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Polypeptides synthesized in vitro by plastids isolated from tobacco cell cultures: purification of the organelles on a silica density gradient
Plant Science Letters, 19 (1980) 181--191
181
© Elsevier/North-Holland Scientific Publishers Ltd.
POLYPEPTIDES SYNTHESIZED IN VITRO BY PLASTIDS ISOLATED FROM TOBACCO CELL CULTURES: PURIFICATION OF THE ORGANELLES ON A SILICA DENSITY GRADIENT
ANNE-MARIE LESCURE Laboratoire de Biochimie Fonctionnelle des Plantes, Ddpartement de Biologic Mol~culaire et CeUulaire, Facultd des Sciences de Luminy, 13288 MarseiUe Cedex 2 (France)
(Received March 1st, 1980) (Revision received May 5th, 1980) (Accepted May 20th, 1980)
SUMMARY
We have reported (Lescure, Cell Differ., 7 (1978) 139) that a series of polypeptides were actively labelled by incorporation of tracer amino-acids into crude chloroplast fractions, prepared from tobacco cell suspensions. It was suggested that the synthesis of some of these polypeptides was controlled by cytokinin. In order to specify the nature of these in vitro labelled polypeptides, chloroplasts capable of protein synthesis were extensively purified from tobacco cells. The SDS-PAGE analyses of polypeptides synthesized either by crude or by purified chloroplasts, show radioactive peaks migrating in similar molecular weight positions. These results are evidence for the chloroplast origin of the labelled polypeptides and will help to their identification with known chloroplast protein subunits.
INTRODUCTION
It is now well established that mature chloroplasts isolated from plants [1--4] or algae [5], are able to incorporate in vitro labelled amino-acids into some of their protein subunits. However little information is available dealing with the changes which might occur in the pattern of these plastidsynthesized polypeptides along the development of the organelles. The only reports related to this problem concern the transition from etioplasts to Abbreviations: CAMP, D-threo-chloramphenicol; BSA, bovine serum albumin; CH, cycloheximide; LHC, light-harvesting chlorophyll a/b-protein complex; PAGE, polyacrylamide
gel electrophoresis; PEG, polyethylene glycol; RUBPCase, ribulose 1,5 bis-phosphate carboxylase (EC 4.1.1.39); SDS, sodium dodecylsulfate; Trieine, N-tris (hydroxymethyl) methyl glycine.
182
chloroplasts [3,4]: these.studies have shown that a 3 2 ~ d a l t o n thylako'/d bound polypeptide, synthesized on plastid ribosomes, increased steadily during the light-induced greening. It is very likely that the transition of proplastids to mature chloroplasts brings about stepwise changes of the plastid-synthesized polypeptide map. To investigate this problem, suspension cultures have been shown to be well adapted [6,7]. In the case of the tobacco AG~4 cell line, grown under constant white light, it was found that the development of mature chloroplasts will n o t occur except in the presence of cytokinin [6]. The morphology of plastids present in the cytokinin starved cells remained similar to that of proplastids. In a previous report [7], we have compared the polypeptides synthesized in vitro by plastids isolated from cells grown either in the presence or in the absence of cytokinin: the relative rate of labelling of the polypeptides was n o t significantly different in both samples, except for two low molecular weight polypeptides (peak F, 16 kdaltons and peak G, 11 kdaltons) which labelling was strongly reduced in the case of cytokinin starved plastids. We are interested to study the nature and the function of these polypeptides which are useful as markers of the response of chloroplasts to cytokinin. However our previous experiments were performed with crude chloroplast preparations and ATP was required as energy source in the dark, to stimulate the protein synthesis of dedifferentiated plastids which were bleached. These experiments did n o t rule out the possibility that some of the peaks labelled in the ATP.driven system might be extra~hloroplastic. Consequently, in order to provide a more rigorous demonstration that the labelled polypeptides and especially the cytokinin markers were synthesized inside plastids, we decided to reexamine the in vitro protein synthesis with purified organelles. MATERIALS AND METHODS
Chloroplast purification The AG14 cell line of Nicotiana tabacum c.v. Wisconsin 38, was grown in liquid suspension in the presence of cytokinin, as previously described [6,7]. Cells were harvested in stationary phase at the 12th or 13th day after transfer and washed as detailed in [7]. All the following operations were performed at 4°C. Washed cells (40 g) were suspended in 60 ml of the ice-cold GR m e d i u m [8], supplemented with 4 mM 2-mercaptoethanol instead of araboascorbic acid. Suspended cells were homogenized for 2 s in a Sorvall omnimixer blendor at maximum speed. The homogenate was filtered through 4 layers of Miracloth and the process was again repeated twice. Pooled filtrates were centrifuged at 2500 g for 10 min: the resulting pellet was called the crude chloroplast fraction. Intact chloroplasts were isolated by isopycnic centrifugation in fortified gradients of Ludox AM, in the presence of PEG and BSA according to [9]. Two millilitres of crude chloroplasts resuspended in GR medium (to about 4 mg protein/ml) were layered on the top of a 15 ml gradient. The centrifugation was performed in a Beckman
183
sW 27.1 rotor, at 8200 g for 20 rain. Two bands of green material were obtained: the lower (about 2 ml) contained intact chloroplasts; it was diluted with 8 ml of ST incubation buffer (0.33 M sorbitol, 50 mM tricineKOH, pH 8.3) and centrifuged for 15 rain at 3000 g. The resulting pellet was called purified chloroplasts.
