Methylation of ribosomal proteins in Tetrahymena pyriformis

Br6ve communication

BIOCHIMIE, 1981, 63, 641-644.

Methylation of ribosomal proteins in Maria Luisa C Y R N E , Claudina Rodrigues P O U S A D A and Donal H A Y E S *

Tetrahymena p'riformis.

Laboratorio de Gen~tica Molecular, lnstituto Gulbenkian de CiOncia, Apartado 14, 2781 Oeiras, Portugal and

(Regue le 6-5-1981, accept[e le 28-5-1981).

* Laboratoire de Chimie Cellulaire, lnstitut de Biologie physico chimique, 13 rue Pierre et Marie Curie, 75005 Paris.

R~sum~.

Summary.

Un procdd~ de double marquage a £t~ utilis~ pour r£vdler la prdsence de protJines m~thyl~es dans les ribosomes cytoplasmiques du protozoaire Tetrahymena pyriformis. Deux des prot~ines basiques et deux des prot~ines acides de la grande sous-unit~ ainsi que deux des protOines basiques de la petite sous-unitO de ces ribosomes contiennent des acides aminds m~thyl~s.

A double labelling technique has revealed the presence of methylated proteins in the cytoplasmic ribosomes o/ the protozoa Tetrahymena pyriformis. Two basic and two acidic proteins o] the large subunit, and two basic proteins of the small rubunit of these ribosomes contain methylated amino acids.

Mots-el~s : Tetrahymena / ribosome / prot~ine / m~thylation.

Key-words: Tetrahymena / ribosome / protein / methylation.

Introduction.

Here we show that Tetrahymena ribosomes contain methylated proteins. Their identification was undertaken as a preliminary to a study of the time course of their formation during ribosome biosynthesis and of their possible role in this process. We show here that four and possibly five proteins of the large subunits and two of the small subunit of the cytoplasmic ribosomes of Tetrahymena pyriformiy are methylated.

In a continuing investigation of ribosome formation in the protozoa Tetrahymena pyri[ormis, we have described the ribosomes [1] and ribosome precursor particles [2] of this eukaryotic microorganism, characterized the protein complements of the mature ribosomes [1, 3] and of their early 80S nuclear precursor particles (in preparation), and proposed a scheme for ribosome biogenesis in Tetrahymena [2]. Ribosomal proteins from eukaryotes are known to be modified by phosphorylation [4, 5] and by methylation [6, 7] and it has been suggested by some authors that these modifications may have important implications for structure - function relationships within the ribosome a n d / o r regulation of ribosome biosynthesis and assembly [8]. Phosphorylation of ribosomal proteins in T. pyri[ormis has already been extensively studied [9, 10] but their methylation has not so far been reported.

© To whom all correspondence should be addressed.

Materials and Methods.

Cultures and labelling. Tetrahymena pyri[ormis CGL was grown as described before [2, 11]. Ribosomes were labelled by adding methyl [3H] methionine (s. act ; 15 Ci/mM ; 4 ~Ci/ml) and [1~C] L-methionine (s. act. 59.2 mCi/mM ; 0.2-0.4 ~Ci/ ml) to the culture medium. Both radioactive products were obtained from Radiochemical Centre, Amersham, U.K. Cells were harvested by centrifugation at 3000 rpm for 15 minutes when the cultures had attained the exponential phase (1,5 × 105 cells/ml).

M . L. Cyrne and coll.

642

Isolation oJ ribosomal subunits.

300 V, 4°C, for both basic and acidic proteins) was carried out at pH 4.6 in gel slabs (165 × 180 × 2 mm) containing 18 per cent polyacrylamide. After electrophoresis, gel slabs were stained in 0.6 per cent Coomassie blue for 45 minutes and destained in a solution containing 5 per cent acetic acid and 15 per cent ethanol. Samples excised from each stained spot were digested with 0.3 ml of 30 per cent (V/V) hydrogen peroxide, at

Ribosomal subunits were isolated as described by Rodrigues-Pousada and Hayes [1]. After sucrose gradient centrifugation, 40S and 60S ribosomal subunits were pooled and precipitated from the appropriate sucrose gradients fractions with 0.7 vol. of ice-cold 96 per cent ethanol in the presence of 8 mM MgC12 [12]. The precipi-

