Plant Physiol. (1977) 60, 532-537

Synthesis of the Small Subunit of Ribulose-1,5-bisphosphateCarboxylase by Soluble Fraction Polyribosomes of Pea Leaves1

Received for publication December 13, 1976 and in revised form May 23, 1977

HARRY Roy2 AND BARRY TERENNADepartment of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181LORETTA C. CHEONGDepartment of Life Sciences, Polytechnic Institute of New York, Brooklyn, New York 11201

ABSTRACT

The products of amino acid incorporation by pea (Pisum sativumL.) leaf soluble fraction polyribosomes in the wheat germ system wereexamined by two-dimensional electrophoresis and fluorography.

There are two isoelectric variants of the small subunit of ribulosebisphosphate carboxylase in this organism, and the more alkaline ofthese is consistently labeled in the cell-free protein-synthesizing system.The more acid variant is also labeled, but often less extensively. Aminority of leaf polyribosomes are recovered from low speed sediment-able fractions. While these appear also to synthesize the small subunits,the patterns of labeling do not indicate a preferential synthesis of thesepolypeptides by the sedimentable fraction polyribosomes.

In the same experiments, labeling of the large subunit spots wassharply below background; these results confirm a cytoplasmic site ofsynthesis for small subunits of ribulose bisphosphate carboxylase.

Ribulose- 1,5-bisphosphate carboxylase (EC 4.1.1.39) inhigher plants and several algae contains polypeptide subunits ofmol wt about 55,000 ("large") and 13,000 ("small") (13, 17,19).

In isolated pea chloroplasts, a protein with electrophoreticmobility and peptide map characteristic of native large subunitwas labeled with a radioactive amino acid by light and ATP-dependent processes (1); no small subunit labeling was observedin these experiments. More recently, it has been shown thatisolated wheat leaf polyribosomes promote the incorporation of[3H]leucine into a protein with electrophoretic mobility identicalto that of small subunit and into small subunit tryptic peptides(22). Since this incorporation occurs in a completely chloram-phenicol-resistant manner, it follows that cytoplasmic ribosomesare responsible for synthesis of the small subunit.

Earlier immunological data had suggested that ribosomesengaged in synthesizing small subunits might be components ofa low speed sedimentable fraction in homogenates of greeningwheat (9).The present experiments were designed to see whether poly-

ribosomes engaged in small subunit synthesis were preferentiallylocalized in low speed sedimentable fractions of pea leaf homog-enates.

MATERIALS AND METHODSPlants. Pisum sativum L. Progress No. 9 seeds (Agway,

I Supported by National Institutes of Health Grant GM-21670 and23353 to H. R.

2 To whom reprint requests should be sent.

Buffalo, N.Y.) were hydrated overnight and grown in vermicu-lite for 2 weeks at 25 C on a 12-hr day-night cycle. Youngleaves, usually less than 1 cm in diameter, were cut and storedin liquid N2for subsequent polyribosome isolation. Older plants'leaves were used for isolation of marker carboxylase.

Chemicals. Creatine phosphate, creatine phosphokinase,ATP, guanosine-5'-triphosphate, DTT, and amino acids wereobtained from Calbiochem. Ultra-pure sucrose, urea, and am-monium sulfate (Schwarz/Mann) were employed throughout.NP-40 detergent was obtained from Particle Data Laboratories,Elmhurst, Ill. All other chemicals were reagent grade or thehighest purity available.

