Synthesis of Cytochrome f by Isolated Pea Chloroplasts

t u r . J . Biochem. 9X, 87-92 (1979)

Synthesis of Cytochrome f by Isolated Pea Chloroplasts Andrew DOHERTY and John C. GRAY

Departmcnt of Botany, University of Cambridge

(Received January 29, 1979)

Chloroplasts, isolated from the leaves of 7-day-old pea seedlings, were incubated in the light with [35S]methionine or [3H]leucine. After extraction from the washed chloroplast membranes using a mixture of ethyl acetate, ethanol and ammonia, cytochromef'was precipitated with a monospecific antiserum and resolved by gradient polyacrylamide gel electrophoresis in sodium dodecylsulphate. The cytochromef'band was identified by its intrinsic fluorescence in ultraviolet light and was shown to be radioactive by autoradiography or fluorography of dried polyacrylamide gels. One-dimensional peptide mapping of the products of papain hydrolysis confirmed that the radioactivity was an integral part of cytochrome .f: The incorporation of ["Slmethionine into cytochrome f 'was inhibited by D(-)three-chloramphenicol but not by cycloheximide and did not occur in the dark. The synthesis was resistant to ribonuclease. It is concluded that cytochrome f'is synthesised in intact isolated pea chloroplasts.

Although chloroplasts are a major site of protein synthesis in the leaves of higher plants, the identity of most of the products is unknown. Intact chloroplasts isolated from pea leaves incorporate radioactive amino acids into at least ninety polypeptides, most of which appear to be soluble [l]. However, only four of these products have been positively identified. These are the large subunit of ribulosebisphosphate carboxylase [2] and three of the five subunits of the chloroplast coupling factor, CF, [3,4]. Ribulosebisphosphate carboxylase is the major stromal protein and the CF1 complex is an extrinsic thylakoid protein which can be released from the membranes by mild treatments not involving detergents. The identity of none of the intrinsic membrane proteins that are synthesised on chloroplast ribosomes is known.

Gregory and Bradbeer [5] found that the accumula- tion of cytochromes ,f; h - 5 5 9 L p and b-563 in greening bean leaves was inhibited by D(-)three-chloramphenicol, suggesting that their synthesis is dependent on 70-S ribosomes. Cytochrome .f; a membrane-bound component of the photosynthetic electron-transfer chain, is the most fully characterised of the chloroplast cytochromes. The availability of a monospecific antiserum raised against pure cytochrome ,f from charlock [6] made possible a direct study of its synthesis. In this paper we report that cytochromefis a product of protein synthesis by intact isolated chloroplasts.

~~

E I I : J , ~ c , . s . Papain (EC 3.4.22.2); ribonuclease A (EC 3.1.4.22); i,-ribulose-l.S-bisphosphate carboxylase (EC 4.1.1.39).

MATERIALS AND METHODS

Plant Material

Seeds of Pisum sativum cv. Feltham First were grown in compost in a greenhouse. Daylight was supplemented with six white fluorescent tubes, 75 cm above the seed trays, for 18 h each day.

Reagents

Cycloheximide, D( - )threo-chloramphenicol, ribonuclease A (type 1-A) and papain (P4762) were obtained from Sigma (Poole, Dorset). Pronase (B grade, nuclease-free) was obtained from Calbio- chem (Bishops Stortford, Herts). ~- [~~S]Methionine and ~ - [ 4 , 5 - ~ H ] leucine were obtained from The Radio- chemical Centre (Amersham, Bucks.).

Proteins used in the determination of the molecular weights of polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate were : bovine serum albumin (Armour Pharmaceuti- cals, Eastbourne, Sussex) ; cytochrome c (BDH, Poole, Dorset) and ovalbumin, ci-chymotrypsinogen A, carbonic anhydrask and myoglobin (Sigma, Poole, Dorset).

An t iserunz

Antibodies to purified cytochromeffrom charlock (Simppis arvensis L.) [6] were raised in rabbits by multiple intramuscular injections of cytochrome ,f

88 Synthesis of Cytochrome ,/ by Isolated Pea Chloroplasts

emulsified with Freund's complete adjuvant. The antisera cross-reacted with cytochrome .f extracted from all higher plants examined.

Chloroplast Isolation and Incubation

Chloroplasts were isolated from 15 g of leaves from 7-day-old pea seedlings as described by Blair and Ellis [2] and were washed once with 100 ml of ice-cold isolation medium. The chloroplasts were resuspended in 0.3 M sorbitol, 50 mM Tricine-KOH (pH 8.0) at a chlorophyll concentration of 0.2 mg . ml- ' . [35S]Me- thionine or [3H]leucine were added in amounts given in the legends to the table and figures and the chloroplast suspension was incubated at 20 "C for 60 min. Illumination was with a 500-W tungsten lamp (Philips No. 2 Photoflood) at 15 cm. Sterile solutions and glassware were used throughout, to minimise bacterial contamination.

