In vitro synthesis of the large subunit of ribulose diphosphate carboxylase on 70 S ribosomes

ARCHIVES

OF BIOCHEMISTRY

AND

174, 216-225

BIOPHYSICS

(1976)

fn Vitro Synthesis of the Large Subunit of Ribulose Carboxylase on 70 S Ribosomes’ RUTH

Departments Davis,

Diphosphate

ALSCHER,’ M. A. SMITH,” L. W. PETERSEN,4 R. C. HUFFAKER,4 AND R. S. CRIDDLE’

of 2Biochemistry and Biophysics California 95616, and “Graduate

and -?4gronomy and Range Science, Section of Biochemistry, Brigham Prouo, Utah 84501

Received

September

University of California, Young University,

30, 1975

Polyribosomes isolated from greening barley leaves were active in directing protein synthesis, using soluble components isolated from Escherichia coli. A peptide of 55,000 molecular weight was a major product of translation activity. This peptide was precipitated by antibody to ribulose 1,5-diphosphate carboxylase (RuDPCase), and comigrated with the large subunit of RuDPCase on sodium dodecyl sulfate-polyacrylamide gels. Cyanogen bromide peptides of the peptide of 55,000 molecular weight also corresponded to the peptides prepared from authentic RuDPCase large subunit. The peptides synthesized were shown by sucrose density gradient sedimentation to be largely associated with 70 S ribosomes.

Complete machinery for protein synthesis in chloroplasts has been known to exist for some time (l), and isolated chloroplasts are capable of protein synthesis. The large subunit of RuDPCase” has been identified as one of the products of amino acid incorporation in isolated pea chloroplasts (2). Hartley et al. (3) recently found that an in vitro heterologous system, containing phenol-extracted RNA from pea chloroplasts and Escherichia coli ribosomes and cofactors, incorporated labeled amino acid into the large subunit of RuDPCase. Except for our preliminary report (4), no published account exists of in vitro synthesis of a particular characterized chloroplast protein on 70 S polyribosomes from plants. However, 70 S ribosomes from chloroplast preparations are active in polyphenylala-

nine synthesis in the presence of polyuridylate (5, 6). Polyribosomes isolated from wheat chloroplasts direct amino acid incorporation into hot-acid-insoluble products (7). Evidence is accumulating that supports the concept that chloroplasts are involved in RuDPCase synthesis. Criddle et al. (8) demonstrated that both chloramphenicol and cycloheximide inhibited the production of RuDPCase in greening barley leaves. The selective inhibition of cytoplasmic and chloroplastic protein synthesis is consistent with the view that the larger of the two constituent subunits of RuDPCase (molecular weight 55,000 (9)) is synthesized on 70 S ribosomes, while synthesis of the smaller (17,000 M,) subunit is completed on 80 S cytoplasmic ribosomes. This preliminary conclusion is supported by several more recent studies in both intact plants and isolated chloroplasts (2, 10, II). fn uivo studies relying on protein synthesis inhibitors for determination of sites of protein synthesis have major interpretational difficulties due to both lack of inhibitor specificity and to incomplete inhibi-

I Supported in part by Grant Nos. GM10017 and lF03GM53192 from the USPHS and a grant from the Donald F. Jones Research Foundation. i Abbreviations used: RuDPCase, ribulose 1,5-diphosphate carboxylase; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; HEPES, N-2-hydroxyethyl piperazine-N’-2 sulfonic acid; PVP-40, polyvinylpyrollidone-40. 216 Copyright

D 1976 by Academic

All

of reproduction

rights

in any

Press, Inc. form

reserved.

IN

VITRO

SYNTHESIS

OF

tion (12, 13). In an in vivo study, chloramphenicol at concentrations as high as 100 pug/ml did not completely inhibit translation on 70 S ribosomes (14). The present study documents those difficulties further in demonstrating the inability of the common inhibitors of cytoplasmic protein synthesis to quantitatively block incorporation of 1‘*C]amino acids on 80 S ribosomes using an in vitro protein-synthesizing system from wheat germ. The above difficulties have been overcome in part by using isolated chloroplasts. Blair and Ellis, for example, found that the large subunit of RuDPCase was one of the products of light-dependent protein synthesis in pea chloroplasts (2). No synthesis of the small subunit was detected. While those results support the conclusion that the large subunit is made on chloroplast ribosomes, a major problem exists in attempting to prepare chloroplasts free from significant amounts of associated 80 S ribosomes. This clouds the interpretation somewhat and, again, requires reliance on results from inhibition of prot.ein synthesis. Roy et al. (15) and Gooding et al. (16) circumvented this problem by examining antibody precipitates as peptidyl-1”Hlpuromycins released from 70 and 80 S ribosomes. Their experiments demonstrated the in vim association of the large subunit with 70 S ribosomes and the small subunit with 80 S ribosomes. To obtain specific in, vitro synthesis of proteins on 70 S ribosomes, advantage can be taken of similarities between organelle and procaryotic ribosomes (17-20), especially the demonstrated ability of organelle ribosomes to utilize bacterial peptide chain initiation and elongation factors (3, 21-25). To analyze in vitro translation products, polyribosomes isolated from greening barley leaves were used in combination with a protein-synthesizing system from E. coli MRE 600 (26). The synthetic products from this heterologous combination have as a major component a peptide identifiable as the large subunit of RuDPCase, and were shown by sucrose density-gradient sedimentation to be largely associated with 70 S ribosomes, presumably of chloroplast origin.