ISOLATION OF RIBULOSE BISPHOSPHATE CARBOXYLASE

Pea leaves (100 g) were homogenized in 300 ml of 0.05 Mtris-0.02 M ascorbate-7 mm mercaptoethanol (pH 8) at 0 to 4 Cin a Waring Blendor and centrifuged at 30,000g for 10 min.The supernatant was rendered 50% saturated in ammoniumsulfate and centrifuged at 10,000g for 10 min. The resultingpellet was dissolved in 15 ml of 0.05 M tris-HCI (pH 7.6) andapplied to a Bio-Gel A5M column (2.5 x 50 cm) and elutedovernight with the same buffer. The carboxylase peak (thesecond major peak in the A-275 profile) was pooled, precipitatedas before with ammonium sulfate, and dialyzed for 2 hr against200 volumes 0.05 M tris-HCI (pH 7.6), and then applied to a 5to 20% sucrose gradient in the same buffer and centrifuged for17 hr at 120,000g, at 4 C in the SW 27 rotor of a Beckman L2-65 ultracentrifuge. The fastest sedimenting peak in the sucrosegradient profile was repurified on a second, identical sucrosegradient, and the final purified carboxylase was stored as ssupension in 50% saturated ammonium sulfate-25 mm tris-HCl(pH 7.6) at 0 to 4 C. The final preparation was 99% homoge-neous in acrylamide gel electrophoresis under nondenaturingconditions, and contained large and small subunits with minordetectable contaminants in SDS-acrylamide gel electrophoresis(Fig. 1).

ISOLATION OF POLYRIBOSOMES

The methods used are based partly on the procedures ofBlobel and Potter (2) used for isolation of "bound" and "free"ribosomes of rat liver, with buffers and salts altered to preserveplant polyribosome integrity (7).Method I. Twenty g of young pea leaves were removed from

liquid N2 and placed in a prechilled ceramic mortar. When thetemperature of the leaves reached -10 C they were groundvigorously with a hand-held pestle in 200 ml of ice-cold 0.4 Msucrose-0.2 M tris-HCI (pH 8.5)-0.01 M Mg acetate-7 mmmercaptoethanol for about 3 min. The homogenate was thengently forced through two layers of Miracloth and centrifugedat 5,000g for 5 min at 0 C. The supernatant was layered

532

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SMALL SUBUNIT POLYSOMES

01

0 5 10LENGTH, CM

FIG. 1. SDS electrophoresis of purified pea ribulose bisphosphatecarboxylase. Two hundred l.g of enzyme prepared as described under"Materials and Methods" (except that only one sucrose gradient run wasmade), was denatured in SDS and electrophoresed on 12% polyacryl-amide, stained, and scanned on a Gilford spectrophotometer equippedwith a linear gel transporter. Direction of electrophoresis: left to right.

directly on step gradients consisting of 1.5 M sucrose-0.2 Mtris HCI (pH 8.5)-0.02 M KCl-0.01 M Mg acetate-7 mm mer-captoethanol (8 ml) on top of 2 M sucrose in the same buffer (6ml) in a polycarbonate centrifuge bottle, sealed, and centrifugedfor 5 hr at 80,000g in the Beckman 30 rotor. The resultingpellet ("soluble fraction polyribosomes") was dissolved in 0.05M HEPES-KOH (pH 7.6)-0.02 M KCI-0.005 M Mg acetate-7mM mercaptoethanol and stored in liquid N2. The green materialfloating in the sucrose pads above this pellet was diluted 5-foldin 0.2 M tris-HCl (pH 8.5)-0.02 M KCl-0.01 M Mg acetate-7mM mercaptoethanol and centrifuged at 30,000g for 30 min.The pellet was resuspended in 30 ml of the same solutioncontaining 1% Triton X-100. This was applied to a second stepgradient of sucrose and centrifuged for 10 hr at 27,000g toobtain a total sedimentation equivalent to that of the firstsucrose step gradient centrifugation. The resulting pellet ("sedi-mentable fraction polysomes") was dissolved in 0.05 MHEPESKOH (pH 7.6)-0.02 M KCl-5 mm Mg Acetate-0.007M mercaptoethanol and stored in liquid N2. Sucrose gradientsshowed that the soluble fraction ribosomes were >90% poly-somes, while sedimentable fraction ribosomes were >75%polysomes (not shown).Method II. Because of the extra time involved in preparing

sedimentable fraction polyribosomes by method I, we carriedout some experiments with sedimentable fraction polyribosomesisolated from a crude 30,000g pellet of a homogenate preparedas described in method I but without sucrose, so as to facilitaterapid pelleting of membranes. This crude pellet was washed,lysed with 2% NP-40, and centrifuged in parallel with the30,000g supernatant. Sedimentable fraction polyribosomes pre-pared by this method were contaminated with green material.Sucrose gradients (not shown) indicated that both soluble andsedimentable fraction ribosomes were >90% polyribosomes.