Radioactive amino-acid incorporations Conditions for light
Electrophoretic fractionation of plastid proteins Polypeptides were fractionated by SDS-PAGE as detailed in [7]. In the present experiments, after radioactive incubations samples for electrophoresis were prepared by direct addition of an equal volume of double-concentrated sample buffer [7] to the incubated chloroplasts. Washings, which may be a source of loss of soluble plastid proteins, were avoided, as all remaining low molecular weight materials were eliminated from gel during electrophoresis. After electrophoresis, the gels were stained with Coomassie blue R and the polypeptide bands were scanned with a PMQ II Zeiss spectrophotometer. Solubilisation and measurements of radioactivity in 2-mm acrylamide gel slices were as in [7]. The molecular weights of polypeptides were determined on PAGE with the following marker proteins: BSA (monomer 67 kdaltons); ovalbumin (45 kdaltons); chymotrypsinogen (25 kdaltons) and cytochrome c (12.4 kdaltons).
Miscellaneous analyses Cytochrome c oxidase activity wasmeasured spectrophotometrically at 550 n m according to [10]. Chlorophyll concentrations were determined according to [ 11 ]. Protein contents were measured by the Lowry m e t h o d [12], after precipitation of the sample by 10% TCA. RESULTS
Purification of intact chloroplasts and analysis of their in vitro protein synthesis After chloroplast centrifugation on silica concentration gradients of Ludox-PEG-BSA, two green zones were observed as described by Morgenthaler et al. [9]. Under the phase contrast microscope, the upper zone
184
OD 650nm Cytochrome c
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Fractions (ml) Fig. 1. Distribution of cytochrome c oxidase activity after centrifugation of crude chloroplasts on linear gradient of Ludox concentration (w/v): dotted line. Black dots: optical density at 650 nm. Open circles: cytochrome c oxidase activity per 1 ml fraction (% of the total activity contained in crude chloroplasts). appeared to contain stripped chloroplasts, and intact chloroplasts were f o u n d in the lower zone. Figure 1 shows the absorbance at 650 nm of the different fractions of such a gradient: peaks I and II correspond to the green zones. The contamination o f the heavier band by mitochondria was found to be very low, as shown by measurements of c y t o c h r o m e c oxidase activity throughout the gradient. Figure 2 shows a typical densitometric scan of total, soluble and membrane, polypeptides of such intact purified chloroplasts. Very similar patterns were obtained after purification of the AG14 cell line chloroplasts on sucrose concentration gradients [Axelos, M. and P~aud-Leno~l, C., submitted]. Among the separated polypeptide bands, the 52 kdalton band has been shown to co-migrate with the LS of RUBPCase and the 25 kdalton peak has been suggested to be a subunit of the LHC apoprotein [Axelos, M. and Pdaud-Leno61, C., submitted]. The large peak of 67 kdaltons shown on Fig. 2 is due to the presence of BSA which was added to the Ludox gradient. Crude chloroplasts and purified chloroplasts were then prepared from the same culture and incubated in the presence of [ 3sS ] methionine under the light-driven protein synthesis conditions: the results of this comparison are given in Table I. Purified intact chloroplasts contained a b o u t a quarter of the a m o u n t of protein in the crude preparation. The a m o u n t of chlorophyll
185 A
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10 Kd Fig. 2. Fractionation of polypeptides prepared from intact chloroplasts. Densitometric tracing at 600 nm of the SDS-PAGE profile, stained by Coomassie Blue. Linear gradient of acrylamide concentration 10--18%. Numbers above stained peaks correspond to the apparent molecular weights. Arrows indicate the position of the in vitro labelled peaks (Fig. 3). LS: large subunit of RUBPCase; LHC: subunit of the light-harvesting chlorophyll a/b complex. TABLE I PURIFICATION OF CHLOROPLASTS Cells were isolated from 13-day-old cultures (75 g of fresh material, 20 ug of chlorophyll/g fresh material). Protein synthesis was performed in the light-driven system, in the presence of [ 3sS] methionine for 30 min. protein content (mg) Crude chloroplasts 20 Purified chloroplasts 5.6
chlorophyll content (~g/mg protein)
protein synthesis effieiency (cpm/mg protein)
15.60 32.14
78 842 380 485
186
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i II t I I I I I III I I I I 10070 50 30 20 10 Kd 70 50 30 20 10 Kd Fig. 3. SDS-PAGE analysis of polypeptides synthesized in the light-driven system by plastids at two steps of their purification. Histogram 1: crude chloroplasts; h i s t o ~ a m 2: purified chloroplasts. Plastids were isolated from 13-day-old cultures. Radioactive incubations were performed in the presence of [ 3~S] methionine, for 35 min. Gel 1 was loaded with 250 ~g protein (18 000 cpm); gel 2 was loaded with 140 ug protein (50 000 cpm). Numbers: calculated molecular weights of radioactive peaks. Insert: separation of peak D region on 10% PAGE.
was twice as high in the purified fraction. Protein synthesis efficiency (cmp/ mg protein) of the purified fraction was 4 times higher than that of crude chloroplasts. Figure 3 shows histograms of the SDS-PAGE fractionation of radioactive polypeptides synthesized by crude or purified preparations. The pattern from the crude chloroplast preparation showed the same radioactive peaks, called A to G in our previous paper [ 7 ]. In the pattern obtained from purified preparations, 7 peaks were also observed, migrating with very similar electrophoretic mobilities. The qualitative similarity of the two histograms is a good evidence for the chloroplast origin of these products. It should be noticed that the amount of radioactivity incorporated into peak B (52 kdaltons) by purified chloroplasts was low when compared with the labelling of the same peak by crude chloroplasts. On the other hand a higher background was observed on the histogram from purified preparations: this background might be due to a large number of weakly labelled polypeptides as reported by Ellis [2]. It might also result from altered protein synthesis, like wrong initiations or early terminations, in the purified preparations.
