Eur. J. Biochem. 99, 133-137 (1979)

Chloroplast Phosphoproteins Phosphorylation of Polypeptides of the Light-Harvesting Chlorophyll Protein Complex

John BENNETT

Department of Biological Sciences, University of Warwick

(Rcceived March 6, 1979)

When isolated, intact chloroplasts of pea (Pisum sativum) are incubated in the light with ["PI- orthophosphate, isotope is incorporated into several polypeptides. Among the most conspicuous phosphoproteins are two which form a very closely spaced doublet on dodecyl sulphate/poly- acrylamide gels and co-electrophorese with the major polypeptide component of the light-harvesting chlorophyll a/b binding complex. Like the light-harvesting polypeptide, the phosphoprotein doublet is bound to thylakoids, sediments with the heavy particles released from thylakoids after digitonin treatment, is soluble in chloroform/methanol and has an apparent molecular weight of about 26000. The doublet also appears in the highly purified light-harvesting chlorophyll a/b binding complex isolated from thylakoids by hydroxylapatite chromatography. I conclude that two polypeptide components of the complex are phosphorylated. One of these components may be the major light-harvesting chlorophyll a/b binding protein.

Pea (Pisum sativum) chloroplasts contain several phosphorylated polypeptides in the molecular weight range 7000- 70 000 [I]. The polypeptides are rapidly labelled in vitro when intact chloroplasts are illumi- nated in the presence of [32P]orthophosphate. Incu- bation in darkness or in the presence of uncouplers inhibits protein phosphorylation and leads to rapid dephosphorylation of the labelled proteins. These results suggest very strongly that chloroplasts contain both a protein kinase (which uses ATP generated by photophosphorylation) and a phosphoprotein phos- phatase.

To understand the significance of chloroplast pro- tein phosphorylation, it is necessary first of all to identify the phosphorylated proteins. Most articles on plant phosphoproteins deal with examples that are also found in animal cells [2]. These include cyto- plasmic ribosomal proteins and nuclear proteins. However, in the case of chloroplast phosphoproteins, there is no analogy with animal cells to help with identification. In this paper, I employ a series of fractionation techniques to establish that the two major chloroplast phosphoproteins derive from the light-harvesting chlorophyll a/b binding complex. The role of this complex in photosynthesis is to absorb photons and then transfer excitation energy to the photocentres of both photosystems [3 - 61.

Abbreviation. Hepes, 4-(2-hydroxyethyl)-l-piperazineethane-

Enzyme. Ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). sulphonic acid.

MATERIALS AND METHODS

Plants

Pea seedlings (Pisum sativum L. var. Feltham First) were grown as described [7]. Leaves were harvested 12 - 20 days after sowing.

Materials

Hydroxylapatite powder (Bio-Gel HTP) was ob- tained from Bio-Rad Laboratories (Richmond, Calif., U.S.A.). Carrier-free [32P]orthophosphate was ob- tained from The Radiochemical Centre (Amersham, Bucks, U.K.). X-ray-sensitive film (Kodirex) was sup- plied by Kodak Limited (Heme1 Hempstead, Herts, U.K.). Miracloth was purchased from Calbiochem (San Diego, Calif., U.S.A.).

Labelling of Pea Chloroplast Phosphoproteins

Chloroplasts were isolated and resuspended in isotonic KCI medium as described previously [7]. A chloroplast suspension (100 pg chlorophyll in 0.5 ml KCI medium) was incubated at 20 "C for 15 min with carrier-free [32P]orthophosphate (100 pCi). Illumina- tion was provided by a single-coil 200-W tungsten lamp (Philips) placed under the glass-bottomed water bath at a distance of 20 cm. After incubation the chloroplasts were diluted with 10 ml of ice-cold 0.35 M sucrose containing 25 mM Hepes/NaOH pH 7.6, 2 mM EDTA and 2 mM sodium D-isoascorbate

134 Chloroplast Phosphoproteins

(buffer A), and centrifuged at 2800 x g for 2 min. The chloroplast pellet was taken up in an appropriate resuspending medium (see below).

