extent of reduction in the light was relatively low compared to that of the thioredoxins (see above) is in agreement with earlier evidence obtained with a reconstituted system (8, 9). The results from both lines of study suggest that the redox potential and turnover of the FTR disulfide group are sufficient to promote the higher level of reduction (about 90%) of
the thioredoxins f and m required for activation of their respective target enzymes, NADP-MDH (8) and FBPase (9). In the future, it will be of interest to measure the redox potential of the FTR disulfide and relate that potential to the value reported for the active disulfide of thioredoxin m (E. = -330 mV) (35). Consistent with observations made with thioredoxins f and m, DTT did not significantly reduce FTR in intact chloroplasts (Fig. 5, treatment 3) but did reduce FTR in lysed chloroplasts (Fig. 5, treatment 4). As expected, the extent of reduction of FTR by DTT under these conditions was less than that observed previously (19) in a reconstituted system incubated anaerobically with a twofold higher DTT concentration and a fourfold longer incubation period (31% vs 60% reduction). An additional noteworthy point concerns the subunit composition of FTR. Pre-
FERREDOXIN/THIOREDOXIN
viously both our laboratory and that of Schiirmann have found that FTR is a dimerit protein consisting of two dissimilar subunits migrating in SDS-PAGE with molecular masses of 13 and 16 kDa (19,39). The 13-kDa (similar) subunit is present in all FTRs examined, whereas the other (variable) subunit (16 kDa in the case of spinach) varies as to the species. In the present study, the catalytically active 13kDa subunit was preferentially precipitated from chloroplast extract together in some experiments with an unidentified 19kDa subunit (Fig. 5, protein treatment 1). Immunoprecipitation of the mBBr-labeled 13-kDa band, as well as an unlabeled 19kDa band just above it, was inhibited by the addition of excess pure unlabeled FTR as in treatment 6. This finding suggests that the 13- and 19-kDa bands are associated and raises the question of their relation to one another. Significantly, we saw no evidence for the 16-kDa variable subunit associated with purified FTR preparations (19) by following this procedure. It is therefore possible that the 16-kDa subunit associated with purified spinach FTR is derived from the 19-kDa component during purification, presumably by proteolysis. Other unidentified larger bands in the 30to 35-kDa range were also recovered from chloroplast extract, but, unlike the 19-kDa subunit, their precipitation was not inhibited by added FTR. Hence, these larger bands appear to have precipitated as a result of nonspecific adsorption. Target Enx ymes The above results are consistent with the conclusion, drawn from enzyme studies, that the ferredoxin/thioredoxin system functions as a regulatory thiol chain, linking noncyclic electron transport to enzyme regulation. Here, photoreduced ferredoxin serves as reductant for the catalytically active disulfide group of FTR, which, in turn, transfers the reducing equivalents to thioredoxins f and m. The terminal member of the chain is a target enzyme, e.g., NADP-MDH or FBPase, which contain a regulatory disulfide site that, when reduced, leads to activation of
SYSTEM
235
catalysis (8, 9). To date, a photoreduction of such a target enzyme has not been reported for an intact chloroplast system. We, therefore, addressed this problem using the procedure described above. Figure 6 illustrates that light increased the incorporation of mBBr label into both NADP-MDH and FBPase. In the case of NADP-MDH, the light-induced increase in fluorescence corresponds to a 33% reduction when compared to a DTT-reduced purified standard for the enzyme (Table I). This increase was the result of a net photoreduction on the basis of two lines of evidence. First, when subjected to the SDS treatment developed for thioredoxinf, the NADP-MDH subunit from light-treated chloroplasts showed a 23% greater fluorescence than its dark counterpart. As expected, the fluorescence background was increased markedly by this SDS treatment, as previously unaccessible sulfhydry1 groups became available to react with mBBr. The second line of evidence for a net photoreduction of NADP-MDH in intact chloroplasts came from application of the reverse labeling technique described above for thioredoxin$ In this case, the chloroplasts were labeled by NEM in both the light and the dark. Thus, any sulfhydryls produced in the light would be derivatized with NEM rather than mBBr. Here, as expected, NADP-MDH from light-treated chloroplasts was found to be 36% less fluorescent when related to dark controls (data not shown). This above value correlates well with the value of 33% calculated from the data of Fig. 6. We estimate these values for extent of reduction to be minimal because of the above-noted difficulty in precipitating reduced enzyme; i.e., reduced NADP-MDH precipitated only about 50% as effectively as the oxidized enzyme. Because of the close proximity of the heavy chain of y globulin to the FBPase subunit, it was not possible to quantitate the light-induced thiol changes in FBPase with the mBBr technique. While scanning of fluorescence was not a problem, the Coomassie blue-stained FBPase could not be resolved from an IgG band. If one makes the assumption that the oxidized and reduced forms of the FBPase precipitate
236
CRAWFORD
ET AL.
