Plant Physiol. (1992) 100, 272-2810032-0889/92/100/0272/1 0/$01 .00/0

Received for publication November 27, 1991Accepted April 20, 1992

Synthesis and Assembly of Large Subunits into RibuloseBisphosphate Carboxylase/Oxygenase in Chloroplast

Extracts1

Alan Hubbs and Harry Roy*

Plant Science Group, Biology Department, and Center for Biophysics, Rensselaer Polytechnic Institute,Troy, New York 12180-3590

ABSTRACT

We have developed a new system for the in vitro synthesis oflarge subunits and their assembly into ribulose bisphosphate car-boxylase oxygenase (Rubisco) holoenzyme in extracts of higherplant chloroplasts. This differs from previously described Rubiscoassembly systems because the translation of the large subunitsoccurs in chloroplast extracts as opposed to isolated intact chlo-roplasts, and the subsequent assembly of large subunits into holo-enzyme is completely dependent upon added small subunits. Aminoacid incorporation in this system displayed the characteristics pre-viously reported for chloroplast-based translation systems. Incor-poration was sensitive to chloramphenicol or RNase but resistantto cycloheximide, required magnesium, and was stimulated bynucleotides. The primary product of this system was the largesubunit of Rubisco. However, several lower molecular weight poly-peptides were formed. These were structurally related to the Rub-isco large subunit. The initiation inhibitor aurintricarboxylic acid(ATA) decreased the amount of lower molecular weight productsaccumulated. The accumulation of completed large subunits wasonly marginally reduced in the presence of ATA. The incorporationof newly synthesized large subunits into Rubisco holoenzyme oc-curred under conditions previously identified as optimal for theassembly of in organello-synthesized large subunits and requiredthe addition of purified small subunits.

Previous investigations have characterized the protein-synthesizing apparatus of the chloroplast. By most criteria,chloroplast ribosomes resemble those of bacteria in both formand function (9, 10). Translation systems derived from Esch-erichia coli, rabbit reticulocyte, and wheat germ can expresschloroplast transcripts (6, 7). Additionally, several laborato-ries have characterized translation systems derived from thechloroplast stroma, finding that they can express endogenousor heterologous genes or transcripts (2, 3, 7). Some of thesesystems have been reported to synthesize Rubisco large sub-units (2, 6).

Rubisco holoenzyme contains eight large subunits, synthe-sized in the chloroplast, and eight small subunits, synthesized

' This work was supported by a grant from the National ResearchInitiative Competitive Grants Program of the U.S. Department ofAgriculture. The work reported here is taken from a thesis to besubmitted by A.H. in fulfillment of the requirements for the Ph.D.degree at Rensselaer.

in the cytosol (9, 28). Our laboratory has described the invitro assembly into holoenzyme of large subunits synthesizedin isolated intact chloroplasts ('in organello') (4, 22, 23).However, there have been no reports that in vitro-synthe-sized large subunits assemble into holoenzyme.

Researchers interested in improving the catalytic activityof this enzyme have emphasized the agricultural importanceof assembling the higher plant enzyme in an easily manipu-lated bacterial source (28). Cloned genes for higher plantRubisco subunits have not yielded detectable Rubisco activitywhen expressed in E. coli (11, 13). Cloned Rubisco genesfrom cyanobacterial sources, on the other hand, did yieldactive Rubisco (12, 30). Considering the similar quaternarystructure of the cyanobacterial and higher plant enzymes, thefailure of the higher plant subunits to assemble in E. coli maybe due to slight differences in the primary structures of thetwo proteins; alternatively, assembly of the higher plantenzyme may be dependent upon some factor (e.g., the higherplant chaperonin, Cpn6014) not available in the bacterialsystem (13), or the higher plant subunits may be sensitive tosome conditions of the foreign synthesis environment. In anycase, critical information leading to the assembly of thisenzyme in bacterial systems may be gained from a study ofits assembly in a native system. Therefore, we sought condi-tions of in vitro protein synthesis that would allow thesubsequent assembly of the translation products into holo-enzyme. We examined the available literature on chloroplast-based in vitro protein-synthesizing systems and chose to useextracts that can complete nascent peptides as a starting point.Previous experience with protein synthesis in intact chloro-plasts suggested that the Rubisco large subunit would be theprimary product of a runoff synthesis reaction. However, wewere unable to observe the assembly of in vitro-synthesizedlarge subunits into Rubisco until we optimized the system tosuit our needs.

Because we required easily detectable amounts of assem-bly-competent large subunits, we selected conditions thatyielded the greatest total accumulation of radioactivity intoprotein, rather than the highest initial synthesis rate. Addi-tionally, we monitored the SDS-PAGE profile of the trans-lation products to optimize recovery of the completed Rubiscolarge subunit polypeptides. This report describes character-istics of the large subunit synthesis reaction as well as thesmall subunit-dependent assembly of the newly synthesizedlarge subunits into Rubisco holoenzyme, under conditions

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IN VITRO SYNTHESIS AND ASSEMBLY OF RUBISCO

previously described as optimal for assembly of in organello-synthesized large subunits.

MATERIALS AND METHODS

Chemicals

We obtained AMP, GTP, ATP, DTT, pyruvate kinase,phosphoenolpyruvate, and creatine phosphokinase fromSigma. We purchased HPRI2 and 1OX biosynthesis reactionmix for rabbit reticulocyte in vitro translation systems (250mM Hepes [pH 7.2], 400 mm KCl, 100 mm creatine phosphate,500 mM amino acids minus leucine or methionine) fromBethesda Research Laboratories (Gaithersburg, MD). Allother chemicals were reagent grade. EN 3Hance was obtainedfrom New England Nuclear Corporation (Waltham, MA).

Growth of Plants and Isolation of Intact Chloroplasts

Pea seedlings (Pisum sativum var. Progress #9, Agway,Rochester, NY) were grown and intact chloroplasts wereisolated as previously described using Percoll gradients (22,23), except that EGTA and MnCl2 were omitted from thechloroplast resuspension buffer (50 mi Hepes-KOH [pH8.15], 2 mm EDTA, 1 mM MgCl2). We determined Chl con-centrations by the method of Arnon (1) as modified by Miloset al. (22, 23).