Protein L 1 Lta L 2 L 3 L 4 L 5 L 6

k ~

L99 Lt0 L 11 L 12 L12a L13 L 14 15 LLI6 L17 L 18 LI9 L20 L21 L 22 L23 L24 L25 L26 L27 L 8

3H

14 C

cpm --

cprn --

2409 3165 4 7 17 2362 1254

913 904 1257 696 410

2381

711 712 360 2273 1374 1202 809 --

3380 2783 3590 1551 -4217 2358 3462 4887 3923 192 3240 227 434

520 781 1219 585 -370 461 1008 1548 1042 I04 917 119 148

L~9

4882

• 31

653

k30

L32 L32a / 3 L 3'~4 L35 LL 57 6 L 38 L 39 L 40 L 41 L42

2171

1106 577 2904 3372 1805 742 1405 306 1457 1748 1 1 2~4

i

I

I

i

I

I

7,8

2516 2413 1338 7803 4907 3628 1948 --

3H/14C

Ratio i

i

,

,

lo

I

I

I

I

+lr~-~--

I I I

5i

12a

l

7 0"4-5

_90 ~a~

~ 8"~101212a

IlC~0 OOol 5 17'~ 20_,%.18% %

-

I

2(3 0 3

1

~5o 28 00~,.~ 3 2 a ~-~

I

2 2 3 % ~ , o027 2 9 L~ ~ v 0 3 1 3 3 u0~ 0 3 5

I

36 ° 0 37 038

I

I

03~)4o

I I

I

1716

041

I

~g

348 211 96 8 976 579

I

/

60

S

042

4289 04

94 497 553 59 156

FIG. 1. - - Methylated basic proteins ol the 60S ribosomal subunit o] Tetrahymena pyriformis. Ribosomal proteins of the 60S subunit were isolated from T. pyriJormis uniformly labelled with [methyl-ZH] methionine and [z4C] methionine. The analysed sample contained 1 100 ~g protein, 1.2 × 106 cpm .~H and 0.6 × 106 cpm ]4C. The 3H/14C ratio of the individual protein components was determined as described in Materials and Methods.

tates were collected by centrifugation at 10 000 g for 30 min. at 4°C, and dissolved in 20 mM Tris HC1, 2.5 mM MgC12, 250 mM KCI and 6 mM i~-mercaptoethanol.

60 ° overnight, and the 3H and 14C radioactivities were then counted [16]. Corrections for the incomplete separation of the spectra were applied.

Extraction and polyacrylamide gel electrophoretic analysis oJ ribosomal proteins. Doubly labelled proteins were extracted from ribosomal subunits by the acetic acid method [13], and concentrated by acetone precipitation [14]. Samples of the protein preparations were dissolved in 8 M urea and analysed by a miniaturised version of the two dimensional electrophoresis method described by Kaltschmidt and Wittmann [15]. First dimension electrophoresis was carried out at pH 8.6 in cylindrical 8 per cent polyacrylamide gels 14 cm long and 4 mm in diameter (100 V, for 16 h followed by 1 h at 200 V and 7 h at 300 V, for basic proteins; 100 V for 20 h followed by 200 V for 4 h, 4°C, for acidic proteins). The second dimension (14 h at 100 V, followed by 2 h at 200 V and 6 h at

BIOCH1MIE, 1981, 63, n ° 7.

Results

and Discussion.

It is a l r e a d y w e l l k n o w n t h a t the s t r u c t u r e of c e r t a i n p r o t e i n s is m o d i f i e d by p o s t - t r a n s l a t i o n a l a l t e r a t i o n s of t h e i r c o n s t i t u e n t a m i n o a c i d s . T h e s e a l t e r a t i o n s i n v o l v e m e c h a n i s m s s u c h as h y d r o x y l a tion, p h o s p h o r y l a t i o n , a c e t y l a t i o n a n d m e t h y l a tion, w h o s e b i o l o g i c a l s i g n i f i c a n c e r e m a i n s u n c l e a r a l t h o u g h e v i d e n c e r e l a t i n g to t h e i r f u n c t i o n has been accumulating. In previous work, we analysed the r i b o s o m a l p r o t e i n s f r o m T e t r a h y m e n a pyriformis by t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s [1] a n d re-

Methylated proteins in Tetrahymena ribosomes.

Protein

3H

14 c

epm

cpm 600

2225 6 7 8 9

,,,8 12 13 14 15 16 17 18 i 19 20 I 20a ~21 23 24

333~ 32

~ S 39

1738 1381 504 374 399 510 1912 326

,13, 18~1

437 2553 2529

4047 1452 3037 794 4404

3H/14C

1

I

I

+ ld

_

~F;2"3 ~5

I

,~o~¢~,6 019~25

I

2 1 0 __ 2 6 ( ~ 0 2 4 ~ 0 27 O 0 ~t ~^ 29 .~ Jz

1208

421 "/83 214 1264

1962 941 970

036a

038

111

1066

17260 a 3515

v ~30 ~ 00035 33 34

C ) 37

200 1562 250

3210343 919 1644

028

036

G6

2515

'8

0 0 __a 12 10 11 ~ ) 0 S 1 5

1

"/59 833

5000 794 2454 1917 161 742 1443 1716

2"/ 28 29 30

I 1

5951 4~60 1575 1172 1354 1504 6862

Ratio

643

9

I

40S

I

483 117 1023 260 462 388 1038 286 639 305 362

I

I

FIG. 2. - - Methylated basic proteins of the 40S ribosomal sttbtmit o] T e t r a h y m e n a pyriformis. T h e analysed sample contained 950 ~xg protein, 0.95 x 10 ~ c p m all, and 0.4 × 10 ~; c p m 14C. See legend to figure 1 for details.