NADP-MDH
Relative Mobility FIG. 6. Effect of light on the incorporation of mBBr into NADP-MDH and FBPase of intact chloroplasts. Densitometric scans of fluorescence in SDS-PAGE gels containing 1 pg each of NADP-MDH and FBPase were made as described under Experimental Procedures.
equally and that the 1 mM DTT control for the enzyme is fully reduced, the FBPase was oxidized in the dark and was 25% reduced under our conditions in the light. Due to the very high fluorescence background, it was not possible to confirm this value by the SDS treatment used above for thioredoxinf, FTR, and NADP-MDH and, for unknown reasons the reverse labeling techniques described above for NADPMDH were unsuccessful. These points aside, evidence recently obtained with a reconstituted enzyme system supports the conclusion that the light-induced increase in incorporation of mBBr into the FBPase is due to a net reduction rather than a conformational change ((9, 40-43); P. Schiirmann, personal communication). It should be noted that controls described above for thioredoxins and FTR were included for both NADP-MDH and FBPase. These revealed (i) inhibition of precipitation of mBBr-labeled enzyme by adding excess unlabeled protein and (ii) inability of purified IgG to precipitate each enzyme. In all cases, the controls confirmed that the proteins precipitated were the indicated target enzymes. Time Course of Thioredoxin Reduction and Enzyme Activation The development of an mBBr technique applicable to intact chloroplasts makes it possible to determine the kinetics of reduc-
tion of thioredoxins f and m relative to the activation of target enzymes. In this connection, we consistently found that reduction of FTR and of thioredoxins f and m preceded activation of target enzymes. In the experiment shown in Fig. 7, the components of the ferredoxin/thioredoxin system were rapidly reduced in the light and the levels of reduced thioredoxin f and m reached steady state after 2 min. Activation of the target enzymes, NADP-MDH and FBPase, occurred more slowly and in this experiment reached steady state after 10 min. When the light was turned off, the activity of NADP-MDH and FBPase decreased less rapidly than oxidation of the thioredoxin and FTR. It should be noted that while the rate of light-mediated enzyme activation varied from one experiment to another (full activation was achieved in the 3 to 10 min time frame), the reduction of FTR and thioredoxinsfand m consistently preceded activation of the target enzymes. Similarly, the oxidation of the regulatory thiol proteins occurred prior to deactivation of the enzymes in the dark. The results thus show a correlation between the reduction of thioredoxins and the activation of NADP-MDH and FBPase that is in accord with previous evidence indicating a central role for the ferredoxin/ thioredoxin system in the light activation of these enzymes (l-5). The regulatory importance of thioredoxins is also illustrated in a recent report describing the thiore-
FERREDOXIN/THIOREDOXIN V//Q&t&C&
LIGHT
THIOL REDUCTION
ENZYME
ACTIVITY
‘ks”,’
i
LIGHT 5
DAR
15
2
FIG. 7. Time course of the light-dependent reduction of components of the ferredoxin/thioredoxin system and concurrent activation of target enzymes in intact chloroplasts. Time of incubation in the light or dark was as indicated. Arrows refer to the time at which the vessel was placed in the dark. For thioredoxin points taken in the light, mBBr was added in the light 2 min prior to placing the vessel in the dark, and the reaction was stopped with 2-mercaptoethanol 3 min later. For points taken in the dark, mBBr was added at the time indicated and the reaction was stopped with 2-mercaptoethanol5 min later. The following amounts were analyzed by the mBBr-SDSPAGE method: thioredoxin m. 4 pg; thioredoxin f. 1 gg; and FTR, 3 pg.
