Planta

Planta 144, 455--462 (1979)

9 by Springer-Verlag 1979

Cell Free Synthesis of some Storage Protein Subunits by Polyribosomes and RNA Isolated from Developing Seeds of Pea (Pisum sativum L.) I. M a r t a Evans, R o n a l d R . D . C r o y , P h i l i p p a H u t c h i n s o n , D o n a l d Boulter*, Peter I. Payne, a n d M a r g a r e t E. G o r d o n * * Department of Botany, University of Durham, Durham DH1 3LE, and Plant Breeding Institute, Huntingdon Road Laboratory, 181A Huntingdon Road, Cambridge CB30DY, U.K.

Abstract. P o l y r i b o s o m e s which have t e m p l a t e activity in the w h e a t germ system have been i s o l a t e d from d e v e l o p i n g p e a seeds. S o m e o f the t r a n s l a t i o n p r o ducts have identical m o b i l i t i e s to the vicilin a n d legurain s u b u n i t s by S D S - P A G E . C e r t a i n p r o d u c t s were specifically i m m u n o p r e c i p i t a t e d with antisera prep a r e d a g a i n s t purified vicilin a n d l e g u m i n fractions. V a r i o u s R N A fractions i n c l u d i n g p o l y A - r i c h R N A have also been isolated f r o m p o l y r i b o s o m e s a n d shown to direct the synthesis o f p o l y p e p t i d e s whose p r o p e r t i e s are similar to the storage p r o t e i n subunits. T h e results are discussed in r e l a t i o n s h i p to o t h e r investigations with seed s t o r a g e p r o t e i n b i o s y n t h e s i s in vitro.

Key words: thesis -

Polyribosomes P r o t e i n synR i b o s o m e s - R N A - S t o r a g e proteins. Pisum

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Introduction The storage p r o t e i n fractions, vicilin a n d legumin, constitute a b o u t 80% o f the t o t a l p r o t e i n o f the mature p e a seed ( R a a c k e , 1957). F u r t h e r m o r e it has been shown t h a t they are f o u n d o n l y in the c o t y l e d o n s a n d e m b r y o n i c axis o f seeds (Millerd, 1975). F o r these reasons, the d e v e l o p i n g pea, as are o t h e r l e g u m e seeds (Beachy et al., 1978; Sun et al., 1975), is a p o t e n t i a l l y v a l u a b l e e x p e r i m e n t a l system for investigating the c o n t r o l s i n v o l v e d in p r o t e i n synthesis. This fact, coup l e d with the i m p o r t a n c e o f legume seeds as a source o f p r o t e i n for m a n a n d his a n i m a l s , has led to an increasing interest in t h e m by scientists. A series o f * To whom reprint requests should be addressed ** Department of Botany and Zoology, Massey University, Palmerston North, New Zealand Abbreviations." DTT = dithiothreitol; SDS-PAGE = SDS-poly-

acrylamide gel electrophoresis; TCA = tricarboxylic acid

investigations have been c a r r i e d out with d e v e l o p i n g pea seeds, p a r t i c u l a r l y in the U S A . by Beevers a n d P o u l s o n (1972) a n d in A u s t r a l i a by M i l l e r d a n d Spencer (1974) (see review by M u n t z , 1978). This p a per r e p o r t s on the i s o l a t i o n o f p o l y r i b o s o m e s f r o m d e v e l o p i n g seeds at a time w h e n they were actively synthesising vicilin a n d legumin. T h e p o l y r i b o s o m e s have t e m p l a t e activity in the w h e a t g e r m cell-free syst e m i n d i c a t i n g the presence o f f u n c t i o n a l m R N A . A p o l y r i b o s o m a l R N A f r a c t i o n has also been shown to be active a n d the t r a n s l a t i o n p r o d u c t s o f these systems have been p a r t i a l l y characterised.

Materials and Methods 1. Developing Seed Material

Seeds of Pisum sativum L. cv. Feltham First (Sunons Seeds Ltd., Reading, Berks, U.K.) were germinated in Alkathenex polyethylene granules (I.C.I. Plastics Division, Welwyn Garden City, Hefts, U.K.) in a dark spray room at 28~ C, with water misting for 5 min in every hour for 4-5 days. Seedlings were then transferred to 21 water culture bottles of nutrient solution and were grown under controlled environmental conditions, as follows: lighting- 16 h total (including morning and evening phases); temperature - day 28~ C, night 23~ C; humidity, 75-80% rei. hum. Pods were harvested 14 days after flowering. The cotyledons of the seeds were separated aseptically from the testas, radicles and hypocotyls and whole seeds or organs were stored at - 7 0 ~ C. 2. Other Materials'