Chloroplast Lysate Preparation

Freshly isolated chloroplasts were used for all experiments.Typically, resuspended chloroplasts were distributed to Corextubes so that 600 to 1500 ,ug of Chl was contained in eachtube. The suspension was then centrifuged at 4000g for about30 s and the supematant was removed with a Pasteur pipette.Various methods of lysis were investigated to determinewhich would yield the most concentrated lysate. The methodfinally adopted was carried out in two steps. First, a verysmall amount of lysis buffer (10 mm Tris, pH 7.8, 5 mm DTT,25 ,ug/mL of creatine phosphokinase, 160 units/mL of HPRI;50-80 ,uL per Corex tube) was added to the pellet, and thiswas stirred into a slurry using a paint brush with severalshort bristles. This step served to disrupt the chloroplasts andremove residual resuspension buffer, which compromisedthe lysis procedure. The slurry was then centrifuged at 8000gfor 2 min and stopped without braking. The supernatant wascompletely removed and saved for later protein determina-tion. The chloroplasts were suspended in 100 to 150 ,uL oflysis buffer per 800 ,g of Chl. Membranes were removed bycentrifugation at 45,000g for 10 min, and the recoveredsupernatant was immediately placed on ice. This supernatantwas then used in the protein-synthesis reactions. This secondlysis of the chloroplasts was found to yield the most concen-

trated extracts, which correspondingly contained the greatesttranslational activity. No loss of translational activity was

observed for lysates held on ice for up to 30 min prior totranslation.

2Abbreviations: HPRI, human placental ribonuclease inhibitor;ATA, aurintricarboxylic acid.

In Vitro Protein Synthesis in Chloroplast Lysates

Most of the added components of the translation systemwere freeze dried in microcentrifuge tubes immediately priorto the experiment. It was important to bring the cocktail todryness to achieve the precalculated conditions in the finalreaction. However, care was taken not to over-dry the sam-ple, especially when [35S]methionine was used as the labeledamino acid, because possible oxidation was considered un-desirable. The components added to the translation reactionin this fashion were included to give final concentrations asfollows: Bethesda Research Laboratories 1Ox biosynthesisreaction mix (50 1LM amino acids minus leucine or methionine,10 mM phosphocreatine, 40 mi KCl, 0.4 mM ATP, 0.3 mMGTP, 8.0 mm Mg acetate, 50 mm K acetate), 1 ,UM [3H]leucine(New England Nuclear, 143.7 Ci/mM), 8 to 16 AM unlabeledleucine (optional), and 25 mm Hepes-KOH (pH 8.0). (TheKOH contributed 11 mm K+ to the system.) When ATA wasused, it was also added at this stage at a final concentrationof 100 ,M. In some experiments, [35S]methionine (New Eng-land Nuclear, approximately 1100 Ci/mM, 0.456 FAM) wasemployed. Protease inhibitors were not included in the re-actions because we did not want to take a chance of inhibitingpossible required proteolytic processing events. As a precau-tion against externally introduced RNase activity, all glass-ware used in the preparation of the lysate was baked at2000C for 2 h.

Assembly of in Vitro-Synthesized Large Subunits

The assembly of large subunits sythesized in vitro wascarried out essentially as described for the assembly of inorganello-synthesized large subunits (4, 22, 23, 25). However,DTT was not added to the assembly reaction because it wasalready present during synthesis; also, lower salt concentra-tions were used. The synthesis lysate represented one-tenthto one-half of the final reaction. The only other componentscritical to assembly (provided synthesis has been carried outfor at least 20 min) were potassium chloride at a final con-centration of 150 mm and small subunits at 0.5 to 1 lM. Weconsidered all sources of ions as contributing to the finalreaction. When ATP stimulation of assembly was to be ob-served, it was important that the chloride concentration bemaintained below 130 mm and magnesium be maintained at5 mM.

Partial Proteolytic Analysis

Protein synthesis was carried out for 1 min in the presenceof [35S]methionine at a final concentration of 500 uCi/mL ofreaction solution. Synthesis was halted by addition of chlor-amphenicol. Multiple samples were separated by SDS-PAGEusing a 12.5% polyacrylamide running gel with an acrylamideto bisacrylamide ratio of 100 to 1 and a 4% polyacrylamidestacking gel with a 50 to 1 acrylamide to bisacrylamide ratio.Electrophoresis was carried out at 20 to 50 V until thebromphenol blue dye front approached the end of the gel.After electrophoresis in the first dimension, gel lanes wereexcised and intact lanes were subjected to partial proteolysisusing a method similar to that used by Lizardi (19) or werestored immediately in the -200C freezer for later analysis.

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Plant Physiol. Vol. 100, 1992

Proteolysis was carried out by incubating the entire length ofa 1-mm wide slice of the first dimension gel lane in SDScocktail containing 30 units/mL of Staphylococcus aureus V8protease (Sigma). Incubations were carried out with gentleshaking for 10 or 20 min. The gel lanes were then placedatop a second SDS-PAGE gel. The running gel contained17% acrylamide. The gel lane being analyzed was placed incontact with the 2-mm stacking gel and was held in place bythe glass plates. The gel lane was layered with the protease-containing SDS cocktail and electrophoresis was carried outuntil the bromphenol dye front reached the running gel.Electrophoresis was then suspended for 10 min to allowadditional proteolysis. Electrophoresis was then continued at20 to 50 V until the bromphenol dye front reached the endof the gel. The gel was then prepared for fluorography, withduplicate first dimension gel lanes placed atop the seconddimension gel in place of the digested gel lane.

Analysis of Products of Protein Synthesis and Assembly

SDS-PAGE (18), nondenaturing electrophoresis, stainingof gels, fluorography, and liquid scintillation counting of acid-insoluble material were carried out essentially as previouslydescribed (22).