Digitonin Fractionation Pea chloroplasts, labelled in vitro with [32P]ortho-

phosphate, were resuspended in buffer B (10 mM Tris pH 8.0, 5 mM 2-mercaptoethanol; 10 ml per 100 pg chlorophyll) and centrifuged at 10000 x g for 10 min to sediment thylakoids. Proteins were precipitated from the supernatant with four volumes of acetone to give a lipid-free preparation of stromal proteins. The thylakoids were taken up in buffer B to give a chlorophyll concentration of 0.5 mg per ml. A 4 % (w/v) digitonin solution in water was added to give a final detergent concentration of 0.5 %. The solution was stirred overnight at 4°C. To half the solution were added four volumes of acetone to precipitate total thylakoid polypeptides, while the remainder of the solution was centrifuged at 30000xg for 30 min to separate light and heavy membrane sub-particles [S]. The proteins of the non-sedimenting or light particles were precipitated by addition of acetone. The pellet of heavy particles was resuspended in the same volume of buffer B and acetone added to precipitate proteins.

Chlorojorm] Methanol E-xtraction The method of Chua and Bennoun 191 was used.

Chloroplasts or washed thylakoids were taken up in a minimal volume of buffer C (0.1 M Na2C03, 0.1 M dithiothreitol; approximately 1 ml per mg chloro- phyll). To 0.5-ml aliquots of the suspension were added 10- 15 ml of freshly prepared chloroform/ methanol (211). After shaking for a few seconds, the extraction was allowed to stand at 4°C for at least an hour. The precipitate which formed was collected by centrifugation and the polypeptides in the super- natant were precipitated overnight at 4°C by the addition of 10- 15 ml of ether. The second precipitate was also collected by centrifugation. The two pellets represented respectively the chloroform/methanol- insoluble and chloroform/methanol-soluble polypep- tides of the chloroplast or thylakoid. For comparison, total chloroplast or thylakoid polypeptides were pre- cipitated from the original organelle or membrane preparation using acetone.

Hydroxyupatite Chromatography

Thylakoids prepared from pea chloroplasts that had been labelled in vitro with [32P]orthophosphate, were fractionated on hydroxyapatite by a modification of the procedure of Kung and Thornber [lo]. Thyla- koids (1.5 mg of chlorophyll) were washed in 50 mM Tris pH 8.0, resuspended in 1.5 ml of the same buffer and solubilized by the addition of sodium dodecyl

sulphate to a final concentration of 1 '%, (w/v). After 30 min at 20 "C, the solution was clarified by centrif- ugation (30000 x g for 10 min) and the supernatant, containing 99 % of the chlorophyll, was stirred with 5 ml of a slurry of hydroxyapatite (1 g, previously washed three times in 50 ml of 10 mM sodium phos- phate pH 7.0). The slurry was centrifuged at 200 x g for 30 s, the supernatant was retained to represent non-adsorbed material, and the pellet was washed successively with 3 x 10 ml of 0.4 M sodium phos- phate pH 7.0, 3 x 10 ml of 0.4 M sodium phosphate containing 1 mM MgC12, 3 x 10 ml of 0.4 M sodium phosphate containing 1 mM MgCl2 and 0.050/0 (w/v) sodium dodecyl sulphate, and 3 x 30 ml of 0.4 M sodium phosphate containing 1 mM MgCI2, 1 % sodium dodecyl sulphate. Washings employing the same buffer were pooled, and visible absorption spectra were taken before and after extraction of the pigments into 15 ml of ether. Proteins were recovered from the delipidated washings by adding solid trichloroacetic acid to a concentration of 10 % (w/v). After standing overnight at 4 "C, the precipitates were collected, taken up in 1 ml of 50 mM Tris, pH 8.0, and reprecipitated by the addition of acetone (4 ml).