Glassware was sterilised by dry heat at 170~ C and buffers were either autoclaved or those containing sucrose were sterilised by filtration through 0.22 gm Millipore filters. 3. Isolation o f Poly A-Rich-RNA from Total R N A

Total RNA was prepared essentially by using the method of Brawerman (1974). The preparation was chromatographed on oligodT-cellulose (grade T-3, Collaborative Research) columns and poly A lacking-RNA and poly A rich-RNA were eluted (Aviv and

0032-0935/79/0144/0455/$ 01.60

456 Leder, 1972). Fractions were then treated and stored as in Gordon and Payne (1976).

4. Extraction of Polyribosomes Total and membrane-bound polyribosomes were prepared by a modification of the method of Larkins and Davies (1975). Frozen cotyledons were homogenised (1:3 w/v) in buffer A (0.2 M TrisHC1, 0.2 M sucrose, 60 m M KC1, 30 mM MgC12, 1 m M dithiothreitol (DTT), pH 8,5 at 2~ C) with a Willems polytron for 3 s, speed setting 8. In preparing total polyribosomes, 0.1 volume of a mixture of 20% (v/v) Triton X-100, 0.25 M sucrose, 10 m M DTT was added. The homogenate was filtered through Miracloth, centrifuged at 500 g for 10 min and the supernatant re-centrifuged at 29,000 g for 10 rain. When total polyribosomes were prepared, the post mitochondrial supernatant Was layered over a 10 ml sucrose pad of 700 mg/ ml sucrose in buffer B and centrifuged for 2 h in an 8 x 35 ml Ti angle rotor at 95,000 gay in a MSE SS65. The polyribosomal pellet was washed twice in sterile water, suspended in the buffer used for " i n vitro" systems, frozen and stored as aliquots in liquid nitrogen. For the preparation of the membrane-bound polyribosomes the 29,000g pellet was resuspended in buffer A, re-centrifuged at 29,000 g and stored as for total polyribosomes. For polyribosome profiles the pellets were resuspended in buffer A + 1% Triton X-100 and layered (200400 gg RNA in 1 ml) over 20 ml linear 15 60% (w/v) sucrose gradients, and centrifuged in a 3 x23 ml swing-out rotor at 95,000 g,v for 2 h at 2 ~ in an MSE SS65. Gradients were scanned with an ISCO Model 640 fractionator equipped with a UA-5 absorbance monitor.

I.M. Evans et al. : Storage Protein Synthesis by Isolated Pea Ribosomes products the incubations were carried out in 40 IA volumes at 30 ~ C for 30 min and the assays centrifuged for 1 h at 100,000 gav at 2 ~ in an MSE SS65 using special adaptors to fit the 10 x 10 ml Ti angle rotor. The adaptors were made of Delrin with a hole of 2 mm diameter and 16 mm length drilled in each (Whyteleafe Manufacturing, Charlwood Road, Charlwood, Surrey, U.K.). The high-speed supernatants thus obtained were used for immunochemical analyses. Hot trichloroacetic acid-insoluble radioactivity in 1 gl aliquots was determined using glass fibre (Whatman GF/C) filter discs. The in vitro products of translation were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a modification of the method of Laemmli (1970). Electrophoresis on a 17% (w/v) polyacrylamide gel at 8 mA was continued for about 16 h. The gel was dried between layers of cellophane under vacuum in a gel drier (GSD-4, Pharmacia Fine Chemicals, Sweden). The labelled translation products were detected by autoradiography using Kodirex X ray film or more usually by fluorography (Bonner and Laskey, 1974) on Kodak X-Omat H film or Fuji Rx Medical film developed in Ilford Phenisol. In our experience, fluorography did not give as good a resolution of radioactive polypeptide bands as with conventional autoradiography (cf. Fig. 3C and 5B) however it was routinely employed for shorter exposure times. The figures are composite, results being assembled from different experiments. This was made necessary partly for convenience of experimentation (it is not possible to assay different preparations simultaneously) and also because different preparations differ in activity, requiring different X-ray film exposure times. Each experiment was conducted using radioactive standards (vicilin and legumin) and the composite alignments are exactly dictated by the position of bands relative to the standards and polyribosome controls on the same slab gel (see Fig. 6).

