Plant Science Letters, 15 (1979) 387--397 ~) Elsevier/North-Holland Scientific Publishers Ltd.

387

THE RIBOSOMAL PROTEINS OF WHEAT GERM. DISTRIBUTION AND PHOSPHORYLATION IN VITRO

M.M. SIKORSKI, D. PRZYBL and A.B. LEGOCKI

Institute of Biochemistry, UniverMty of A~iculture 60-637 Poznu~ WolyrisI~ 35 (Poland)

W. KUDLICKI and E. GASIOR

Department o f Molecular Biology, Maria Curie Sklodowska University, 20-033 Lublin, Akademicka 19 (Poland)

J. ZALAC and T. BORKOWSKI

Department o f Physiological Chemistry, Medical School 20-123 Lublin, Lubartowska 85 (Poland) (Received December 14th, 1978) (Revision received March 21st, 1979) (Accepted March 21st, 1979)

SUMMARY

Ribosomal proteins from wheat germ were analysed by two-dimensional polyacrylamide gel electrophoresis. The numbers of proteins estimated were 35 in the active 40 S subunit and 44 in the active 60 S subunit. Three hetero- logous kinases from yeast and rabbit brain differing in their substrate specificity were used to phosphorylate in vitro both wheat germ ribosomal subunits. Nine of the ribosomal proteins. $6, $8, $10, $13, S17, L2, L8, L l l and L16 were phosphorylated using yeast protein kinase 3 whereas in the case of rabbit brain protamine kinase almost all basic ribosomal proteins from both subunits were labelled.

INTRODUCTION

The wheat germ cell-free system has become one of the most popular plant systems in the field of eukaryote protein biosynthesis for two reasons. Firstly, it proved to be very efficient for the translation of various synthetic and natural template RNAs. Secondly, it is considered a representative model of higher plants in the study of the overall mechanism of protein assembly and nucleic acid .metabolism. Therefore a detailed characterisation of the individual components of wheat translational apparatus is important for understanding its funcitonal properties and for ascertaining the degree of similarity of protein synthesis in higher plants and other eukaryotes.

388

Our interest in protein synthesis ink plants has led us to undertake a systematic analysis of the ribosomal proteins in wheat germ using two- dimensional polyacryiamide gel electrophoresis. In this paper we present a separation and enumeration of wheat germ individual proteins from both ribosomal subunits using the Sherton and Wool numbering system [1,2]. We al~o report here the results of studies on in vitro phosphoryiation of ribosomal proteins using two protein kinases from yeast [3] and protamhle kinase from rabbit brain (J. Zajac, unpublished data). The phosphorylation in vitro of ribosomal proteins has been reported for a number of eukaryote systems [4--9]. These studies however, have not been reported for higher plants. It was therefore of interest to test the accessibility of wheat ribosomes for phosphorylation by different kinases of heterologous origin and to identify the phosphorylated proteins.

MATERIALS AND METHODS

Isolation of ribosomes and ribosomal subunits Ribosomes were prepared from wheat germ (General Mills, Valiejo, CA,

U.S.A.) by the procedure described earlier [10] with the following modifi- cations: 63 g of wheat germ floated previously with carbon tetrachloride/ cyclohexane (4:1 by vol.) was blended at low speed for 60 s (six blendings of 10 s duration) in 420 ml 50 mM Tris--acetate buffer (pH 8.2) 50 mM KCI, 5 mM Mg-acetate and 5 mM 0-mercaptoethanol containing 0.25 M sucrose. The homogenate was centrifuged twice for 15 rain at 21 000 g and adjusted to pH 7.6 with untreated 1 M Tris. The postmitochondrial super- natant was layered over two 4 ml cushions of 1 M and 0.5 M sucrose in the grinding buffer and centrifuged for 5 h at 35 000 rev./min in a 42.1 Beckman rotor. The ribosomal pellet was dissolved in a dissociation buffer: 20 mM Tris--acetate (pH 7.6), 150 mM KCI, 0.75 mM Mg-acetate, 5 mM 0-mercap- toethanol and 5% sucrose (v/v). The ribosomes were dissociated into sub- units by dialysis against the above buffer for 3 h at 4°C. The subunits were then separated by zonal centrifugation in a Ti14 Beckman rotor using 10--30% linear sucrose gradient in dissociation buffer. After 4 h of centri- fugation at 45 000 rev./min the gradient was fractionated into 10 ml fractions. Figure I shows the absorbance profile of a typical separation of wheat germ ribosomes int~) subunits. The indicated fractions were pooled and the sub- units recovewd by centrifugation at 35 000 rev./min for 12 h in a Beckman 42.1 rotor. The subunit preparations were suspended in a buffer of 50 mM Tris-acetate (pH 7.6) 50 mM KC1, 5 mM Mg-acetate, 3 mM DTT and 10% glycerol (v/v).