Purification of Rubisco Small Subunits

Small subunits were isolated from purified native Rubiscoas described by Roy et al. (25). Purified Rubisco in a solutionof 10 mm sodium phosphate, 500 mm sodium chloride, and1 mM EDTA was brought to pH 5.1 on ice. The solution wasincubated for 1 h, and the resulting precipitate was removedby centrifugation. A proportion of the small subunits associ-ated with Rubisco remain in the supematant fraction underthese conditions. This solution was recovered and brought topH 7.8 using a 1-M solution of Tris base.

RESULTS

General Characteristics of Amino Acid Incorporation

Lysates prepared as described in 'Materials and Methods'incorporated [3H]leucine or [35S]methionine in amounts thatwere readily analyzed by liquid scintillation counting as wellas by fluorography of polyacrylamide gels. We used [3H]-leucine for most experiments. However, when protein syn-thesis was to be carried out for short periods, or when thelevel of detection was a consideration, we used [35S]methio-nine. We optimized the incorporation of [3H]leucine for eachof the components added to the translation reaction exceptfor the lOx biosynthesis reaction mixture, which we includedas recommended by the manufacturer (Bethesda ResearchLaboratories). The lysates in combination with this reactioncocktail and extemally supplied magnesium were the onlycomponents strictly required to attain [3H]leucine incorpora-tion into protein. However, to obtain optimal incorporationwe included both an RNase inhibitor and an ATP-regener-ating system in all reactions.

In view of the known dependence of protein-synthesizingsystems on extemally supplied magnesium and potassium,we studied the effects of several extemally supplied ions.

Sulfate, chloride, and acetate salts of magnesium all affectedtranslation. However, sulfate inhibits creatine kinase (accord-ing to the Sigma Chemical Corporation data sheet) andchloride inhibits translation by the rabbit reticulocyte system(17). Therefore we generally used magnesium acetate. Figure1A shows the relative incorporation of radioactive amino acidversus the concentration of added magnesium acetate. Wecarried out this experiment at several dilutions of lysate toassess the possibility that the optima drift under more diluteconditions. Such a drift might result if the contribution ofendogenous magnesium by the lysate is significant. We foundthat negligible amounts of radioactivity appeared in proteinwhen the translation reaction contained less than 3 mmmagnesium. We observed maximum incorporation with ad-dition of 7 to 8 mm magnesium. Although the drift in themagnesium optimum across this range of lysate concentra-tions was not obvious, we found an approximately 1-mMdifference in the threshold concentration required to attainleucine incorporation. Examination of the SDS-PAGE profileof the translation products as a function of added magnesiumdemonstrated that the distribution of radioactivity into prod-ucts of different mol wts is affected by the magnesiumconcentration (data not shown). We observed that the pro-portion of completed Rubisco large subunits was highestbetween 8 and 9 mm added magnesium. Therefore, wegenerally carried out protein synthesis at 8 mm addedmagnesium.

Potassium chloride (40 mm) was provided primarily by theBethesda Research Laboratories biosynthesis reaction mix-ture. A small amount of potassium (11 mM) was supplied asKOH to adjust the pH of the reaction mixture. The furtheraddition of KCl did not enhance accumulation of labeledamino acids into protein. However, added potassium acetatedid increase the amount of radioactivity incorporated. Figure1B demonstrates that amino acid incorporation increased asthe concentration of added potassium rose from 50 to 110mM using potassium acetate, whereas KCI showed little stim-ulatory effect. We also found that NaCl served only to reduceincorporation but that sodium acetate could be added to afinal concentration of 60 mm before inducing any reductionin the incorporated radioactivity (Fig. 1C). Calcium chloride,on the other hand, yielded a 50% loss of incorporation at a1-mM concentration, and a 78% loss of incorporation at 5mm. The adverse effects of calcium addition could be reversedby including an excess of EGTA (data not presented).The time course of incorporation under optimal ionic con-

ditions varied depending upon the concentration of the lysateused and the temperature. The optimal temperature for theaccumulation of [3H]leucine into protein was between 24 and280C (data not shown). Temperatures below 240C gave lowersynthesis rates, whereas temperatures above 300C gave highinitial rates with a lower amount of accumulated radioactivityat times greater than 5 min. Figure 1D shows the time courseof incorporation at 260C. In this experiment, radioactivitycontinued to accumulate into acid-insoluble material for atleast 25 min (@). Addition of excess unlabeled amino acid at10 min suppressed further incorporation (0). Lysates ofhigher concentration displayed a greater initial rate of in-corporation, but this was compensated by a lower rate ofincorporation from 10 to 20 min, yielding similar amounts of

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IN VITRO SYNTHESIS AND ASSEMBLY OF RUBISCO

total incorporated radioactivity in the completed reaction(data not presented).

Characterization of Amino Acid IncorporationUsing classic inhibitors, we demonstrated that amino acid

incorporation into acid-insoluble material was due to mRNA-dependent protein synthesis (Table I). When we includedRNase in the reaction, we observed almost no incorporationof amino acids, even without any preincubation period toallow for RNA digestion. Chloramphenicol, an inhibitor of

70S ribosomal function, inhibited incorporation by 80 to 92%.In contrast, not more than a 5% reduction in acid-insolubleradioactivity was observed at cycloheximide concentrationsas high as 100 .g/mL. Puromycin also strongly inhibitedincorporation. However, inhibition was only 75% when pu-romycin was added simultaneously with the radioactiveamino acid. The residual incorporation appeared on SDS-PAGE gels as low mol wt products (data not shown). Theresults obtained with [35S]methionine were similar to thoseobtained with [3H]leucine.