5. Isolation of Poly A rich-RNA from Polyribosomes m R N A was isolated from polyribosomes by two different methods. The first method was based on that of Krystosek (1975). A polyribosome pellet was resuspended in 50 m M Tris-HC1, 10 m M EDTA, 0.5% (w/v) SDS, (pH 7.4 at 2 ~ C) heated in a water bath at 60 ~ C for 2 min and cooled down in ice to room temperature. The sample was then made 0.5 M with respect to NaC1 and applied to an oligo-dT-cellulose column. Poly A rich-RNA was eluted with saltfree buffer, made 0.2 M with respect to sodium acetate pH 5.5, and mixed with 2 vol of ethanol at - 2 0 ~ C. Storage at - 2 0 ~ C for 3 days ensured a quantitative precipitation of poly A-rich RNA. The R N A was then collected by centrifugation, washed three times with 80% cold ethanol to remove SDS, dissolved in 10 mM TrisHC1 pH 7.4 and reprecipitated by adding 0.1 vol of 2 M potassium acetate pH 5.6 and 2 volumes of cold ethanol. The second method of isolating m R N A from polyribosomes was adapted from Evans and Lingrel (1969). Polyribosomes were resuspended in 5 m M Tris-HC1 pH 7.4 (at 2 ~ C) and clarified by centrifugation at 12,000g for 10 rain at 4 ~ C. The supernatant was adjusted to an R N A concentration of 6 8 mg ml- 1 (1 mg ml A260 = 10), made 0.5% (w/v) with respect to SDS and incubated at 37~ for 5 rain. The solution was then immediately layered over a 21 ml linear 10-35% (w/v) sucrose gradient in the buffer without SDS and centrifuged at 2~ for 21 h at 90,000 g~v in the MSE 3 x 23 ml swing-out rotor. The separation of R N A species was checked using E. coli rRNAs (16S and 23S) and tRNA (4S).

6. In vitro Protein Synthesis' The cell-free protein synthesising system prepared from wheat germ was that described by Gordon and Payne (1976) with minor modifications. For immunochemical characterisation of the translational

7. Isolation and Radioactive Labelling of Storage Proteins from Pea Seeds Total protein, vicilin and legumin fractions were prepared from dried, hexane extracted meal from mature seed essentially by the established methods (Wright and Boulter, 1974; Scholz et al., 1974) and were used to raise antisera in rabbits as described later. Vicilin and legumin were further purified by ion-exchange chromatography and hydroxylapatite chromatography (R. Croy, unpublished results) and tritium-labelled vicilin and legumin standards (gift of Dr.J. Gatehouse) were prepared by acylation with ([all]acetic anhydride (500 mCi mmol x)) according to the method of Fraenkel-Conrat (1957). After exhaustive dialysis they were freezedried, weighed and dissolved in a minimal amount of 2% (w/v) SDS, and stored in liquid nitrogen. Specific radioactivity was calculated to be 2.5 x 106 ct min-1 mg-1 and 4.3 x 106 ct rain-1 rag- 1 for vicilin and legumin, respectively.

8. Preparation of Antibodies Antibodies against total protein extract, vicilin and legumin were raised in New Zealand white rabbits ( > 3 kg) by standard protocols (e.g. Harboe and Ingild, 1973). Specificity of the relevant antisera was tested using immunoelectrophoresis of total protein extract. Antibodies were prepared from the antiserum by precipitation at 0 ~ C with 40% relative saturation of ammonium sulphate at pH 7.5 The precipitate was recovered by centrifugation, redissolved, precipitated twice more with ammonium sulphate and redissolved in 50 mM borate buffered saline pH 8.0. Further purification of the antibodies was effected by the dialysis method of Harboe and Ingild (1973), before sterile filtration (0.22 jam Millipore filters) and storage at 4 ~ C in the presence of 0.05% sodium azide and

1.M. Evans et al. : Storage Protein Synthesis by Isolated Pea Ribosomes 50 units of aprotinin per ml of antiserum (Trasylol, Bayer U.K. Ltd.) as described by Bjerrum et al. (1975, 1977). Monospecific goat anti-rabbit IgG antibodies (Miles Yeda Ltd., Israel) were used without further purification.

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0.5

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9. [mmunoprecipitation Conditions for maximum immunoprecipitation by this double immunoprecipitation system were established in preliminary titration experiments. 2 gl of the appropriate rabbit antibody preparation, containing about 40-50 gg of total immunoglobulin protein 1 mg ml-l~A280=i.46, was added to 40 gl of the high speed supernatant from each "in vitro" sample and incubated for 1 h at 37~C. The appropriate amount of goat anti-rabbit IgG antibodies to give maximum immunoprecipitation of the rabbit antibodies, was then added and incubation continued at 37~ for a further 60 min at 37~ C, and then overnight at 4~ C. Immunoprecipitates were recovered by centrifugation at 4~ C and washed at least three times with ice-cold 50 mM borate buffer, 0. I5 M sodium chJoride pH 8.0. The final washed precipitate was dissolved and incubated at 105~ C for 10rain in 10% (w/v) SDS, 5% (v/v) mercaptoethanol in 0.I25 M Tris-HC1 pH 6.8 for SDS-PAGE. Unequivocal identification by coincidence of product and standard subunits was made difficult because of band distortion due to overloading of the gel tracks with immunoglobulin protein; this is inherent in the method.