Routinely, 300 A260 unit of 40 S subunit and 580 A2~0 units of 60 S subunit were recovered from 1500 A260 units of 80 S ribosomes. As seen from Fig. 1 A and B, the isolated 40 S and 60 S particles sedimented as single, symmetric peaks when analysed on analytical gradients. Both ribosomal subunits were active after recombination in polyphenylalanine polymerisation test.

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Fig. 1. Separation of wheat germ ribosomal subunits by zonal centrifugation. The isolation of ribosomes and centtifugation in the Beckman Ti14 zonal rotor are described in text. The direction of sedimentation is to the right. Fractions 30 to 39 (40 S) and 47 to 56 (60 S) were pooled and the ribosomal particles were recovered by centrifugation as de- scribed. Inset: Sedimentation profiles of separated 40 S (A) and 60 S (B) particles to determine their purity. Six A2~0 units of each subunit from zonal centrifugation were centrifuged in a 10-30% linear sucrose gradient in a Beckman SW 40 rotor at 40 000 rev./min for 210 min. The absorbance prof'fles were recovered continuously with an ISCO density gradient fractionator.

Extraction of ribosomal proteins and two-dimensional gel electrophoresis Ribosomal proteins were extracted from the subunit preparations,

dialysed and lyophilised according to the procedure of Hardy et al. [ 11]. The fraction of acidic proteins of 80 S wheat ribosomes was isolated accord- ing to Richter and MSller [12]. The proteins were separated by two~limen- sional gel electrophoresis according to Kaltschmidt and Wittmann [ 13 ] with minor modifications. For the first dimension, 4% acrylamide in 6 M urea (pH 8.2) was used and for the second dimension, 16% acrylamide in 6 M urea (pH 4.0) was applied. Polyacrylamide gels were stained with Coomassie

390

brilliant blue G-250 and photographed after destaining with acetic acid. For autoradiography of s2P-labelled proteins, the gel plates were placed on What- man No. 1 chromatography paper and dried under vacuum. The dried gel plates were autoradiogzaphed on X-Ray film (Foton, Poland) for 2--8 days.

Protein kinases preparation and in vitro phosphorylation of ribosomal proteins

Kinases I and 3 from Saccharomyces cerevisiae were prepared and purified as described previously [3]. The preparation of protamine kinase from rabbit brain will be published elsewhere (J. Zajac, unpublished). Phosphorylation of ribosomal subunits was carried out in a 100 ~1 reaction volume containing 50 mM Tris--HCl (pH 7.5)15 mM Mg-acetate, 100--150 mM KCI as indicated in figure legends, 6 mM ~-mercaptoethanol, 52 #g ribosomal subunits or 300 ~g caseine or 100/~g pmtamine in control experiments, 5--8 ~g of yeast protein kinases or 0.25/~g of rabbit kinase and 2 nmol of [7-32P] ATP (Amersham-Searle, spec. act. 280 cpm/pmol). The reaction was initiated by adding protein kinase and continued for 20 min at 30°C. Incubation was terminated by the addition of 0.2 ml of 10% trichloracetic acid and the samples were counted for radioactivity as described earlier [7]. For separation on polyacrylamide gels s2P-labelled proteins, approx. 5 mg of each subunit were submitted to phosphorylation in the presence o£ 0.2 mmol of [~.S2p] ATP and 0.5 mg of protein kinase from yeast, or 25 I~g of kinase from rabbit, for 20 rain at 30°C. Labelled subunits were precipitated with 0.5 volume of cold ethanol, resuspended in 20 mM Tris--HCl (pH 7.5) containing 0.1 M Mg-acetate and ribosomal proteins were extracted with 67% acetic acid, dialysed and lyophilised according to the procedure of Hardy et al. [11].