AMINO ACID INCORPORATION

The procedures described in (22) were followed, except thata mixture of 15 "4C-amino-acids (Schwarz/Mann) and fiveunlabeled amino acids were employed. Generally five A2,,, ofsoluble fraction polysomes, and as little as one A.,,6, of sediment-able fraction polysomes were incubated per 0.3 ml of totalreaction mixture containing 2.5 mm HEPES- KOH, 0.1 M KCI,S mM Mg acetate, 2 mM DTT, 1 mM ATP, 2 to 4 x 10' Maurintricarboxylic acid, and the amino acids. Incubation wascarried out for 40 min at 25 C, during which the incorporationof "4C into acid-precipitable material as a function of time waslinear.

ANALYSIS OF PRODUCTS OF PROTEIN SYNTHESIS

A modification of the method of O'Farrel (18) was employed.The completed proteins were collected from a 150,000g

supernatant of the reaction mixture, mixed with 30 ,ug Ru-BPCase,: and dried by a stream of N2 or lyophilization, dissolvedin 30 ,tl 9.5 M urea-2.5% NP-40-2% Ampholines (LKB, pH3.5-10)-5% mercaptoethanol. They were then applied to isoe-lectric focusing gels made up as described by O'Farrel containing8 M urea, 2% NP-40, but with pH 3.5 to 10 Ampholinesreplacing the pH 5 to 7 Ampholines. Isoelectric focusing wascarried out overnight until v x hr ¢ 5,000, and the gels werestored with or equilibrated with 10 ml 2.3% SDS-5% mercap-toethanol-0.0541 M tris * H.,SO (pH 6.1)-20% glycerol-0.005% bromophenol blue, for 2.5 hr. The gels were thenapplied to the top of a stacking gel in a Studier-type apparatus(Bob Dillingham, Aqueboque Machine and Repair Shop, LongIsland) using 0.5 ml of molten 1 agarose in 0.1% SDS, 0.0541M tris H2SO4 (pH 6.1) as an adhesive. For many of theexperiments, the gradient slab gel method of Chua and Bennoun(5) was substituted for that described by O'Farrel. The currentwas kept at 10 mamp or less until the tracking dye entered thepolyacrylamide gradient, and then was set at 17.5 mamp untilthe tracker dye was 1 cm from the bottom of the gradient gel.The method of O'Farrel, however, gives comparable results(18) and was used for the experiment in Figure 5.The gel was fixed overnight in 50% ethanol-7% acetic acid to

remove staining Ampholines, and then stained by the methodof Weber and Osborn (24) and photographed. The gel was thensoaked in dimethylsulfoxide and PPO and fluorographed asdescribed by Bonner and Lasky (4).

RESULTS

The data in Table I show the recovery of polyribosomesisolated from soluble and 30,000g sedimentable fractions asdescribed under "Materials and Methods." There are minorvariations in the recovery of sedimentable polyribosomes, andin their apparent integrity, depending on the exact proceduresused: the proportion of recovered polyribosomes obtained fromthe sedimentable fraction varies from 3 to 15% and the propor-tion of these sedimenting faster than 80S varies from 75 to 90%.The data in Table II indicate the extent of incorporation of

various amino acids by several typical polyribosome prepara-tions from both the soluble and sedimentable fractions. In thecase of [35S]methionine, less than one methionine residue wasincorporated/polysomal ribosome, while with a mixture of "4C-amino-acids, from 1.9 to 11.3 amino acids were incorporated/polysomal ribosome. The sedimentable fraction polyribosomesappeared to have a slightly lower over-all activity in some cases,while yielding a higher proportion of released counts than thesoluble polysomes. This apparently higher yield of solublecounts appeared to be due to endogenous synthesis of an

3Abbreviations: RuBPCase: ribulose-1,5-bisphosphate carboxylase.

