in vitro protein synthesis plastids vulgaris formation ... · in vitro protein synthesis by...

Plant Physiol. (1968) 43, 504-514

In Vitro Protein Synthesis by Plastids of Phaseolus vulgarisIII. Formation of Lamellar and Soluble

Chloroplast Protein" 2

Maurice M. Margulies and Francesco Parenti3Radiation Biology Laboratory, Smithsonian Institution, Washington, D.C. 20560

Received November 9, 1967.

Abstract. Chloroplasts from leaves of plants which had been grown in the dark, and thenilluminated for 12 hours were isolated, and allowed to incorporate 14C-leucine into protein,and the products of this incorporation were studied. Lamellar and soluble proteins are theprincipal p'roducts, and are formed in about equal amounts. Only some of the soluble proteinsbecome heavily labeled. Those with highest specific activity have a molecular weight of theorder of 140,000, while the higher molecular weight Fraction I protein has a much lowerspecific activity. The soluble protein as a whole does not serve as a precursor for thelamellar protein, and vice-versa, al,though a precursor-product relationship between a minorcomponent of the soluble fraction and the lamellar fraction has not been ruled out. Therelative protein synthesizing oapabilities of chloroplasts and mitochondria are discussed withreference to the data presented.

It has been reported that chloroplasts have thematerials necessary for genetic autonomy. Theycontain ribosormes which would be necessary toconvert the information in their DNA into proteins(8). A previous report from this laboratoryshowed that ch'loroplasts, in the crude chi4oroplastpreparation tunder investigation, were responsiblefor the amino acid incorporation into protein thatwas observed in vitro (17). Amino acid incor-poration into protein by chilorolplasts ha's been re-ported from a number of laboratories, as reviewedin the first publication in this series (17). Thepresent work reports further studies of the systemdescribed previously, and was carried o-ut to findout the nature of the prodtucts formed.

Materials and Methods

Plant Material. Chloroplasts of Phaseolus vul-garis L. var. Black Valentine were obtained fromprimary leaves as already described (17). Leaveswere obtained froim plants grown in the dark for6 days, and then illuminated with white light for0.5 day.

1 Research was supported in part by funds providedby the United States Atomic Energy Commission Con-tract No. AT(30-1)2373.

2 Published with the approval of the Secretary of theSmithsonian Institution.

3 The work reported in this com,munication was car-ried out while Dr. Parenti was holder of a National Re-search Council Visiting Research Associateship at theSmithsonian In,stitution. Present address: Departmentof Biochemi'stry, Yale University, New Haven, Con-iiecticut.

504

Incorporation Assay. Incorporation of uni-formly labeled 14C-L-leucine was carried out at achloroplast concentration which contained 16 to50 jug ch'lo'rophylll/ml of reaction mixture. One ,ugchlorophyll is equivalent to 10 to 20 pg protein.Within the range u'sed, incor,poration is a linearfunction of chloroplast concentration. Incorpora-tion was carried out in the high osmotic strengthHonda medium to which 0.25 volume of other re-agent solutionis was added (17,23), except wherestated. The contribution of bacteria to incorpo,ra-tion was evaluated only occasionally wi-th the aidof the nonionic detergent Tri;ton X-100 (17). Tri-ton scolubi'lizes chloroplasts without affeceting bac-teria, making it possible to separate chl-oroplastcon-tents from bacteria by differential centriftuga-tion. It had been found previously, in experimen'tsconducted over a period of a year, that bacteria inthe chioroplast preparation contained less than 10 %of the radioactivity incorporated into protein. Spotchecks carried ou't during the course o'f the experi-ments presented here confirmed this o'bserva!tion.

14C-4deucine was obtained from New EnglandNuclear Corporation, Boston, Massachusetts, andhad a specific radioactivity of about 250 mc/mmole.Pyrtivate kina'se, type II from muscle (E.C. 2.7.1.40),and phosphoenolpyruvate, sodi.um salt, were ob,tainedfrom Sigma Chemical Corporation, St. Lquis.Miss-oturi.