CARBOXYLASE

217

SUBUNIT

MATERIALS

AND

METHODS

Barley plants (Hordeurn vulgare L. var. mar”) were grown in the dark at 80°C and relative humidity for 7 days. Plants were transferred to light (40,000 Ix) for 6 h prior to vest.

“Nu55% then har-

Isolation of Polyrihosomes Seven-day-old dark-grown barley plants were illuminated for 6 h under conditions described previously (27). The terminal 10 cm of the leaves were excised, weighed, sliced with a sharp razor blade, and frozen in a stainless-steel container surrounded by a jacket of dry ice-acetone. The frozen leaves were crumbled and transferred to a precooled mortar packed in dry ice, and ground to a finely divided powder. This was transferred to a Waring Blendor and homogenized for 30 s in 2 ml of 20 mM Tris-HCl buffer, pH 8.5, per gram of tissue, containing 50 mM magnesium acetate, 20 mM potassium chloride, 10 mM dithiothreitol, and heparin at 100 @g/ml. The homogenate was filtered through four layers of cheesecloth and centrifuged at 35,000g for 30 min to remove cell debris. The supernatant phase was filtered through Kimwipes to remove a lipid layer at the surface. The filtrate was then layered over 5 ml of 1.2 M sucrose, in the original grinding buffer, and centrifuged in a Beckman SW27 rotor for 3 h at 27,000 rpm. Following centrifugation, the supernatant fraction was removed and discarded. Centrifuge tubes containing the polyribosome pellets were drained upside down for a few minutes, and the sides above the pellet were wiped dry with Kimwipes. The ribosomal pellets were resuspended in water or stored at 4°C for up to 3 days before use. in Vifro Protein Synfhesis CA) What grrnl-barley polyribosome system. Procedures described by Marcus and his co-workers 128, 29) and Klein ut al. (30) were used for extraction and preparation of soluble factors from wheat germ. Reaction mixtures (0.5 ml) contained 50 rnM Tris-acetatc, pH 8.0. 4.4 mM dithiothreitol, 2 ITEM ATP, 0.05 mM GTP, 8.6 mM creatine phosphate (disodium salt), 20 pg creatine phosphokinase, 24.8 mM KCl, 2.84 rnM Mg acetate, 6 x 10 ii mM of each of the 20 common amino acids, 2 FCi of ( ‘%jamino acid mixture” (algal hydrolysate), 80 ~1 of wheat germ high” New England Nuclear NEC-445, Lot No. 71% 066. L-[V-‘%]Amino acid mixture (in millicuries per millimole): L-alanine, 129; L-arginine, 256; n-aspartate, 172; L-glutamate, 215; glycine, 93; t-histidine, 258; L-isoleucine, 258; L-leucine, 258; L-lysine, 258; Lphenylalanine, 414; L-proline, 210; L-serine, 129; Lthreonine, 172; L-tyrosine, 387; L-valine, 215.