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ROY, TERENNA, AND CHEONG

Table I Pellets of Leaf Homogenates Centrifuged on Sucrose Pads

Soluble and sedimentable fraction polyribosomes were prepared asdescribed in Method I, except for experiment IV. In experiment IV,sucrose was omitted from the homogenization medium, and the Miracloth-filtered homogenate was centrifuged at 30,000g for 20 min. The pelletwas resuspended, recentrifuged, suspended, lysed in detergent andcentrifuged on sucrose step gradients in parallel with 55% of the first30,000g centrifugation. Although the polysome pellet from the 30,000gsupernatant was colorless, the polysomes from the washed 30,000g pelletwere visibly green. The A260 recovered was therefore determined byintegrating under polysome profiles of sucrose gradients of the twopreparations. The data reflect this correction for experiment IV, andthe correction for the fact that only 557. of the 30,000g supernatantwould fit in the rotor. In all other experiments, A260 was determineddirectly from aliquots of redissolved polysomes after clarification bya 5000g centrifugation. In control experiments 907. of all solublefraction ribosomes sedimented faster than 8OS; sedimentable fractionribosomes prepared as described in experiment IV were of equivalentintegrity, while those prepared by Method I were 25% monosomes, 75%polysomes.

Fraction of 1st Step Total %SolubleGradient Used Polysomal A260 Fraction Polysomes

I Pellet 1.00 90 92Green Band about 0.90 6.8

II Pellet 1.00 34 97Green Band about 0.90 1.2

III Pellet 1.00 11.6 89Green Band about 0.90 1.4

Fraction of 30,OOOgCentrifugation Used

IV Supernatant 0.55 76 85Pellet 1.00 13

Table II Incorporation of Amino Acids by Soluble and SedimentableFraction Polysomes

Polyribosomes were prepared as described in Method I, except forexperiment I, in which polyribosomes were prepared by Method II. Aminoacid incorporation was as described in Materials and Methods, except that35S-methionine (3.3 Ci/_ole), was used in experiment I. The % releasedcpm reflects acid insoluble material left in the 150,000g supernatantof the reaction mixture. The number of amino acids incorporated perpolysomal ribosome was calculated from the average specific radioactivityof the amino acids, uncorrected for isotope dilution by endogenous aminoacids, a value og 13 A260/mg/ml ribosomes, an assumed ribosomal molecularweight of 4 x 10 , and counting efficiencies of 100% and 50% for 35S and14C respectively. ND means not determined.

gradient gels was high (5), there were so many low mol wtspecies present that small subunits could not be resolved.

In order to resolve small subunit from among all of thelabeled proteins in the low mol wt region, we employed two-dimensional electrophoresis essentially as described by O'Farrell(18). For standards, purified small subunit (23), or RuBPCasewere employed. When either of these proteins was subjected totwo-dimensional electrophoresis, there were always just twolow mol wt spots corresponding to small subunit, always ofapproximately equal staining intensity; when the carboxylasewas used, there were in addition three to five intensely staininghigh mol wt spots, clustered together, and a variety of minorcontaminants which cannot be seen in the print (Fig. 3b).Analysis of proteins from wheat germ supernatant showed thatthe large and small subunit spots were not present (Fig. 3a).Thus, when carboxylase and wheat germ proteins are mixedand analyzed in the same sample, the positions of large andsmall subunit can be identified unambiguously in the same gel.The 14C-labeled proteins from soluble fraction polyribosomes

incubated in the wheat germ system were thus mixed withRuBPCase and subjected to two-dimensional electrophoresis;the gel was stained, saturated with PPO, dried, and exposed tox-ray film. Figure 4 shows the distribution of the radioactivematerial recorded by the x-ray film. About 80 different spotscan be counted on the print. More importantly, the collectionof low mol wt proteins is substantially resolved into severaldiscrete species which stand out detectably from the back-ground.