187
Sensitivity to protein synthesis.inhibitors A n o t h e r piece of evidence t h a t the protein synthesis observed with crude preparations occurred on chloroplast ribosomes, was provided by the differential effect of inhibitors, specifically active against 70S or 80S ribosomesupported protein synthesis. Crude chloroplasts were incubated with the radioactive amino-acids in the ATP-driven system. The different incubation conditions are described in the legend to Fig. 4. The polypeptide structure o f products was shown by their digestion with pronase, except peak G which migration was c o m m o n to rapidly moving peptides. When incubations were performed in the presence of CH, a selective inhibitor of protein synthesis on 80S ribosomes, none of the labelled bands of the map was reduced. This result suggested t h a t even the crude preparations were n o t significantly c o n t a m i n a t e d with active cytosol ribosomes. On the contrary, in the presence of CAMP, a specific inhibitor of translation mediated by 70S ribosomes, the synthesis of all the radioactive peaks was strongly inhibited.
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Fig. 4. Effect of protein synthesis inhibitors on the synthesis of radioactive polypeptides by crude chloroplasts; sensitivity of these products to pronase digestion. Plastids were isolated from 12-day-old cultures. Radioactive incubations were performed in the ATPdriven system, in t h e p r e s e n c e of [z4C]amino-acid mixture, for 30 min. CH: i n c u b a t i o n in the presence of 25 /Jg/ml cycloheximide. CAMP: i n c u b a t i o n in t h e presence of 25 /~g/ml of D-threo-chloramphenicol. Pronase: sample digested by pronase, (500 pg/ml, for 60 min at 20°C) after radioactive i n c u b a t i o n . Labelled polypeptides were analysed o n SDS-PAGE. Each gel was loaded with 250/Jg protein.
188
Protein synthesis by purified chloroplasts in the A TP-driven conditions We have previously reported [ 7 ], that t h e addition of exogenous ATP to intact crude chloroplasts incubated in the ST incubation buffer, resulted in a poor stimulation of the labelled amino-acid incorporation. As we mentioned, the incorporation was n o t improved by the use of the KCl-medium described by Siddell and Ellis [13]. Therefore to operate protein synthesis in the dark with tobacco cell chloroplasts, it was first necessary to induce ruptures of the outer plastid envelopes by an osmotic shock, according to [8], in order to allow the penetration of ATP. When the same procedure was applied to chloroplasts purified on Ludox gradients, the pattern of labelled polypeptides appeared to be altered: some radioactive peaks scarcely emerged from a high continuous background (not shown). Purified organelles appeared to be very sensitive to the change of osmolarity: indeed, resuspension of the purified chloroplasts in the hypotonic conditions caused immediate lysis as observed by phase~ontrast microscopy; in the same conditions crude chloroplasts were stripped from envelopes without disruption of the internal structure [ 7]. This situation complicates studies of polypeptides synthesized by dedifferentiated plastids, after purification on Ludox, since the ATP. driven system is the only one which may be used, in the absence of chlorophyll.
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I I I I I ,I I I I I I I 1OO 50 25 10 Kd 100 5 0 25 10 Kd 10 Kd 1OO 50 25 Fig. 5. SDS-PAGE analysis of polypeptides synthesized by purified plastids in the lightdriven system, after various periods of incubation. Effect of chase by isotopic dilution of the radioactive marker. Plastids were purified from 13-day~)ld cultures. Radioactive incubations were performed in the presence of [sSS]methionine (0.1 ~M) during (1) 10 rain; (2) 35 min; (3) 35 rain, with an isotopic dilution by cold methionine (0.2/zM) after 10 min. Each gel was loaded w i t h 56 ~g protein.
189
Analysis of radioactive polypeptides synthesized by purified chloroplasts in the light-driven system, after different periods of incubation In the heretofore described experiments, the products of protein synthesis were analyzed after a 30-rain incubation. The gross incorporation of radioactivity into TCA-insoluble fraction has been shown to increas during the incubation period [ 7 ]. This did not indicate whether the kinetics of synthesis of each of the radioactive peaks were similar. Furthermore some of the observed radioactive peptides might have resulted from the splitting of larger molecular weight polypeptides by endogenous proteases during the incubation. In order to answer these questions, polypeptides synthesized by purified chloroplasts were analyzed after 10 rain (Fig. 5, histogram 1) and 35 rain (histogram 2) incubations in the presence of [3SS]methionine. A chase was realized in a third sample by the addition of an excess of unlabelled methionine after 10 rain in the course of a 35 rain incubation (histogram 3). Comparison of histograms 1 and 3 showed that the pattern of products synthesized during the first part of the incubation was similar to that obtained after the chase: therefore the observed peaks were n o t due to a proteolytic activity during the incubation. Comparison of histograms 1 and 2 showed that the radioactivity incorporated into the major peaks observed after 10 rain, increased during the second part of the incubation: for peaks A, B, D, E and F this increment corresponded to about 50% of the radioactivity measured after 10 rain. Only peaks C and G were stationary during the second part of the incubation. Peak G was very small in this experiment. DISCUSSION The purification of leaf chloroplasts is now well documented. In contrast, the purification of plastids from cell suspension cultures is still a source of problems. Compared with leaves, chloroplasts are smaller and their number is reduced in the cell cultures. The contamination of organelles with polysaccharides is also troublesome. Axelos and Pdaud-Leno61 (submitted) obtained highly purified plastids from tobacco cells by sucrose concentration gradient centrifugation. However, these plastids were inactive for protein synthesis. As shown by Morgenthaler et al. [9], the use of silica concentration gradients provided functional organelles. By this m e t h o d we were able to isolate intact chloroplasts from tobacco cells: on account of the exacting purification conditions, the yield was of the order of 10%. When radioactive precursors were incorporated by purified plastids in the light-driven system, the electrophoresis map showed a series of labelled bands, with well characterized positions. Apparent molecular weights of these polypeptide bands agreed with the previous reported molecular weights of polypeptides synthesized by crude chloroplast preparations [7]. T h e major peak labelled by purified chloroplasts, was peak D which, according to its molecular weight, fits with the 32 kdalton thylako'/d peptide synthe-