C

2

cpm x 104

1-

3 4 5 6 7 8 9 10 11 12 0 30 60 90 120 150

Added Mg , mMAdded Na, mM

D 4.0

3.0

cpm x 104 -

2.0 -

1.0

50 80 110 140 170 200

Added K*, mM4 8 12 16 20 24

Thim, mh

Figure 1. Optimization of translation. A, Magnesium effects. Chloroplast lysates were prepared as described in "Materials and Methods," as

were the freeze-dried translation cocktails. Translation cocktails were prepared containing different amounts of magnesium acetate to yieldfinal added magnesium concentrations from 3 to 12 mm in 1-mM increments. Portions of the lysate were diluted by addition of lysis bufferto yield extracts representing 100, 75, 66, and 50%-strength lysates. Translation was initiated and halted as described in "Materials andMethods." Translation was carried out for 15 min with no chase period. Samples (equivalent to 1 qL) from each reaction were then assayedfor acid-insoluble radioactivity as described in "Materials and Methods." The A260/A280 of each lysate was as follows: 100% strength (0), 0.903/0.753; 75% (0), 0.612/0.475; 66% (U), 0.491/0.393; and 50% (O), 0.386/0.294. B, Incorporation of leucine into acid-insoluble material as a

function of added potassium as acetate or chloride. Chloroplast lysates were prepared as described in "Materials and Methods," as were thefreeze-dried translation cocktails. Each cocktail contained different amounts of potassium chloride (U) or potassium acetate (0) to yield thefinal concentrations of acetate or chloride as indicated in the figure. Translation was initiated and halted as described in "Materials andMethods." Translation was carried out for 10 min. Samples (equivalent to 1 gL) of all reactions were assayed for acid-insoluble radioactivity.C, Incorporation of leucine into acid-insoluble material as a function of added sodium as chloride or acetate. This experiment was conductedas in B above except that sodium chloride (-) or sodium acetate (0) were used to supply the anions of interest. D, The time course ofincorporation at 26°C with a 0°C control sample. Chloroplast lysate and translation cocktail were prepared as described in "Materials andMethods." Translation was initiated simultaneously in eight individual reactions as described in "Materials and Methods." One reaction was

held on ice for the duration of the experiment; all others were placed at 26°C. Reactions were conducted in the absence of unlabeledleucine (0) or were supplemented with unlabeled leucine to a final concentration of 2 mm after 10 min (M). At predetermined times, synthesiswas halted in each individual reaction by rapid freezing. Samples (equivalent to 1 ,uL) were then analyzed for acid-insoluble radioactivity as

described in "Materials and Methods." The range of the data samples is shown.

A 35

30-

cpm x10

B3-

cpmx 104 2-

1*

u

n

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Plant Physiol. Vol. 100, 1992

Table I. The Effect of Inhibitors of Ribosomal Function on theIncorporation of Leucine or Methionine into Acid-Insoluble MaterialThe combined results from four different experiments are shown.

The experimental conditions tested in each experiment overlappedbut were not identical. Two of the experiments (A and B) testedthe effects of inhibitors on leucine incorporation exclusively,whereas one experiment examined only methionine incorporation(C) and one tested the effects upon leucine or methionine incor-poration (D). The methods and analysis employed for each of theseexperiments were similar. The chloroplast lysates and the freeze-dried translation cocktails for each experiment were prepared asdescribed in "Materials and Methods." In each experiment, precal-culated amounts of a variety of inhibitors of translation were addedto each cocktail as follows: no addition (A, B, C, D), 20 gg/mL ofRNase A (A and C), 200 gM chloramphenicol (A and C), 100 Mmchloramphenicol and 20 ,ug/mL of RNase A (A, C, and D), 100 Mmcycloheximide (A), 50 Mm puromycin (C), 20 Mm puromycin (B), 0°C(D). Translation was conducted for 12 min (A, C, and D) or 15minutes (B). All reactions were analyzed for incorporation of radio-activity into acid-insoluble form. Duplicate samples were analyzedin each experiment. The results of each experiment were pooledby first averaging the duplicate samples, then expressing them asthe percentage of incorporation observed for the control samplefor that experiment. Any duplicate experiments were then aver-aged. Additional data collected from dissimilar experiments sup-ported these results but were not included in this analysis.

Radiotracer

Sample [3H]Leucine [35S]MethioninePercent SD Percent SDcontrol control

Control 100 4 100 6RNase 0.2 0.02 1.5 0.7Chloramphenicol 18 0.04 8 1RNase and chloram- 1.3 0.07 1.2 0.06

phenicolCycloheximide 97 0.8 N/A N/APuromycin 12 0.03 13 0.20°C 4 0.4 3 0.1

The fact that ATA affects the synthesis of many polypep-tides with lower mol wt than the Rubisco large subunitimplies that these polypeptides represent in vitro initiationproducts. However, it remained unclear whether these poly-peptides represent incomplete large subunit peptides or un-related proteins. To distinguish between these possibilities,we carried out second-dimension SDS-PAGE analysis ofproteins synthesized for a short period after partial proteolyticdigestion of intact first-dimension SDS gel lanes (Fig. 4). Thepattern of radioactive newly synthesized protein generated

A Z.

2.0cpm x104

1.6

1.2

0.8

0.4

I1

B100

80

In , %

40

20

100 200 300 400 500

ATA, IM

5 10 15

We also studied the effects of ATA, a well-characterizedinhibitor of initiation. We found that as the ATA concentra-tion increased from 5 to 100 Mm, incorporation fell sharply(Fig. 2A). At ATA concentrations above 100 Mm, incorporationcontinued to fall gradually. Figure 2B shows the time courseof [3H]leucine incorporation in the presence or absence of100 Mm ATA. We observed a significant reduction in totalincorporation by 4 min translation, with 30 to 50% reductionin total radioactivity incorporated by 15 min. We confirmedthis decrease in leucine incorporation by examining the SDS-PAGE profile of the translation products. Figure 3 shows thatalthough the radioactivity comigrating with Rubisco largesubunit only slightly decreased in the presence of ATA,incorporation of radioactivity in the lower mol wt polypep-tides substantially declined when ATA was added. Of theconcentrations employed, 200 Mm ATA gave the greatest ratioof completed large subunits to smaller polypeptide chains(data not shown). However, we preferred 100 Mm ATA inpractice because this concentration eliminates protein synthe-sis in initiation-dependent chloroplast systems (8).