Radioactivity incorporated into protein was de-termined in a proportional counter after sampleshad been extracted with hot trich'loroacetic acid,an'd col,lected on Mililipore filters (17). However,in the experiment presented in figture 5, radio-activity in hot trichloroacetic acid insol,tible materialwas determined by scinitillation couniting (15).

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MARGULIES AND PARENTI-CHLOROPLAST PROTEIN SYNTHESIS 505

FIG. 1. Electron micrograph of lamellar fraction. X 49.000. See 'Methiods for details. Arrows indicate granacross sections.

Insert



MARGULIES AND PARENTI-CHLOROPLAST PROTEIN SYNTHESIS

Chlorophyll was determined spectrophotometrically,and protein of the chloroplas!t preparations was

determined by Kjeldahl digestion and nesslerization(17).

Preparation of "Lamnellar" Material Labeledwith 14C-Leucine for Analysis -in Density Gradients.Chloroplasts were incubated for 60 minutes with14C-letucine, as described above, at which time tun-labeled letucine was added, and the reaction mixtutrechilled to 00. The reaction mixture was then cen-

triftuged at 6000 X g X 20 minuites. The peliletwas resuspended in a small vo,lume o,f Honda solu-tion, and layered on a density gradient, as describedbelow, -or the pellet was washed once or twice inHonda soflution, by resuspension in a voilume equalt,o thait of the reaction mixture, fo.llowed bv cen-

trifugation at 6000 X g X 20 minutes, and thenlayered on a density gradient.A stepwise gradient was prepared by layering

solutions of 0.05 M phosphate (pH 7.0) which con-

tained different amounits of stucrose, or in the easeof the highest density solu,tion used, sucrose and sor-

bose. The densities and voilumes of the solutionsu-sed to prepare the gradient were: p = 1.320, 3.0 ml;p = 1.305, 3.0 ml; p = 1.282, 3.0 ml; p = 1.260,3.0 ml; p - 1.230, 3.0 mil; p = 1.215, 4.0 ml;p = 1.170, 4.0 ml; p- 1.155, 4.0 ml. The gradientwas allowed to smooth for abo-ut 8 houtrs, and then3.0 ml of a suspension of labeled chloroplast lameliarmaterial in Honda meditum (p = 1.05) was layeredon top. The gradients were centrifuged at 25,000RPM for 1 hour in a Spinco SW 25.1 rotor. Thetulbes were punctured ait the level of the interfaceabove the densest layer (whicth had been markedwhen the gradients were prepared), and 1 ml sam-

ples were collected. These were dilul!ted to 2.0 mlwith water, and samples taken for determination ofincorporated activity, and chlorophyll.

Preparationt of "Soluible" Proteint Labeled wzeith'4C-Leucine for Analysis int Dentsity Gradients.Chiloroplasts were prepared as described in thepreceding section, except that plastds were sus-

penided in Honda meditum from which Ficoll anddextran were omitted, and the s,ucrose concentrationwas raised to 0.5 Mi (suicrose mediulm). Thesesubstitutions were made to avoid the need to remove

Fico'll and dextran before concentrating the proteincontainting chloroplast extract. The rates of incor-poration of plastids suispended in sutcrose mediuimwere less than the rates of plastids suispended inHonda mediutm. After 60 minutes incuibation, un-

labeled leuicine was added to the reaction mixture,and it was chilled to 00. The reaction mixtture was

then centrifuged at 30,000 X g X 30 minuites, andthe suipernatant liquid carefully decanted. Thepellet was resuspended in a vo,lume of 1 mM GSHthat was one-fifth that of the originall incorporationreaction mixtutre, and incubated at 0° for 30 mintiteswith occasional stirring. It was then centrifug-edat 30,000 X g X 30 minuites, and the suipernatanitliquid was decanted, and comibined with the first.

This extract was dialyzed for about 12 hours against100 volumes of 10 % carbowax 6000 (polyethyleneglycol, Union Carbide Corporation, New York, NewYork) in 50 mm potassium matleate, 1 mM GSH(,pH 7.0). The dialysis served to concentrate theextraot, and to remove enough of the sucrose in itso that the dia!lyzed extract coulid be floated on5 % stucrose.