Protein S 2 $14 $20 S20a

3H

14 c

cpm 260 173 309 I 55

cpm 53 44 71 37

Ratio L

=

3H/14C

5 I

=

10

=

I

I I J !

2O

20o0

14°

20a

40S Protein L 1 L la L 13 L 14 L 19

3H

14 C

cpm 718 I 36 577 267 442

cpm 197 44 70 68 61

i

I

i

I

5 i

i

i

i

i

10 i

I I

I

l a ~> 130

14%

2d _~

19

60 S FIG. 3. - - Methylated acidic proteins isolated #ore T. pyriformis

40S and 60S ribosomal subunits. Analysed samples contained : 40S ; 1150 ~g protein, 1.5 × 106 c p m 8H, 0.6 × 106 c p m 1~C , 60S, 1350 Ixg protein, 1.2 × 106cpm3H,0.6 × 1 0 6 c p m xaC.

BIOCH1MIE, 1981, 63, n ° 7.

644

M . L. Cyrne and coll.

ported the presence of 31 proteins in the 40S subunit, and 35 proteins in the 60S subunit. Recently, we revised these values to 39 and 42, respectively, after reanalysis of protein preparations, using different electrophoresis conditions, showed that some of the originally reported spots contained multiple proteins [3]. In the present report, in which we study ribosomal protein methylation in Tetrahymena, the revised results are used. We have employed a double labelling procedure introduced by Chang et aI. [15] in which the cells are labelled with [*H-CH3] methionine and [14C] methionine and thus the ratio 3H/14C is higher in methylated than in non-methylated proteins. Figure 1 shows the aH/14C ratio for each of the 60S ribosomal subunit proteins which are positively charged at p H 8.6. It can be seen that two proteins, L15 and L20 have a aH/14C ratio much higher than the average value and therefore contain methylated aminoacids. A third protein, L21, is probably also methylated although its 3H/14C ratio is only slightly higher than the average value. Data concerning the methylated proteins extracted from the 40S subunits are shown in figure 2. Proteins $36 and $36a display a very high ~H/14C ratio, and are clearly heavily methylated. Figure 3 shows the results obtained for the acidic 40S and 60S ribosomal proteins. None of the acidic proteins of the small ribosomal subunit is methylated but two acidic proteins of the large subunit, L13 and L19, contain methylated aminoacids. However, it is worth noting that incorporation of radioactivity into the acidic ribosomal proteins was considerably lower than into basic proteins. This may be due to a low content of methionine in the acidic proteins. These results have been reproduced in six different experiments. They obviously do not exclude the presence of additional slightly methylated ribosomal proteins since the sensitivity of the double labelling method depends upon the number of metbionine residues in a protein and cannot reliably detect the presence of a small number of methyl groups in a protein containg several methionine residues. Our findings are more compatible with those obtained on the methylation of the ribosomal subunit proteins in eukaryotic than in prokaryotic cells. Methylated ribosomal proteins occur mainly in the large subunit of E. coq ribosomes [16] whereas in H e L a cells [7] and yeast [8, 17, 18], as in Tetrahymena, both ribosomal subunits contain several methylated proteins. However, in these two eukaryotic ceils, no methylated acidic proteins were detected, possibly due to the low sensibility of the double-labelling procedure. It is interesting to note that more recent results BIOCH1MIE, 1981, 63, n ° 7.

(Rodrigues Pousada e t a ! . , in preparation) have shown that the methylated ribosomal proteins, L 15 and L20, are present in the 80S nuclear ribosomal precursor particle, but that atl other methylated proteins, including the acidic proteins are absent in this precursor particle. Methylated proteins are thus inserted into the ribosome at different stages during its assembly. Identification of the methylated aminoacids present in the proteins of the mature cytoplasmic ribosomes of T e t r a h y m e n a and in their nuclear precursors is in progress.

Acknowledgement. This work financed in part by N.A.T.O. Research Grant n ° 1670. M.L. Cyrne thanks the Gulbenkian Foundation for fellowship support.

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