218

ALSCHER

speed supernatant, and 20 A,,,, units of barley polyribosomes. Incubation was at 30°C for 30 min. Reactions were terminated by addition of 0.04 ml 55% trichloroacetic acid. An additional 3 ml of 5% trichloroacetic acid containing unlabeled amino acids was added, and the resulting precipitates were washed with 5% trichloroacetic acid, collected by centrifugation, resuspended, heated for 20 min at 9O”C, cooled over ice, and collected on type-HA Millipore filters. Filters were subsequently washed, respectively, with 5% trichloroacetic acid, 90% ethanol, and 95% ethanol:ether (l:l), air-dried, and then counted, using toluene-base scintillation fluid. (Bi Heterologous E. coli-barley polyribosome system. The reaction mixtures contained 50 mM Trisacetate, pH 8.0, 100 mM NH,Cl, 16 rnM MgCI,, 1 mM dithiothreitol, 15.5 mM phosphoenolpyruvic acid, 2 mM ATP, 0.5 mM GTP, 0.1 mM of each of the twenty common amino acids, 2 FCi l’4Clamino acid mixture,” 0.2 mM folinic acid, 1 mgiml E. coli B tRNA, 30 ~1 of S-100 (supernatant proteins from E. coli MRE-600, Strain C6, NClB 9270, NCTC 8165) (26, 31), and 20 A,,;<, units of barley polyribosomes. The total volume of the reaction mixture was 0.2 ml. Reactions were carried out at 30°C for varying times. After the incubation period, reaction mixtures were: (a) assayed for incorporation of L’4C]amino acids into hot-trichloroacetic-acid-insoluble material; (b) assayed for incorporation of label into RuDPCase protein by precipitation with a specific antibody; (c) treated with RNAse T, and layered on to sucrose density gradients; or (d) prepared for SDS-urea polyacrylamide gel electrophoresis. (a) Trichloroacetic acid: Reactions were terminated by addition of 55% trichloroacetic acid, and the precipitate was washed, heated, cooled, filtered, and counted as described under (A) above. (b) Assay for RuDPCase specific-antibody-precipitable protein: Preparation of anti-RuDPCase was as described previously (32). Reaction mixtures were centrifuged briefly as described above to remove rapidly sedimenting material, and 50 A of RuDPCase-specific antiserum or control nonimmune serum was added to the reaction mixture. After 15 min at 37”C, carrier RuDPCase was added, and incubation was continued for 1 to 2 h. Samples were stored at 5°C overnight, and precipitates were collected, washed, heated, filtered, and counted as described previously (27, 33). (c) Sucrose density gradients: Reaction mixtures were treated with RNAse T,’ at 1 pg/ml for 20 min at YC, followed by the addition of heparin at 75 kg/ml to inhibit further hydrolysis, clarified in a clinical centrifuge, layered onto exponential 10 to 34% sucrose gradients in 10 mM Tris-HCl, 15 mM KCl, and 10 mM Mg acetate, pH 7.5, and centrifuged ’ RNAse Worthington

T,, 300,000 Biochemical

units/mg; Corp.

obtained

from

ET

AL.

for Q’/z h in a Beckman SW27 rotor at 27,000 rpm. After centrifugation, gradients were monitored at 260 nm and the radioactivity of selected fractions was determined in Aquasol scintillation fluid, using a Beckman LS scintillation counter. (d) Polyacrylamide gel electrophoresis was carried out by the methods of Weber and Osborn (34) as modified by Neville et al. (35). Gels were prepared from solutions containing 8 M urea and 0.1% SDS. Coomassie blue (0.25% in 7% acetic acid) was used to stain protein bands in certain reference gels containing authentic markers. When simultaneous location of radioactivity and protein was required, gels were soaked in 5% TCA and the position of opaque precipitin bands was determined. These gels were subsequently cut in slices, dissolved in 10% hydrogen peroxide at 6o”C, and counted in a scintillation counter using Aquasol scintillation fluid. Preparation

of :
RuDPCase

Subunits

The top 10 cm of 7-day-old etiolated barley seedlings were placed in a 1”Hlamino acid mixture (New England Nuclear)” and illuminated for 6 h as described previously (8, 27). “H-labeled RuDPCase was isolated as described earlier (27). Sodium dodecyl sulfate was added to a final concentration of 1% and the subunits of RuDPCase were separated by chromatography on Sephadex G-100 by the procedure of Rutner and Lane (9). Analysis of Cyanogen Bromide Fragments of RuDPCase The product of a 30-min bacterial-plant l’4C]amino acid incorporating system was precipitated with anti-RuDPCase antibody and collected as described above. :‘H-labeled large subunits of RuDPCase were added to the precipitate, and the mixture was hydrolyzed with cyanogen bromide in 70% formic acid (36). The hydrolysis products were lyophilized, suspended in I% sodium dodecyl sulfate, and separated by electrophoresis on 15% polyacrylamide gels in urea-SDS using the buffer system of Neville et al. (35). Gels were sliced into 6-mm sections, dissolved in H,O,, as above, and counted in a scintillation counter to determine “H and lqC in each section. Similar gels were run with cyanogen bromide fragments of unlabeled large subunits of RuDPCase. These gels were stained with Coomassie blue as above, for comparison with the labeled fragments. * New England Nuclear NET-250, Lot No. 640010. L-amino acid “HCG mixture (in millicuries per millimole): L-alanine, 1.113; t-arginine, 26.4; L-aspartate, 26.0; L-glutamate, 0.161; glycine, 11.1; Lhistidine, 2.44; L-isoleucine, 1.25; L-leucine, 36.6; Llysine. 3.0; L-phenylalanine, 15.7; L-proline, 4.8; Lserine, 2.23; L-threonine, 1.82; L-tyrosine, 43.8; Lvaline, 2.14.