Extraction and Immunoprecipitation of Cytoclzrome f

After incubation, the chloroplasts were lysed by adding 25 ml of ice-cold wash buffer (50 mM potas- sium phosphate buffer, pH 7.2) and the membranes were collected by centrifugation at 38 000 x g for 5 rnin at 0°C. The pellet was washed three times with 25 ml of cold wash buffer and then resuspended in 2.5 ml of the same buffer. Cytochromefwas extracted by adding a freshly prepared mixture of 7.5 ml ethyl acetate, 2.5 ml ethanol and 38 pl ammonium hydroxide(specif- ic gravity 0.88) at - 20°C [7] and blending for 30 s using a Polytron homogeniser. The resulting unstable emulsion was allowed to settle at - 20 "C for 10 min, after which the bulk of the upper organic phase was poured off. After centrifuging at 20000 x g for 10 rnin at - 5 "C, material remaining in the aqueous phase was precipitated by adding 1.1 vol. of acetone at - 20 "C. The precipitate was collected by centrifugation and extracted three times with 50 p1 l % (v/v) Triton X-100, 0.9 % (w/v) NaC1. The pooled extracts were clarified by centrifugation at 20000 x g for 5 min at 0 ° C and then mixed with 0.1 ml of rabbit antiserum against cytochrome f. The immunoprecipitate which developed after incubation for 30 min at 37°C and then overnight at 4 °C was collected by centrifugation and washed three times with 0.2 ml 1 :( Triton X-100, 0.9 NaCl.

Determination of Radioactivity

Radioactivity incorporated into total chloroplast proteins and the washed membrane fraction was determined by mixing samples with an equal volume of a saturated solution of methionine or leucine (whichever had been used as the labelled amino acid) and two volumes of 10 yd (w/v) trichloroacetic acid.

The precipitate that formed overnight at 4 'C was washed with 0.4 ml 5 '%, trichloroacetic acid and resuspended in 0.4 ml 5 % trichloroacetic acid. This suspension was heated at 90°C for 15 min and the precipitate was collected by filtration on a Whatman GF/A disc. The precipitate was washed with ethanol, ethanol/diethyl ether (1 : 1, by volume) and finally diethyl ether and dried for 15 rnin at 60 'C. Radio- activity was measured by liquid scintillation spectro- metry at counting efficiencies of 70% for 35S and 22 76 for 3H.

Radioactivity in immunoprecipitates was determined by washing the precipitate three times with 0.2 ml 1 Triton X-100, 0.9% NaCl and then twice with 0.2 ml 0.9% NaCl. The washed precipitate was resuspended in 0.4 ml 5 % trichloroacetic acid, 25 '%, saturated with unlabelled methionine or leucine. This suspension was heated, filtered and counted as described above.

Polyacrylamide Gel Electrophoresis

Gradient polyacrylamide gel electrophoresis in sodium dodecylsulphate was performed as described by Chua and Bennoun [8] using a 10 - 20 % acrylamide gradient gel (10-cm long) and a 5 % acrylamide stacking gel (2-cm long). Electrophoresis was for 16 h at 95 V. Proteins were stained by immersing the gel for 2 h in 0.25% (w/v) Coomassie brilliant blue R250 in 50 % methanol, 7 'x acetic acid. Excess dye was removed by repeated washings in 5 % methanol, 7 % acetic acid. Marker proteins used for estimating molecular weights were bovine serum albumin (68 000), ovalbumin (46000), carbonic anhydrase (29 000), a-chymotrypsinogen A (25 700), myoglobin (1 7 200) and cytochrome c (1 2 400).

The intrinsic fluorescence of cytochrome J was detected by illuminating unstained gels with a Camag type 29200 ultraviolet lamp set on 366 nm [9]. Proteins labelled with [35S]methionine were detected by autoradiography. Proteins labelled with t3H]leucine were detected by fluorography at - 70 'C [lo] using pre- exposed film [ll].