Dodecyl SulphatelPolyacrylamide Gel Electrophoresis

Proteins that had been precipitated from an organic solvent were dried under a stream of nitrogen and were thoroughly dispersed in buffer C (protein equiva- lent to 1 mg of chlorophyll requiring about 1 ml of buffer C). To this was added an equal volume 4 % (w/v) sodium dodecyl sulphate, 15 % (v/v) glycerol and 0.1 % (w/v) bromophenol blue and heating to 70 "C for 4 min. Exponential 10 - 30 (w/v) polyacrylamide slab gels were formed as described [ I l l , loaded with 10 - 100 p1 of solubilized protein solution, and electro- phoresed until the tracker dye had reached the bottom of the gel (Fig. 1 and 2) or 50 % longer (Fig. 4). After electrophoresis, the gels were stained with Coomassie Brilliant blue, destained, dried and autoradiographed using X-ray-sensitive film.

Chlorophyll Determination

acetone was performed by the method of Arnon [I21 and in ether by the method described by Strain et al. [13].

Chlorophyll determination in 80

RESULTS

Location of Phosphoproteins within the Chloroplast

Isolated intact chloroplasts incorporate ["PI- orthophosphate largely into thylakoid polypeptides (Fig. 1). Relatively little isotope is incorporated into stromal proteins. As none of the stromal phospho-

J . Bennett

a b c d e f g h 135

Fig. 1 . Sub-chloroplust locution q/phosplzoryluted polypeptides. Intact pea chloroplasts were labelled in virro with [32P]orthophosphate and then fractionated into four reactions: stroma(a, h), total thylakoids (d, e), heavy particles released from thylakoids by digitonin (b, 8). and light particles released from thylakoid by digitonin (c. t). Dodecyl sulphate/polyacrylamide gel electrophoresis was used to analyze the polypeptides of these fractions. Tracks a-d, stained gel. Tracks e-h, autoradiogram. The five bars between tracks d and e indicate, from top to bottom, the following apparent molecular weights: 56000, 54000, 26000, 12000 and 9000

proteins has the same electrophoretic mobility on dodecylsulphate/polyacrylamide gels as the large (54000-MI) and small (12000-MI) subunits of ribulose- 1,5-bis-phosphate carboxylase, the most abundant chloroplast protein is not phosphorylated. Either the chloroplast protein kinase is quite specific for its pro- tein substrates or the carboxylase lacks a suitable phosphorylation site.

The most conspicuous phosphoproteins of thy- lakoids include a 9000-M, polypeptide and two 26000-M, polypeptides which are difficult to resolve electrophoretically [I]. I shall refer to the two proteins of very similar mobility as the 26000-MI phospho- protein doublet. Of the thylakoid phosphoproteins, only this doublet co-migrates with a known stained polypeptide. The doubled co-migrates with the most abundant thylakoid polypeptide which is known to be a component of the light-harvesting chlorophyll a/b binding complex of the photosynthetic membrane.

Digitonin fractionation separates thylakoids into heavy particles enriched in the light-harvesting com- plex and light particles enriched in the coupling factor. Most of the phosphoproteins, including the 26000-M, doublet, are recovered in the heavy particle fraction, strengthening the idea that the light-harvesting pro- tein and the 26000-MI doublet are related. The two 56000-MI subunits of the coupling factor are not phosphorylated.

Solubility in ChloroforrnlMethanol

The light-harvesting polypeptide is known to be highly hydrophobic and to be soluble in chloroform/

methanol solution [9, 141. Fig. 2 shows that the 26 000-MI phosphoprotein doublet is also soluble in this solvent. When 32P-labelled chloroplasts are ex- tracted with chloroform/methanol, the large subunit of the carboxylase is insoluble, while the small sub- unit and the light-harvesting polypeptide appear in the organic phase, along with the 26000-M, phospho- proteins. The hydrophobicity of the complete poly- peptide is sufficient to counteract the hydrophilicity of the phosphorylated amino acid, known from the first paper in this series to be a threonyl residue [l]. Thus, in sub-chloroplast location, in hydrophobicity and in apparent molecular weight, the 26000-MI doublet closely resembles the light-harvesting chloro- phyll a/b binding protein.

