220

Biochimica et Biophysica Acta, 4 7 9 ( 1 9 7 7 ) 2 2 0 - - 2 3 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99046

METHYLATION OF RIBOSOMAL PROTEINS IN HeLa CELLS

C A R L O S J. G O L D E N B E R G a n d G E O R G E L. E L I C E I R I

Department o f Pathology, St. Louis University School o f Medicine, St. Louis, Mo. 63104 (U.S.A.) (Received March 28th, 1977) ( R e v i s e d m a n u s c r i p t received M a y 2 5 t h , 1 9 7 7 )

Summary The in vivo post-translational methylation of individual ribosomal proteins has been studied. Methylation was detected by: (1) 3H labeling of cells incubated with a mixture of [Me-3H]methionine plus [~SS]methionine, the incorporation of the 3H-labeled amino acid methionine being corrected by the asS uptake, (2) 3H incorporation from [Me-3H]methionine during inhibition of protein synthesis with cycloheximide, and (3) amino acid analysis. A minimum of seven methylated ribosomal proteins were detected. The levels of methylation differed among several proteins (and possibly between two sites of the same protein) in relationship to suppression of protein synthesis or ribosome formation. The methylation of some ribosomal proteins (most of Nos. 30, 62, 63 and part of 18 and 42) occurred on ribosomes that were being processed, i.e., it was inhibited when ribosome formation was suppressed with actinomycin D. The methylation of other ribosomal proteins occurred in mature ribosomes (protein No. 38 and apparently another site of 42), as it was not suppressed by actinomycin D. In another experiment cells were both pulsed with [Me-3H]methionine and chased with an excess of non-radioactive methionine during suppression of protein synthesis (in the presence of cycloheximide); when protein synthesis was allowed to recover protein 18 was preferentially labeled (methylated), in the continued presence of an excess of non-radioactive methionine. The level of methylation of protein 30 as well as one site of protein 42, both apparently methylated during ribosome processing, seemed to increase in the

Abbreviations used: 60-S, large cytoplasmic ribosomal subunit; 40-S, small cytoplasmic ribosomal subunit: 55-S nucleolar ribosomal precursor, precursor to the large cytoplasmic ribosomal subunit.

221 absence of protein synthesis. The methylation of protein 42 in mature ribosomes did not appear to be affected by suppression of protein synthesis. In general, the ribosomal proteins which appeared to be methylated in nascent ribosomes contained methyllysine, while methylarginine was found in those methylated in mature ribosomes.

Introduction The presence of methylated ribosomal proteins in bacterial ribosomes has been reported by Terhorst et al. [12], Alix and Hayes [3] and Chang et al. [4]. An analogous phenomenon has more recently been indicated for mammalian cells. Labeling HeLa cells with a mixture of [3SS]methionine plus [Me3H] methionine, Vandrey et al. [5] observed that some ribosomal proteins showed high 3H/3SS ratios, suggesting that they were methylated. Chang et al. [6] found that the methylated amino acids in unfractionated ribosomal proteins from HeLa cells were mainly N G, NG-dimethylarginine, e-N-trimethyllysine, and e-N-dimethyllysine. In this report we have continued our study of the methylation of individual ribosomal proteins in HeLa cells, including their different dependence on ribosome maturation and protein synthesis. Materials and Methods The conditions of cell culture and labeling, isolation of ribosomes and ribosomal subunits, and extraction of ribosomal proteins were as previously described [5], except that the puromycin incubation was carried out in 0.5 M KC1, 2 mM magnesium acetate, 50 mM Tris • HC1, pH 7.4, 1 mM dithiothreitol, and 1 mM puromycin. When indicated, the ribosomal subunits were not separated by sucrose gradient centrifugation, but were pelleted through a cushion of 0.5 M KC1 and 5 mM MgC12 [7]. The ribosomal proteins were separated by the two-dimensional gel electrophoresis method of Mets and Bogorad [8]. After electrophoresis, gel slabs were stained and destained [9], and individual spots were cut out with a scalpel. These gel pieces were then completely digested in sealed glass vials, using 0.5 ml of 30% hydrogen peroxide and ammonium hydroxide (99 : 1) per vial at 55°C overnight [10]. Each digested gel piece was counted in a liquid scintillation counter after addition of 10 ml of scintillation mixture containing a 3/2 ratio of toluene and ethylene glycol m o n o m e t h y l ether [11], with a final concentration of 0.4% Omnifluor (New England Nuclear). The numbering system used to identify the various ribosomal proteins, using the gel electrophoresis system of Mets and Bogorad [8], was as previously shown [12]. The conditions for elution of ribosomal proteins from two-dimensional gel electrophoresis, dialysis, lyophilization, hydrolysis with HC1, and amino acid analysis by paper electrophoresis in a buffer containing pyridine/acetic acid/ water (25 : 1 : 225), pH 6.5 [13], were as indicated by Chang and Chang [14]. 55-S nucleolar ribosomal precursor particles were isolated as described by Warner and Soeiro [15].