Peptide Mapping of' Cytochrome f Peptide mapping was performed using a slight

modification of the procedure described by Cleveland et al. [12]. Cytochromefwas immunoprecipitated and then resolved by gradient gel electrophoresis in sodium dodecylsulphate as described above. The gel was stained for 30 min and destained for 45 min. The cytochromefband was cut out, soaked for 30 min in stacking gel buffer containing 1 mM EDTA and then pushed to the bottom of a sample well in a second gel. This gel had been prepared according to Chua and Bennoun [8], except that all solutions contained 1 mM

A. Doherty and J. C. Gray

EDTA. The gel slice was overlayed with 10 pl stacking gel buffer containing 1 mM EDTA and 20% glycerol and then with 10 p1 stacking gel buffer containing 1 mM EDTA, loo/, glycerol and 10 pg papain. Electrophoresis was performed at 95 V for 16 h at room temperature, except that the current was switch- ed off for 30 min just before the bromophenol blue reached the separating gel. After electrophoresis, the gel was stained for 2 h and destained fully. Labelled proteins were detected by autoradiography or fluorography as described above.

RESULTS Estraction and Immunoprecipitation of Cytochrome f

Homogenisation of washed chloroplast membranes (Fig. 1, lane a) in ethyl acetate/ethanol/ammonia extracted cytochrome f with an efficiency of 45 %. After precipitation with acetone, cytochrome f was recovered in low yield (about 12%) but with high specificity. The extract contained only two pre- dominant polypeptides (Fig. 1, lane b). The larger of these had a molecular weight of 37300 and was specifically precipitated by antiserum against cytochrome ,f (Fig. 1, lane c). The antiserum also caused the quantitative precipitation of cytochrome .f as determined from the quinol-reduced minus ferri- cyanide-oxidised difference spectrum of the super- natant [7]. Serum from a rabbit which had not been immunised against cytochromef(non-immune serum) did not precipitate either the 37 300-Mr polypeptide or spectrally determined cytochrome,f. The identifica- tion of this protein as cytochromefwas confirmed by the orange-red fluorescence, characteristic of c-type cytochromes [9], which the band emitted when an unstained gel was exposed to ultraviolet light at a wavelength of 366 nm (Fig. 1, lane d).

Labelling ojCytochvome f in vitro

Several preliminary experiments demonstrated that the isolated chloroplast system used in this study had similar characteristics to that used by Blair and Ellis [2]. When the chloroplasts were incubated in the light, incorporation of [35S]methionine into material which was insoluble in hot trichloroacetic acid was complete in 60 min. Incorporation did not occur in the dark unless the chloroplasts were supplied with ATP. D(-)threo-Chloramphenicol (50 pg . ml- ') inhibited the incorporation almost completely, whereas cycloheximide (0.1 mg . m1-I) had no effect. In the presence of ribonuclease A (30 pg . rnl-'), labelling of the trichloroacetic-acid-insoluble fraction was 72 7; of that in a control incubation.

When chloroplasts were incubated in the light with [35S]methionine, the radioactivity incorporated into immunoprecipitated cytochrome f was about

89

a b C d e

Fig. I . Light-driven incorporation of [35S]methionine into c.j'loc./?ronw f hy isolated pea chloroplasts. Chloroplasts ( 1 mg chlorophyll) were incubated in the light for 60 min with 0.136 mCi ["S]methionine (810 Ci . mmol-') and cytochrome/was extracted as described in Materials and Methods. Samples from different stages in the isolation of cytochromef were subjected to electrophoresis on gradient gels of 10-200/, acrylamide. (a) Stained gel of washed chloroplast membranes. (b) Stained gel of aqueous extract containing cytochrome f. (c) Stained gel of immunoprecipitated cytochrome ./. (d) Unstained gel as (c) illuminated with ultraviolet light of 366 nm. (e) Autoradiograph of (c)

0.1 of the incorporation into the washed membrane fraction and about 0.02% of the incorporation into total chloroplast protein (Table 1). Immunoprecipitat- ed cytochrome f from chloroplasts labelled with [3H]leucine contained about 0.5 % of the radioactivity in the washed chloroplast membranes and about 0.16 "/, of that in total chloroplast protein (Table 1).

Following polyacrylamide gel electrophoresis of the immunoprecipitate, autoradiography of the dried slab gel showed a single band of radioactivity exactly coincident with cytochromef'(Fig. 3 , lane e). A similar result was obtained when cytochrome f ' was prepared from chloroplasts labelled with 13H]leucine. Both the radioactivity and the staining cytochromefband were completely digested to low molecular weight material on incubation of the immunoprecipitate with pronase for 4 h at 37°C before electrophoresis. When the small precipitate that was obtained with non-immune serum was analysed by polyacrylamide gel electrophoresis, no staining material or radioactivity was detected in the region of the gel corresponding to cytochrome j ;