This soluble protein preparation was analyzedon a linear density gradient consisting of 50 mMpotassium maleate, 1 mm GSH (pH 7.0) (24), inwhich the stucrose concentration was varied from5 to 20 %. The volume of 'the gradient was 4.5 ml.Five-tenths ml oif soluble protein (0.2-0.4 % pro-tein) was layered on it, and the gradient centrifugedat 40,000 rpm for 6.0 hours in a Spinco SW 39rotor. Ttubes were puncttured at the bottom, andtheir contents were collected in 15 drop fractions.This procedure yielded 27 fractions per gradient.Any residue in the bottom of the tube was resus-pended in water. The fractions were analyzed forradioactivity present in protein, and for protein.The latiter was determined by a modified Folinprocedure (13), using 1 cm path length microcellsand a Rodder microcell positioner (MicrotechServices Co., Lo;s Altos, Cailifornia). Fraction Iprotein from spinach (14) was used as a referencematerial in replicate density gradien(t centrifugations.

Preparation of Chloroplast Structural Protein.Chloroplast structural protein was prepared by theprocedure of Criddle (5). After chIloroplasits hadbeen incubated for 1 hotur with labeled letucine, asusual, the reaction mixture was centrifuged at5000 X g X 30 minutes. The pellet was resus-pended in an equal voluime of water, foillowed bycentrifuigation at 30,000 X g X 30 minutes. Thewashed peltlet was resuspended in a volume of wa,terhalf thalt of the original reaction mixture, and wasthen sonicalted with cooling on ice for 4 15-secondperiods at 2 to 3 minuiite intervals. For this pro-cedture, a Branson 75 W machine was operated at50 % of fulll power using a 0.5 inch horn. Theresulting suspension was centrifuged at 1000 X g X10 minutes, ancd the pellet discarded. The super-natanit was then centrifuged at 140,000 X g X 10minutes, and the pellet was resuspended in one-qtuarter the original reaction mixture volume ofwater, and stored frozen. Unla'beled 140,000 X gpellet, which was uised as carrier in subsequentsteps, was prepared a!s above, except that the chloro-plast suspension was in-cubaited withotut the additionof reagents used to carry out incorporation. One-quarter volume of water was added to the chloro-plast stuspension instead. After determination ofradioactivity in protein, anid protein, the labeledand unlabeled preparations were pooled, and struc-tuiral protein prepared from it using sodiuim dodecylsulfate, and urea as solubilizing agents (5). Radio-activity in protein was determ,ined as tusual, and thequantity of protein by Kjeldah,l digestion andnesslerization ( 17).

507



I5LANT PHYSIOLOGY

IEetron M-icr."coAy. Chiloroplasts were incu-bated in Hondia medium plus 0.25 voltume o,f w&-terfor 1 hour a,t 250. They were then collected bycentrifugation at 6000 X g X 0.5 hour, and washedonce by resuspension in water anid recentrifugationat 30,000 X g X 0.5 hour. This pelilet was resuis-pended in a smalll volume of Hondia medium whichlacked mercaptoethanoll, and conftained phosphateinstead of tris. Then the suspension was fixed in2 % gluttaraldehyde, postfixed in 1 % OS04, em-bedded, and examined as ailready described (16).

Results

When the reaction mixture was fractionated bydifferentiail cenitrisfugation at the enid of 1 hour ofincorporation, the results presented in table I wereobtalined. Forty-two percent of radiloactivity in-conporated inito protein was sedimentted alt 6000 X gX 20 mintutes. Only 11 % of radioactivity in pro-tein was sedimented at forces above 6000 X g. Ofthis fraction, only 4 % was sedimented above30,000 X g. The remaining 50 % of radioactivityin protein is found in the supernatant liiquiid from140,000 X g X 60 minutes centrifugation. A por-tion of the radioadtive protein in the 6000 X gpe]Adt was not removed by washing. After 1 wash-ing, the radioactive protein in the 6000 X g pelfletremains essentialtly constant (table II). This resultwas obtained when the washing solution was Hondamedium, or a much lower osmotic strength buffersolution.