190
sized by leaf chloroplasts. The synthesis of this polypeptide has been shown to be light dependent [3,13]. In tobacco cell cultures, light is not ratelimiting: in any case, the synthesis of polypeptide D did not seem to be regulated by cytokinin [7]. According to our chase experiment, in contrast with some claims [2], polypeptide D did not appear to be submitted to any noticeable turn-over. In fact, peak D included a wide labelled zone of the PAGE (Fig. 3), suggesting that more than one labelled polypeptide were mixed in this peak. On a 10% PAGE, peak D was splitted into two radioactive bands of 32 and 27 kdaltons (Fig. 3, insert). Peak B corresponded to the 52 kdaltons polypeptide of the densitometric pattern, which migrated with the LS of the stromal protein RUBPCase [Axelos, M. and P~aud-Leon~l, C., submitted]. In purified chloroplasts, peak B was never labelled to the extent observed with crude chloroplast fractions, when peak D was taken as a reference. This observation suggested that during chloroplast purification, a loss of factors necessary for stroma protein synthesis might have occurred. It is relevant to mention the work of Tao et al. [14] showing that during cell fractionation, intact chloroplasts lost free ribosomes whereas the amount of membrane-bound ribosomes remained constant. In addition to these two major peaks, labelled histograms showed that a large amount of radioactivity (peak A) did not migrate into the gel: this peak was not observed when plastid polypeptides were labelled in vivo with the same precursors and the organelles were further purified and analyzed by the same techniques (not shown). It is not clear whether this peak corresponded to the synthesis of high molecular weight polypeptides or whether it resulted from the formation of aggregates in the in vitro conditions. The minor labelled peaks previously described, using crude preparations incubated in the ATP-driven system, have been repeatedly observed with defined electrophoretic mobilities, when purified chloroplasts were incubated in the light-driven conditions. Therefore these polypeptides did appear to be synthesized on chloroplast ribosomes. We tried to identify these minor labelled polypeptides with defined peaks of the densitometric tracing (Fig. 2). This identification was uncertain on account of the number of nearby coloured bands on the gel: polypeptide C migrated at the level of the 45 kdalton stained peak; in fact, this peak was split on the gel in two or three stained bands. Peak E was always found coincident with a small stained band of 22 kdaltons. Peak F was located inside the wide 18 kdalton stained region, which was composed of several non-separated polypeptides. Peak G migrated at the level of low molecular weight polypeptides digested by pronase and was not coincident with any defined stained band. As we already mentioned, the rate of synthesis of peak F was found to be controlled by cytokinin [ 7]. The apparent molecular weight of this material (16 000--17 000) does not fit with any known plastid-synthesized polypeptide, such as coupling factor subunits or cytochrome f [15,16]. In their studies on Chlamydornonas chloroplasts, Chua et al. [ 17] have reported that
191 t w o low molecular weight thylako'id polypeptides, were synthesized on chloroplast ribosomes. According t o our preliminary experi m ent s, pol ypeptide F also ap p ear ed t o be a thylako'id c o m p o n e n t and its apparent molecular weight (17 000) suggested t h a t it could be equivalent t o t h e s e Chlamydomonas polypeptides.
ACKNOWLEDGEMENTS I a m indebted to Dr C. Pdaud-Leno~l for useful discussions during this work. I thank Dr. M.M. Smith for reading the English manuscript. I a m grateful to Mrs. M.C. Durand for her technical assistance and help with the cell cultures. This work benefited from financial support of the Centre National de la Recherche Scientifique, France (Contracts E R 104 and A T P 2815). REFERENCES 1 J.J. Morgenthaler and L. Mendiola-Morgenthaler, Arch. Biochem. Biophys., 172 (1976) 51. 2 R.J. Ellis, Biochim. Biophys. Acta, 463 (1977) 185. 3 A.E. Grebanier, K.E. Steinback and L. Bogorad, Plant Physiol., 63 (1979) 436. 4 N.W. Gillham and J.E. Boynton, Genetic Control of Chloroplast Proteins, in D.R. Sanadi and L.P. Vernon (Eds.), Current Topics in Bioenergetics, Vol. 8 part B, Acad. Press N.Y., San Francisco, London, 1978, p. 211. 5 A.C. Vasconcelos, Plant Physiol., 58 (1976) 719. 6 P. Seyer, D. Marty, A-M. Lescure and C. P~aud-Leno~l, Cell Differ., 4 (1975) 187. 7 A-M. Lescure, Cell Differ., 7 (1978) 139. 8 W. Bottomley, D. Spencer and P.R. Whitfeld, Arch. Biochem. Biophys., 164 (1974) 106. 9 J.J. Morgenthaler, C.A. Price, J.M. Robinson and M. Gibbs, Plant Physiol., 54 (1974) 532. 10 T. Yonetani, in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. X, Acad. Press N.Y. and London, 1967, p. 332. 11 L.P. Vernon, Anal. Chem., 32 (1960) 1144. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R. Randall, J. Biol. Chem., 193 (1951) 265. 13 S.G. Siddelland R.J. Ellis,Biochem. J., 146 (1975) 675. 14 K.L.J. Tao and A.T. Jagendorf, Biochirn. Biophys. Acta, 324 (1973) 518. 15 L.R. Mendiola-Morgenthaler, J.J.Morgenthaler and C.A. Price,FEBS Lett.,62 (1976) 96. 16 A. Doherty and J.C. Gray, Eur. J. Biochem., 98 (1979) 67. 17 N.H. Chua and N.W. GiUham, J. Cell Biol.,74 (1977) 441.