TOP

BOTTOM

Fig. 1. Sucrose density gradient sedimentation of polyribosomes. 430 gg of polyribosomes (1 mg ml = A26o = I0), was sedimented on a 15-60% (w/v) linear sucrose gradient and monitored at 254 nm. Polyribosomes were untreated ( - - ) or treated (.... ) with 0.1 gg of pancreatic ribonuclease A per ml at 30~ for 5 rain. Centrifugation was performed at 95,000 g~, for 2 h at 2~C

Results A=I

A=2

1. Plant Material 18S

A spray r o o m was used to give a n excellent percentage g e r m i n a t i o n (Croy 1977) a n d growth in water culture u n d e r controlled c o n d i t i o n s e n s u r e d rapid, u n i f o r m a n d highly r e p r o d u c i b l e s y n c h r o n i s e d development. G e r m i n a t i o n to flowering t o o k a b o u t 28 days a n d flowering to m a t u r i t y (onset of d r y i n g - o u t phase) another 20 days (Millerd a n d Spencer, 1974). Selection of the time at which to harvest the seeds was based o n the results o f several " i n v i v o " labelling experiments, results to be p u b l i s h e d ; these indicated that the seeds were actively synthesising b o t h the storage p r o t e i n fractions by day 14 after flowering.

25S

i

TOP

2. Isolation and Characterisation of Total RNA, Polyribosomes, Polyribosomal RNA and Poly A-rich RNA from Pea Cotyledons 1 g of fresh cotyledons (c. 0.5 g/cotyledon) yielded a b o u t 0.8 m g of total R N A (1 mg m l - 1 ~ A 2 6 0 = 25) or a b o u t 1.2 mg o f total p o l y r i b o s o m e s ; of the latter approx. 67% were recovered as m e m b r a n e b o u n d polyribosomes. Poly A-rich R N A a c c o u n t e d for a b o u t 1% of total R N A . T o t a l p o l y r i b o s o m e s were p r e p a r e d from peas f r o m which the testa a n d e m b r y o axis h a d been removed. M e m b r a n e - b o u n d p o l y r i b o s o m e s were p r e p a r e d from whole seeds. As j u d g e d by ratio of Az6o/A28 o a n d A26o/A235 the pu-

FRACTION NO.

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Fig. 2. Sucrose density gradient sedimentation of polyribosomal RNA. 1 mg RNA was sedimented on a 10-35% (w/v) linear sucrose gradient and monitored at 254 nm. 1.2 ml fractions were collected, pooled and designated fractions A, B and C as shown. Bands of RNA were identified by E. coli rRNAs (16S and 23S) and tRNA (4S) separated on equivalent gradients. Centrifugation was performed at 90,000 g~, for 21 h at 2~ C

rity of different p r e p a r a t i o n s was the same with values of 1.86_+0.04 a n d 1.62_+0.04 respectively. 3 0 m g of p o l y r i b o s o m e s yielded by direct p u r i f i c a t i o n approx. 250 gg o f poly A-rich R N A . W h e n poly A-rich R N A was purified from fraction-B p o l y r i b o s o m a l R N A , the fraction f o u n d to be m o s t active in the wheat germ system, the equivalent yield was 65 gg.

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1.M. Evans et al. : Storage Protein Synthesisby Isolated Pea Ribosomes

Sucrose gradient profiles of polyribosome preparations before and after treatment with pancreatic ribonuclease are given in Fig. 1. These show up to 13 ribosomes in the undegraded polyribosome preparation as compared to aggregates of 1-3 ribosomes in the RNase-treated preparation. Figure 2 shows an absorption profile (A260) of polyribosomal R N A separated on a sucrose density gradient. Pooled fractions designated at A, B and C were assayed for their synthetic activity in the wheat germ system. Fraction B, which contained 18S rRNA, exhibited most of the translational activity and was subsequently used for the isolation of poly A richRNA.