222 Results

General assay for methylation Table I shows an experiment like that of Vandrey et al. [5], in which cells were incubated for 24 h with a mixture of L-[Me-3H]methionine and L-[3sS] methionine, except that in this case the ribosomal subunit proteins were analyzed by the gel electrophoresis method of Mets and Bogorad [8]. At least three proteins (numbers 18, 30, and 62) have higher 3H/3SS ratios than the rest of the ribosomal proteins.

TABLE I INCORPORATION OF 3H AND 35S INTO INDIVIDUAL RIBOSOMAL PROTEINS FROM SEPARATED RIBOSOMAL SUBUNITS OF HeLa CELLS INCUBATED FOR 24 h WITH L-[Me-3H]METHI ONINE AND L-[ 35 S] METHIONINE Hela cells were resuspended at 4 • 106 eells/ml in methionine-free Joklik modified minimum essential medium (Grand Island Biological Co., Grand Island, N.Y.). A mixture of 2.5 mCi of L-[Me-3H]methionine (11 Ci/mmol) and 0.5 mCi of L-[35S]methionine (166 Ci/mmol) per 100 ml of culture was added and the cells were incubated for 15 rain at 37°C. Then, 0.I vol. of regular medium was added, 150 rain later the cells were diluted to 2 • 10 S ceils/ml with regular medium and they were harvested 24 h later. The procedures in the remaining steps are indicated in Materials and Methods. The radioactivity recovered from the spots cut from the second dimension gel slabs represented 40--60% of the input radioactivity loaded on the first-dimension gels, in this and the following experiments. Spot

3H

35S

3H/35S

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33

Spot

3H

35S

3H/35S

805 715 6523 2623 2080 2344 1476 2456 1384 2665 2018 440 500 328 548 563 1132 438 118 224 574 164 4357 1419 1215 131 439 1654 1264 606 1658

122 114 952 396 221 314 197 328 207 405 309 59 74 50 82 89 167 59 21 38 89 22 619 219 177 20 73 248 57 75 237

6.5 6.2 6.8 6.6 9.4 7.4 7.4 7.4 6.6 6.5 6.5 7.4 6.7 6.5 6.6 6.3 6.7 7.4 5.6 5.8 6.4 7.4 7 6.4 6.8 6.5 6 6.6 22 8 6.9

No. 1256 187 459 2308 121 1701 2769 2022 1303 812 3238 334 412 4243 349 480 641 9115 1411 1447 2018 1871 1561 151 525 574 750 427 305 355 716 1313

123 22 60 336 17 256 388 295 197 123 472 47 69 563 48 77 107 411 619 210 274 267 244 19 81 88 119 64 23 64 114 199

i0 8.5 7.6 6.8 7.1 6.6 7.1 6.8 6.6 6.6 6.8 7.1 6.9 7.5 7.2 6.2 5.9 22.1 7.7 6.8 7.3 7. 6.3 7.9 6.1 6.5 6.3 6.6 13 5.5 6.2 6.5

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

223

Amino acid analysis Chang et al. [6] have reported that the methylated amino acids in unfractionated ribosomal proteins from HeLa cells are mainly N G, NG-dimethylarginine, e-N-trimethyllysine, and e-N-dimethyllysine. We incubated cells with L-[Me-3H] methionine, and the extracted ribosomal proteins were then separated by twodimensional gel electrophoresis. Individual ribosomal proteins were then