90

Table 1 , Ligh/-tlriwri iircoiportr/ioii of /3sS]niethioriinr arid [ 3 H / l c ~ l ~ - cirie ii7to total chloroplast protc,in, chloroplast rnernhruries und y v o - chromr f Pea chloroplasts were incubated in the light for 60 min with ["S]- methionine (1.48 mg chlorophyll with 0.1 mCi) or [3H]leucine (1 mg chlorophyll with 0.2 mCi). Radioactivity incorporated into each fraction was determined as described under Materials and Methods

Fraction Radioactivity incorporated -~ ~ __ ~- -

["'Slmethionine [3H]leucine

dis.jmin ~ ~~ -~

1. Total chloroplast protein 14 190000 7 608 000 2. Washed chloroplast membranes 3433000 2 31 3000 3. Precipitate obtained with anti-

serum against cytochromej 3937 12 690 4. Precipitate obtained with

non-immune serum 906 29 1

Pep t ide h if upp ing

To confirm that the radioactivity co-migrating with cytochromef was an integral part of the protein and not due to some minor contaminating polypeptide, the immunoprecipitated material was analysed using the one-dimensional peptide mapping technique of Cleveland et al. [12]. Papain was selected as the proteolytic enzyme since, in the presence of dodecylsulphate, pure cytochromef from tobacco is resistant to digestion by Staphylococcal V8 protease, cc-chymo- trypsin, trypsin or pepsin (J. C. Gray, unpublished results).

Immunoprecipitated cytochrome ,f was prepared from chloroplasts which had been incubated in the light with ["Slmethionine or [3H]leucine. After sep- aration by polyacrylamide gel electrophoresis, a slice of gel containing cytochromeJ'was placed in a sample well of a second gel and overlayed with papain. Proteolysis took place in the stacking gel to generate a pattern of four major polypeptides (Fig.2, lanes b and d). Two of these bands were derived from papain (Fig. 2, lane a). The largest and the smallest of the four major fragments result from the limit digestion of cytochrome f: In the digestion of cytochrome ,f from chloroplasts labelled with [3H]leucine (Fig. 2, lane b) several partial proteolytic fragments are also visible in the centre of the track. Fluorography of this gel showed that all of the cytochrome f fragments were radioactive (Fig. 2, lane c). Proteolysis of cytochrome ffrom chloroplasts labelled with [35S]methionine produced only one radioactive polypeptide (Fig. 2, lane e) which corresponded with the smaller of the two major cytochrome ffragments (Fig.2, lane d). This was not unexpected, since methionine is a comparatively un- common amino acid and cytochromef'from charlock has been shown to contain only one methionine

a

Synthesis of Cytochrome,f' by Isolated Pea Chloroplasts

b C d e

Fig. 2. P q t i d e aria1j.si.s of' cytochronrc f f iom chloroplc~sts l(~hcllrd in vitro with /35S]methioriiiie o r (3H]I~~uc ine . Immunoprecipitated cytochrome ,f' was obtained from chloroplasts which had been incubated with [35S]methionine (0.9 mg chlorophyll with 65 pCi at 660 Ci . mmol-') or [3H]leucine (2.2 mg chlorophyll with 0.4 mCi at 105 Ci . mmol-I). Peptide mapping was performed as described under Matcrials and Methods. (a) Stained gel of proteolytic fragments produced on self-digestion of papain. (b) Stained gel of proteolytic fragments of cytochrome f from chloroplasts labelled with [3H]leucine. (c) Fluorograph of (b). (d) Stained gcl of proteolytic fragments of cytochrome f' from chloroplasts labelled with [35S]methionine. (e) Autoradiograph o f (d)

residue [6]. These observations indicate that the labelled amino acids are being incorporated into the polypeptide chain of cytochromef:

Effects of' Protein-Synthesis In11ibitor.s

A variety of incubation conditions were used to establish that the observed incorporation of [35S]methionine into cytochromej'was occurring in intact chloroplasts and was not due to contamination with bacteria or cytoplasmic polysomes. Radioactivity was not incorporated into immunoprecipitated cytochrome J; or any other discrete polypeptide, when the chloroplasts were incubated in the dark. The synthesis is therefore light-driven and is unlikely to be occurring in bacteria or on cytoplasmic polysomes. Also, Blair and Ellis [2] have shown that broken chloroplasts are unable to use light energy to drive protein synthesis, suggesting that the chloroplasts responsible for the observed synthesis of cytochrome fwere intact. This was supported by the finding that when ribonuclease A was added to the chloroplast incubation at 30 pg . ml-', labelling of immunoprecipitated cytochrome f was 68% of that in a control incubation without ribonuclease. A closed membrane system, such as the envelope of an intact chloroplast, will act as a barrier to ribonuclease [2].