181
© Elsevier/North-Holland Scientific Publishers Ltd.
POLYPEPTIDES SYNTHESIZED IN VITRO BY PLASTIDS ISOLATED FROM TOBACCO CELL CULTURES: PURIFICATION OF THE ORGANELLES ON A SILICA DENSITY GRADIENT
ANNE-MARIE LESCURE Laboratoire de Biochimie Fonctionnelle des Plantes, Ddpartement de Biologic Mol~culaire et CeUulaire, Facultd des Sciences de Luminy, 13288 MarseiUe Cedex 2 (France)
(Received March 1st, 1980) (Revision received May 5th, 1980) (Accepted May 20th, 1980)
SUMMARY
We have reported (Lescure, Cell Differ., 7 (1978) 139) that a series of polypeptides were actively labelled by incorporation of tracer amino-acids into crude chloroplast fractions, prepared from tobacco cell suspensions. It was suggested that the synthesis of some of these polypeptides was controlled by cytokinin. In order to specify the nature of these in vitro labelled polypeptides, chloroplasts capable of protein synthesis were extensively purified from tobacco cells. The SDS-PAGE analyses of polypeptides synthesized either by crude or by purified chloroplasts, show radioactive peaks migrating in similar molecular weight positions. These results are evidence for the chloroplast origin of the labelled polypeptides and will help to their identification with known chloroplast protein subunits.
INTRODUCTION
It is now well established that mature chloroplasts isolated from plants [1--4] or algae [5], are able to incorporate in vitro labelled amino-acids into some of their protein subunits. However little information is available dealing with the changes which might occur in the pattern of these plastidsynthesized polypeptides along the development of the organelles. The only reports related to this problem concern the transition from etioplasts to Abbreviations: CAMP, D-threo-chloramphenicol; BSA, bovine serum albumin; CH, cycloheximide; LHC, light-harvesting chlorophyll a/b-protein complex; PAGE, polyacrylamide
gel electrophoresis; PEG, polyethylene glycol; RUBPCase, ribulose 1,5 bis-phosphate carboxylase (EC 4.1.1.39); SDS, sodium dodecylsulfate; Trieine, N-tris (hydroxymethyl) methyl glycine.
182
chloroplasts [3,4]: these.studies have shown that a 3 2 ~ d a l t o n thylako'/d bound polypeptide, synthesized on plastid ribosomes, increased steadily during the light-induced greening. It is very likely that the transition of proplastids to mature chloroplasts brings about stepwise changes of the plastid-synthesized polypeptide map. To investigate this problem, suspension cultures have been shown to be well adapted [6,7]. In the case of the tobacco AG~4 cell line, grown under constant white light, it was found that the development of mature chloroplasts will n o t occur except in the presence of cytokinin [6]. The morphology of plastids present in the cytokinin starved cells remained similar to that of proplastids. In a previous report [7], we have compared the polypeptides synthesized in vitro by plastids isolated from cells grown either in the presence or in the absence of cytokinin: the relative rate of labelling of the polypeptides was n o t significantly different in both samples, except for two low molecular weight polypeptides (peak F, 16 kdaltons and peak G, 11 kdaltons) which labelling was strongly reduced in the case of cytokinin starved plastids. We are interested to study the nature and the function of these polypeptides which are useful as markers of the response of chloroplasts to cytokinin. However our previous experiments were performed with crude chloroplast preparations and ATP was required as energy source in the dark, to stimulate the protein synthesis of dedifferentiated plastids which were bleached. These experiments did n o t rule out the possibility that some of the peaks labelled in the ATP.driven system might be extra~hloroplastic. Consequently, in order to provide a more rigorous demonstration that the labelled polypeptides and especially the cytokinin markers were synthesized inside plastids, we decided to reexamine the in vitro protein synthesis with purified organelles. MATERIALS AND METHODS
Chloroplast purification The AG14 cell line of Nicotiana tabacum c.v. Wisconsin 38, was grown in liquid suspension in the presence of cytokinin, as previously described [6,7]. Cells were harvested in stationary phase at the 12th or 13th day after transfer and washed as detailed in [7]. All the following operations were performed at 4°C. Washed cells (40 g) were suspended in 60 ml of the ice-cold GR m e d i u m [8], supplemented with 4 mM 2-mercaptoethanol instead of araboascorbic acid. Suspended cells were homogenized for 2 s in a Sorvall omnimixer blendor at maximum speed. The homogenate was filtered through 4 layers of Miracloth and the process was again repeated twice. Pooled filtrates were centrifuged at 2500 g for 10 min: the resulting pellet was called the crude chloroplast fraction. Intact chloroplasts were isolated by isopycnic centrifugation in fortified gradients of Ludox AM, in the presence of PEG and BSA according to [9]. Two millilitres of crude chloroplasts resuspended in GR medium (to about 4 mg protein/ml) were layered on the top of a 15 ml gradient. The centrifugation was performed in a Beckman
183
sW 27.1 rotor, at 8200 g for 20 rain. Two bands of green material were obtained: the lower (about 2 ml) contained intact chloroplasts; it was diluted with 8 ml of ST incubation buffer (0.33 M sorbitol, 50 mM tricineKOH, pH 8.3) and centrifuged for 15 rain at 3000 g. The resulting pellet was called purified chloroplasts.