Various features on the film can be correlated directlv withstained regions on the dried gel to which it has been exposed,or with cracks on the gel, etc. For example: (a) the column oflabeled bands on the right corresponds with a relatively muchfainter stained column on the gel, and represents proteins which,possibly because they were insoluble during electrophoresis inthe first dimension, accumulated at the alkaline end; however,in the second dimension, solubilized by SDS, they migrated

Experiment Fraction Input A260 Reaction Incorporation Labeled AminoVolume Total Released(%) Acids Incorp.1 cpa/5 jul Per Ribosome

I SolubleSedimentable

11a Solubleb Soluble

IIIa Solubleb Sedimentable

IV SolubleSedimentable

2.40.772411.410.53.3S

74 409274 2095

300 16,500300 13,800150 7101150 1332300 5265300 373

NDNDNDND

2759(39)543(41)847 (16)218(58)

0.240.336.511.331.99.93.5

approximately 20,000 dalton protein by the wheat germ systemalone (Fig. 2). Even allowing for as much as a 4-fold isotopedilution by endogenous amino acids, it is evident that on theaverage, each polypeptide chain was extended by no more thanabout 10 to 40 amino acid residues. This corresponds essentiallyto a "run-off" synthesis (22) in which no more than one or twopolypeptide chains are completed and released/ribosome. Thus,the distribution of released chains corresponds to the distribu-tion of polyribosomes engaged in their synthesis.The data in Figure 2 show that the mol wt distributions of

[35S]methionine-labeled proteins released in vitro by solubleand sedimentable fraction polyribosomes were similar, althoughextents of labeling differed. Note that two proteins, of 20,000and less than 10,000 daltons, are synthesized by the wheatgerm system alone, and that their synthesis is not enhanced bythe addition of exogenous polyribosomes. These results wereindependent of the method used for isolation of the polyribo-somes, hence independent of variations in polyribosome integ-rity over the range 75 to 90% polyribosomes (unpublishedexperiments). Although the resolution of labeled proteins on

A: B

FIG. 2. Polyacrylamide gel electrophoresis of products of[35S]methionine incorporation by pea polyribosomes. Polyribosomesprepared as described in Table II, experiment I were incubated with[35S]methionine as described for experiment I, and 30-M1I aliquots of the100,000g supernatants of the reaction mixtures were applied to gradientgels of polyacrylamide and electrophoresed. Standards were included inparallel slots. The gel was stained, soaked in PPO and DMS0, andexposed to SB-54 x-ray film for 3 weeks and developed. A: Two samplesof products of soluble polysomes; B: two samples of products of sedi-mentable polysomes; C: two samples of products of wheat germ systemalone.

Plant Physiol. Vol. 60, 1977534

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FIG. 3. Absence of pea RuBP carboxylase markers from wheat germ supernatant proteins. Ten ;LI wheat germ supernatant (a) and 30 Ag RuBPcarboxylase of pea leaves (b) were lyophilized, dissolved in urea, and subjected to two-dimensional electrophoresis. A comparison of the two gelsshows that RuBP carboxylase has a few high mol wt spots (from large subunit), and just two low mol wt spots (from small subunit). These spots arenot present in the wheat germ pattern. Left to right: direction of increasing pH from 3.5 to 10; top to bottom: direction of decreasing mol wt insecond (SDS) dimension. Arrows (S) point toward small subunit, while arrow (L) points toward large subunits (b). Similar unlettered arrows indicatethe locations where those proteins migrate when present (a).

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ROY, TERENNA, AND CHEONG

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FIG. 4. Two-dimensional electrophoresis of 14C-amino-acid-labeledproducts of soluble fraction polyribosomes. Thirty Al of the 150,000gsupernatant of Table It, experiment Ilb was mixed with 30 Ag of purifiedRuBPCase and subjected to two-dimensional electrophoresis as de-scribed under "Materials and Methods," and fluorographed with KodakRP/54 film at -65 C for 2 weeks. The developed film was copied onto anegative by direct contact exposure, and a print made from the negative.Left to right corresponds to an increase in pl from 3.5 to 10; thedownward direction corresponds to decreasing mol wt in the SDS direc-tion. Arrows (S) point toward positions corresponding to stained smallsubunit spots visible on the dried gel; arrow (L) points toward a cluster ofclear areas (above the small subunit spots) which correspond exactly withlarge subunit spots.

straight down. (b) In the high mol wt region, there is a clusterof areas where the exposure is sharply below that of thesurrounding background; these "ghosts" correspond to the largesubunit spots visible on the dried gel. These and other landmarkscan be used to align the film with the gel. When this is done, itcan be seen that two spots on the x-ray film correspond exactlyin size, form, and location to the two visible small subunit spotson the dried gel. It should be noted that the pattern of stainedspots on the dried gel was equivalent to the pattern expected ifone were to superimpose Figure 3a and b.