Thme, mln

Figure 2. Effects of the initiation inhibitor ATA. A, Dose sensitivitycurve for ATA. The chloroplast lysate and the freeze-dried transla-tion cocktails were prepared as described in "Materials and Meth-ods." Precalculated amounts of ATA were added to each cocktailbefore freeze drying to yield final concentrations of ATA as follows:0, 0.1, 1, 5, 10, 15, 20, 50, 100, 500 AM. Protein synthesis wasconducted as.described in "Materials and Methods." Synthesis wascarried out for 15 min with no chase period. Samples (equivalentto 1 MAL) were analyzed by liquid scintillation counting of acid-insoluble radioactivity. SDS-PAGE analyses of selected samples arepresented in Figure 3. B, The time course of leucine incorporationin the presence (U) or absence (0) of 100 AM ATA. Data werecombined from four experiments in which the time course ofaccumulation of acid-insoluble radioactivity was examined. Exper-iments utilizing [35S]methionine as well as [3H]leucine are included.The raw data for each experiment were normalized such that theincorporation at 15 min was considered to be 100%. The data fromeach experiment were then placed on a single graph. Chloroplastpreparation and protein synthesis were conducted as described in"Materials and Methods." The range of the normalized data pointsis shown on the graph.

I

276 HUBBS AND ROY

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IN VITRO SYNTHESIS AND ASSEMBLY OF RUBISCO

in the first dimension appears at the top of the gel. Eachpolypeptide can be aligned with a set of polypeptides gen-erated in the second dimension after the partial digestionreaction. By examining the second-dimension digestion frag-ments below each parent polypeptide, one can see that mostof the first-dimension SDS-PAGE polypeptides migratingwith molecular masses between 30 and 55 kD produce onlydigestion fragments that also occur in the digestion patternderived from the completed radioactive large subunit poly-peptide. (The pattern of polypeptides observed on the fluo-rograms after digestion of the completed radioactive largesubunits matched the pattern of stainable large subunit frag-ments produced during the digestion.) This analysis showsthat at least 8 of the 12 most intensely radioactive polypep-tides observed on fluorograms represent incomplete largesubunits. No conclusion concerning the remaining polypep-tides could be drawn from this analysis. However, dataobtained from immunoprecipitation experiments (to be pub-lished elsewhere) verified large subunit identity with all butone of the remaining polypeptides. This polypeptide, whichdoes not appear to be related to Rubisco large subunit, islabeled in Figure 4 with a minus sign, whereas polypeptidesnot identified by the proteolytic procedure, which werenevertheless identified as having large subunit immunorelat-edness, are labeled with a plus sign.

Assembly of in Vitro-Synthesized Large Subunitsinto Rubisco

Chloroplast extracts used in the in vitro protein-synthesiz-ing reaction were tested for incorporation of newly synthe-sized large subunits into Rubisco holoenzyme using the as-sembly assay previously described by this laboratory (4, 22,23). The conditions for assembly in this system are similar tothose used for the assembly of in organello-synthesized largesubunits. Figure 5A shows a fluorogram prepared from anondenaturing gel demonstrating the incorporation of radio-

,MATA 0 10 20 50100

- 66 kDaLS

- 45 kDa

- 29 kDa

ORW 4W-am - 20.1 kDaSs -

-4- 14.2 kDa

Figure 3. The SDS-PAGE profile of translation products in thepresence of ATA. All samples generated by the experiment de-scribed in Figure 2 were analyzed by SDS-PAGE and fluorography.Selected samples (0, 10, 20 50, 100 FM ATA) are depicted here.

L s-.-............... -

SeconddimensionSDS-PAGE(after partialproteolysis)

T.

'f.

First dimension SDS-PAGE

Figure 4. Second-dimension SDS-PAGE analysis after partial pro-teolytic digestion of intact first-dimension SDS gel lanes. Chloroplastpreparation and translation conditions were as described in "Ma-terials and Methods." Translation was carried out for 1 min in theabsence of ATA to improve the ability to observe low-mol wtpolypeptides. Translation was halted with chloramphenicol, andthe reaction was subjected to SDS-PAGE. After separation in thefirst dimension, gel slices were subjected to partial proteolyticanalysis as described in "Materials and Methods." Polypeptides thatcould not be identified by this method but were identified byimmunoprecipitation as having identity with the large subunit arelabeled in this figure with a plus sign. A polypeptide that could notbe identified as large subunit related is marked with a minus sign.The marks above the diagonal were caused by bubbles in thesecond-dimension gel.

activity into Rubisco after synthesis in the presence or absenceof protein-synthesis inhibitors. In this experiment, aliquots ofthe chloroplast lysate were incubated under protein-synthe-sizing conditions for 20 min. Aliquots of these reactions werethen incubated for an additional 90 min in the presence ofsmall subunits and added salts (conditions previously de-scribed as optimal for Rubisco assembly), followed by non-denaturing electrophoresis and fluorography. In the absenceof inhibitors, a strong radioactive signal comigrated withRubisco (Fig. 5A, lane 1). On the other hand, we observedno incorporation of radioactivity into Rubisco after carrying

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Plant Physiol. Vol. 100, 1992

out the synthesis reaction in the presence of RNase (Fig. 5A,lane 2), chloramphenicol (lane 3), or both (lane 4). However,a small amount of radioactivity comigrated with the molec-ular chaperone or Cpn6014 complex when chloramphenicolalone was included in the synthesis reaction (Fig. 5A, lane3). These results are in line with the observation that chlor-amphenicol alone did not completely block protein synthesisin this system. Although the presence of 100 lM ATA reducedthe incorporation of radioactivity into acid-insoluble materialin this experiment by 34%, we found no reduction in theextent of Rubisco assembly (Fig. 5A, lane 5). This proves thatthe incorporation of radioactivity into Rubisco is not due toassociation of the low-mol wt products (whose production issensitive to ATA) with Rubisco. The presence of cyclohexi-mide in the synthesis reaction did not affect the amount ofradioactivity incorporated into Rubisco during the assemblyreaction (Fig. 5A, lane 6). The combined results demonstratethat radioactivity only comigrates with native Rubisco aftersuccessful protein synthesis.We also confirmed that incorporation of radioactivity into

Rubisco depends upon small subunits (Fig. 5B). After 15 minsynthesis, aliquots of the reaction were incubated at 260C for60 min in the absence (lane 1) or presence (lane 2) of smallsubunits under conditions previously identified as optimalfor the assembly of in organello-synthesized large subunits.In the presence of small subunits, a strong Rubisco band was

formed. In the absence of small subunits, we could detect noradioactive Rubisco.To confirm that the radioactivity comigrating with Rubisco

in the nondenaturing gels is in large subunits, we conducteda two-dimensional electrophoretic analysis (nondenaturingfollowed by SDS-PAGE) (Fig. 5C). The results show thatalthough there are a few incomplete polypeptides associatedwith the Cpn6014 complex, the bulk of radioactivity comi-grated in the second SDS dimension with completed largesubunits, whether it was derived from the Cpn6014 complexor from Rubisco.