IN Isolation

VITRO

SYNTHESIS

OF

CARBOXYLASE

SUBUNIT

219

of Chloroplasts

Barley plants were grown 6 days in dark and then exposed to light for 8 h. Leaf tissue was chopped with multiple rotating blades, using an apparatus designed by D. Branton of Harvard University, in 0.05 M HEPES buffer, pH 7.2, containing 0.33 M sorbitol, 0.01 M Ca(NO:,12, 0.0005 M MnCl,, 0.01 M MgCl,, 0.0005 M K,HPOI, and bovine serum albumin at I mg/ml. Three milliliters of grinding buffer were used per gram of tissue. The homogenate was filtered through eight layers of cheesecloth and then centrifuged at 400g for 5 min at 4°C in an International centrifuge. The supernatant fraction was then sedimented at 2lOOg for 5 min through a 75ml layer of 3% polyvinylpyrrolidone-40 (PVP-40) in grinding buffer, and onto a 50-ml pad of 4% polyacrylamide gel at pH 7.6. The sedimented material was washed from the surface of the gel, suspended in 15% of the original grinding buffer, and recentrifuged at 2lOOg for 7 min over a 50-ml layer of 3% PVP-40 in grinding buffer, pH 7.6, and a 50-ml polyacrylamide gel (4%) pad, pH 7.6. The chloroplast pellet was resuspended in 1.5% of the original grinding volume, and ribosomes were prepared as above.

FIG. 1. Rate of 1“C lamino acid incorporation into hot trichloroacetic acid-precipitable material by the heterologous E. coli-barley synthesizing system. The reactions were run at 30°C and contained 1 mgi ml E. coli B tRNA, 30 ~1 of S-100 supernatant proteins from E. coli MRE-600, and 2OA,,,, units of barley polyribosomes in addition to the other components listed in Materials and Methods. Total volume was 0.2 ml. Reactions were carried out in the presence of 16 mM Mg” to optimize yields 140, 41).

precipitation with anti-RuDPCase (Table I). About 40% of the total trichloroacetic We have shown in previous work that acid-precipitable radioactivity was precipiradioactive amino acids are rapidly incortated by anti-RuDPCase. Treatment with porated in uiuo into the large and small nonimmune control serum demonstrated the specificity of the antibody reaction, subunits of RuDPCase in greening barley since only 510% of the trichloroacetic acid leaves (8, 33). Incorporations were linear for several hours and were simultaneously counts were precipitated. accompanied by a major increase in size Polyacrylamide gel electrophoresis was used to further characterize the antibody distribution and total amount of radioactive polyribosomes. This suggested the precipitation products. Following amino acid incorporation and precipitation with possibility of isolating polyribosomes, some of which may contain RuDPCase trichloroacetic acid or antibody, urea-SDS large subunit messenger, for use in in ui- was added to solubilize and dissociate the various components. Four different samtro amino acid incorporation studies. It was reasoned that specificity for 70 S-asso- ples were electrophoresed simultaneously: ciated amino acid incorporation might be (a) trichloroacetic acid precipitate; (b) antigreatly enhanced by using barley polyriboRuDPCase precipitate; (c) authentic purisomes and bacterial peptide chain initiafied RuDPCase stained with Coomassie blue; and (d) supernatant, following pretion and elongation factors (21-25). Figure 1 shows the time course of a typical in vitro cipitation by anti-RuDPCase. Following electrophoresis, the distribution of radioamino acid incorporation, using polyribosomes isolated during maximum Ru- activity among successive slices was deterDPCase synthesis in uiuo and E. coli su- mined (Fig. 2). Several peaks of radioactivpernatant fraction. Reaction rates were ity were obtained. The tot.al activity in usually linear for about 20 min and yielded each peak varied with individual reaction 4000-6000 cpm. mixtures and different polyribosome prepSeveral criteria were used for establisharations. The trichloracetic acid-precipitaing that RuDPCase large subunit was syn- ble material gave a rather complex distrithesized in the heterologous in vitro sys- bution of radioactivity, as did the supernatem. One of these involved the specific tant following antibody precipitation. In RESULTS

220

ALSCHER TABLE OF I’C~AMINO

INCORPORATION

RuDPCASE-PRECIPITABLE Experiment

1

-

2 3

Antibody ___ Trichloroacetic acid precipitate -_____ 6909 8120

5517

precipitation AB precipitate

2779 -

-

I ACIDS

INTO

ANTI-

PROTEIN” of / “Clprotein _____ NonimAB premune cipitate precipifollowtate ing nonimmune precipitation ---__ 697

478 432

ET

AL.

sponded to the R, of small subunit. Such a peak is clearly seen in supernatant fractions obtained from in viva experiments (33). The reason for the absence of small subunit of RuDPCase is the lack of 80 Sassociated translation, as described later. The similarity of Fig. 2A to the sum of Fig. 2B plus 2D is striking evidence of antiRuDPCase specificity. !A) Tr~cnloroacet~c