doxin-linked photoactivation of FBPase in a reaction in which thylakoids were replaced by a heterogeneous photochemical system consisting of EDTA as electron donor and acridine orange or proflavin as photocatalyst (44). In recent interesting work on the structure of light-activated chloroplast enzymes, the regulatory reduction sites have been identified for NADP-MDH (6), FBPase (7), and PRK (45). In the case of
SYSTEM
237
NADP-MDH and, probably FBPase, the regulatory site consists of a redox-active group containing two cyst(e)ines separated by four or five amino acids that, by analogy with these enzymes from other sources, is not part of the active site. The results of Fig. 8 provide experimental evidence on this point. When added to illuminated intact chloroplasts just prior to turning off the light, mBBr maintained the activated state of NADP-MDH and FBPase in the dark (Fig. 8, right). This result supports the conclusion that the regulatory dithiol groups of both enzymes are separate from the active site. A similar conclusion was earlier reached for FBPase on the basis of kinetic data (46). A different situation exists for PRK. Here, the two regulatory cyst(e)ines are separated by 39 amino acids (45) and one of the cyst(e)ines has been concluded to be a part of the active site (47). The present results support this conclusion. In contrast to NADP-MDH and FBPase, mBBr rapidly inactivated PRK upon transfer to dark conditions (Fig. 8, right). Furthermore, PRK was also rapidly inactivated when mBBr was added to chloroplasts kept in the light. Results similar to those of Fig. 8 were obtained with NEM for PRK as well as FBPase and NADP-MDH (data not shown). CONCLUSIONS
The present results provide evidence that the ferredoxin/thioredoxin system functions in the light-mediated activation of enzymes of isolated intact chloroplasts in the manner established with reconstituted enzyme assays. The data suggest that photoreduction of the thiol-active components of the ferredoxin/thioredoxin system precedes reductive activation of target enzymes as would be expected for the reaction sequence derived from experiments with the isolated pure components, light + chlorophyll + ferredoxin + FTR + thioredoxin f or m + target enzyme. In addition to documenting the function of the ferredoxin/thioredoxin system as a regulatory thiol chain, the current work
238
CRAWFORD ET AL. I
~
I
I
150
.z .-> C :
a, 100 > L= 0
a,
K
50
15
20
25
30
15
20
25
30
Minutes FIG. 8. Effect of mBBr on the light-induced activity of NADP-MDH and FBPase target enzymes after change from light to dark conditions. The left panel shows the time course of activation by light. The other panels show the enzyme activities after the vessels were placed in the dark, either minus (center) or plus mBBr (right). Monobromobimane (1 mM) was added 2 min prior to turning off the lights.
supports recent conclusions on the regulatory reduction sites of chloroplast FBPase, NADP-MDH, and phosphoribulokinase. Our data also give new information on the relation between the regulatory and active sites for each of these thioredoxin-linked enzymes, which can now be compared to CF,-ATPase, another thioredoxin-linked enzyme which has regulatory cysteines separated from its catalytic site (48,49). ACKNOWLEDGMENT This work was supported by a grant from the National Science Foundation. REFERENCES 1. BUCHANAN,B. B. (1980) dnnu. Rev. Plant Physiol. 31,341-374. 2. CS$KE, C., AND BUCHANAN,B. B. (1986) Biochinz. Biophys. Acta 853.43-63. 3. JACQUOT,J.-P. (1984) Physiol. Veg. 22,487-507.
4. EDWARDS,G. E., NAKAMOTO,H., BURNELL,J. N., AND HATCH, M. D. (1985) Annu. Rezl. Plant PhysioZ. 36,255-286.
5. LEEGOOD,R. C., WALKER, D. A., AND FOYER, C. (1985) in Topics in Photosynthesis (Barker, J., and Baker, N. R., Eds.), Vol. 6, pp. 189-258, Elsevier/North-Holland, Amsterdam.
6. DECOTTIGNIES,P., SCHMITTER,J.-M., MIGINIACMASLOW,M., LE MARECHAL,P., JACQUOT,J.-P., AND GADAL, P. (1988) J. Biol. Chem. 263, 11,780-11,785.
7. MARCUS,F., MOBERLY,L., AND LATSHAW, S. P. (1988) Proc. Natl. Acud. Sci. USA 85,5379-5383.
8. DROUX, M., MIGINIAC-MASLOW, M., JACQUOT, J.-P., GADAL, P., CRAWFORD,N. A., KOSOWER, N. S., AND BUCHANAN,B. B. (1987) Arch Bie them. Biophys. 256,372-380.