RESULTS AND DISCUSSION

Patterns and general number of wheat germ ribosomal proteins Recently, several studies have been carried out on numbering of individual

ribosomal proteins from different eukaryote sources [1,2,6,14--17]. The general number of proteins separated by two-dimensional polyacx~,lamide gel electrophoresis seems to be similar but not identical. For example, in" rat liver 30 proteins in the 40 S subunit and 39 proteins in the 60 S subunit have been detected [1,15]. In yeast 8accharomyces cerevisiae, the presence of 34 proteins i~l the 40 S subunit and 42 proteins in the 60 S subunit have been demonstrated by Grankowski et al. [14] whereas 29 and 38 proteins, respectively were found by Zinker et al. [6]. These numbers for HeLa cells were 35 and 47 proteins [17].

The total number of ribosomal proteins separated from both purified wheat germ ribosomal subunits is 7"9 (Fig. 2). Thirty five proteins were found to be constituents of the 40 S subunit whereas 44 proteins were derived from 50 S. Under the slightly modified system of Kaltschmidt-Wittmann [13], all the protein spots were well separated, revealing 10 anodicaUy-

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391

migrating, acidic proteins (3 from 40 S and 7 from 60 S). The proteins L43 and L44 are the fastest-migrating proteins and under standard electro- phoretical conditions they run to the cathodic buffer vessel. These proteins are visible when the time of electrophoresis is shortened in the second dimension (from standard 12 h to 8 h).

Although a high degree of evolutionary conservation exists among the ribosomes of different species [ 16] an exact comparison of two-dimensional gel patterns of ribosomal proteins from one organism with those from another organism is difficult as long as the function of the individual proteins remains unknown. Consequently, it is not possible now to unify the nomen- clatu~ for ribosomal proteins from different eukaryote sources. This is partially due to the lack of univocal criteria for identification of 'ribosomal' proteins. For the purposes of this investigation we assume that the purifi- cation of 80 S ribosomes and the procedure of isolation of both sub-units

392

must be based on their funct ional i ty . Therefore, we call "ribosomal' proteins only those proteins which remain part of the ful ly active r ibosome (sub- unit) under rigorous washing condi t ions (i.e. when washing with the buffer containing the highest possible KCI concentrat ions still yields active parti- cles). The funct ional test for the biological activity of the 40 S and 60 S subunits was the polymerisat ion of p-henylalanine plus, in the case o f 60 S, formation of N-Ac-pheny|~l*nyl-puromycin.

The addi t ional difficulties in comparison of the ribosomal proteins from one source wi th those f rom ano ther organism emerge also from the fact tha t the electrophoreticai methods used to analyse the proteins vary signifi- cant ly f rom one laboratory to another . The first a t t empt to un i fy the nomencla ture of rn~mmalian r ibosomal proteins was made during the EMBO Ribosome Workshop in Salamanca, Spain in 1978, the report f rom which is now being prepared by Dr. E.H. McConkey (Boulder, CO, U.S.A.). Similarly, the adopt ion of a uniform nomenclature for r ibosomal proteins f rom higher plants would be o f great importance for more direct analysis of r ibosome structure and genetics.

Phosphorylation of ribosome subunits The initial objective of in vitro phosphoryla t ion of wheat germ ribosomal

subunits was to determine whether the addit ional ribosomal proteins, no t visible af ter staining, can be detected. This could apply first of all to acidic

TABLE I

PHOSPHORYLATION OF WHEAT GERM RIBOSOMAL SUBUNITS USING HETERO- LOGOUS KINASE PREPARATIONS

Incubation mixtures contained in 100 td: 50 mM Tris--HCi (pH 7.5); 15 mM Mg-acetate, 100 mM KCI, 6 mM ~-mereaptoethanol, 52/~g of ribosomal subunit or 9/~g of 80 S acidic proteins, 2 nmol of [*/-s~] ATP and varying amount of kinase preparations: 5//g of yeast kinase 1, 8/~g of yeast kinase 3 or 0.25/~g of rabbit brain kinase. In the controls, 300 llg of caseine or 100/~g of protamine were present. The kinase preparations were added as the last constituents and incubation was carried out for 20 rain at 30°C.