Ac,d

Ppt

3150 2025

‘I Reactions were carried out as described in Fig. 1. Following the 30-min incubation period, an excess of unlabeled amino acids were added to the reaction mixtures, which were then cooled on ice and centrifuged for 5 min in a clinical centrifuge. Fifty microliters of RuDPCase specific antiserum or nonimmune serum was added, and the mixture was incubated for 15 min at 37°C. After addition of carrier XuDPCase, incubation was continued for 1 to 2 h. Samples were stored at 5°C overnight, and resulting precipitates were collected and counted as previously described. In the case of nonimmune serum, the remaining supernatant was reincubated with immune serum. Further treatment of the antibodyantigen precipitates is described in Materials and Methods. Reaction mixtures precipitated with trichloroacetic acid were as described in Materials and Methods. AB, antibody.

the trichloroacetic acid-precipitable material, a major peak of radioactivity had an R, identical with that of RuDPCase large subunit, while the antibody supernatant following anti-RuDPCase precipitation (D) did not. A single major peak of radioactivity was obtained from the anti-RuDPCase precipitate, and this peak also had an R,,, identical with that of authentic large subunit of RuDPCase. The skewing of this single major peak of radioactivity toward lower molecular weights is expected because of the presence of significant amounts of incomplete large subunit polypeptide chains. This is in contrast to results from in. uivo studies where anti-RuDPCase was added after polyribosome removal, giving a much more symmetrical peak (33). Another striking difference was the obvious absence of a radioactive peak in the anti-RuDPCase precipitate from the heterologous in vitro system which corre-

600

(B)Antl-RbDPcase

I r

I

Ppt

I Front j

Large Subunit 400

iDi Antibody

I

25

] (Cl

Small Subunit

5

75

Sup

IO

D~sfonce fromTopicm1

FIG. 2. SDS-urea gel electrophoresis profiles of anti-RuDPCase-precipitable material obtained from in vitro incorporation of [14Clamino acids by E. coli supernatant and barley polyribosomes. Following electrophoresis, gels were cut into 6-mm sections, dissolved in 10% H,O, at 60°C for 4 h, and counted in Aquasol scintillation fluid. (A) Distribution of radioactivity in 5% trichloroacetic acid precipitate. (B) Distribution of radioactivity of anti-RuDPCase precipitate. Carrier RuDPCase was added to the reaction mixture to aid antibody precipitation. (C) Distribution of Coomassie blue staining components from anti-RuDPCase precipitate of part (B). Carrier RuDPCase is the predominant staining component and serves as an internal marker far comparison with R,n of in vitro synthesis products. (D) Distribution of radioactivity in supernatant fraction following removal of anti-RuDPCase-precipitable components.

IN

VITRO

SYNTHESIS

OF

Products of the heterologous in vitro protein-synthesizing system were compared with the in viva synthesis of large subunit in barley leaves by cyanogen bromide cleavage of proteins containing methionine. Antibody-precipitable ‘“C-labeled protein from the heterologous system was combined with the large subunit of RuDPCase synthesized in viuo in barley leaves and labeled with tritium. The large subunit was separated from the small subunit by gel filtration and the peak fractions massed (8, 33). A mixture of large subunit and antibody precipitate was dissolved in 70% formic acid, hydrolyzed with cyanogen bromide, and then lyophilized. Lyophilate was solubilized in 1% SDS and peptide fragments separated by electrophoresis on 15% acrylamide gels (Fig. 3). The distribution of 14Cradioactivity from the in vitro amino acid incorporation corresponds closely to the distribution of “Hlabeled peptides from authentic large subunit of RuDPCase, and also with the staining of cyanogen bromide peptides obtained from authentic RuDPCase large subunit. Variation in ratios of “H and ‘“C radioactivity in these peaks was attributed to differences in the specific activities of amino acids in the preparations used for the labeling experiments (see footnotes 6 and 8). Having established that the RuDPCase large subunit was synthesized in vitro in the heterologous barley E. coli proteinsynthesizing system, it was important to

CARBOXYLASE

221

SUBUNIT

determine whether synthesis was carried out on 70 S-associated polyribosomes. Considerable evidence has implicated such polyribosomes in this synthesis (2, 8, 10, 11, 16, 37). The effect of various inhibitors of protein synthesis on the incorporation of ]14C]amino acids is shown in Table II. Cycloheximide and emitine had little, if any, effect on the incorporation of [ “Clamino acids into acid-insoluble material, while chloramphenicol was very inhibitory (73%). This strongly suggests specific stimulation of 70 S-ribosome-associated protein synthesis in the heterologous system. This is consistent with our failure to observe TABLE EFFECT AND

OF CHLORAMPHENICOL, EMITINE ON THE

[ “CIAMINO INSOLUBLE

ACIDS MATERIAL BARLEY

Additions

None Chloramphenicol

II CYCLOHEXIMIDE, INCORPORATION OF

INTO TRICHLOROACETIC BY E. COLI SUPERNATANT POLYRIBOSOMES”

Trichloroacetic acid precipitation of 1’ ‘Clprotein Incorporation (cpm) .~ ~ .-~ 6957 (100 gg/ 1878

ACIDAND

Inhibition (% I

~~ 73

ml) Cycloheximide (50 pglml) Emitine (50 pgiml)

6748 6470

” Reactions were carried out for 30 min using about 20 A,,;,, units of polyribosomes co/i supernatant, as described in Fig. 1.