Radioactive amino-acid incorporations Conditions for light
Electrophoretic fractionation of plastid proteins Polypeptides were fractionated by SDS-PAGE as detailed in [7]. In the present experiments, after radioactive incubations samples for electrophoresis were prepared by direct addition of an equal volume of double-concentrated sample buffer [7] to the incubated chloroplasts. Washings, which may be a source of loss of soluble plastid proteins, were avoided, as all remaining low molecular weight materials were eliminated from gel during electrophoresis. After electrophoresis, the gels were stained with Coomassie blue R and the polypeptide bands were scanned with a PMQ II Zeiss spectrophotometer. Solubilisation and measurements of radioactivity in 2-mm acrylamide gel slices were as in [7]. The molecular weights of polypeptides were determined on PAGE with the following marker proteins: BSA (monomer 67 kdaltons); ovalbumin (45 kdaltons); chymotrypsinogen (25 kdaltons) and cytochrome c (12.4 kdaltons).
Miscellaneous analyses Cytochrome c oxidase activity wasmeasured spectrophotometrically at 550 n m according to [10]. Chlorophyll concentrations were determined according to [ 11 ]. Protein contents were measured by the Lowry m e t h o d [12], after precipitation of the sample by 10% TCA. RESULTS
Purification of intact chloroplasts and analysis of their in vitro protein synthesis After chloroplast centrifugation on silica concentration gradients of Ludox-PEG-BSA, two green zones were observed as described by Morgenthaler et al. [9]. Under the phase contrast microscope, the upper zone
184
OD 650nm Cytochrome c
oxidose (% of total)
ff
50
1.0 lI
|
% Ludox
/'/ SI
0.5
1
25 _20 _10
5
10
15
Fractions (ml) Fig. 1. Distribution of cytochrome c oxidase activity after centrifugation of crude chloroplasts on linear gradient of Ludox concentration (w/v): dotted line. Black dots: optical density at 650 nm. Open circles: cytochrome c oxidase activity per 1 ml fraction (% of the total activity contained in crude chloroplasts). appeared to contain stripped chloroplasts, and intact chloroplasts were f o u n d in the lower zone. Figure 1 shows the absorbance at 650 nm of the different fractions of such a gradient: peaks I and II correspond to the green zones. The contamination o f the heavier band by mitochondria was found to be very low, as shown by measurements of c y t o c h r o m e c oxidase activity throughout the gradient. Figure 2 shows a typical densitometric scan of total, soluble and membrane, polypeptides of such intact purified chloroplasts. Very similar patterns were obtained after purification of the AG14 cell line chloroplasts on sucrose concentration gradients [Axelos, M. and P~aud-Leno~l, C., submitted]. Among the separated polypeptide bands, the 52 kdalton band has been shown to co-migrate with the LS of RUBPCase and the 25 kdalton peak has been suggested to be a subunit of the LHC apoprotein [Axelos, M. and Pdaud-Leno61, C., submitted]. The large peak of 67 kdaltons shown on Fig. 2 is due to the presence of BSA which was added to the Ludox gradient. Crude chloroplasts and purified chloroplasts were then prepared from the same culture and incubated in the presence of [ 3sS ] methionine under the light-driven protein synthesis conditions: the results of this comparison are given in Table I. Purified intact chloroplasts contained a b o u t a quarter of the a m o u n t of protein in the crude preparation. The a m o u n t of chlorophyll
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10 Kd Fig. 2. Fractionation of polypeptides prepared from intact chloroplasts. Densitometric tracing at 600 nm of the SDS-PAGE profile, stained by Coomassie Blue. Linear gradient of acrylamide concentration 10--18%. Numbers above stained peaks correspond to the apparent molecular weights. Arrows indicate the position of the in vitro labelled peaks (Fig. 3). LS: large subunit of RUBPCase; LHC: subunit of the light-harvesting chlorophyll a/b complex. TABLE I PURIFICATION OF CHLOROPLASTS Cells were isolated from 13-day-old cultures (75 g of fresh material, 20 ug of chlorophyll/g fresh material). Protein synthesis was performed in the light-driven system, in the presence of [ 3sS] methionine for 30 min. protein content (mg) Crude chloroplasts 20 Purified chloroplasts 5.6
chlorophyll content (~g/mg protein)
protein synthesis effieiency (cpm/mg protein)
15.60 32.14
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186
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i II t I I I I I III I I I I 10070 50 30 20 10 Kd 70 50 30 20 10 Kd Fig. 3. SDS-PAGE analysis of polypeptides synthesized in the light-driven system by plastids at two steps of their purification. Histogram 1: crude chloroplasts; h i s t o ~ a m 2: purified chloroplasts. Plastids were isolated from 13-day-old cultures. Radioactive incubations were performed in the presence of [ 3~S] methionine, for 35 min. Gel 1 was loaded with 250 ~g protein (18 000 cpm); gel 2 was loaded with 140 ug protein (50 000 cpm). Numbers: calculated molecular weights of radioactive peaks. Insert: separation of peak D region on 10% PAGE.
was twice as high in the purified fraction. Protein synthesis efficiency (cmp/ mg protein) of the purified fraction was 4 times higher than that of crude chloroplasts. Figure 3 shows histograms of the SDS-PAGE fractionation of radioactive polypeptides synthesized by crude or purified preparations. The pattern from the crude chloroplast preparation showed the same radioactive peaks, called A to G in our previous paper [ 7 ]. In the pattern obtained from purified preparations, 7 peaks were also observed, migrating with very similar electrophoretic mobilities. The qualitative similarity of the two histograms is a good evidence for the chloroplast origin of these products. It should be noticed that the amount of radioactivity incorporated into peak B (52 kdaltons) by purified chloroplasts was low when compared with the labelling of the same peak by crude chloroplasts. On the other hand a higher background was observed on the histogram from purified preparations: this background might be due to a large number of weakly labelled polypeptides as reported by Ellis [2]. It might also result from altered protein synthesis, like wrong initiations or early terminations, in the purified preparations.
