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397 Biochimica et Biophysica Acta, 418 (1976) 397--403 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 98507 PHOSPHORYLATION OF RIBOSOMAL PROTEINS FROM EUKARYOTES IN HOMOLOGOUS AND HETEROLOGOUS CELL-FREE SYSTEMS Ts. VASSILEVA GRANCHAROVA *, T. ARGIROVA GETOVA and T. KRUMOV NIKOLOV Department of Biochemistry, Sofia University,Sofia (Bulgaria) (Received July 28th, 1975) Summary The phosphorylation of ribosomal proteins from eukaryotes in homolo- gous and heterologous cell-free systems has been studied. The ribosomes and protein kinases from yeast (Saccharomyces cerevisiae, strain Bu), wheat (Triti- cum vulgare) and rabbit (Orystolagus cuniculus) have been used. It has been found that five ribosomal proteins incorporate 7 -3 2p from ATP during the incubation of wheat ribosomes with wheat protein kinase. When the phosphorylation of isolated wheat ribosomal proteins was examined more phosphoproteins were detected. These data confirm the suggestion that the ribosomal structure affects the phosphorylation. Probably some ribosomal proteins remain hidden for the action of protein kinase. The results from the crossed experiments show that there is no barrier for phosphorylation of yeast ribosomes with liver protein kinase, of wheat ribo- somes with yeast and liver protein kinases and of liver ribosomes with yeast and plant protein kinases. The wheat protein kinase does not phosphorylate the yeast ribosomes under these experimental conditions. Some differences in the set of phosphoproteins obtained with various protein kinases have been detected. These data suggest that the ribosomal protein phosphorylation is not highly species specific although it is not universal. Introduction The phosphorylation of enzymic and nonenzymic proteins has been impli- cated as an important mechanism for the biochemical regulation [1--3]. The phosphorylation of ribosomal proteins has been studied in detail in mammals * Present address: Department of Chemistry and Biochemistry, Sofia Medical Faculty, Medical Academy, Sofia, Bulgaria.

Phosphorylation of ribosomal proteins from eukaryotes in homologous and heterologous cell-free systems

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Biochimica et Biophysica Acta, 418 ( 1 9 7 6 ) 3 9 7 - - 4 0 3 © Elsevier Scient i f ic Publ i sh ing C o m p a n y , A m s t e r d a m - - P r in t ed in The N e t h e r l a n d s

BBA 9 8 5 0 7

PHOSPHORYLATION OF RIBOSOMAL PROTEINS FROM EUKARYOTES IN HOMOLOGOUS AND HETEROLOGOUS CELL-FREE SYSTEMS

Ts. VASSILEVA GRANCHAROVA *, T. ARGIROVA GETOVA and T. KRUMOV NIKOLOV

Department of Biochemistry, Sofia University, Sofia (Bulgaria)

(Received July 28th, 1975)

Summary

The phosphorylation of ribosomal proteins from eukaryotes in homolo- gous and heterologous cell-free systems has been studied. The ribosomes and protein kinases from yeast (Saccharomyces cerevisiae, strain Bu), wheat (Triti- cum vulgare) and rabbit (Orystolagus cuniculus) have been used.

It has been found that five ribosomal proteins incorporate 7 -3 2p from ATP during the incubation of wheat ribosomes with wheat protein kinase. When the phosphorylation of isolated wheat ribosomal proteins was examined more phosphoproteins were detected. These data confirm the suggestion that the ribosomal structure affects the phosphorylation. Probably some ribosomal proteins remain hidden for the action of protein kinase.

The results from the crossed experiments show that there is no barrier for phosphorylation of yeast ribosomes with liver protein kinase, of wheat ribo- somes with yeast and liver protein kinases and of liver ribosomes with yeast and plant protein kinases. The wheat protein kinase does not phosphorylate the yeast ribosomes under these experimental conditions. Some differences in the set of phosphoproteins obtained with various protein kinases have been detected. These data suggest that the ribosomal protein phosphorylation is not highly species specific although it is not universal.

Introduction

The phosphorylation of enzymic and nonenzymic proteins has been impli- cated as an important mechanism for the biochemical regulation [1--3]. The phosphorylation of ribosomal proteins has been studied in detail in mammals

* Present address: Department of Chemistry and Biochemistry, Sofia Medical Faculty, Medical Academy, Sofia, Bulgaria.

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[4--10]. There are no data to indicate that it occurs in vivo in prokaryotes. Traugh and Traut report the phosphorylation of Escherichia coli ribosomal proteins but it was achieved in vitro with rabbit liver muscles protein kinase ]11,12]. The process does not appear to have been studied in plants except by Trewavas who examined the phosphorylat ion of Lemna minor ribosomal pro- teins in vivo [13]. Recently, two distinct protein kinases, a ribosome associated and one in the ribosome-free supernatant have been detected in yeast [14]. It has been suggested that the ribosomal proteins phosphorylation is widely spread among the eukaryotes and may function in the control of protein syn- thesis [4,7,15].