Fractionation of Hydroxyapat it e

Direct evidence for association of the 26000-M, phosphoprotein doublet with the light-harvesting complex comes from purification of the complex by hydroxyapatite chromatography [3,10]. When thy- lakoids labelled with 32P are solubilized in dodecyl sulphate and bound to hydroxyapatite, batch-wise elution results in substantial recovery of applied chlorophyll (76 o/,) and enrichment for the light- harvesting chlorophyll a/b binding complex (chloro- phyll alchlorophyll b ratio = 1.55) (Table 1).

The buffer which brings off this particular complex contains 0.4 M sodium phosphate, 1 mM MgC12 and 0.05 sodium dodecyl sulphate. The complex can be eluted, but in less pure form, if the 0.4 M sodium phosphate wash (Table 1) is followed immediately by

Chloroplast Phosphoproteins

0.4-

0.3 - W " . c ; 02- 0

Ll Q

01 -

136

a b c d e f

Fig. 2. Soluhility oJ~c~hloro~~lu,st polypeptides in chlorofbrmjnzerhunol. Intact pea chloroplasts were labelled in vitro with [3ZP]orthophos- phate and then were extracted with chloroform/methanol. Dodecyl sulphate/polyacrylamide gel electrophoresis was used to analyze the polypeptide composition of total chloroplasts (a, b), chloro- form/methanol-insoluble fraction (c, d), and chloroform/methanob soluble fraction (e,f). Tracks a , c and e, stained gel. Tracks b, d and f, autoradiogram. The 26000-M, light-harvesting polypeptide is marked with an arrow

Table 1. Hydroxyaputite chromatography of dodecyl-sulphate-treuted thylukoids A total of 1330 pg of chlorophyll (chlorophyll u/chlorophyll b = 2.39) was stirred into a slurry of hydroxyapatite (1 g) and eluted batchwise a sequence of buffers containing 0.4 M sodium phos- phate, pH 7.0. Recovery amounted to 1005 pg of chlorophyll (767")

Eluting buffer Chloro- Chloro- phyll phyll a/

chloro- phyll b

Pg - Unbound 10

0.4 M sodium phosphate 318 1.79 + 1 mM MgClz 31 ~

+ 1 mM MgCIz + 0.05% sodium

+ 1 mM MgClz + 1 dodecyl sulphate 344 1.55

dodecyl sulphate 302 2.56 sodium

a The ratio could not be determined accurately.

a wash with 0.4 M phosphate, 0.05 % sodium dodecyl- sulphate. Elution can occur in high salt and low detergent and does not require MgC12. However, the intervening wash with 0.4 M phosphate, 1 mM MgClz is retained to enhance the purity of the complex. If higher detergent concentrations are employed to elute the complex, purity is again reduced.

The visible absorption spectrum of the light- harvesting chlorophyll a/h binding complex as pre- pared by this batch procedure is shown in Fig.3.

0 1 LOO 500 600 700

Wavelength (nm)

Fig. 3. Visible absorption spectruni o j light-hurvrsting chlorophyll a/b binding complex. 3ZP-labelled pea thylakoids were treated with dodecyl sulphate and fractionated on hydroxyapatite. A fraction eluted by 0.4 M sodium phosphate pH 7, 1 mM MgClz and 0.05 "/, sodium dodecyl sulphate, gave the indicated visible absorption spectrum in the same buffer. The arrowed peak (at 675 nm) and shoulders (at 653 nm and 485 nm) indicate the presence of chloro- phyll u, chlorophyll h and cdrotenoids, respectively

a b C d

Fig. 4. Polypeptide composition qJ pcu th~lukoicl .~ und the light- hurvesting chlorophyll a/b binding complex. Dodecyl sulphate/poly- acrylamide gel electrophoresis was used to analyze 32P labelled thylakoids (a, b) and 3ZP-labelled light-harvesting complex (c, d) prepared from thylakoids by hydroxyapatite chromatography. Tracks a and c, stained gel. Tracks b and d, autoradiogram. Tracks a and c each contained the polypeptides equivalent to 5 pg chlorophyll h