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Fig. 1 . P a p e r e l e c t r o p h o r e s i s p a t t e r n s of a m i n o acids f r o m H C l - h y d r o l y z e d i n d i v i d u a l r i b o s o m a l p r o t e i n s f r o m H e L a cells i n c u b a t e d w i t h L - [ M e - 3 H ] m e t h i o n i n e . Cells w e r e i n c u b a t e d for 24 h w i t h [ M e - 3 H ] m e t b i o n i n e as in T a b l e I, a n d the s a m p l e s w e r e t a k e n u p to t h e gel e l e c t r o p h o r e s i s step as i n d i c a t e d in Materials a n d M e t h o d s . T h e i n d i v i d u a l s p o t s were e l u t e d , d i a l y z e d , l y o p h i l i z e d , h y d r o l y z e d w i t h HC1 and a n a l y z e d b y p a p e r e l e c t r o p h o r e s i s in a b u f f e r c o n t a i n i n g p y r i d i n e / a c e t i c a c i d / w a t e r ( 2 5 : 1 : 2 2 5 ) , p H 6.5 [ 1 3 ] , as d e s c r i b e d by C h a n g a n d C h a n g [ 1 4 ] . T h e m i g r a t i o n of lysine ( L ) , arginine ( A ) , a n d m e t h i o n i n e (M), c o e l e c t r o p h o r e s e d w i t h e a c h u n k n o w n s a m p l e , is s h o w n . I n a d d i t i o n , t h e f o l l o w i n g m e t h y l a t e d a m i n o acid s t a n d a r d s w e r e c o e l e c t r o p h o r e s e d in (a): e - N - t r i m e t h y U y s i n e ( T M L ) , e - N - m o n o m e t h y l l y s i n e ( M M L ) , e - N - d i m e t h y l l y s i n e ( D M L ) , N G, N G - d i m e t h y l a r g i n i n e ( u D M A ) , N G, N ' G - d i m e t h y l a r g i n i n e ( s D M A ) , a n d 3 - m e t h y l h i s t i d i n e ( 3 - M H ) . T h e s o u r c e a n d h a n d l i n g of these s t a n d a r d s were as i n d i c a t e d b y C h a n g a n d C h a n g [ 1 4 ] . In the g r a p h s f o r p r o t e i n s p o t s 1, 18 a n d 38, t h e scale o n t h e right side applies to the b r o k e n line p o r t i o n of t h e graph. T h e n u m b e r inside e a c h s e c t i o n i n d e n t i f i e s the i n d i v i d u a l p r o t e i n s p o t a n a l y z e d .

224 eluted, dialyzed, lyophilized, hydrolyzed, and their amino acids analyzed by paper electrophoresis, as described by Chang and Chang [ 14]. Fig. 1 shows that all the ribosomal proteins which had a high 3H/3SS ratio in Table I contained 3H peaks whose relative electrophoretic mobility was either compatible with being methylarginine (spots 1 and 38) or methyllysine (spots 30 and 62), while both peaks appeared in protein spot 18. Protein spot 63, which did not have a high 3H/3SS ratio, nevertheless contained a 3H band migrating as methyllysine. No methylated amino acids could be detected in the protein spots with low 3H/3SS ratios that were tested.

Metabolic stability Table II shows that the 3H/~SS ratios in individual ribosomal proteins did not differ appreciably between cells incubated briefly with this isotope mixture, and then chased for 95 min and 24 h with an excess of non-radioactive methionine, indicating metabolic stability of these methylations. Relationship to ribosome formation Methylation in nucleolar precursor particles. If some ribosomal proteins are methylated as part of the process of ribosome maturation, it might be possible to find them methylated at the stage of nucleolar ribosomal precursor particles. The precursor to the large ribosomal subunit, 55-S nucleolar particle, was prepared as indicated by Warner and Soeiro [15] from cells incubated briefly with the mixture of [Me-3H]methionine and [3SS]methionine. It was then mixed with non-radioactive mature large subunits, their proteins were extracted, and then analyzed by gel electrophoresis. The stained non-radioactive proteins were cut out, digested and counted. Only large subunit spot 30 showed a high 3H/3SS ratio, as demonstrated in Table III, suggesting that at least it was already methylated at the stage of the 55-S nucleolar ribosomal precursor.