During incubation of chloroplasts in the light, labelling of immunoprecipitated cytochrome f ' was unaffected by the presence of cycloheximide at a con-

A. Doherty and J . C. Gray 91

centration of 0.1 mg . ml-’. In contrast, incorporation of [35S]methionine into cytochrome ,f was inhibited by 94 in the presence of D(-)thveo-chloramphenicol at 50 pg . ml-’.

DISCUSSION

Cytochrome ,f has been positively identified as a product of protein synthesis by isolated chloroplasts even though the radioactivity incorporated into immunoprecipitated cytochromefaccounted for as little as 0.02 of the total incorporation of [35S]methionine into chloroplast proteins. This very small fraction of the incorporation explains the failure of Eaglesham and Ellis [13], using less sensitive techniques, to detect the synthesis of cytochrome ,f by isolated pea chloro-

Greater incorporation of radioactivity into immunoprecipitated cytochrome ,f was obtained on in- cubating chloroplasts with [3H]leucine. However, even this incorporation (0.16 ‘i: of the total) is undoubtedly an underestimate of the true incorporation of radioactivity into cytochromef; because of the low recovery (1 2 x) of cytochromef from the membranes. In addi- tion, it is not clear what proportion of the newly synthesised cytochrome ,f is correctly incorporated into the thylakoid membrane and so would be extracted by the methods used. Grebanier et al. 1141 have shown that isolated maize chloroplasts are not able to fully integrate several newly synthesised proteins into the membrane structure. The newly synthesised

and p subunits of coupling factor CFI were not fully extractable by methods which normally solubilise the functional enzyme from thylakoid membranes. They have also shown that isolated maize chloroplasts synthesise a membrane-associated protein of molecular weight 34500, which appears by peptide mapping to be a precursor of a 32000-M, thylakoid protein, but the isolated chloroplasts fail to process this precursor. The reason for the failure in processing is not known but the extended amino acid sequence of the precursor may be related to its penetration of a lipid bilayer.

There are now several examples of chloroplast proteins which are synthesised as precursors of higher molecular weight and this is probably related to the need for these proteins to cross membrane systems. The small subunit of ribulose bisphosphate carboxylase, the light-harvesting chlorophyll a/h binding protein and ferredoxin have all been shown to be synthesised as higher molecular weight precursors in the cytoplasm before being transported into the chloroplast [15- 171. There is some evidence that cytochrome f may be located on the inner side of the thylakoid membrane [18] and hence its integration into the thylakoid would involve movement across at least part of the membrane. However, there was no indication from the present study that cytochrome

plasts.

f is produced as a higher molecular weight precursor. Newly synthesised cytochrome,f had the same molecular weight as cytochromef that was present in the chloroplasts before isolation.

It is not clear whether or not isolated chloroplasts are able to attach haem to the newly synthesised apoprotein of cytochrome J The one-dimensional gel electrophoresis system used in this study would not have resolved the complete cytochrome ,f molecule from apocytochromef.

Ellis [4] has suggested two principles for the control of protein synthesis in chloroplasts and mitochondria. The first of these is the multisubunit completion principle. This states that complete proteins are not synthesised on organellar ribosomes but instead, that the role of chloroplast and mitochondria1 ribosomes is to synthesise some of the subunits of multimeric proteins with the other subunits being produced in the cytoplasm. The results presented here seem to contradict this principle since cytochrome f consists of a single polypeptide species [6,19]. However Nelson and Neumann [20] have isolated a discrete membrane particle of molecular weight 120000 from lettuce chloroplasts which contains both cytochrome .f’ and cytochrome h,. Cytochrome ,f’ may therefore be regarded as a subunit of this particle, or indeed of some larger structure in the thylakoid membrane. Because of the difficulty in defining the limits of a subunit complex within a membrane, the multisubunit completion principle may be restricted in its usefulness to proteins which are soluble or only loosely attached to an organelle membrane. Ellis’ second principle is the cytoplasmic control principle which states that cytoplasmic products control organellar protein synthesis but that the converse does not occur. It will be of interest to examine which, if any, cytoplasmic products control the synthesis of cytochromef:

We are extremely grateful to Professor R. J . Ellis and Dr T. a p Rees for their helpful discussion and advice. This work was supported by a grant from the Science Research Council.

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92 A. Doherty and J. C. Gray: Synthesis of Cytochrome f b y Isolated Pea Chloroplasts

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A. Doherty and J . C.Gray, Department of Botany, University of Cambridge, Downing Street, Cambridge, Great Britain, CB2 3EA

Synthesis of Cytochrome f by Isolated Pea Chloroplasts

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