DISCUSSION

The work reported here demonstrates for the first time thein vitro synthesis and assembly of large subunits into pea

Rubisco holoenzyme. We have described some characteristicsof protein synthesis in this system and noted conditionsrequired for the subsequent assembly of newly synthesizedlarge subunits. We optimized the synthesis reaction to achievethe greatest accumulation of radioactivity and the greatestproportion of completed newly synthesized Rubisco largesubunits. In this way, we hoped to maximize the chance ofobserving the incorporation of large subunits into Rubisco.These results should not be considered a determination ofrate optima. It is important to make this distinction becauseour measurements clearly represent the net result for a set ofprocesses that include synthesis but may also include proc-

essing or even degradation.The addition of ATP and GTP to pea chloroplast extracts

stimulated amino acid incorporation into protein. However,incorporation did take place without externally supplied nu-

cleotides provided there was an ATP regenerating system(data not shown). This indicates that a significant amount of

these nucleotides remained in the extracts after preparationby hypotonic lysis. Although we found that incorporationwas maximal at an ATP concentration near 1 mm, we rou-tinely added only 0.4 mm ATP. This concentration gavegreater than 90% of the maximal incorporation, and we feltthat using the lower ATP concentration might help to avoidproblems from ATP-dependent proteolytic pathways (20, 21).The dependence of this system upon added magnesium

agrees with reports of magnesium dependence for other invitro protein-synthesizing systems (2, 3, 8). Unlike manyothers,'however, we prepared our lysates without addedmagnesium. Thus, we might expect that enzyme systems thatare associated with magnesium in intact chloroplasts wouldgradually lose magnesium to the solvent before lysis (27).However, we have observed routinely that nascent proteinchains (presumably initiated in vivo) can be completed uponsupplementing these lysates with magnesium. Additionally,we observed no loss of translational activity after incubatingthe lysate at 0°C for 30 min or more, and inclusion ofmagnesium in the lysis buffer did not increase incorporation.Therefore, we conclude that any loss of magnesium fromribosomes is negligible, provided the extracts remain at lowtemperatures.The drift in the magnesium optimum across a 2-fold range

of lysate concentration was not significant. However, if weassume that the drift in the threshold concentration of addedmagnesium required for protein synthesis is a consequenceof the reduction in magnesium concentration caused by di-lution of the lysate, then we can estimate the basal contri-bution of magnesium in a typical lysate. The data in Figure1 suggest that the magnesium concentration in a lysate withA260 = 109 is 1.96 ± 0.03 mm. This is 7- to 10-fold moredilute than reported magnesium concentrations in intact il-luminated chloroplasts (16) and probably reflects magnesiumcontributed by the chloroplast stroma and any residual mag-nesium from the resuspension buffer. The response of thissystem to externally supplied magnesium is similar to thatpreviously observed for amino acid incorporation in brokenchloroplasts in the dark (5).Although we examined incorporation with respect to sev-

eral added salts, it is difficult to assign the resulting changesin incorporation to any specific stimulatory or inhibitoryeffects. We speculate that we are measuring a combinationof effects that may include stimulation by potassium andinhibition by chloride, and inhibition by high ionic strength.This information is useful because many investigators intro-duce these ions routinely into the system. The addition ofcalcium severely inhibits the accumulation of radioactivityinto acid-insoluble material. Addition of EGTA can preventthese effects, and other divalent ions did not display suchdramatic inhibitory effects (data not shown).We demonstrated that the incorporation of both [3H]leucine

and [35S]methionine into acid-insoluble material was the re-sult of mRNA-directed protein synthesis (Table I). Puromycinand RNase A each blocked incorporation of radioactiveamino acids into acid-insoluble material. Additionally, chlor-amphenicol reduced incorporation by greater than 80%,whereas cycloheximide did not greatly reduce incorporation.This indicates that protein synthesis was taking place on 70Schloroplast ribosomes and that contamination by cytoplas-