T A B L E II I N C O R P O R A T I O N OF 3H A N D 35S I N T O I N D I V I D U A L R I B O S O M A L P R O T E I N S F R O M H e L a CELLS PULSED WITH L-[Me-3H]METHIONINE AND L-[35S]METHIONINE AND THEN CHASED F O R 9 5 rain A N D 24 h W I T H A N E X C E S S O F N O N - R A D I O A C T I V E M E T H O N I N E A n a l i q u o t o f the cells in t h e e x p e r i m e n t in T a b l e I was h a r v e s t e d 1 1 0 rain a f t e r i s o t o p e a d d i t i o n . T h e 3 H / 3 5 S r a t i o s of the 24 h a l i q u o t f r o m T a b l e I are s h o w n h e r e again. P r o t e i n s f r o m s e p a r a t e d s u b u n i t s were analyzed. Spot No.

1 18 30 38 42 53 60 62

24 h

95 min

3H

35 S

3H/35 s

3H135S

443 2087 350 426 536 74 122 695

48 110 25 51 77 10 19 28

9.2 18.9 14.0 8.3 5.9 7.4 6.4 24.8

10.0 22.1 13.2 9.4 6.6 5.8 6.0 22.1

225 T A B L E III I N C O R P O R A T I O N O F 3 H A N D 35S I N T O I N D I V I D U A L R I B O S O M A L P R O T E I N S I N 55-S N U C L E O LAR RIBOSOMAL PRECURSOR PARTICLES FROM CELLS INCUBATED WITH L-[Me-3H]METHI ONINE AND L-[35SlMETHIONINE Cells w e r e i n c u b a t e d as in T a b l e I e x c e p t t h a t t h e y w e r e h a r v e s t e d 4 5 rain a f t e r i s o t o p e a d d i t i o n . n u c l e o l a r r i b o s o m a l p r e c u r s o r p a r t i c l e s w e r e i s o l a t e d e x a c t l y as d e s c r i b e d b y W a r n e r a n d S o e i r o t h e y w e r e t h e n m i x e d w i t h n o n - r a d i o a c t i v e 60-S m a t u r e c y t o p l a s m i c s u b u n i t s , t h e e x t r a c t e d p r o t e i n s a n a l y z e d b y t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s a n d t h e gel slabs w e r e s t a i n e d a n d d e s t a i n e d . T h e g i v e n by t h e m a t u r e s u b u n i t w e r e c u t o u t , d i g e s t e d a n d c o u n t e d . Spot

3H

35S

3H/35S

812 1133 1669 972 984 495 325 950 776

130 176 233 145 152 28 50 145 118

6,2 6,4 7,1 6.5 6.4 18.0 6.5 6.5 6.5

55-S [15]; were spots

No. 6 7 11 22 23 30 42 43 44

Effect of its suppression. One would also expect that if some ribosomal proteins are methylated during ribosome processing, their methylation would be suppressed when ribosomal RNA synthesis, and therefore ribosome formation, is inhibited with actinomycin D. Such an experiment is shown in Table IV, where the protein spots which had low aH/3SS ratios in the control had marked decreases in incorporation of both 3H and 3sS in the presence of actinomycin D (spots 10, 24, 57). Protein 18 had a high 3H/3SS ratio in the control, while with

TABLE IV EFFECT OF ACTINOMYCIN D ON THE RELATIVE INCORPORATION OF 3H AND 35S INTO INDIVIDUAL RIBOSOMAL PROTEINS FROM CELLS INCUBATED WITH L-[Me-3H]METHIONINE AND L-[35S]METHIONINE One half of the cells was incubated with aetinomycin D (0.1 }ag/ml) for 1 h at 37°C, and the other half without it. [Me-3H]Mcthionine and [35S]methionine were added at half the concentration of the experiment in Table I; 15 min later non-radioactive methionine was added up to the final concentration of the regular medium, and 150 rain later the cells were harvested. Proteins from separated ribosomal subunits were analyzed. Spot No.