'Once washeod 6000 X g pelilet consists largelyof chlioroplast memibranes (ifig 1). Cross sectionsof Stacks of grana in which the thylakoid compart-ments are swollen are clearly recognizable, andocctr frequently. Tangenti,al sectiions of granumthylakoids are allso clearly recognizable. Stromacontents cannot be dlearily distinguished. In con-trast, freshly prepared ch'loroplasits, which aare usedin these stu'dies, are largely intact, containing boththe internal membrane system anid stroma contents(16). During the incorporation incubation periodchl'oroplia9ts lose their stroma conitenits. Thesecohloroplasts appear to differ from washed 6000 X g

Table I. Distributtionl of RadioactiVity Incorporated inito

Table II. Partial Removal of Radioactivity in Proteit,of 6000 X g Pellet Fraction by Repeated WashingsChloroplasts were incubated 1 hour,- a*id replicate

samples taken. To some trich(loroacetic aci.d was addedimmediately. Others were chilled to 00, and centrifugedfor 20 minutes at 6000 X g. Supernatant fluids weredecanted and trichloroacetic acid was added to them, andalso to the residues which were first resuspended inwater, or the residues were resuspended in washingmedium. The latter were centrifuged at 30,000 X g X30 minu.tes, and the supernatant liquid was pooled withthe 6000 X g supernatant liquid. After the appropriatenumber of washings, trichloroacetic acid was added tothe pellets which had been resuspended in water, andto the pooled washings. The isotonic medium wasHonda solution (23). The hypotonic medium contained10 mM tris-H'CI (pH 7.8); 10 mM MgC12; and 4 mmmercaptoethanol. There were 458 cpm in the unfrac-tionated reaction mixture in the experiment with iso-tonic medium and 392 cpm in the experiment with hypo-tonic medium.

Fraction

Amount of total incorporated radio-activity remaining in pellet after

washing with

Isotonic medium Hypotonic medium

Total incorporation6000 X g pelletPellet, 1 X wash,edPellet, 2 X washedPellet, 3 X washed

100644136

10064423535

pellet in that starch grains are sti!ll contained withinthe chiloroplast menrbrane system (16). The pres-ence of radioa,ctive protein in a preparation whichmainly conXssists of chlioroplast lamelilae ;suggests thatleucine was incorporated in the protein of theselamell.ae.

Additional experimenits confirm this conclusion.When 6000 X g pellet is washed twice and examinedin a den,sity gradient, the chlorophyll sedimenXtsforming 1 peak. The prin,cipa;l peak of radioactiveprotein is almost isuperimposable on the peaqk ofchlorophyll (Ifig 2C). With unwashed, or oncewashed 6000 X g pdllet, although a peak of radio-active protein is present in the same region as the

Chloroplast Proteini .Among Centr-itfugal F1actioIIs ofJlixtutre

At the end of 1 hour inicubation the 12 ml of reaction mixture was centrifuged at 6000 X g X 20 minutes,antd the supernatant liquid was decanted, and centrifuged at the next higher cenitrifugal force. Each pellet was re-susspended in 12 ml of water, and replicate samples of resuspended pellets, anid the final stipernatant liquid weretaken for determiniationi of radioactivity in protein. Dispersion is standard de-iation for 3 replicate samples.

Fraction

Total6000 X g X 20 miii pellet10,OOX g X 30 min pellet30000 X g X 30 nmin pellet140,000 X g X 60 min pellet140,000 X g X 60 mimi supernatant

CPM/fraction

790037405301804343960

4-

-4-

340224101618

360

% Of total

10042

450

508



MARGliLIES AND PARENTI-CHLOROPLAST PROTEIN SYNTHESIS

C

a

Fa

b-

a

-C0'C

-

2400

1800

C

0

1200 0l-

E

600

0

509

500

375

250

125c

0

0

E0

Traction ( density -) Fraction (density-)

FiC. 2. Density gradient centrifugationi of lamellar fraction prepared from labeled chloroplasts. A) Unwashed.-B) Washebd once with Honda medium. C) Washed twice with Honda medium. Solid circles represent the radio--activity in protein of the fractions. Opetn circles represent the chlorophyll contents of the fractions.