3. Optimal Conditions for Protein Synthesis in vitro on Polyribosomes, Polyribosomal RNA-fraction B and Poly A rich-RNA The efficiency of different preparations of the cell-free wheat germ system was checked using a standard R N A preparation. Also the translation efficiency of each different type of m R N A used, was investigated with regard to the incubation components. The optimum concentration of Mg 2+ for translation on polyribosomes was found to be 3.0-4.0 m M and with translation assays containing 3.0 m M Mg 2+ , the optimum concentration for potassium was accepted as 80 raM. The translation products obtained after incubation in the reaction mixtures with increasing concentration of Mg 2+, showed qualitatively the same subunit pattern on SDS-PAGE (data not shown). The pattern of the polypeptide products released from the polyribosomes after centrifugation at 100,000 gav for 1 h did not show a specific enrichment of any completed polypeptide products, and therefore centrifugation of incubations after completion of assays was omitted. The polyribosomal RNA-fraction B was active in the wheat germ system whereas fraction A showed none and fraction C showed little incorporation above the endogenous level. Poly A rich-RNA and poly A lacking-RNA isolated from i) total RNA, ii) polyribosomal RNAfraction B and iii) total polyribosomes, were also investigated for their template activity in the wheat germ system. The Mg 2+ and K + optima for these R N A preparations were similar to those for the polyribosomes. Poly A lacking-RNA showed a much lower level of radioactive incorporation than poly A rich-RNA. A time course for the translation of poly A rich polysomal-RNA demonstrated a continued activity up to 180 rain. It is possible that with increasing time some of the m R N A was being degraded.

Fig. 3. Comparison of in vitro labelled polypeptideproducts driven by different preparations of polyribosomes analysed by SDSPAGE. Tracks A, B, D, [3H]leucinelabelled translation products from differentpreparations of polyribosomes.Track C, [aSS]methionine labelled translation products from total polyribosomes. Track E, [3H]leucinelabelledtranslation products from membranebound polyribosomes(undissociated). Vic = Tritiated vicilin; Leg = Tritiated legumin; L+V = Mixture of tritiated vicilin and legumin. Mol. wt. of standard subunits are given in the right hand column Using optimal concentrations of Mg 2+ and K +, concentrations of polyribosomes and polyribosomal RNA-fraction B giving maximal incorporation of label were 90 ~tg and 2 gg, respectively. The corresponding optimal value for poly A rich-RNA was 1 gg R N A per assay.

4. Identification of Total Translational Products from PoIyribosomal and RNA Fractions When total polyribosomes were used to drive the wheat germ system a very complex pattern of labelled polypeptide subunits was produced (Fig. 3). A comparison of the subunit patterns of these translation products with those of storage proteins tritiumlabelled in vitro showed the presence of subunit bands with mobilities coincident with the major subunits of vicilin and legumin; other subunits were also present. The translation products from different polyribosomal preparations showed some variation in their subunit patterns (Fig. 3 A-D). Translation products from membrane-bound polyribosomes, undissociated from the membranes, gave subunit patterns which were also complex, but showed an enrichment of the 51,000 mol. wt. subunit (cf. vicilin 51,000 subunit) as well as showing subunits with mobilities coincident with the other major vicilin and legumin subunits (Fig. 3 E).

1.M. Evans et al. : Storage Protein Synthesis by Isolated Pea Ribosomes

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Fig. 4. [3H]leucine labelled translation. Products from various RNA preparations analysed by SDS-PAGE as follows. Track A = total polyribosomes; B = fraction B from polyribosomal RNA; C = poly A rich-RNA from fraction B ; D - anti-vicilin immunoprecipitated products from poly A-rich RNA from fraction B translation products; E = poly A rich-RNA isolated directly from polyribosomes; L+V = Mixture of tritiated vicilin and legumin

Wheat germ translation products of polyribosoreal RNA-fraction B and poly A rich-RNA isolated from it showed correspondence with vicilin and legumin subunits (Fig. 4B, C). Poly A rich-RNA isolated directly from polyribosomes gave a pattern (Fig. 4E) qualitatively identical with that from poly A-rich fraction B (Fig. 4 C). Poly A lacking-RNA had a low level of template activity; the banding pattern of the radioactive polypeptide products were very faint, but qualitatively, essentially the same as those of poly A rich-RNA translation products (results not shown). Comparison of the polyribosomal translation products (Fig. 4A) with those produced by polyribosoreal RNA-fraction (Fig. 4B) and poly A rich-RNA isolated from (Fig. 4C) showed that both the latter templates generated a higher proportion of subunits of lower mol. wt. than did polyribosomes. In comparison with the wheat germ system, the reticulocyte lysate system directed by poly A richR N A isolated from the total cellular R N A , produced a simpler polypeptide pattern which lacked some of the lower mol. wt. subunits (Fig. 5). This can be clearly seen although the translation products were analysed in separate laboratories using slightly different gel systems. Other subunits are present in addition to those found on most product gels especially 2 prominent high mol. wt. subunits (between 60 and 70,000). These are also seen on SDS gels of products of polyribosome driven systems but not in those from RNA.