1 10 18 24 30 38 42 57 63

Actinomycin D

Control

3H

35 S

852 71 369 74 97 1198 348 67 73

169 14 43 14 4 204 15 14 9

3H/35 S 5.0 8.6

5.8 23.0

3H

35 S

3H/35S

2293 2284 4564 2367 1198 7903 2414 4632 1651

490 597 554 593 121 1645 407 1090 353

4.6 3.8 8.23 4.0 9.9 4.8 5.9 4.2 4.67

226 actinomycin D, where isotope incorporation was markedly decreased, a similar ratio was maintained, suggesting that most of the methylation of spot 18 occurs in newly made ribosomes. The marked decrease in 3H incorporation into spot 30 in the presence of actinomycin D also suggests that most of its methylation occurs in newly synthesized ribosomes. The disproportionately higher decrease in 3sS incorporation than 3H incorporation into spot 42 in the presence of actinomycin D would imply that some methylation may be occurring under these circumstances, that is, methylation in previously made ribosomes. A small, but reproducible, increase in the 3H/3SS ratio in the sample of protein 38 from actinomycin D-treated cells suggests the existence of methylation of 38 in previously completed ribosomes. The limited decrease in [3SS]methionine incorporation into protein spot 1 in the presence of actinomycin D reflects the resistance to actinomycin D of protein I incorporation into ribosomal particles, which we have recently described [ 12 ]. A rather similar situation appears to occur with protein 38.

Effect of inhibition of protein synthesis In the absence of protein synthesis (e.g. cells exposed to cycloheximide), 3H labeling of ribosomal proteins after incubation with [Me-3H]methionine would reflect methylation only, and apparently as a modification independent of protein synthesis. If ribosome formation is also suppressed (with actinomycin D), 3H labeling of ribosomal proteins would indicate that this methylation is occurring in previously made ribosomes. In the next experiment cells were labeled for 24 h with ~4C-labeled amino acids to correct later for variations in yields of individual proteins recovered, and then pulsed with [Me-3H]methio nine and chased with an excess of non-radioactive methionine, without drugs, in the presence of actinomycin D, cycloheximide, or actinomycin D plus cycloheximide. Table V shows that in the presence of cycloheximide 3H incorporation could be detected in the methylated spots (spots 1, 30, 38, 62, 63), while it decreased to background levels in the nonomethylated spots (spots 8, 21 -23 25, 28, 37, 44, 60). Three proteins that did not show methylated amino acids in Fig. 1 (19, 43 and particularly 42) still incorporated 3H in the presence of cycloheximide, suggesting methylation at least in the absence of protein synthesis. When both protein synthesis and ribosome formation, were suppressed by the simultaneous addition of cycloheximide plus actinomycin D, a marked decrease in the 3H/14C ratio with respect to that with cycloheximide alone could be seen in proteins 30 and 62, suggesting that their methylation occurs mainly in newly made ribosomes. A marked difference between these two ratios could also be seen in protein 42, but a significant number of 3H counts were still incorporated in the presence of cycloheximide plus actinomycin D, suggesting that protein 42 has two types of sites, one which is methylated in previously made ribosome and another one that is methylated in new ribosomes. The methylation site of protein 42 is previously made ribosomes did not seem to be affected by suppression of protein synthesis, as the 3H/~4C ratios were similar in the presence of actinomycin D alone and cycl0"heximide plus actinomycin D. From the 3H/35S ratios in protein 18 vs. most of the non-methylated ribosomal proteins in a series of experiments like that in Table I, it can be esti-

D AND

CYCLOHEXIMIDE

ON THE

LABELING

OF

INDIVIDUAL

RIBOSOMAL

PROTEINS

IN CELLS

INCUBATED

WITH

63

1 8 18 19 21 22 23 25 28 30 37 38 42 43 44 60 62

Spot No.