-peak of chlorophyll, the curves are consistently not

sWperimposable in the lighter density regions of the:gradient. The discrepancy is greatest with theiunwashed material. Here, the ratio off radioPactiveprotein to chlorophyll is much higher than in theregion of greatest chlorophyll concentration. Sincethis does not occur with pellets thaft have beenwashed twice, it is proba-bly due to removal ofra4ioactive protein from the chlorophyil containingparticles in the suspended pellet, during resuspen-sion, or gradient centrifugaition, or both.

Chloropla,st structtural protein (5) was preparedfrom 6000 X g pellet and was found to be radio-

active. The specific radioactivity of the structuralprotein equalled that of the protein of the chloro-pl,asts from which it was prepared (table III).

The results presented above strongly suggestthat the system tunder study is making protein whichis incorporated inito the lamellar structure of thechloroplast. However, the possibility exists thatincorporation into the 6000 X g fraction representsformation of incomplete protein chains bound toribosomes or other ribonucleoprotein attached to thelamellae, and these serve as precursors of stromaproteins. Evidence presented below indicates thatradioaotive leucine in the hot trichloroacetic acid

Table III. Associatioii of Iiicorporated Radioactivity weith Chloropl(ast Strulcttlrall ProteintDetails of procedure are given in 'Methods. Specific radioactivity of the acetone powder, and specific radio-

activity cof structural protein are corrected for dilution of 140,000 X g pellet with unlabeled carrier. A simi'larcorrection is applied to values of jug N! fraction, and cpm in fractions.

CPM/ ,g N!Prepara,tioni stage fraction fraction CPM/,g N

Reactioni mixture after 1 hr incorporation 72,500 2290 326000 X g pellet 48 000 1430 346000 X g pellet, 1 X washed 20,300 765 27140,000 X g X 10 min pellet of soniicatcd

1 X washed 6000 X g pellet 13,200 505 26Sodium dodecv1lsulfate-urea extract of acetonlepowder of 140,000 X g pellet 6600 161 41

Structural protein 2890 82 35



PLANT PHYSIOLOGY

Table IV. Time Independence of Relative Incorpora-tion of Amwino Acid into Lamellar and Soluble

Protein FractionsIncorporation was stopped at the indicated times

by chilling samples rapidly to 00, and unlabeled leucinewas added. The samples were centrifuged at 6000 X g,and the supernatant fluids decanted. Pellets were re-suspended in water, and recentrifuged at 30,000 X g.The resultant supernatant fluid wa's pooled with the first(solluble fraction). The pellet (lamellar fraction) wasresuspended in water, and radioactivity in protein of it,and soluble fraction were determined. Percent radio-activitv in lamellar fraction = (radioactivity in lamellarfraction)/ (radioactivity in lamellar fraction + radio-activity in soluble fraction). Values followed by (a)are the average of 3 experiments, and those followedby (b) are the average of 5.

Amount of tatal incorporatedradioactivity in protein of

Min lamellar fraction

51 + 1 (a)52 7 (a)541 8 (b)46 + 13 (b)49 + 10 (b)

inso,luble portion of 6000 X g fraction is a stableend prodtuct of the incorporation reaction, probablyprotein. First, no change in the proportion ofradioactiv,ity incorporated into protein of the6000 X g fraction as a function of time of incor-poration is observed ('table IV). That is, the ratioof radioactivity in protein of the 6000 X g pellet,to radio,activity in totajl pro,tein, is constant. If theradioactive protein in the 6000 X g pellet fraction

1600 A T

1800 00So U4

0 15 30

MINUTES

45 60

represented precursor of soluble protein, it mightbe expected that the proportion of radioactive pro-tein in this fraction would reach a maximum valuewhich would then decrease with time. Second, theamounit oif radioactivity in protein of soiluble orinsolulble fract ons remains essentially constant afteraddition of unilabeled leucine, even though, in theabsence o;f addition of unilalbeled leucine, inicorpora-tion into each fraction continues (fig 3). For thisexperimenit washed 6000 X g pellet is defined asthe insoluble fraction, and the 6000 X g super-natant pooled with the washing is defined as thesoluble fraction.