Fig. 5. Comparison by SDS-PAGE of the [35S]methionine labelled translation products of total poly A-rich RNA synthesised in the wheat germ and reticulocyte lysate cell, free systems. Products detected by autoradiography. A = Products from the reticulocyte lysate system; B = Products from wheat germ system

5. Characterisation of Translation Products by Selective Immunoprecipitation Immunoprecipitation of the labelled translation products using total polyribosomes in the wheat germ system by anti-total protein (Fig. 6E) and by antivicilin antibodies (Fig. 6C) gave specific enrichment of two polypeptide subunits of approx, mol, wt. 51,000 and 47,000; polyribosome controls are A, D and G on the same gel slab (i.e. a non-composite Fig.). This shows the correspondence of the two immunoprecipitated bands with those of polysomes translation products not always so evident (see e.g. Fig. 4C and D) due to compression of the bands by unlabelled I g G protein. A similar enrichment of these two subunits was not observed using non-specific antibodies (Fig. 6F) or anti-legumin antibodies (Fig. 6B). There was no conclusive enrichment of any legumin subunits in immunoprecipitates of the translation products of any assay. Sometimes however (see Fig. 6B), a subunit band of about 60,000 was observed, which could correspond to undissociated legumin 40,000 plus 20,000

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I.M. Evans et al. : Storage Protein Synthesisby Isolated Pea Ribosomes

Fig. 6. SDS-PAGE analysisof immunoprecipitatedproducts from the total translated products from total polyribosomes using [3H]leucine. Track A = total polyribosomes; B = anti-legumin; C = anti-vicilin;D = total polyribosomes;E = anti-totalprotein; F = non specific antibodies; G = total polyribosomes; L+V = mixtureof tritiated leguminand vicilin

subunits (Wright and Boulter, 1974). The immunoprecipitation by anti-legumin of some vicilin 51,000 and 47,000 subunits (Fig. 6B) was shown to be due to the presence of a small amount of cross-contamination (vicilin) in the legumin antibody preparation used, since these subunits were not found on product gels after immunoprecipitation with anti-legumin preparations which had been purified by passage through a vicilin-sepharose 4B column (results not shown). When polyribosomal RNA-B (results not shown) and poly A-rich RNA prepared from it (Fig. 4D) were used to drive the wheat germ system and anti-vicilin immunoprecipitated products were analysed, the two vicilin subunits were again observed to be present. Other subunits were seen in immunoprecipitates, especially on prolonged exposure of the gels to the X-ray film, but conclusive identification of these was not possible in the absence of any specific enrichment (Fig. 6). Discussion

Several groups, working with seed materials different to those used here have reported the successful translation of storage protein subunits using polyribosomal preparations to programme the wheat germ cell-free system. Sun et al. (1975) showed the 'in vitro' synthesis of the subunits of G1 globulin or glycoprotein II (Pusztai and Watt (1970)) of Phaseolus vulgaris. They were unable to show the synthesis of these subunits from RNA preparations isolated from polyribosomes.

Similarly, using polyribosomes from developing soyabean seeds, Beachy et al. (1978) reported the 'in vitro' synthesis of several subunits of mol. wt. corresponding approximately to those of purified soyabean 7S and 11S protein subunits on SDS-PAGE. In addition they showed that poly A-rich R N A from their polyribosomes gave the same products. Neither of these groups used characterisation methods for ' in vitro' products other than the coincidence of the mobilities of polypeptide subunits of translation products and standards on SDS-PAGE. Using the ethanol-solubility of zein as a specific selective method for identifying the major storage protein in Zea and in addition by using the coincidence of its subunits on SDS-PAGE, Larkins et al. (1976a) were able to show that zein was translated with polyribosome preparations from developing corn seed. This group also showed zein subunit synthesis with poly A-rich R N A from corn polyribosomes (Larkins et al., 1976b). Muntz and Co-workers (Muntz 1978) have incubated free and membrane bound polyribosomes isolated from immature seeds of Vicia faba and immunoprecipitation with specific antibodies followed by SDS-PAGE was used to show that polypeptides of both vicilin and legumin were preferentially synthesised in the cell-free system by membrane bound (but dissociated) polysomes. Luthe and Peterson (1977) working with developing oat groats (Arena sativa L.) were able to show the in vitro synthesis of oat globulin polypeptide subunits from isolated polyribosomes. They used a combination of coincidence of mobilities on SDSPAGE of intact and cyanogen bromide cleaved labelled product polypeptide subunits and standard oat globulin polypeptide subunits. Their results also indicated preferential synthesis of the oat globulin polypeptides on membrane bound polyribosomes. In this paper it is shown that vicilin of about 51,000 subunit mol. wt. is synthesised in a cell-free wheat germ system by polyribosomes and messenger R N A preparations isolated from them and from total R N A of developing pea seeds. SDS-PAGE followed by fluorography, detected polypeptides corresponding to all three major vicilin subunits (i.e. 51,000, 33,000 and 17,000) in the translational products, but only the 51,000 subunits were definitely shown to be specifically immunoprecipitated, other subunits were not apparently enriched. This may be due to a preferential synthesis in the vicilin (mol. wt. c. 180,000) fraction injected rabbits, of anti-bodies against the major vicilin polypeptide (51,000 subunit mol. wt.) to the exclusion of synthesis of those against the vicilin proteins/polypeptides of 33,000 and 17,000 subunit tool. wt. (Derbyshire et al., 1976).