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IN VITRO SYNTHESIS AND ASSEMBLY OF RUBISCO

A 1 2 3 4 5 6 B SS

CpnRuBisCO - _p

Cpn *..IRuBisCO - is

CRuBisCO (Cpn 60)14 LS

66 _ Cpn 6045- *LS36 -29

0 to 20.1

66 A, =>~~~Cpn 60... ..

0 to20.1-:mas>Figure 5. Assembly of large subunits into Rubisco. A, Fluorogram prepared from a nondenaturing gel demonstrating the incorporation ofradioactivity into Rubisco under a variety of conditions. Protein synthesis reactions were performed as described in 'Materials and Methods"except that inhibitors of protein synthesis were included in some reactions. Additions to each sample were as follows: lane 1, no addition;lane 2, 20 Mg/mL of RNase A; lane 3, 200 AM chloramphenicol; lane 4, 100 AM chloramphenicol + 20 Mg/mL of RNase A; lane 5, 100,AM ATA;and lane 6, 100 MM cycloheximide. Protein synthesis was conducted for 20 min, and each reaction was assayed for incorporation of newlysynthesized large subunits into Rubisco using the standard assembly assay previously used to test for the assembly of in organello-synthesizedlarge subunits (9). The chloroplast lysate was diluted 10-fold with lysis buffer (10 mm Tris, pH 7.8, 5 mm DTT, 25 Ag/mL of creatinephosphokinase, 160 units/mL of HPRI) in the presence of 220 mm potassium chloride. All samples were held at 26"C in the presence ofadded small subunits at an approximate concentration of 1 ,M for 90 min. Each reaction was then loaded onto nondenaturing gel lanes andanalyzed as described in "Materials and Methods." Cpn, the position of migration of the stainable chaperonin 60 oligomer. B, The effect ofsmall subunit addition on incorporation of radioactivity into Rubisco. Protein synthesis was carried out for 15 min as described in "Materialsand Methods." The protein synthesis reaction was then assayed for the incorporation of newly synthesized large subunits into Rubisco in theabsence (-) or presence (+) of externally supplied small subunits. Small subunits used for this purpose were isolated from purified Rubiscoas described in "Materials and Methods." The final concentration of added small subunits was not determined for this experiment but isestimated to be between 0.1 and 0.5 AM as determined in similar experiments. C, Two-dimensional analysis of assembled Rubisco. Usingprocedures similar to those described in A and B above, a sample was subjected to nondenaturing electrophoresis. The strip of gel containingthis material was then soaked in SDS cocktail and electrophoresed in a second SDS slab gel. The gel was stained (upper) and fluorographed(lower). Rubisco and the Cpn6014 complex positions are marked in the nondenaturing dimension, the positions of the two subunits (a, ,) ofthe Cpn6O complex and the large subunit of Rubisco (LS) are shown in the SDS dimension. In the nondenaturing dimension, there is sometrailing of Rubisco holoenzyme, resulting in staining at the large subunit position between the peak of Rubisco and the chaperonin peak.This is a not-infrequent phenomenon apparently caused by Rubisco precipitation during the electrophoresis period and does not interferewith the analysis. In the SDS dimension, the small subunits of Rubisco comigrate with the dye front, due to the modified cross-linking of thegel in this experiment that was designed to resolve the two subunits of the Cpn6O complex. There are also some radioactive polypeptidesthat comigrate with the dye front.

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Plant Physiol. Vol. 100, 1992

mic ribosomes was not a significant source of incorporatedradioactivity.More than 50% of the incorporated radioactivity comi-

grated with Rubisco large subunits under a wide range ofconditions. However, additional radioactive polypeptides mi-grating more rapidly than large subunits during SDS-PAGEwere clearly visible in all experiments, provided the fluoro-graphic exposures were sufficiently long. These polypeptidesbecame radioactive at very early time points (e.g. 30 s). Thissuggested to us that they had been initiated in vitro. To testthis possibility, we studied the effects of ATA on amino acidincorporation and the distribution of radioactivity into prod-ucts of varying sizes. ATA is a known inhibitor of protein-nucleic acid interaction (15) and is believed to inhibit pref-erentially initiation of translation over elongation (14, 29). Itwas found that ATA increased the relative proportion ofradioactivity associated with completed Rubisco large sub-units compared with other polypeptides. This clearly suggeststhat the low-mol wt polypeptides are derived at least partiallyfrom in vitro-initiated protein chains. The results of compar-isons between reactions carried out in the absence or presenceof 100 Mm ATA indicate that as much as 50% of the radio-activity incorporated in the absence of ATA enters proteinchains that have been initiated in vitro. Furthermore, as muchas 90% of the radioactivity incorporated in vitro can beincorporated into completed Rubisco large subunits wheninitiation is suppressed.We suspected that most of these newly initiated peptides

represented incomplete large subunits, for several reasons.First, these polypeptides became radioactive about as rapidlyas the completed large subunit within the first 30 s of proteinsynthesis (data not shown), but little radioactivity accumu-lated in them when initiation was suppressed (Fig. 3). Duringpulse-chase experiments, the low-mol wt polypeptides de-creased in total radioactivity and large subunit radioactivityincreased, and most of these polypeptides co-sedimentedwith ribosomes in sucrose gradients, indicating that synthesisor release of the chains from the ribosomes was not complete(data not presented); finally, some of these polypeptides wereobserved to comigrate with the high-mol wt form of thechaperonin 60 protein (Cpn6014, or the Rubisco subunit-binding protein), as is true for completed large subunits (Fig.5C). They do not represent degradation products, becausethey do not accumulate in the presence of ATA (Fig. 3). Theyalso do not accumulate during continued synthesis in thepresence of nonradioactive amino acids (data not presented).To test the possibility that these polypeptides represented

incomplete large subunits, we performed partial proteolyticanalysis. Lizardi used this method previously to identify in-complete polypeptides produced during in vitro synthesis ofsilk fibroin (19). The results of our studies indicated that mostof these polypeptides represented incomplete large subunits.This conclusion follows because the digestion fragments gen-erated from most of the lower-mol wt polypeptides corre-spond to fragments generated by digestion of completed largesubunits. However, we also carried out immunoprecipitationexperiments to identify unambiguously the polypeptidesof molecular mass less than 30 kD, because the resolutionof digestion fragments generated from these polypep-tides was suboptimal. The results of the immunoprecipitation

studies, summarized in Figure 4 along with the results of theproteolytic analysis, confirmed the interpretation that thesepolypeptides are structurally related to large subunits. Theseshort polypeptides probably represent large subunit 'pausedtranslation intermediates' that have been described by Mul-let's group (24). Paused translation products also have beenobserved during the in vitro synthesis of silk fibroin (19). Itremains to be seen what the reasons are for the transientaccumulation of these discrete partial Rubisco large subunits.