Analysis of soluble ch'loroplast protein, preparedas described in Methods, shows that durting incor-poration in vzitro the ch'loroplasts make some solubleproteins in preference to others (fig 4). The ctirverepresenting distribution of protein in the gradientshows 2 distinct peaks. The faster sedimentingpeak is probably Fraction I protein, since a prep-aration of Fraction I protein from spinach (14)has the same location in the density gradienit whenit is ruin in a separate replic'ate gradient. Usingthis protein as reference, an'd assuming a sedimen-tation coefficient of 18 S for it (24), it is calcu-lated that 'the sl.ower sedimeniting peak has a sedi-menitation coefficient of 4 S. The pyruvate kinaseadded to the reaction mixture conitributes only 5 %to the protein in the extracit which is analyzed bydensity gradient centrifugation. Dis9tri'bution ofpro'tein in denslity gradien'ts is the same whetherextracts were prepared from chl'oropl'asts stuspendedin a meditum containing pyruvate kinase or lackingit. The cuirve representing distribtition of incor-porated radioactivity shows a single peak in the

C.

0 15 30 45 60 0 15 30

MINUTES MINUTES

FIG. 3. Effect of addition of unlabeled leucine during amino acid incorporation on the time dependence ofincorporation into lamellar and soluble chloroplast fractions. Radioactivity incorporated into protein: A) total;B) soluble fraction; C) lamellar fraction. Reaction mixture was incubated at 250. After samples were taken at30 minutes, a portion of the reaction mixture was transferred to a vessel containing 1 pmole unlabeled leucine perml of reaction mixture. The undiluted reaction mixture contained 5 m,umole leucine/ml. Incubation was continuedat 250, and samples were taken from the vessel which contained undiluted amino acid (@), and from the vesselto which unlabeled amino acid was added (0). Samples were fractiolnated into lamell3r and soluble fractions asdescribed in table IV.

515304560

510




100

75z0-

c)

zIu-0c-

50

3 e0-2

, o

25 R / \ 50'I~~~~~~~~''0 00 5 10 I5 20 25

FRACTION (density-)

FIG,. 4. Density gradient centrifugation of labeledsoluble proteins from chloroplasts (30,000 X g super-natant. See Methods for details). Protein per frac-tion, *. Radioactivity in protein, 0. Each pointrepresenits an average value for 3 replicate densitygradients.

vicinity of the slower sedimenting protein peak, butsomewhat displaced in the direction of the bottomof the density gradient tube. It has a calcul,atedsedimentation coefficient o,f 7 S. A number ofenzyrmes of the chloroplast photosynthetic carbonreduction cycle have simiilar sedimentation coeffi-cients (5), corresponding to a molecu-lar weight,in the ca-se of spinach chloroplast aldolase, of140,000. Protein amounting to 5 % of the totalapplied to the density gradient, and radioactivityamounting to 10 %, is found sedimented at thebottom of the ttube. This probably represents pro-tein anid radioactivity in menmbrane fragments, andin Trilbosomes, since the protein preparation examinedwas obtained by removal of particles sedimentingat forces of 30,000 X g.

The assymetry of the peak of radioactivity inthe density gradient presenTted in figure 4 suggeststhat some labeled Fraction I protein may be formedduring incorporation, even though no peak of radio-acti'vity is found in the Fraction I region. AInenrichment for Fraction I protein was made bycentrifuging labeled soluble protein at 140,000 X gX 4 hours. When the pelilet obtained was redis-solved and analyzed in a density gradient, the major

protein peak corresponded to Fraction I protein,and there was a clear indication of a peak of radio-active protein associated with it (fig 5).

Discussion

The protein in the 140,000 X q supernatantliquids, obtained from incorporation reaction mix-tures, probably consists largely of chloroplast stromaproteins, and may conta;in some easily solubilizableportions of the internal membrane system, and thespaces this membrane system encloses (18). Thecalculated sedimentation coefficients of 18 S, and4 S, based on a comparison with known Fraction Iprotein, correspond to the sedimentation coefficientsof Fractions I and II proteins (18). Since densitygradient analyses were run with 30,000 X g super-na'tanit, rather than 140,000 X g supernatant liquid,initact ribosomes and ribosome fragments would alsobe presen-t in the soluble protein fraction that wvasanalyzed. However, intact ribosomes wotlld be ex-pected at the bottom of the density gradient tutbe,

40

30

z0

10

< 20

LL-.z

LU

I-0a-

00 5 10 15 20

FRACTION (density-)25

160

120

z0

"I..C.