In other experiments, we found that the more acidic of thesetwo small subunit spots was less intensely labeled than its morealkaline counterpart (Fig. 5). This was the case with both thesoluble fraction polyribosomes and sedimentable fraction poly-ribosomes in the experiment shown. Superimposition of theoriginal x-ray films in the local area of the small subunitsshowed that the spots coincided very closely in shape andarrangement; similarly close agreement in any other comparablearea on the two films was also observed. Since it is practicallyimpossible to ensure that the gels on which the samples areelectrophoresed are physically identical, one does not expectany closer matching of the two films than that which is indeedfound; every resolvable spot on film B has a resolvable counter-part on film A.

DISCUSSION

It has been shown before that amino acid incorporation bypolyribosomes added to the wheat germ system is sufficient forthe completion of one or perhaps two polypeptides/polyribo-some (22). The capacity for reinitiation in wheat germ extractsis not employed under these conditions, primarily becauseelongation is rate-limiting, and secondarily because of the delib-erate addition of aurintricarboxylic acid. The variations in aminoacid-incorporating ability by different preparations of polyribo-somes are therefore of minor importance: all reactions observedwere of the run-off type. Thus, the distribution of released,discrete labeled chains roughly corresponds to the distributionof polyribosomes engaged in their synthesis.The technique of two-dimensional electrophoresis is clearly

of high resolution when applied to intact proteins (18). In our

hands, the properties of in vitro synthesized proteins appearless favorable, in that these tend to stick to the top of theelectrofocusing gels, especially when over 40 ug of protein areapplied to the gels. This leads to a lower resolution of labeledproteins. Whether this is a "carrier" effect or reflects a differ-ence in the solubility properties of recently synthesized proteinscompared to native proteins is still not clear. Despite thisproblem, many labeled proteins do behave well, and focusthemselves at specific loci in the gels.We were led to attempt these experiments because of immu-

nological evidence suggesting that small subunit-synthesizingribosomes were localized in a cell fraction which could beisolated by low speed sedimentation (9). The results we ob-tained, however, demonstrate that small subunit-synthesizingpolyribosomes are proportionately represented among the poly-ribosomes of both the low speed sedimentable fraction and thesoluble fraction of the homogenates. Because almost all of therecoverable polyribosomes seem to be derived from the solublefraction, and the patterns of proteins labeled by polyribosomesof both fractions seem to be the same, we conclude that thebulk of small subunit-synthesizing polyribosomes are in thesoluble fraction of pea leaf homogenates.

. I

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FIG. 5. Two-dimensional electrophoresis of 14C-amino-acid-labeledproducts of soluble and sedimentable fraction polyribosomes. Aliquots(30 ,ul) of the 150,000g supernatants of reactions Illa and Itlb (TableII) were subjected to two-dimensional electrophoresis and fluorographyas described in Figure 4. A: Soluble fraction products; B: sedimentablefraction products; C: contrast-enhanced enlargement of low mol wtregion for sedimentable fraction products. Significance of arrows is thesame as in Figure 4.

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SMALL SUBUNIT POLYSOMES

Published electron micrographs of bean, barley, and pea

leaves (3, 6, 10-12) strongly suggest that "free" cytoplasmicribosomes far outnumber those which are bound to membranes.If one makes the assumption that the solutions required forpolyribosome isolation (7) have no effect on ribosome-mem-brane interaction, one would expect to recover more ribosomesfrom the soluble fraction of leaf homogenates than from thesedimentable fraction. Our data indicate that such is the case.