1252 406 1482 1543 506 1254 1124 348 546 315 1217 1932 674 1566 1450 482 324 895

35 28 30 52 38 71 37 20 25 20 40 51 31 31 36 18 20 47

16 19

38 22 51

16

49 30 13

36

23 95

43

35 42 111

52 78 78 111 96 193 108 43 47 28 106 105 60 74 22

14 C

D

1617 67 213 259 145 74 219 41 5 71 76 301 216 194 95

3H

3H/14 C

3H

14 C

Aetinomycin

Control

0.8 1.8 1.2

2.8 3.6 2.6

2.5

0.95

2.7 2.3 1.5

31.0

3H/14 C 186 0 84 136 27 0 0 0 41 584 43 172 562 123 4 0 207 140

3H 35 95 68 104 115 159 102 41 71 34 111 68 57 71 58 37 20 104

14 C

Cyeloheximide

10.0 1.34

2.5 9.8 1.7

17.0

1.2 1.3

5.3

3H/14 C

1045 17 83 265 38 19 11 7 22 132 47 446 515 70 46 0 91 254

3H

134 189 78 166 238 328 208 88 109 42 254 213 138 141 163 84 48 203

14 C

Actinomycin D + cycloheximide

1.8 1.2

2.1 3.7 0.5

3.1

1.0 1.6 0.15

7.7

3H/14 C

Cells were incubated for 15 rain at 4 - 106 cells]ml with a 14C-labeled amino acid mixture (NEC-445, New England Nuclear, Boston, Mass.) at 3 ~Ci/ml in Joklik modified medium lacking those amino acids and supplemented with 7% dialyzed horse serum. Then, 0.05 vol. of complete medium was added and 105 rain later the cells w e r e d i l u t e d t o 2 • 1 0 5 c e l l s / m l w i t h c o m p l e t e m e d i u m . 2 4 h l a t e r t h e c e l l s w e r e r e s u s p e n d e d a t 4 • 1 0 6 c e l l s / m l in m e t h i o n i n e - f r e e J o k l i k m e d i u m s u p p l e mented with 7% dialyzed horse serum. Then one aliquot was incubated for 1 h with actinomycin D (0.1 ~g/ml) followed by the addition of cycloheximide (150 pg] ml) and 10 rain of further incubation, while other aliquots had similar preincubations with actinomycin D only, cycloheximide only, and without drugs (control). [Me-3H]Methionine ( 1 2 . 5 / a C i / m l ) w a s t h e n a d d e d , 1 5 r a i n l a t e r t h e m e t h i o n i n e l e v e l w a s b r o u g h t t o t h e f i n a l c o n c e n t r a t i o n o f t h e r e g u l a r m e d i u m b y a d d i t i o n o f non-radioactive methionine and the cells were harvested 150 rain later. Proteins from dissociated subunits pelleted through a cushion were analyzed (see Materials and Methods).

EFFECT OF ACTINOMYCIN L-[Me-3H]METHIONINE

TABLE V

to to --4

228 TABLE

VI

EFFECT OF CYCLOHEXIMIDE ON THE RELATIVE INDIVIDUAL RIBOSOMAL PROTEINS FROM CELLS PLUS L-J35S]METHIONINE

INCORPORATION INCUBATED WITH

OF 3H AND 35S INTO L-[Me-3H]METHIONINE

T h i s e x p e r i m e n t is l i k e t h e o n e in T a b l e I V , e x c e p t t h a t o n e h a l f o f t h e cells w a s p r e i n c u b a t e d w i t h c y c l o heximide (150 pg/ml) for 10 rain and the other half was preincubated without drugs (control). Cycloheximide was left in the culture during the pulse and chase. Proteins from dissociated subunits pelleted through a cushion were analyzed. Spot No.

1 6 7 9 18 19 21 30 38 42 62

Cycloheximide

Control

3H

35 S

425 0 0 0 90 121 0 604 502 322 386

S 1 3 0 6 7 5 9 8 2 3

3H/35S

3H

35 S

3H/35 S

1437 1275 2921 973 4427 2295 1194 630 3118 735 388

101 102 217 70 187 180 92 23 198 48 14

14.2 12.5 13.4 13.9 23.6 12.7 12.9 27.0 15.7 15,0 27.7

mated th at ab o u t 1/2--2/3 of the 3H counts in 18 were in m et hyl groups, and the rest in methionine residues. Then, from the 1482 3H counts for spot 18 in the control column in Table V, 7 4 0 - 9 9 0 counts would be in m et hyl groups. Correcting the 3H counts in the actinomycin D column to a similar recovery of I4C counts gives 82 3H counts in the actinomycin D sample. A decrease from 740--990 counts in m et hyl groups in the control to 82 counts in spot 18 in the presence o f actinomycin D would indicate that most of the m e t h y l a t i o n in 18 in untreated cells occurs in new ribosomes. It can be seen in Table VI that when cells were incubated with [Me-3H] methionine and [3SS]methionine in the presence of cycloheximide, a significant n u m b e r o f 3H counts, but not of 35S counts, were incorporated in the m e t h y l a t e d spots. Protein 42, which did n o t show m e t h y l a t e d amino acids in the absence of cycloheximide in Fig. 1, but incorporated 3H from [ M e ) H ] methionine in the presence of cycloheximide in Table V, had a significant incorporation o f 3H but n o t of 3sS in Table VI, also suggesting m e t h y l a t i o n of protein 42 during inhibition of protein synthesis. Warner et al. [16] and Willems et al. [17] have shown a decreased appearance of new cytoplasmic ribosomal RNA in the presence of cycloheximide. We wanted to quantitate the inhibition of cytoplasmic appearance of new ribosomal subunits under our experimental conditions. Analyzing ribosomal subunits by gel electrophoresis as shown in Fig. 2, we found a 4 to 6-fold decrease in the exit o f new large subunits and could n ot detect any new small subunits in the presence of cycloheximide. Similar results were obtained analyzing phenol-extracted RNA. In experiments like the one in Table II, protein 30 had a 3H/3SS ratio approximately 2-fold higher than the majority o f the non-m et hyl at ed ribosomal