187
Sensitivity to protein synthesis.inhibitors A n o t h e r piece of evidence t h a t the protein synthesis observed with crude preparations occurred on chloroplast ribosomes, was provided by the differential effect of inhibitors, specifically active against 70S or 80S ribosomesupported protein synthesis. Crude chloroplasts were incubated with the radioactive amino-acids in the ATP-driven system. The different incubation conditions are described in the legend to Fig. 4. The polypeptide structure o f products was shown by their digestion with pronase, except peak G which migration was c o m m o n to rapidly moving peptides. When incubations were performed in the presence of CH, a selective inhibitor of protein synthesis on 80S ribosomes, none of the labelled bands of the map was reduced. This result suggested t h a t even the crude preparations were n o t significantly c o n t a m i n a t e d with active cytosol ribosomes. On the contrary, in the presence of CAMP, a specific inhibitor of translation mediated by 70S ribosomes, the synthesis of all the radioactive peaks was strongly inhibited.
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Fig. 4. Effect of protein synthesis inhibitors on the synthesis of radioactive polypeptides by crude chloroplasts; sensitivity of these products to pronase digestion. Plastids were isolated from 12-day-old cultures. Radioactive incubations were performed in the ATPdriven system, in t h e p r e s e n c e of [z4C]amino-acid mixture, for 30 min. CH: i n c u b a t i o n in the presence of 25 /Jg/ml cycloheximide. CAMP: i n c u b a t i o n in t h e presence of 25 /~g/ml of D-threo-chloramphenicol. Pronase: sample digested by pronase, (500 pg/ml, for 60 min at 20°C) after radioactive i n c u b a t i o n . Labelled polypeptides were analysed o n SDS-PAGE. Each gel was loaded with 250/Jg protein.
188
Protein synthesis by purified chloroplasts in the A TP-driven conditions We have previously reported [ 7 ], that t h e addition of exogenous ATP to intact crude chloroplasts incubated in the ST incubation buffer, resulted in a poor stimulation of the labelled amino-acid incorporation. As we mentioned, the incorporation was n o t improved by the use of the KCl-medium described by Siddell and Ellis [13]. Therefore to operate protein synthesis in the dark with tobacco cell chloroplasts, it was first necessary to induce ruptures of the outer plastid envelopes by an osmotic shock, according to [8], in order to allow the penetration of ATP. When the same procedure was applied to chloroplasts purified on Ludox gradients, the pattern of labelled polypeptides appeared to be altered: some radioactive peaks scarcely emerged from a high continuous background (not shown). Purified organelles appeared to be very sensitive to the change of osmolarity: indeed, resuspension of the purified chloroplasts in the hypotonic conditions caused immediate lysis as observed by phase~ontrast microscopy; in the same conditions crude chloroplasts were stripped from envelopes without disruption of the internal structure [ 7]. This situation complicates studies of polypeptides synthesized by dedifferentiated plastids, after purification on Ludox, since the ATP. driven system is the only one which may be used, in the absence of chlorophyll.
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I I I I I ,I I I I I I I 1OO 50 25 10 Kd 100 5 0 25 10 Kd 10 Kd 1OO 50 25 Fig. 5. SDS-PAGE analysis of polypeptides synthesized by purified plastids in the lightdriven system, after various periods of incubation. Effect of chase by isotopic dilution of the radioactive marker. Plastids were purified from 13-day~)ld cultures. Radioactive incubations were performed in the presence of [sSS]methionine (0.1 ~M) during (1) 10 rain; (2) 35 min; (3) 35 rain, with an isotopic dilution by cold methionine (0.2/zM) after 10 min. Each gel was loaded w i t h 56 ~g protein.
189
Analysis of radioactive polypeptides synthesized by purified chloroplasts in the light-driven system, after different periods of incubation In the heretofore described experiments, the products of protein synthesis were analyzed after a 30-rain incubation. The gross incorporation of radioactivity into TCA-insoluble fraction has been shown to increas during the incubation period [ 7 ]. This did not indicate whether the kinetics of synthesis of each of the radioactive peaks were similar. Furthermore some of the observed radioactive peptides might have resulted from the splitting of larger molecular weight polypeptides by endogenous proteases during the incubation. In order to answer these questions, polypeptides synthesized by purified chloroplasts were analyzed after 10 rain (Fig. 5, histogram 1) and 35 rain (histogram 2) incubations in the presence of [3SS]methionine. A chase was realized in a third sample by the addition of an excess of unlabelled methionine after 10 rain in the course of a 35 rain incubation (histogram 3). Comparison of histograms 1 and 3 showed that the pattern of products synthesized during the first part of the incubation was similar to that obtained after the chase: therefore the observed peaks were n o t due to a proteolytic activity during the incubation. Comparison of histograms 1 and 2 showed that the radioactivity incorporated into the major peaks observed after 10 rain, increased during the second part of the incubation: for peaks A, B, D, E and F this increment corresponded to about 50% of the radioactivity measured after 10 rain. Only peaks C and G were stationary during the second part of the incubation. Peak G was very small in this experiment. DISCUSSION The purification of leaf chloroplasts is now well documented. In contrast, the purification of plastids from cell suspension cultures is still a source of problems. Compared with leaves, chloroplasts are smaller and their number is reduced in the cell cultures. The contamination of organelles with polysaccharides is also troublesome. Axelos and Pdaud-Leno61 (submitted) obtained highly purified plastids from tobacco cells by sucrose concentration gradient centrifugation. However, these plastids were inactive for protein synthesis. As shown by Morgenthaler et al. [9], the use of silica concentration gradients provided functional organelles. By this m e t h o d we were able to isolate intact chloroplasts from tobacco cells: on account of the exacting purification conditions, the yield was of the order of 10%. When radioactive precursors were incorporated by purified plastids in the light-driven system, the electrophoresis map showed a series of labelled bands, with well characterized positions. Apparent molecular weights of these polypeptide bands agreed with the previous reported molecular weights of polypeptides synthesized by crude chloroplast preparations [7]. T h e major peak labelled by purified chloroplasts, was peak D which, according to its molecular weight, fits with the 32 kdalton thylako'/d peptide synthe-