3 7 at 30°C and E.

FIG. 3. Electrophoretic fingerprint ofcyanogen-bromide-treated mixture of 1’“Clamino acid, anti-RuDPCase-precipitable incorporation products (-0-j and :‘H authentic (in viva labeled) RuDPCase large subunit (-0-). Hydrolysis products were separated on 15% poiyacrylamide gels in urea-SDS. Data points are taken from scintillation counts of successive 2-mm sections. Further details in Materials and Methods.

222

ALSCHER

RuDPCase small subunit, as described above, which is presumably synthesized on 80 S-associated ribosomes. The level of inhibition by chloramphenicol and cycloheximide varied considerably in repeated experiments (36-73 and 3-40%, respectively). This has also been observed by other workers (12-14). Table III shows the effect of chloramphenicol and cycloheximide on the synthesis of anti-RuDPCase precipitable protein. In general, 35-40% of the total trichloroacetic acid-precipitable radioactivity was precipitated by anti-RuDPCase. Chloramphenicol significantly inhibited incorporation into trichloroacetic acid and antibodyprecipitable material, while further addition of cycloheximide had little effect, if any. Since the major component of antibody-precipitable material was RuDPCase large subunit, and since chloramphenicol had a major inhibitory effect on its synthesis, 70 S-associated ribosomes were Iikely involved in the synthesis of large subunit

ET AL TABLE

III

EFFECT OF CHLORAMPHENICOL AND CYCLOHEXIMIDE ON THE INCORPORATION OF [14C]A~r~~ ACIDS INTO ANTI-RuDPCASE-PRECIPITABLE PROTEIN” Additions

None Chloramphenicol Chloramphenicol cycloheximide

Antibody CAB) precipitation of [‘dC]protein Trichloroacetic acid precipitate

AB precipitate

Nonimmune precipitate

6909 2517 2700

2779 1154 941

697 380 -

+

” Experiments were carried out as described in Table I and Fig. 1, except for the addition of inhibitors. 71

in vitro.

More direct qualitative evidence consistent with these findings was obtained by analysis of nascent protein products of the heterologous protein-synthesizing system by sucrose gradient centrifugation. Incorporation reaction mixtures containing [‘4C]amino acids were treated with RNAse T, and centrifuged for 9liz h to separate 70 and 80 S monosomes and associated labeled nascent peptides from unincorporated amino acids, which remained largely at the top of the gradient (Fig. 4). Clearly, most of the nascent proteins were associated with the 70 S fraction. The 80 S monosomes showed very little radioactivity, if any. Assuming a Gaussian distribution of radioactivity, and subtracting counts associated with unincorporated amino acids which tail down from the top of the gradient, approximately 80% (5000 cpm) of the acid-precipitable counts trichloracetic (6337) are accounted for under the 70 S monosome peak. This provides convincing evidence that 70 S ribosomes were involved in the synthesis of RuDPCase large subunit. In contrast, when polyribosomes isolated from greening barley leaves were combined with the wheat germ protein

Fraction

Number

FIG. 4. Sucrose gradient velocity sedimentation profile of leaf polyribosomes following [‘“Clamino acid incorporation in the heterologous protein-synthesizing system. Following a 30-min reaction, RNAse T, was added to reduce the polyribosome preparation to predominantly monosomes, and sedimentation was carried out in a 10 to 34% sucrose gradient for 91/z h. A,,,, and radioactivity were determined as described previously. E. coli ribosomes were used to determine the position of 70 S material.

synthesizing system and analyzed similarly almost all of the radioactivity was 80 S-associated. This is consistent with results of others. The possibility of using purified chloroplast ribosomes in the wheat germ and E. coli protein-synthesizing systems was considered. Although chloroplast purification resulted in polyribosome preparations highly enriched in 70 S monosomes, these were always contaminated with significant amounts of 80 S ribosomes (Fig. 5).

IN

VITRO

FIG. 5. Sucrose gradient leaf, and purified chloroplast centrifugation was for 4 h.