The presence of chlorophyll a, chlorophyll b and carotenoids is indicated by the absorption peaks or shoulders at 675 nm, 653 nm and 485 nm, respec- tively. The spectrum closely resembles that recently reported by Anderson et al. [I51 for an electrophore- tically purified light-harvesting chlorophyll a/b binding complex from spinach, although the chlorophyll a/ chlorophyll b ratio reported here for the pea complex is higher than the ratio reported for spinach (a value of 1.28).

J. Bennett 137

Does this preparation of the complex contain the phosphoprotein doublet '? Fig. 4 shows the polypeptide and phosphoprotein composition of total thylakoids and the purified complex. In both cases, the gel is loaded with a volume of solubilized protein equivalent to 5 pg of chlorophyll b. Thus any staining polypeptide or any phosphoprotein which co-purifies with chloro- phyll b during hydroxyapatite chromatography should be equally abundant in the two tracks.

The complex contains five staining polypeptides (Fig. 4). On a chlorophyll b basis, these polypeptides are as abundant in the complex as in the original thylakoid membrane. The most abundant is the 26000-M, polypeptide. Of the other polypeptides, one possesses a slightly higher molecular weight than the major band while the other three have molecular weights in the 20000-25000 range. The complex also contains the two elements of the 26000-M, phos- phoprotein doublet. Again, the equal labelling of these components in the two tracks argues strongly that they are true constituents of the complex. One phos- phoprotein co-electrophoreses with the major poly- peptide while the second phosphoprotein migrates with the less abundant 25 000-M, polypeptide.

DISCUSSION

The results presented in this paper establish that the 26000-M, phosphoprotein doublet of the chloro- plast is derived from the light-harvesting chloro- phyll a/b binding complex. The doublet is bound to thylakoids, is recovered in the heavy digitonin mem- brane sub-particles, is soluble in chloroform/methanol, and has the same electrophoretic mobility on dodecyl sulphate/polyacrylamide gels as the polypeptide com- ponents of the light-harvesting complex. Moreover, light-harvesting complex purified from thylakoids by hydroxyapatite chromatography contains both phos- phoproteins. The results also suggest very strongly that one of the two 26000-M, phosphoproteins is the major polypeptide component of the complex while the other phosphoprotein is a less abundant poly- peptide of the complex and has a very slightly lower molecular weight.

The polypeptide composition of the light-har- vesting complex is controversial. There is no con- sensus as to the number of polypeptides in the complex, their apparent molecular weights, and their relative abundance [3,16,17]. Moreover, the numbers of mole-

cules of chlorophyll a , chlorophyll b and carotenoiti\ per complex are also in dispute [3,15,16]. As differcnt research groups study different species of plant, isolate the complex by different methods and employ different electrophoretic procedures to estimate molecular weights, the lack of agreement is not unexpected. The results presented above indicate that the light-har- vesting complex of pea, when isolated by rapid hydroxyapatite chromatography, consists of five poly- peptides, of which two are phosphorylated.

The first paper in this series [l] reported that phos- phorylation of the 26000-M, doublet is reversible, at least in vitro. Although new examples of protein phos- phorylation are regularly being discovered, especially in animal cells, it is impossible to assess the physio- logical significance of these reactions until the cellular functions of the polypeptides in question have been established. The results presented in this paper, having identified the 26000-M, phosphoprotein doublet as being derived from the light-harvesting apparatus of the photosynthetic membrane, suggest that we should begin to look for effects of protein phosphorylation on excitation transfer and electron transport.

1 thank Nick Lengden and Richard Williams for technical assistance.

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S . Bennett, Department of Biological Sciences, University of Warwick, Coventry, Warwickshire, Great Britain, CV4 7AL