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3

4

cm from origin Fig. 2. E f f e c t of c y c l o h e x i m i d e on [ 3 H ] u r i d i n e i n c o r p o r a t i o n into r i b o s o m a l s u b u n i t s , a n a l y z e d b y 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 s i s . Cells w e r e i n c u b a t e d w i t h [ 1 4 C ] u r i d i n e (2 n C i / m l , 53 C i / m o l ) for 24 h. T h e cells w e r e t h e n r e s u s p e n d e d in m e t h i o n i n e - f r e e J o k l i k m e d i u m , c y c l o h e x i m i d e was t h e n a d d e d ( 1 5 0 p g / m l ) , 10 rain l a t e r [ 3 H ] u r i d i n e was a d d e d ( 1 0 p C i / m l , 28 C i / m m o l ) ; a f t e r 15 m i n the m e t h i o n i n e c o n c e n t r a t i o n w a s b r o u g h t to t h e final c o n c e n t r a t i o n of the r e g u l a r m e d i u m , a n d t h e cells w e r e h a r v e s t e d 1 5 0 m i n l a t e r as i n d i c a t e d p r e v i o u s l y [ 1 8 ] . T h e p o s t m i t o c h o n d r i a l s u p e r n a t a n t w a s c e n t r i f u g e d for 1 h a t 50 0 0 0 r e v . / m i n in a B e c k m a n 50 Ti r o t o r (Palo A l t o , Calif.) to pellet t h e r i b o s o m e s . T h e r i b o s o m a l subu n i t s w e r e a n a l y z e d by e l e c t r o p h o r e s i s in p o l y a c r y l a r n i d e gels of u n i f o r m c o n c e n t r a t i o n u n d e r t h e c o n d i t i o n s of M i r a u l t and S c h e r r e r [ 1 9 ] , a n d gels w e r e sliced, t r e a t e d , a n d c o u n t e d as p r e v i o u s l y i n d i c a t e d [20]. • •, 3H cpm;© . . . . . . ©, 14C c o m .

proteins, suggesting that about one half of its 3H counts were in the amino acid m e t h io n in e and the ot he r half in m e t h y l groups. It can then be estimated that in the e x p e r i m e n t in Table V a b o u t 157 o u t of 315 3H counts in the control column of protein 30 were in m e t h y l groups, in comparison to 344 3H counts in the cycloheximide column, after correcting with the 14C counts for recovery variations. The data in Tables IV and V suggest that most of the m e t h y l a t i o n of protein 30 occurs in newly made ribosomes, while experiments like the one in Fig. 2 indicate that in the presence of cycloheximide there was a 4 to 6-fold decrease in cytoplasmic appearance of new 60-S subunits. Assuming homogeneity in the ribosome population and correcting by an average factor of 5 to compare equal numbers of new 60-S subunits appearing in the cytoplasm, there would be 3 4 4 X 5 = 1 7 2 0 c p m in the cycloheximide-treated sample vs. 157 cpm in the control, a higher than 10-fold increase in m e t h y l a t i o n in protein 30 in the absence of protein synthesis. With the same rationale one can estimate a marked increase in the level of m e t h y l a t i o n of the proposed methylation site of large subunit protein 42 in nascent ribosomes during suppression o f protein synthesis.