The successful experiments on phosphorylat ion in vitro of ribosomal pro- teins from [7- 3 2 p] ATP by a cyclic adenosine 3': 5 ' -monophosphate-dependent protein kinase [5,7] allowed the specificity of this process to be studied. Delaunay et al. have not found any tissue specificity [16]. The species speci- ficity of the ribosomal protein phosphorylation in particular has not been examined.

We have studied the phosphorylation of ribosomal proteins in homologous and heterologous cell-free systems using ribosomes and protein kinases from yeast, wheat roots and rabbit liver. The species have been selected from the different regions of the evolutionary scale of eukaryotes. Since there are no available data on phosphorylat ion of plant ribosomes phosphorylation in vitro of wheat root ribosomal proteins has been more thoroughly investigated.

Materials and Methods

Preparation of ribosomes Yeast ribosomes. Yeast was grown on defined medium (0.3% yeast ex-

tract, 5% glucose, 0.1% (NH4)2 SO4, 0.94% sodium citrate and 0.37% citric acid). The cells were harvested during the logarithmic phase of growth by centrifugation, suspended in 1 : 2 (w/v) 0.63 M sorbitol, citrate/phosphate buf- fer, pH 5.8, 30 mM 2-mercaptoethanol, 0.4 mM EDTA and broken by digestion of the cell walls [17]. Protoplasts were collected by centrifugation and lysed in ice-cold dilute salt buffer: 10 mM Tris • HC1, 10 mM MgCl~, 6 mM 2-mercapto- ethanol, pH 7.5. After 30 min the crude lysate was centrifugated at 15 000 X g for 15 min. Ribosomes were isolated from the supernatant by centrifugation at 105 000 X g for 2 h.

Wheat root ribosomes. 60 g root tips from 72oh ethiolated wheat seedlings were ground with 2.5 vol. (w/v) of ice-cold medium containing 500 mM su- crose, 50 mM KC1, 5 mM MgC12 5 mM 2-mercaptoethanol, 100 mM Tris • HC1 buffer, pH 7.5. The homogenate was filtered through a cot ton cloth and spun at 26 000 X g for 30 min. The postmitochondrial supernatant was adjusted to 2% with respect to Triton X-100. For the pelleting of the ribosomes 5 ml of it were layered over 2 ml 1 M sucrose, 500 mM KCI, 5 mM MgC12 5 mM 2-mer- captoethanol, 100 mM Tris • HC1 buffer, pH 7.5 and centrifugated at 105 000 X g for 150 min.

Rabbit liver ribosomes were isolated according to the procedure of Kirsch et al. [18].

The ribosomal preparations were purified by centrifugation through a

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sucrose cushion consisting of 1 M sucrose, 500 mM KC1, 5 mM MgC12,50 mM Tris • HC1, pH 7.5 at 105 000 × g for 2 h.

Preparation of protein kinases The postmitochondrial supernatant (from yeast, wheat roots or rabbit

liver) centrifugated at 105 000 × g for 2 h and passed through a Sephadex G-25 column (equilibrated with 50 mM Tris • HC1, 5 mM 2-mercaptoethanol, pH 7.5) was used as a source of protein kinase. Protein concentration was measured by the method of Lowry et al. [19] with serum albumin as a standard. In some experiments protein kinase from rabbit liver purified by DEAE-cellulose chro- matography has been used [20].

Cell-free system for phosphorylation of ribosomal proteins The incubation mixture for phosphorylation of ribosomal proteins con-

tained in the total volume of 0.4 ml: 0.1 pmol [7-32P]ATP, 0.1 mM ATP, 1 mM dithiothreitol, 50 mM Tris • HCI, 5% glycerol, 4 mg ribosomes (or 2 mg ribosomal proteins) and protein kinase corresponding to 0.4 mg protein. Uni- formly, cyclic adenosine 3':5'-monophosphate was added in all experiments to 1 #M final concentration in spite of the findings [21] that it did not affect the activity of the plant protein kinases, pH was adjusted to 7.5. After 30 min of incubation at 37°C the test tubes were placed in ice and the ribosomes were sedimented at 105 000 × g for 2 h.

Extraction and purification of ribosomal proteins Ribosomal proteins were extracted with 67% CH3 COOH [22] containing

33 mM MgC12 [23], dialyzed for 24 h against distilled water and freeze-dried. The lyophilized proteins were purified from contaminations of RNA and lipids [24].