Finally, we attempted to observe the incorporation of thein vitro-synthesized large subunits into the Rubisco holoen-zyme. The comigration of radioactivity with native Rubiscodoes not result from artifactual labeling of preformed Rubiscobecause no radioactivity comigrated with preformed Rubiscoin the absence of large subunit synthesis. The radioactivematerial comigrating with preformed Rubisco during nonde-naturing gel electrophoresis also comigrated with Rubiscoduring sucrose gradient sedimentation (data not presented).Using two-dimensional, nondenaturing and SDS-PAGEanalysis, we confirmed that the radioactive signal comigratingwith Rubisco in the nondenaturing dimension comigrateswith large subunits in the SDS dimension (Fig. 5C). Moreover,we showed that the appearance of radioactive Rubisco hol-oenzyme w#s completely dependent upon externally suppliedsmall subunits (Fig. 5B). This extends the observation thatsmall subunits stimulate the incorporation of in organello-synthesized latge subunits into Rubisco (25). In experimentsto be described elsewhere, we have observed the incorpora-tion of radioactive small subunits into Rubisco in this system(A. Hubbs, unpublished data). The fact that some assemblyof in organello-synthesized large subunits takes place withoutadded small subunits (22, 23), but little or no assembly of invitro-synthesized large subunits takes place without addedsmall subunits, may reflect the additional period betweendilution of the 'stroma and assembly that is required for invitro translation and assembly. Moreover, the strong depend-ence of Rubisco assembly upon added small subunits sup-ports the idea that the endogenous supply of free smallsubunits is low. This observation agrees with previous reportsthat small subunits are found in very low concentrations inchloroplasts and that they are easily degraded in the absenceof assembly (26). The dependence of holoenzyme labelingupon added small subunits indicates that the appearance ofradioactive Rubisco does not result from exchange of newlysynthesized large subunits into preexisting Rubisco, nor doesit result from nonspecific adhesion of radioactive large sub-units to preexisting Rubisco. Finally, the failure of ATA toinhibit the incorporation of radioactivity into Rubisco holo-enzyme indicates that the incorporation is not due to nascentpolypeptides binding to preformed Rubisco holoenzyme. Thisis also confirmed by the results in Figure 5C. Under theconditions reported here, no in vitro-initiated chains areincorporated into Rubisco.

In summary, we have demonstrated the in vitro completionand small subunit-dependence of assembly of large subunitsinto pea Rubisco. We have also confirmed the occurrence ofpaused translation intermediates of Rubisco large subunits.

LITERATURE CITED

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IN VITRO SYNTHESIS AND ASSEMBLY OF RUBISCO

2. Bard J, Bourque DP, Hildebrand M, Zaitlin D (1985) In vitroexpression of chloroplast genes in lysates of higher Plantchloroplasts. Proc Natl Acad Sci USA 82: 3983-3987

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10. Ellis RJ, Blair GE, Hartley MR (1973) The nature and functionof chloroplast protein synthesis. Biochem Soc Symp 38:137-162

11. Gatenby AA (1988) Synthesis and assembly of bacterial higherplant Rubisco subunits in Escherichia coli. Photosynth Res 17:145-157

12. Gatenby AA, Van Der Vies SM, Bradley D (1985) Assemblyin E. coli of a functional multi-subunit ribulose bisphosphatecarboxylase from a blue-green alga. Nature 314: 617-620

13. Gatenby AA, Van Der Vies SM, Rothstein SJ (1987) Co-expression of both the maize large and wheat small subunitgenes of ribulose-bisphosphate carboxylase in Escherichia coli.Eur J Biochem 168: 227-231

14. Goldstein ES, Reichman ME, Penman S (1974) The regulationof protein synthesis in mammalian cells. VI. Soluble andpolyribosome associated components controlling in vitro poly-peptide initiation in HeLa cells. Proc Natl Acad Sci USA 71:4752-4756

15. Gonzolez G, Haxo RS, Schleich T (1980) Mechanism of actionof polymeric aurintricarboxylic acid, a potent inhibitor ofprotein-nucleic acid interactions. Biochemistry 19: 4299-4303

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Barber, ed, The Intact Chloroplast. Elsevier, Amsterdam,p 156

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18. Laemmli UK (1970) Cleavage of structural proteins during as-sembly of the head of bacteriophage T4. Nature 227: 680-685

19. Lizardi PM (1979) Genetic polymorphism of silk fibroin studiedby two-dimensional translation pause fingerprints. Cell 18:581-589

20. Liu X, Jagendorf AT (1984) ATP-dependent proteolysis in peachloroplasts. FEBS 166: 248-252

21. Malek L, Bogorad L, Ayers AR, Goldberg AL (1984) Newlysynthesized proteins are degraded by an ATP-stimulated pro-teolytic process in isolated pea chloroplasts. FEBS 166:253-257

22. Milos P, Bloom M, Roy H (1985) Methods for assembly ofribulose bisphosphate carboxylase. Plant Mol Biol Rep 3:33-42

23. Milos P, Roy H (1984) ATP-released large subunits participatein the assembly of RuBP carboxylase. J Cell Biochem 24:153-162

24. Mullet JE, Klein RR, Grossman AR (1986) Optimization ofprotein synthesis in isolated higher plant chloroplasts: identi-fication of paused translation intermediates. Eur J Biochem155: 331-338

25. Roy H, Chaudhari P. Cannon S (1988) Incorporation of largesubunits into ribulose bisphosphate carboxylase in chloroplastextracts: influence of added small subunits and of conditionsduring synthesis. Plant Physiol 86: 44-49

26. Schmidt GW, Mishkind ML (1983) Rapid degradation of un-assembled ribulose 1,5-bisphosphate carboxylase small sub-units in chloroplasts. Proc Natl Acad Sci USA 80: 2632-2636

27. Sheird B, Miall SH, Peacocke AR, Walker IO, Richards RE(1967) Proton magnetic relaxation studies of the binding ofmanganese ions to Escherichia coli ribosomes. J Mol Biol 28:389-402

28. Sommerville CR (1986) Future prospects for genetic manipula-tion of Rubisco. Phil Trans R Soc Lond B 313: 459-469

29. Stewart ML, Grollman AP, HuangM (1971) Aurintricarboxylicacid: inhibitor of initiation of protein synthesis. Proc Natl AcadSci USA 68: 97-101

30. Tabita FR, Small CL (1985) Expression and assembly of activecyanobacterial ribulose-1,5-bisphosphate carboxylase/oxy-genase in Escherichia coli containing stoichiometric amountsof large and small subunits. Proc Natl Acad Sci USA 82:6100-6103

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