I.M. Evans et ai. : Storage Protein Synthesis by Isolated Pea Ribosomes Vicilin as isolated from mature pea seeds appears to have only a single subunit 51,000 on S D S - P A G E (Fig. 3) but two and sometimes more, specifically immunoprecipitated radioactive subunits of 51,000 mol. wt. were constantly observed to be translated with polyribosomes and R N A preparations. This result is strongly indicative that the lower mol. wt. subunits represent populations of partially completed vicilin 51,000 polypeptides but which still possess specific vicilin antigenic determinants. The fact that a discrete population of incomplete subunits appears to be synthesised, indicates premature termination at unique sites on the 51,000 subunit m R N A . The probability of such an occurrence in cell free systems has been reported by Boime and Leder (1972) from their work on the translation of viral m R N A . While subunits apparently coincident with the legumin standard appear to be synthesised from both polyribosome and R N A preparations we were unable to show their specific immunoprecipitation by the methods adopted in this study. The reason for this is as yet not known, but may be due to lack of legumin synthesis in significant quantities in a form recognisable by the antibody system, e.g. subunits which lack antigenic determinants of intact legumin. The reduced amounts of lower mol. wt. translation products found in the reticulocyte lysate system as compared to the wheat germ when similar R N A preparations were used to drive them may be accounted for by incorrect initiation and/or premature termination. It should be noted that these translation products are produced in large and reproducible amounts. A less likely alternative explanation is that the differences noted above arise from the greater breakdown of m R N A in the wheat germ system. Higgins and Spencer (1977) recently reported their findings on the translation of products in vitro using polyribsomes and polyribosomal R N A from developing seeds of Pisurn sativum c.v. Greenfeast. The translation products were characterised on sucrose gradients, by single immunoprecipitation followed by detection by fluorography after separation on SDS-PAGE, and by peptide mapping of tryptic digests of the TCA-precipitated translation products. Immunoprecipitation showed that only a small proportion, if any, of the cell-free products carried the same antigenic determinants as did storage proteins from mature seeds. The pattern of immunoprecipitates analysed on gels was similar to the supernatants, but with an occasional enrichment of the polypeptides in the 50,000 region. Most products were different for the two systems when analysed on S D S - P A G E and fluorography. Tryptic fingerprints showed 67% and 52% of the radioactive peptides associated with ninhydrin-positive storage protein peptides for the

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polyribosomal and R N A systems respectively. Only 40% and 42% of the total number of storage protein peptides resolved were radioactive for the two systems respectively and it is doubtful if peptide mapping yields much conclusive evidence when applied to so complicated a product as pea storage protein. In contrast to the present finding albeit with a different variety of Pisum sativum, Higgins and Spencer (1977) were unable to conclude that their system made vicilin and legumin subunits in vitro. It is of interest that the m e m b r a n e bound polyribosomes give an enrichment of 51,000 vicilin subunit relative to the total polyribosomal R N A pattern (Fig. 3 E) in view of the evidence presented by Bailey et al. (1970) that storage proteins are synthesised on R E R [see also Muntz, 1978; Luthe and Peterson (1977) and Larkins et al. (1976)]. We wish to thank Drs. C.J. Leaver, U.E. Loening, N. Harris, J.A. Gatehouse for helpful demonstrations and advice on various techniques, Dr.T. Hunt for carrying out the reticulocyte experiment and R. Swinhoe for technical assistance. References