80 E

40

0

FIG. 5. Density gradient centrifugation of crudeFraction I protein prepared from labeled chloroplastsoluble protein. Protein per fraction, 0. Radioactivityin protein, 0. The data represent the analysis of asingle density gradient. Crude Fraction I protein wasobtained as follows. Thirty thousand X g supernatantwas centrifuged at 140,000 X g X 4 hours. The pelletwas dissolved in 50 mm potassium maleate (pH 7.0)and residual insoluble material removed by centrifuga-tion at 1000 X g.

511



PLANT PHY'SIOLOGY

since they have a sedimentation coefficient of 70 S(2, 3). Chloroplast ribosome subunits of sedimen-ta'tion coefficients 35 S and 50 S are observed atlow concentrations of magnesium (2, 3). ThesesuQbtuniits, if present, would be expected at a positionin -the density gradient reasonably well removedfrom the 18 S Fraction I protein peak, probably aspart of the ribosomal pellet at the bobttolm of thetuibe. For these rea,sons it is felit that the radio-activity in hot trichloroacetic acid precipitaible ma-teriail, which is found throughout the gradient, isin free protein chains, and does not represent pro-tein bound to ribosomes, or ribosomall subunits.

Analysis by density gradient centrifugation ofthe soluible protein fraction extracted from ch'loro-plasits shows that the distribution of radioactivityin protein differs firom the distribution of protein(fig 4). This suggests that on'ly some chlloroplastproteins are being syntthesized by this (system, orthat some are being synthesized more acLtively thanothers. This difference in distPibution of protein,and radioactivity in protein represents the syntheticcapabilities of the ch.loroplasts in vivo at this stageof developmenjt, rather than an artifact resultingfrom chloroplast isolaltion. When leaves, instead ofchloropl'asits, are incubated with radioactive leucine,and chiloroplasts isolated, extract prepared, andanallyzed, it is found that the relaitive distr.butionsof protein and radioactivity in protein are the sameas those presented in figure 4 (unpublished results).Radioactive Fraction I protein, although ma'sked byradioactive 7 S protein (fig 4), is formed in vitro( fig 5). S ince Fraction I protein is primarilyri'bulose-1,5-diP carboxylase (24), an enzyme local-ized in the ohloroplasts (11), the results presentedare regarded as prel:m-nary evidence that chloro-plasts can synthesize this chloroplast protein.

The products formed by the chl'oroplast aminoacid incorporating system being studied differquantitatively from products formed with otherchloroplast systems. Spencer (22) found that in-corporation into soluble protein accounted for 25 %of ithe product formed in his system, -compared tothe 50 %, oir more, found in ithe present work.Spencer (22) ailso found indications that "nascent"protein, probably on ribosomes, accounted for 43 %of the amino acid incorpora'ted into protein. Incontras1t, only 5 % of our radioactive iprotein productis presenit in the pellet sedimenting between 30,000and 140,000 X g, where one would expect ribosomes.Further, the experiments 'presented in figure 3indicate that only a smaill portion of the radioactiveprotein in the lamellar fraction is "naiscent" protein.

The soluble protein is a ilarge fraction of theradioactive protein prodtuct formed by wheat chloro-plasts in viitro (1). The proportion of solubleprotein product of this syistem more neatlly corre-sponds to that of the bean chilloropl'ast system. Withwheait the percent radioactivity in protein in thesoluble protein fraction increases with time, andstggests that the insoltuble component acts as pre-

ctursor of the soluble. This resuilt is not obtainedwith the 'bean chloropl'ast system (table IV). Thiisdifference may be explained by the differences incondiition!s used ito measure incorporation. Withwheat, 'chtloroplasts were suspended in a low ionicstrength medium which probalbly reosulted in break-age of chiloroplasts, and release of ribosomes intothe medium. Thus the wheatt chiloroplast systemcan be con!sidered 'a crude chlorlopla,st ribosomepreparation, in contrast to the bean system in whichchIloropla,sts are initact at the start of incorporation.In contrast to the large fraction of soluabile proteinformed by the wheat and bean systems, the productof amino acid incorporation by ribosomes fromEtuglenat chloroplasts was ailmost en,tirely boutnd tothe rilbosomes (7). Since little radioactivity isincorporated into the ri(bosome fraction, or into"nascent" protein, the bean chloroplast amino acidincorporating system mu,st be completing proteinchains anid releasintg them from ribosomes.