190
sized by leaf chloroplasts. The synthesis of this polypeptide has been shown to be light dependent [3,13]. In tobacco cell cultures, light is not ratelimiting: in any case, the synthesis of polypeptide D did not seem to be regulated by cytokinin [7]. According to our chase experiment, in contrast with some claims [2], polypeptide D did not appear to be submitted to any noticeable turn-over. In fact, peak D included a wide labelled zone of the PAGE (Fig. 3), suggesting that more than one labelled polypeptide were mixed in this peak. On a 10% PAGE, peak D was splitted into two radioactive bands of 32 and 27 kdaltons (Fig. 3, insert). Peak B corresponded to the 52 kdaltons polypeptide of the densitometric pattern, which migrated with the LS of the stromal protein RUBPCase [Axelos, M. and P~aud-Leon~l, C., submitted]. In purified chloroplasts, peak B was never labelled to the extent observed with crude chloroplast fractions, when peak D was taken as a reference. This observation suggested that during chloroplast purification, a loss of factors necessary for stroma protein synthesis might have occurred. It is relevant to mention the work of Tao et al. [14] showing that during cell fractionation, intact chloroplasts lost free ribosomes whereas the amount of membrane-bound ribosomes remained constant. In addition to these two major peaks, labelled histograms showed that a large amount of radioactivity (peak A) did not migrate into the gel: this peak was not observed when plastid polypeptides were labelled in vivo with the same precursors and the organelles were further purified and analyzed by the same techniques (not shown). It is not clear whether this peak corresponded to the synthesis of high molecular weight polypeptides or whether it resulted from the formation of aggregates in the in vitro conditions. The minor labelled peaks previously described, using crude preparations incubated in the ATP-driven system, have been repeatedly observed with defined electrophoretic mobilities, when purified chloroplasts were incubated in the light-driven conditions. Therefore these polypeptides did appear to be synthesized on chloroplast ribosomes. We tried to identify these minor labelled polypeptides with defined peaks of the densitometric tracing (Fig. 2). This identification was uncertain on account of the number of nearby coloured bands on the gel: polypeptide C migrated at the level of the 45 kdalton stained peak; in fact, this peak was split on the gel in two or three stained bands. Peak E was always found coincident with a small stained band of 22 kdaltons. Peak F was located inside the wide 18 kdalton stained region, which was composed of several non-separated polypeptides. Peak G migrated at the level of low molecular weight polypeptides digested by pronase and was not coincident with any defined stained band. As we already mentioned, the rate of synthesis of peak F was found to be controlled by cytokinin [ 7]. The apparent molecular weight of this material (16 000--17 000) does not fit with any known plastid-synthesized polypeptide, such as coupling factor subunits or cytochrome f [15,16]. In their studies on Chlamydornonas chloroplasts, Chua et al. [ 17] have reported that
191 t w o low molecular weight thylako'id polypeptides, were synthesized on chloroplast ribosomes. According t o our preliminary experi m ent s, pol ypeptide F also ap p ear ed t o be a thylako'id c o m p o n e n t and its apparent molecular weight (17 000) suggested t h a t it could be equivalent t o t h e s e Chlamydomonas polypeptides.
ACKNOWLEDGEMENTS I a m indebted to Dr C. Pdaud-Leno~l for useful discussions during this work. I thank Dr. M.M. Smith for reading the English manuscript. I a m grateful to Mrs. M.C. Durand for her technical assistance and help with the cell cultures. This work benefited from financial support of the Centre National de la Recherche Scientifique, France (Contracts E R 104 and A T P 2815). REFERENCES 1 J.J. Morgenthaler and L. Mendiola-Morgenthaler, Arch. Biochem. Biophys., 172 (1976) 51. 2 R.J. Ellis, Biochim. Biophys. Acta, 463 (1977) 185. 3 A.E. Grebanier, K.E. Steinback and L. Bogorad, Plant Physiol., 63 (1979) 436. 4 N.W. Gillham and J.E. Boynton, Genetic Control of Chloroplast Proteins, in D.R. Sanadi and L.P. Vernon (Eds.), Current Topics in Bioenergetics, Vol. 8 part B, Acad. Press N.Y., San Francisco, London, 1978, p. 211. 5 A.C. Vasconcelos, Plant Physiol., 58 (1976) 719. 6 P. Seyer, D. Marty, A-M. Lescure and C. P~aud-Leno~l, Cell Differ., 4 (1975) 187. 7 A-M. Lescure, Cell Differ., 7 (1978) 139. 8 W. Bottomley, D. Spencer and P.R. Whitfeld, Arch. Biochem. Biophys., 164 (1974) 106. 9 J.J. Morgenthaler, C.A. Price, J.M. Robinson and M. Gibbs, Plant Physiol., 54 (1974) 532. 10 T. Yonetani, in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. X, Acad. Press N.Y. and London, 1967, p. 332. 11 L.P. Vernon, Anal. Chem., 32 (1960) 1144. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R. Randall, J. Biol. Chem., 193 (1951) 265. 13 S.G. Siddelland R.J. Ellis,Biochem. J., 146 (1975) 675. 14 K.L.J. Tao and A.T. Jagendorf, Biochirn. Biophys. Acta, 324 (1973) 518. 15 L.R. Mendiola-Morgenthaler, J.J.Morgenthaler and C.A. Price,FEBS Lett.,62 (1976) 96. 16 A. Doherty and J.C. Gray, Eur. J. Biochem., 98 (1979) 67. 17 N.H. Chua and N.W. GiUham, J. Cell Biol.,74 (1977) 441.