The apparent absence of any labeled protein specific forsedimentable fraction polyribosomes leaves open the possibilitythat these two fractions do not represent biologically differentpopulations. The operations used, however, make it very un-

likely that the recovery of sedimentable fraction polyribosomesis merely due to volumetric trapping in the membrane-contain-ing pellets. These pellets represent about 1% of the volume ofthe extracts from which they are sedimented, and are washedonce before detergent treatment, so that polyribosome yields of5% are well above what would be reasonably attributed tocross-contamination. Although one infers that the ribosomesare attached to membranes, it is difficult to imagine why thisshould be biologically important if all of the proteins they makeare also made in the cytosol.

It is known that from 15 to 30% of chloroplast ribosomes inpeas are engaged in synthesis of the large subunit of RuBPCase(1, 9). Our data show that there is no detectable labeling oflarge subunit polypeptides in the wheat germ extract supple-mented with leaf polyribosomes. Inasmuch as these preparationsprobably contain chloroplast polyribosomes, the absence oflarge subunit labeling confirms the specificity of the wheat germ

system for cytoplasmic ribosomes (16, 22). The data presentedhere therefore confirm the in vitro evidence that small subunitsof higher plant RuBPCase are synthesized in the cytoplasm (22).The heterogeneity of large and small subunits revealed by

two-dimensional electrophoresis resembles that observed withcarboxymethylated large and small subunits of tobacco Ru-BPCase (14). The detection of this heterogeneity requires theuse of techniques which are sensitive to differences in netcharge on the denatured polypeptides; by all other analyticalcriteria, the protein is pure RuBPCase, divisible into two subunitclasses by denaturation in urea or SDS (23). While the largesubunit heterogeneity varies from one preparation to another,giving three to five or even more spots, the small subunits are

always found in the same two places. This result suggests that atleast the small subunit heterogeneity reflects a characteristic ofthe enzyme in vivo, rather than a partial modification introducedduring isolation or analysis of the enzyme.

Since the isoelectric focusing step is begun right after disrup-tion of the enzyme in mercaptoethanol and high concentrationsof urea, it is unlikely that the heterogeneities are due toconformational differences among the polypeptides. They are

probably due to charge differences, which reflect either aminoacid sequence differences or side group modifications, such as

acetylations.The fact that each of the small subunit spots is labeled when

polyribosomes function in the cell-free heterologous systemraises the question whether there are two different messages forsmall subunit in peas. It is possible that only one mRNA type ispresent, however, and that prior side chain modification in vivoor even post-translational modification in the cell-free systemcan occur, leading to the appearance of the two small subunitspots. The variability in labeling of the spots could be related tosuch a phenomenon, or could reflect different concentrations oftwo different small subunit messenger RNAs in vivo, or it couldbe an analytical artifact.

The possible existence of a 20,000 dalton precursor of thesmall subunits is not excluded by these data, since immunologi-cal criteria were not used in selecting the polypeptides from thereaction mixture, and since numerous heterologous protein-synthesizing systems are known to be capable of carrying outappropriate post-translational modifications of the proteins theymake (8, 15, 22).* Our data do not support a model in which native smallsubunits are preferentially synthesized on membranes of theendoplasmic reticulum (9, 20), but favor one in which solublepolyribosomes release small subunits (or their precursors) intothe cytosol, whence they travel by some unknown path to someunknown site of assembly.

Acknowledgments -The authors thank L. Mader and L. Rueckert for technical assistance.

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319: 223-2342. BLOBEL G, VR POTTRE 1967 Studies on free and membrane-bound ribosomes in rat liver.

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9. GOODING LR, H Roy, AT JAGENDoRF 1973 Immunological identification of nascent

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15. LODISH H 1976 Translational control of protein synthesis. Annu Rev Biochem 45: 39-7216. MARcus A, JO BEWLEY, DP WEEKS 1970 Aurintricarboxylic acid and initiation factors of

wheat embryo. Science 167: 1735-173617. NISHIMURA M, T TAKABE, T SUGIYAMA, T AKAZAWA 1973 Structure and function of

chloroplast proteins. XIX. Dissociation of spinach leaf ribulose-1,5-diphosphate carbox-ylase by p-mercuribenzoate. J Biochem 74: 945-954

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5 37Plant Physiol. Vol. 60, 1977

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