8ubstrate 3~p incorporated (pmole/mg protein substrate)

Kinase 1 Kinase 3 Protamine from yeast from yeast kinase from

rabbit brain

Casein Protamine 40 S subunit 60 S subunit Acidic fraction of 80 S ribosomal proteins

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393

proteins which usually are weakly stained. Moreover, it was also interesting to examine the specificity of three highly purified kinases from yeast and rabbit brain in the phosphorylation of wheat germ ribosomal proteins.

Table I presents the specificity of yeast kinases and protamine kinase from rabbit brain when wheat ribosomal subunits were used as the substrates. It is seen that the subunits are efficiently phosphorylated only with kinase 3 from yeast and kinase from rabbit brain. Kinase 1 demonstrates poor activity although, as it was shown previously, it phosphorylates the yeast ribosomes to a similar extent as kinase 3 [3]. Additionally, kinase 3 strongly phos- phorylates the fraction of acidic proteins extracted from 80 S ribosomes.

Incubation of ribosomal subunits with [732P]ATP in the presence of yeast kinase 3 and brain rabbit protamine kinase resulted in the progressive incorporation of radioactivity into trichloroacetic acid-precipitable material for approx. 15 min (Fig. 3). The extent of phosphorylation to both subunits was proportional to the amount of the enzyme used in the incubation, and with yeast kinase 3 the maximal phosphorylation of each subunit (52 ,g) was observed when 9 ~g of the enzyme was used in the assay (Fig. 4). The reaction was slightly dependent on potassium concentration showing the phosphorylation optimum of the 40 S subunit at 100 mM KCI and 150 mM KCI for 60 S. The yield of phosphorylation was dependent on the amount of [7-32P] ATP used in the reaction (Fig. 5). With the conditions under which the most extensive phosphorylation of the substrate subunits was observed

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(molar ratio ATP:60 S = 80, ATP:40 S = 57), 5.5 pmol of 32p was incor- porated per pmol of 60 S subunits and 2.7 pmol of 32p was incorporated per pmol of 40 S subunit. The apparent Km values for 40 S subunit substrate was 7.7 X 10 -6 M ATP and for 60 S 5.0 X 10 -6 M ATP.

In order to identify the phosphorylated proteins, each subunit was sub- mitted to phosphorylation, then proteins were extracted and radioauto- graphed after separation by two-dimensional gel electrophoresis. Autoradio- graphy of the ribosomal proteins phosphorylated with protein kinase 3 re- vealed 7 protein labelled spots from 40 S (three aidic and 4 basic) and 6 spots from the 60 S subunit (4 acidic intense and two basic faint). (Fig. 6A and B). The exact number of 40 S phosphoproteins has not been settled since the autoradiogram reveals two additional, presumably nonribosomal, radioactive spots (numbered as I and 2) which are not visible on gel after staining, even when the gels were overloaded with 40 S proteins. The location of phosphoprotein $6 may suggest that this protein is equivalent to the phosphorylated protein $6 in mammals [18,19]. This suggestion, however, should be tested by SDS-polyacrylamide gel electrophoresis. It should be noted here that all intense labelled proteins of the 60 S subunit are acidic: L2, Ls, L~ and L~6. The fast migration in the first direction on the poly- acrylamide gel of some of these proteins (L~, LI~, L~6) and their location on the gel slab seem to indicate that two of them might correspond to bacterial L7 and L12 proteins. It has been shown recently that these strongly acidic proteins are phosphorylated animal [20--22] and yeast [6,7] ribosomes. In general, the phosphorylation pattern of wheat germ ribosomal proteins is similar to the one previously shown for yeast ribosomal proteins in the

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presence of yeast kinase (ref. 7 and Kudlicld, unpublished). The yeast basic ribosomal proteins were, however, less intens/vely labelled than the wheat germ basic proteins. It is quite obvious that the estimation of the exact number of phosphorylated ribosomal proteins should be derived from in vivo labelling since phosphorylation in vitro strongly depends on the specificity of the kinase used for the labelling. Brain rabbit protamine kinase, which phosphorylated almost all basic proteins of both ribosom,! subunits (Fig. 6C and D), appeared to be less specific to wheat ribosomes than yeast kinase 3.