Fractionation and hydrolysis of ribosomal proteins For one-dimensional polyacrylamide gel electrophoresis the freeze-dried

ribosomal proteins were dissolved in potassium acetate/acetic acid buffer con- taining 8 M urea and 15 mM 2-mercaptoethanol. 2 mg protein per origin were applied on 5% spacer gel. Separation was performed in 15% gel plate (25/15/0.3) at 200 V, 18--20 mA for 72 h in sodium acetate cathode buffer and glycine/acetic acid anode buffer, pH 3.9. Before applying the samples the gel plates were subjected to 6 h pre-electrophoresis.

Two-dimensional electrophoresis was carried out according to the proce- dure of Kaltschmidt et al. [25] slightly modified for small plates (10/10/0.3).

Th e gels were stained with Amido Black 10 B and destained in 10% acetic acid. They were packed into plastic foils, covered with X-ray films and exposed for 6 to 8 weeks. The one-dimensional electrophoretic patterns were traced.

Samples of the lyophilized ribosomal proteins were hydrolyzed for 12 h in 6 M HC1 at 102°C in evacuated ampules. They were then freeze-dried, dissolved in 50% ethanol and fractionated by ascending paper chromatography on Whatman No. 1 strips in solvent consisting of n-butanol/acetic acid/water (4 : 1 : 1, by vol.) [26]. O-Phosphoserine and O-phosphothreonine were used as markers. The chromatograms were subjected to radioautography for 8 weeks

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and were stained with ninhydrin or isatin. The satisfactory chromatographic separation of O-phosphoserine and O-phosphothreonine was achieved after four times development of the Chromatograms. All amino acids rose to the top of the paper strips under these conditions without being well separated while the phosphoamino acids left behind and separated for each other. The low Rf- values (i.e. higher partition coeficient) of O-phosphonoserine and O-phospho- threonine as compared with other amino acids might be the result of their increased hydrophylity. For the identification of the phosphorylated amino acids the fact that they did not stain with isatin was used.

In the experiments [7 -32p] ATP 1700 mCi/mM from Amersham, O-phos- phonoserine and O-phosphothreonine from Koch-Light and cyclic adenosine 3':5'-monophosphate from Harthen were used.

Results and Discussion

The two-dimensional electrophoretic pattern of proteins from wheat root ribosomes is demonstrated in Fig. 1. When the ribosomes were incubated in cell-free system with [7- 32p] ATP and wheat root cell sap five of these proteins became labelled (Fig. 2a). If the ribosomes have been replaced by isolated ribosomal proteins, three proteins in addition incorporate the phosphate (Fig. 2b). The greatest number of phosphoproteins however has been obtained by incubation of isolated plant ribosomal proteins with heterologous protein kinase (from rabbit liver) (Fig. 2c).

Trewavas [13] who examined the in vivo phosphorylation of ribosomal proteins from a sterile higher plant, Lemna minor, detected at least one strong- ly labelled ribosomal protein. We have not examined the in vivo phosphoryla- tion of wheat ribosomal proteins. It is unlikely that the great number (five} of

~ - - ÷

B

Fig. 1. T w o - d i m e n s i o n a l e lec trophoret ic pattern of r ibosomal prote ins f rom wheat roots . Electrophores i s was carried out according to procedure o f Kaltschmidt et al. [ 2 5 ] but o n small plates ( 1 0 / 1 0 / 0 . 3 ) . A corresponds with the schemat ic representat ions in Fig. 2; B is f r o m a different exper iment but has better reso lut ion and does n o t co inc ide c o m p l e t e l y wi th the schemat ic representat ions.

401

~o ~ °°o i o% o 0 ¢ t o% o ° ° o

o o o 0 0 ~ ' 0 o ° , o ~,

0 0 •

Fig. 2. Schematic representat ion o f labelled r ibosomal proteins obtained after incubat ion o f (a), whole wheat ribosomes w i th wheat roo t prote in kina~e and [ ~ - 3 2 p ] A T P in a cell-~ree system (see tex t for details); (b), isolated wheat roo t r ibosomal proteins w i th wheat roo t prote in ldzmse; (e), isolated wheat roo t r ibosomal proteins w i th rabbi t l iver prote in ]dnase. Dark spots represent the labelled proteins.

phosphoproteins observed in our experiments is due to the loosely associated contaminants of non-ribosomal origin since the washing with 0.5 M KC1 re- duces the protein content [4] and even causes loss of some labelled proteins [13]. The available data indicate that in mammals some of the products of cell-free labelling with [7.3 2p] ATP are similar to the products of whole cell labelling with o-[ 3 2p] phosphate but the number of strongly labelled phospho- proteins after injecting o-[ 3 2p] phosphate is reduced [4,7,13].