9. DROUX, M., CRAWFORD,N. A., AND BUCHANAN, B. B. (1987) C R. Acd Sci. Paris 305,335-341. 10. DROUX,M., JACQUOT,J.-P., SUZUKI, A., AND GADAL, P. (1984) in Advances in Photosynthesis Research (Sybesma, C., Ed.), Vol. 3, pp. 533536, Nijhoff/Junk, Dordrecht/The Netherlands. 11. SCHEIBE,R. (1981) FEBS Lett. 133,301-304. 12. DARLING, A. J., ROW~L, P., AND STEWARD,W. D. P. (1986) Biochim. Biophys. Acta 850,116-120. 13. TSUGITA, A., MAEDA, K., AND SCH~RMANN,P. (1983) Biochem. Biophya Res. Ccwnmun. 115, l-7. 14. KOSOWER,N. S., AND KOSOWER,E. M. (1987) in Methods in Enzymology (Jakoby, W. B., and Griffith, 0. W., Eds.), Vol. 143, pp. 76-84, Academic Press, San Diego. 15. REEVES,S. G., AND HALL, D. 0. (1980) in Methods in Enzymology (San Pietro, A., Ed.), Vol. 69, pp. 85-101, Academic Press, San Diego.
FERREDOXIN/THIOREDOXIN 16. LEECOOD, R. C., AND MALKIN, S. (1986) Photosynthesis Energy Transduction: A Practical Approach (Hipkins, M. F., and Baker, N. R., Eds.), pp. 9-26, IRL Press, Oxford. 17. ANDERSON, D. J., AND BLOBEL, G. (1983) in Methods in Enzymology (Fleischer, S., and Fleischer, B., Eds.), Vol. 96, pp. 111-120, Academic Press, San Diego. 18. CRAWFORI), N. A., YEE, B. C., HUTCHESON, S. W., WOLOSIUK, R. A., AND BUCHANAN, B. B. (1986) Arch. Biochem. Bivphys. 244,1-15. 19. DROUX, M., JACQUOT, J. P., MIGINIAC-MASLOW, M., GADAL, P., HUET, J. C., CRAWFORD, N. A., YEE, B. C., AND BIJCHANAN, B. B. (1987) Arch. Biochem. Biophys. 252,426-439. 20. JACQUOT, J.-P., GADAL, P., NISHIZAWA, A. N., YEE, B. C., CRAWFORD, N. A., AND BUCHANAN, B. B. (1984) Arch. Biochem. Bioph,ys. 228, 170178. 21. Bio-Rad Laboratories, Bulletin 1059. 22. TIJSSEN, P. (1987) ill Laboratory Techniques in Biochemistry and Molecular Biology (Burdon, R. H., and van Knippenberg, P. H., General Eds.), p. 105, Elseveier, New York. 23. LEEGOOD, R. C., AND WALKER, D. A. (1980) Bicchim. Biophys. Acta 593,362-370. 24. PORTER, M. A., MILANEZ, S., STRINGER, C. D., AND HARTMAN, F. C. (1986)Arch. Bioch,em. Biophys. 245,14-23. 25. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248254. 26. ARNON, D. I. (1949) Plant PhysioL 24,1-15. 27. NISHIZAWA, A. N., YEE, B. C., AND BUCHANAN, B. B. (1982) in Methods in Chloroplast Molecular Biology (Edelman, M., Hallick, R. B., and Chua, N.-H., Eds.), pp. 707-714, Elsevier Biomedical, New York. 28. MAYHEW, S. G. (1971) Anal B&hem. 42,191-194. 29. JACQUOT, J.-P., DROUX, M., MIGINIAC-MASLOW, M., JOLY, C., AND GADAL, P. (1984) Plant Sci. Lett. 35,181-185. 30. CRAWFORD, N. A., KOSOWER, N. S., AND BuCHANAN, B. B. (1987) in Progress in Photosynthesis Research (Biggins, J., Ed.), Proceedings, Vllth International Congress on Photosynthesis, August 10-15, 1986, Provi-
31. 32. 33.
34. 35. 36. 37. 38. 39.
40.
41. 42. 43.
44.
45. 46. 47. 48. 49.
SYSTEM
239
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