Protein kinases associated with ribosomes have already been detected in higher plants [ 23--25]. This is consistent with previously described phosphory- lation of plant ribosomal proteins in vivo [ 26]. Our preliminary experiments indicate that the acidic proteins from wheat germ ribosomes are phos- phorylated in vitro with the protein kinase from the same source to a much smaller extent than with the kinases of heterologous origin. This is probably due to the fact that, as we have estimated separately by digestion with alka- line phosphatase, acidic proteins from wheat germ ribosomes are already significantly phosphorylated in vivo (W. Rychlik et al., unpublished results).

Summarising the above data on phosphorylation in vitro of wheat germ ribosomal proteins in heterologous systems, we believe that, despite some differences, there are several similarities in phosphorylation accessib'flity and patterns among eukaryote ribosomes. Both assayed protein kinases may be very useful for further studies of protein topography in providing a convenient method for labelling of definite protein. Although it is now difficult to determine the physiological role of ribosomal phosphorylation, its conservation during evolution strongly suggests the biological significance of this process.

ACKNOWLEDGEMENTS

This investigation was carried out as Project 09.7 of The Polish Academy of Sciences.

REFERENCES

1 C.C. Sherton and I.G. Wool, J. Biol. Chen~, 247, (1972) 4460. 2 C.C. Sherton and I.G. Wool, J. Biol. Chem, 249 (1974) 2258. 3 W. Kudlicki, N. Grankowski and E. Gasior, Eur. J. Biochem., 64 (1978) 493. 4 C. Ell and I.G. Wool, Biochem. Biophys. Res. Commun., 43 (1971) 1001. 5 C.D. Ashby and S. Roberts, J. Biol. Chem., 250 (1975) 2546. 6 S. Zinker and J.R. Warner, J. Biol. Chem., 251 (1976) 1799. 7 W. Kudlicki, N. Grankowski and E. Gasior, Mol. Biol. Rep., 3 (1976) 121. 8 I. Horak and D. Schiffmann, Eur. J. Biochem., 79 (1977)375. 9 J. H~rbert, M. Pierre and J.E. Loeb, Eur. J. Biochem., 72 (1977) 167.

10 B. Golifiska and A.B. Legocki, Biochim. Biophys. Acta, 324 (1978) 156. 11 J.S. Hardy, G.G. Kurland, P. Voynon and G. Mora, Biochemistry, 8 (1969) 2898. 12 D. Richter and W. M6iler, in D. Richter (Ed.) Lipmann Symposium Energy,

397

Regulation and Biosynthesis in Molecular Biology, Walter de Gruyter, Berlin, 1974, pp. 524--533.

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(1976) 341. 15 H. Welfe, J. Staid and H. Bieika, Biochhn. Biophys. Acta, 243 (1971) 416. 16 C. Gualerzi, H.G. Janda, H. Passow and G. St/iffler, J. Biol. Chem., 249 (1974) 3347. 17 D. Schiffmann and I. Horak, Eur. J. Biochem., 82 (1978) 91. 18 A.M. Grassner and I.G. Wool, J. Biol. Chem., 249 (1974) 6917. 19 D.P. Leader and A.A. Coia, FEBS Lett., 90 (1978) 270. 20 O.-G. Issinger, Biochin~ Biophys. Acta, 477 (1977) 185. 21 D.P. Leader and A.A. Coia, Biochem. J., 162 (1977) 199. 22 A.J. Van Agthoven, J.A. Maassen and H. M~ller, Biochim. Biophys. Res. Commun.,

77 (1977) 989. 23 R.A.B. Keates and A.J. Trewavas, Plant Physiol., 54 (1974) 95. 24 G. Carratu, L.A. Manzocchi, G.A. Lanzani and M. Giannattasio, Plant Sci. Lett., 3

(1974)313. 25 A. Trewavas and B.R. Stratton, in L. Bogorad and J.H. Weft (Eds.), Nucleic Acids

and Protein Synthesis in Plants., Plenum Press, New York, 1977, pp. 309--319. 26 A.J. Trewavas, Plant Physiol., 51 (1973) 760.