SYNTHESIS

OF

CARBOXYLASE

sedimentation profiles extracts. Sedimentation

Chloroplast purification resulted in depolymerization of associated polyribosomes, even in the presence of heparin. In addition, low chloroplast and subsequent ribosome yields made this an unrealistic approach for identifying specific 70 S translation products. Shown for comparison is the distribution of total polyribosomes isolated from greening barley leaves. This distribution varied with growth conditions and time of light exposure. Thus, the heterologous E. coli-barley protein system was superior to other techniques and provided an analytical tool for studying 70 S translation products from mixed populations of 70 S- and 80 S-associated polyribosomes.

of polyribosomal was carried

223

SUBUNIT

preparations out as in Fig.

from whole 5 except that

anti-RuDPCase-precipitable acids into large subunit protein. Detailed analysis of the products of this protein-synthesizing system showed that precipitation by anti-RuDPCase accounted for nearly half of the trichloroacetic acidprecipitable counts. Electrophoretic analysis of the antibody precipitate yielded a single major peak migrating with an R,, identical to that of RuDPCase large subunit. Furthermore, cyanogen bromide fingerprints of “H-authentic large subunit and the [ 14Clanti-RuDPCase-precipitable counts from the in vitro system were identical. Differences in the ratios of “H and ‘“C counts in various fractions from those fingerprints may be attributable to differences in specific activities of amino acids DISCUSSION in each of the two radioactive preparaA major contribution of this work is the tions.“, x specific synthesis in vitro of 70 S translaHaving demonstrated that the in vitro tion products from mixed populations of 70 system is active in the synthesis of Ruand 80 S ribosomes. The difficulties inherDPCase large subunit, it was important to ent in the use of “specific” inhibitors of show whether the E. coli translation sysprotein synthesis, or the direct isolation of tem specifically catalyzes protein synthehighly polymerized polyribosomes from sis on 70 S-associated ribosomes in mixed chloroplasts in sufficient yields, were over- populations of 70 and 80 S ribosomes. Sevcome by using a total barley polyribosome eral workers have reported that the E. coli preparation in combination with E. coli protein-synthesizing system is active with supernatant. This protein-synthesizing chloroplast and mitochondrial ribosomes system provides a technique for the pre- but much less so with the 80 S class of dominant incorporation of [ “Clamino ribosomes (20, 25). Our results are consist-

224

ALSCHER

ent with those conclusions since the products of the heterologous system were predominantly associated with 70 S ribosomes. Furthermore, chloramphenicol significantly inhibited incorporation of [‘4Clamino acids into such products. One might argue that a small fraction of the 80 S ribosomes remained extremely active and were turning over at a rate sufficient to account for all the synthesis of RuDPCase large subunit. However, that would also require rapid release and accumulation of the products in the supernatant fraction. Since the majority (about 80%) of the radioactivity incorporated into protein (as indicated by counts in acid-insoluble products) remained associated with the 70 S monosomal peak at the end of the incubation period (Fig. 4), that interpretation is not consistent with our results. Furthermore, much evidence has accumulated implicating 80 S ribosomes in the synthesis of the small subunit (8, 11, 14, 16). It is significant that we did not observe RuDPCase small subunit synthesis in the heterologous system using polyribosomes isolated from greening barley leaves during a period of linear synthesis in. L&O of both RuDPCase small and large subunits (33). The involvement of chloroplastic 70 S and cytoplasmic 80 S ribosomes in RuDPCase synthesis is consistent with genetic indications of nuclear control over the direction of synthesis of this enzyme (38), and with polypeptide compositions of RuDPCase from parasexually hybridized plants (37). Our results demonstrated that only a small fraction of the proteins produced during in vitro synthesis were released from the polyribosomes. The reasons for this are not clear; however, similar results were noted by other workers when studying in vitro synthesis on the eukaryotic type ribosomes of plants (39). Although the possibility of translation of RuDPCase large subunit on 70 S mitochondrial ribosomes has not been ruled out, there is no good evidence tending to support that interpretation, while much evidence is available supporting the synthesis of large subunits by isolated chloroplasts (2) and by mRNA isolated from pea chloroplasts (3).

ET

AL.