Aviv, H., Leder, P. : Purification of biologically active globin messenger RNA by chromatography on oligothymidylicacid - cellulose. Proc. Natl. Acad. Sci. USA 69, 1408-1412 (1972) Bailey, C.J., Cobb, A., Boulter, D. : Cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of the seed storage protein of Vicia faba. Planta 95, 103-118 (1970) Beachy, R.B., Thompson, J.F., Madison, J.T. : Isolation of polyribosomes and messenger RNA active in in vitro synthesis of Soyabean seed proteins. Plant Physiol. 61, 139-144 (1978) Beevers, L., Poulson, R. : Protein synthesis in cotyledons of Pisum sativum L. Plant Physiol. 49, 476-481 (1972) Bjerrum, O.J., Ramlau, J., Clemmeson, I., Ingild, A., Bog-Hansen, T.C.: An artefact in quantitative immunoelectrophoresis of spectrin caused by proteolytic activity in antibody preparations. Scand J. Immunol. 4, Supp. 2, 81-88 (1975) Bjerrum, O.J., Bog-Hansen, T.C.: Immunochemical gel precipitation techniques for the analysis of membrane proteins In, "Membrane Methods" Maddy A.H., ed. London: Chapman and Hall 1977 Boime, I., Leder, P. : Protein synthesis directed by Eucephalomyocarditis virus in RNA III. Discrete polypeptides translated from a Monocistronic Messenger in vitro. Arch. Biochem. Biophys. 153, 706-713 (1972) Bonner, W.M., Laskey, R.A. : A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88 (1974) Brawerman, G. : The isolation of messenger RNA from mammalian cells. Methods in Enzymol. 30, Part F, 605-612 (1974) Croy, R.R.D. : Ph.D. Thesis, University of Aberdeen, Localisation of the major proteins and Proteolytic enzymes in Phaseolus vulgar& L. at Germination 1977 Derbyshire, E., Wright, D.J., Boulter, D.: Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 15, 3-24 (1976) Evans, M.J., Lingrel, J.B. Hemoglobin mRNA synthesis of 9S and rRNA during erythroid cell development.: Biochem J. 8, 3000-3005 (1969)

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Fraenkel-Conrat, H.: Methods for investigating the essential groups for enzyme activity. Methods in Enzymology 4, Section I, 11,251 (1957) Gordon, M.E., Payne, P.I.: In vitro translation of the long-lived messenger ribonucleic acid of dry seeds. Plant 130, 269 273 (1976) Harboe, N., Ingild, A. : Immunisation, isolation of immunoglobulins, Estimation of Antibody Titre. Scand. J. Immunol.2, Supp 1, 161-164 (1973) Higgins, T.J.V., Spencer, D.: Cell-free synthesis of pea seed proteins. Plant Physiol. 60, 655-661 (1977) Krystosek, A., Cawthorn, M.L., Kabat, D.: Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. J. Biol. Chem. 250, 6077-6084 (1975) Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage. T4. Nature 277, 680-685 (1970) Larkins, B.A., Bracker, C.E., Tsai, C.Y. : Storage protein synthesis in maize. Plant Physiol. 57, 740-745 (1976a) Larkins, B.A,, Jones, R.A., Tsai, C.Y. : Isolation and in vitro translation of Zein Messenger Ribonucleic Acid. Biochemstry 15, 5506-5510 (1976b) Larkins, B.A., Davis, E.: Polyribosomes from peas. An attempt to characterise the total, free and membrane-bound polysomal population. Plant Physiol 55, 749-756 (1975) Luthe, D.S., Peterson, D.M.: Cell-free synthesis of globulin by developing oat (Avena sativa L.) seeds. Plant Physiol. 59, 836-841 (1977) Millerd, A. : Biochemistry of legume seed proteins. Ann. Rev. Plant Physiol. 26, 53-72 (1975)

Millerd A, Spencer, D. : Changes in RNA synthesising activity and template activity in nuclei from cotyledons of developing pea seeds, Aust. J. Plant Physiol. 1, 33-1341 (1974) Muntz, K. : Cell Specialisation Processes during biosynthesis and storage of proteins in plant seeds in ' Regulation of developmental processes in plants.' p. 70-97. Schutte H.R., Gross, D., eds. Jena: VEB Gustav Fischer 1978 Pelham, H.B.R., Jackson, R.J.: An efficient mRNA-dependent translation system for reticulocyte lysates. Eur. J. Biochem. 6% 247-256 (1976) Pusztai, A., Watt, W.B. : Glycoprotein II. The isolation and Characterisation of a major antigenic and non-haemagglutinating Glycoprotein from Phaseolus vulgaris L. Biochim. Biophys. Acta. 207, 413-431 (1970) Raacke, I.D.: Protein synthesis in ripening pea seeds. J. Analysis of whole seeds. Biochem. J. 66, 101-110 (1957) Scholtz, G., Richter, J., Manteuffel, R. : Studies on seed globulins from legumes I. separation and purification of legumin and vicilin from V. faba L. by zone precipitation. Biochem. Physiol. Pflanzen 166, 163 172 (1974) Sun, S.M., Buchbinder, B.U., Hall, T.C.: Cell-free synthesis of the major storage protein of the bean Phaseolus vulgaris L. Plant Physiol. 56, 780-785 (1975) Wright, D.J., Boulter, D. : Purification and subunit structure of legumin of Viciafaba L. (Broad bean). Biochem. J. 141, 413M18 (1974)

Received 9 August; accepted 20 September 1978

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.).

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products hav...
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