Spencer (22) presented eviden,ce tha't some ofthe protein formed by a 'spinach diloroplast aminoacid incorporating system is incorporated into pro-tein of the insoluble .lamel1lar fraction. Likew'ise,the bean ch'loroplast system is incorporating aminoacid into lamellar protein. First, chlorophyl.l andradioactivity in protein of washed Ilamelilar fractionlsediment together in a density gradient. Thesewashed lamel'lar fractions appear to consist pri-marily of internal chloroplast membranes. No at-tempt was m'ade to rule out the possihtility thatanother particle fraction, lacking chlorophyll, sedi-ments in the same position as the chlorophyll con-taining lamel'lae. However, previous work indicatesthat radioactivity in mitochondria, the most likelycanididate for a contaminant of similar density,contributes onily a smalil portion to the total radio-active protein (17). Seconnd, no more th,an a small.amount of 'radioactivity in the lamellflar fraction isremoved from it when chloroplasts are inculbatedwith unlabeled leucine under conditions necessaryfor incorporation (fig 3). If the radioactivity inthe 'lamel,lar fraction wa,s an intermediate in theformation o'f the radioactive protein fotund in thesoluible fraction, removall of label. f'ro'm the lamellarfraction would be expected uinder these conditions.Third, structurall protein which is radioactive canbe prepared from chloroplasts that have incorpo-rated amino acid. The interpretation of this ex-perimen't depends on what "structural proltein"preparation's represent. They aire prepare'd fromthe insoilutble fractions of mitochondria (6), an!dchIoroplasts (5). II t'he case of the former, thereis some evidence that the preparation consists of asingle protein compoilent (6).

TIhe so'ltible protein fraction, as a whole, doesnoit seem to be aIn intermediate in the formation oflamellar protein, and(l vice-versa. This conclusionis drawn from the observation that no appreciablelabeil is transferred from the 'protein of the soluiblefract'on to the lamellar fraotion, and vice-vzesC.

5 1 2)




Interpretation, however, is scomewhat complicateddue to the fact that the Ilamelilar fraction that was

used was 6000 X g pellet, an,d not 140,000 X gpelilet. However, since only a smalil portion of theradioactivity in protein sediments between 6000 and140,000 X g, it is felt that the difference betweenthese 2 procedures would not have beeni significantfor ithe experiment considered. Among the possi-bilities to be considered for the formation oflamelilar protein are the folilowing. A small fractionof the -soluble protein might act as precursor forthe lamellar protein. Alternatively, ch'loroplastribasomes, or other ribonucleoprotein aittached tothe '1amellae might be the sites of lamePl'ar lproteinformation, in view of the observation that RNA isassociated with -the lamelilar system (10). Stillanother possibility is that rilbosomes in the stromasynthesize lame'll1ar protein, but direct contact be-tween ithe ribosome botund protein chain and thelamelilae is necessary for incorporation into thelamelilar system. This 'possibility is analogous tothe hypothesis ,proposed to explain transfer ofprotein from cytoplasmic microsomes to mitoclhon-dria, which may be involved in the synthesis of thesoluble proteins of the mitochondria, and the pro-teins which are loosely bouind to mitochondrialmembranes (12).

The protein synthesis which is carried otit byintact ch'oroplasts differs from that carried otut bvmito,chondrial systems. Unlike chloroplasts, mito-chondria form only insoluble protein in vitro(19, 20, 21, 25). This observation seems to be ex-

plained by reports which indicate that the sioluble,or readily extractable proteins of the mitochondriaare synthesized by cytoplasmic ribosomes and are

then transferred to the mitochondria (4, 9, 12).This difference may indicate a higher degree ofgeneitic autonomy for *the chloroplast.

Acknowledgment

The authors are indebted to Dr. E. Gantt for thepreparation of the electron micrograph which appearsa!s a figure in this article.

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