Apparently, ribosomal protein phosphorylation takes place in plants, but maybe there are some differences in the number of phosphoproteins obtained in vivo and in vitro. In plants as well as in mammals [9] ribosomal structure affects phosphorylation and some ribosomal proteins remain hidden for the action of the protein kinases.

In order to study the species specificity of the ribosomal protein phos- phorylation, crossed in vitro experiments have been carried out with the fol- lowing combinations of ribosomes and protein kinases:

A0, Yeast ribosomes without protein kinase AI, Yeast ribosomes + yeast protein kinase A2, Yeast ribosomes + wheat root protein kinase A3, Yeast ribosomes + rabbit liver protein kinase B0 Wheat ribosomes without protein kinase S l

B2 B3 Co Ci C2 C3

Wheat ribosomes + yeast protein kinase Wheat ribosomes + wheat protein kinase Wheat ribosomes + rabbit liver protein kinase Rabbit liver ribosomes without protein kinase Rabbit liver ribosomes + yeast protein kinase Rabbit liver ribosomes + wheat protein kinase Rabbit liver ribosomes + rabbit liver protein kinase.

The results from these experiments are summarized in Fig. 3. The pho~ phorylated proteins are indicated with arrows on the ground of the ribosomal protein pattern. No detectable phosphorylation has been found in controls without protein kinases (see A0, B0 and Co in Fig. 3).

It can be seen from Fig. 3A that the yeast ribosomes undergo phosphory-

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B C

0 0

2 2

3 3

Fig. 3. P o l y a c r y l a m i d e gel e l e c t r o p h o r e t i c p a t t e r n s o f p r o t e i n s e x t r a c t e d f r o m (A) y e a s t r i b o s o m e s , (B) w h e a t r o o t r i b o s o m e s a n d (C) r a b b i t l iver r i b o s o m e s . The r i b o s o m e s were i n c u b a t e d in cel i - f ree s y s t e m in t h e p r e s e n c e o f "y-32p- labeUed p h o s p h a t e (see M e t h o d s ) . 0, W i t h o u t p r o t e i n k inase ; 1, y e a s t p r o t e i n k inase ; 2, w h e a t r o o t p r o t e i n k ina se ; 3, r a b b i t l iver p r o t e i n k inase . O n e - d i m e n s i o n a l e l e c t r o p h o t e s i s was p e r f o r m e d o n ve r t i ca l 15% p o l y a c r y l a m i d e p l a t e s ( 2 5 / 1 5 / 0 . 3 c m ) f o r 72 h a t 2 0 0 V, 1 8 - - 2 0 m A in g l y c i n e / a c e t i c b u f f e r ( a n o d e ) a n d s o d i u m a c e t a t e / a c e t i c ac id b u f f e r ( c a t h o d e ) , p H 3.9 . A r r o w s i n d i c a t e t he p h o s p h o r y l a t e d b a n d s .

lation under the action of homologous (yeast) and heterologous (rabbit liver) proteinkinases. We could not succeed in the phosphorylation of yeast ribo- somes with wheat protein kinase under any experimental conditions.

The phosphorylation of higher plant ribosomal proteins (group B) is demonstrated on Fig. 3B. All the three protein kinases (yeast, wheat and rabbit liver) phosphorylate the proteins in wheat ribosomes.

An incubation of rabbit liver ribosomes (group C) with the protein kinases from all the tested sources leads to phosphorylation (Fig. 3C).

Comparison of patterns 1 and 3 in Fig. 3A, 1, 2 and 3 in Fig. 3B and 1, 2, 3 in Fig. 3C reveals differences in the phosphoprotein set of the tested ribo- somes obtained with homologous and heterologous protein kinases. The num- ber of strongly labelled bands is greater when rabbit liver cell-sap (or protein kinase) was used. The chromatographic and radioautographic analysis of the

403

labelled proteins show a radioactivity concentrated in O-phosphonoserine and O-phosphonothreonine.

It may be deduced from the presented results that the ribosomal protein phosphorylation is not a highly species specific process: ribosomes from yeast, higher plant and liver could be phosphorylated by heterologous protein kinases. The limitations observed for phosphorylation of yeast ribosomes with plant protein kinase need confirmation.

The number and electrophoretic mobility of the phosphorylated bands vary for yeast, wheat and liver ribosomes. These results correlate with the differences in the ribosomal protein patterns of these organisms. According to Delaunay et al. [27] ribosomal proteins strongly vary among eukaryotes be- longing to genetically very distinct species, but they appear to be remarkably similar over a wide range of species within the same class.

As far as the number of phosphorylated proteins depends on the organiza- tion of the proteins in the ribosomal particles, probably the ribosomal structure exposes only a limited number of available protein serines and threonines to the enzyme action.

The species determined differences detected in these experiments could perhaps provide some explanation when the biological role of ribosomal pro- tein phosphorylation becomes clear.

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