It appears clear from these studies that messenger RNA for RuDPCase large subunit is directly associated with 70 S chloroplast ribosomes. It is not likely that this messenger also may be translated on 80 S ribosomes, but that remains to be determined. Further studies also will be necessary to prove whether synthesis of mRNA for large subunit of RuDPCase is directed by chloroplast DNA, nuclear DNA, or both. It appears that in tobacco, the large subunits of the protein are coded by chloroplast DNA, whereas the small subunits are coded by nuclear DNA (37). REFERENCES 1. KIRK, J. T. 0. (1971)Ann. Reu. Biochem. 40, 161. 2. BLAIR, G. E., AND ELLIS, R. J. (1973) Biochim. Biophys. Acta 319, 223. 3. HARTLEY, M. R., WHEELER, A., AND ELLIS, R. J. (1975) J. Mol. Bid. 91, 67. 4. ALSCHER, R., SMITH, M. A., PETERSON, L. W., HUFFAKER, R. C., AND CUDDLE, R. S. (1974) Plant Physiol., Suppl., 8. J. L., AND WILDMAN, S. G. (1970) 5. CHEN, Biochim. Biophys. Acta 209, 207. N., TUCKER, E. B., 6. JONES, B. L., NAGABHASHAN, AND ZALIK, S. (1973) Canad. J. Biochem. 51, 686. 7. HADZIYEV, D., AND ZALIK, S. (1970) Biochem. J. 116, 111. 8. GRIDDLE, R. S., DAU, B., KLEINKOPF, G. E., AND HUFFAKER, R. C. (1970) Biochem. Biophys. Res. Common. 41, 621. 9. RUTNER, A. C., AND LANE, M. D. (1967) Biochem. Biophys. Res. Commun. 28, 531. 10. BAMJI, M. S., AND JAGENDORF, A. T. (1966)Pkznt Physiol. 41, 764. 11. ELLIS, R. J. (1973) Comment. Pl. Sci. 4, 29. 12. LARK, K. G., AND LARK, C. (1966) J. Mol. Biol. 20, 9. 13. LEVINE, A. J., AND SINSHEIMER, R. L. (1968) J. Mol. Biol. 32, 567. 14. VAMBUTAS, V. K., AND SALTON, M. R. J. (1970) Biochim. Biophys. Acta 203, 83. 15. ROY, H., GOODING, L. R., AND JAGENDORF, A. T. (1973) Arch. Biochem. Biophys. 159, 312. 16. GOODING. L. R.. ROY, H., AND JAGENDORF, A. T. (1973) Arch. Biochem. Biophys. 159, 324. 17. STUTZ, E., AND NOLL, H. (1967) Proc. Nat. Acad. Sci. USA 57, 774. 18. HOOBER, J. K., AND BLOBEL, G. (1969) J. Mol. Biol. 41, 121. 19. LOENING, U. E., AND INGLE, J. (1967) Nature (Londonl 215, 363. 20. BOARDMAN, N. K., FRANCKI, R. I. B., AND WILD MAN, k-3. G. (1966) J. Mol. Biol. 17, 470. 21. SWANSON, R. F. (1973) Biochemistry 12, 2142.

IN

VITRO

SYNTHESIS

OF

22. GRIVELL, L. A., AND GROOT, G. S. P. (1972)FEBS Lett. %, 21. 23. SALA, F., AND KUNTZEL, H. (1970) EUF. J. Biothem. 15, 280. 24. EISENSTADT, J. M., AND BRAWERMAN, G. (1966) Biochemistry 5, 2777. 25. RICHTER, D., AND LIPMANN, F. (1970) Biochemistry 9, 5065. 26. WADE, H. E., AND ROBINSON, H. K. (1966) Biothem. J. 101, 467. 27. KLEINKOPF, G. E., HUFFAKER, R. C., AND MATHESON, A. (1970) Plant Physiol. 46, 416. 28. MARCUS, A., LUGINBILL, B., AND FEELEY, J. (1968) Proc. Nat. Acad. Sci. USA 59, 1243. 29. MARCUS, A. (1970) J. Biol. Chem. 245, 955. 30. KLEIN, W. H., NOLAN, C., LAZAR, J. M., AND CLARK, JR., J. M. (1972) Biochemistq 11, 2009. 31. GOLD, L. M., AND SCHWEIGER, M. (1969) hoc. Nat. Acad. Sci. USA 62, 892. 32. KLEINKOPF, G. E., HUFFAKER, R. C., AND MATHESON, A. (1970) Plant Physiol. 46, 204.

CARBOXYLASE

SUBUNIT

225

33. SMITH, M. A., CRIDDLE, R. S., PETERSON, L. W., AND HUFFAKER, R. C. (1974) Arch. Biochem. Biophys. 165, 494. 34. WEBER, K., AND OSBORN, M. (1969) J. B&l. Chem. 244, 4406. 35. NEVILLE, D. M., JR. (1971) J. Biol. Chem. 246, 6328. 36. STEERS, JR., E., CRAVEN, G. R., ANFINSEN, C. B., AND BETHUNE, J. L. (1965) J. Biol. Chem. 240, 2478. 37. KUNG, S. D., GRAY, J. C., WILDMAN, S. G., AND CARLSON, P. S. (1975) Sciences 187, 353. 38. KAWASHIMA, N., AND WILDMAN, S. G. (1972) Biochim. Biophys. Acta 262, 42. 39. DAVIES, J. W., AND SAMUEL, C. E. (1975) Biothem. Biophys. Res. Commun. 65, 788. 40. AVADHANI, N. G., AND BUETOW, D. E. (1972) Biochem. J. 128, 3.53. 41. CHUA, N-. H., BLOBEL, G., AND SIEKEVITZ, P. (1973) J. Cell Biol. 57. 798.