Removal of cycloheximide Assuming h o m o g e n e i t y in the ribosome population, these apparent increases in the levels of m e t h y l a t i o n of proteins 30 and 42 in the absence o f protein

230 synthesis could be due to increased methylation or increased metabolic stability of the methylations in the absence of protein synthesis. Therefore, we proceeded to incubate cells first with I4C-labeled amino acids for 24 h, then cycloheximide was added, followed by a pulse with [Me-~H]methionine which was chased for 150 min with an excess of non-radioactive methionine, cycloheximide was then removed, and the cells were allowed to recover. Table VII shows (columns a and c) that 3H counts were not lost from proteins 30 and 42 when protein synthesis was allowed, to recover, suggesting that the higher levels of methylation of protein 30 and 42 were not due to decreased demethylation in the absence of protein synthesis. The small ribosomal subunit methylated protein 18 normally showed low levels of 3H incorporation in the presence of cycloheximide, which would be expected as we could not detect any new 40-S subunits appearing in the cytoplasm in the presence of cycloheximide, as shown in Fig. 2, and the data in Tables IV and V suggest that the methylation of protein 18 in untreated cells occurs mainly, if not only, in newly made ribosomes. An unexpected finding TABLE VII EFFECT OF REMOVAL OF CYCLOHEXIMIDE ON THE LABELING OF VARIOUS RIBOSOMAL P R O T E I N S IN C E L L S W H I C H H A D B E E N P U L S E D W I T H L - [ M e - 3 H ] M E T H I O N I N E AND CHASED WITH AN EXCESS OF NON-RADIOACTIVE M E T H I O N I N E , IN T H E P R E S E N C E O F C Y C L O H E X IMIDE C e i l s w e r e i n c u b a t e d w i t h 1 4 C - l a b e l e d a m i n o a c i d s f o r 2 4 h as i n T a b l e V. T h e c e l l s w e r e t h e n c o n c e n t r a t e d , p r e i n e u b a t e d w i t h c y e l o h e x i m i d e ( 1 5 0 p g / m l ) f o r 1 0 r a i n , [ M e - 3 H ] m e t h i o n i n e w a s t h e n a d d e d as i n T a b l e V, a n d 1 5 r a i n l a t e r t h e m e d i u m w a s b r o u g h t t o t h e m e t h i o n i n e c o n c e n t r a t i o n o f t h e r e g u l a r m e d i u m . A f t e r 1 5 0 m i n o n e a l i q u o t w a s h a r v e s t e d (a), o n e a l i q u o t w a s i n c u b a t e d f o r 2 h m o r e (b) a n d a third aliquot was spun down, washed three times with regular medium without eyeloheximide, and then i n c u b a t e d f o r 2 h i n c o m p l e t e m e d i u m w i t h o u t c y c l o h e x i m i d e (c). T h e t h r e e e x p e r i m e n t s are s i m i l a r , e x c e p t t h a t i n E x p t s . 1 a n d 2 t h e d i s s o c i a t e d s u b u n i t s w e r e n o t s e p a r a t e d , a n d i n E x p t . 3 t h e y w e r e sepa r a t e d a n d p r o t e i n s p o t s f r o m t h e 4 0 S s u b u n i t are s h o w n . Spot No.

a

b

3H

14 C

Expt. 1 6 7 9 14 18 21 30 63

28 51 14 26 77 47 738 84

65 142 96 41 57 82 24 70

Expt. 2 1 6 9 21 42 62

2802 91 83 44 2461 1315

453 584 516 412 347 112

Expt. 3 18 20 21

3H/14 C

1.4 31.0 1.2

6.1

7.0 11.7

3H

14 c

21 50 12 48 164 58 1297 163

70 125 87 68 75 75 41 66

797 8 0 15 1176 612

113 265 235 200 129 45

5 3 4

125 62 58

3H/14 c

2.2 32.0 2.5

7.0

9.1 13.0

3H

14 C

12 44 2 54 351 15 616 109

31 68 25 20 42 42 21 50

997 0 13 31 789 375

128 146 173 149 120 29

294 5 12

123 71 41

3H/14 c

8.4 29.0 2.2

7.7

6.5 13.0 2.4

231 TABLE VIII EFFECT OF REMOVAL OF CYCLOHEXIMIDE ON THE LABELING OF VARIOUS P R O T E I N S IN C E L L S W H I C H H A D B E E N P U L S E D W I T H L - [ M e - 3 H ] M E T H I O N I N E METHIONINE AND CHASED WITH AN EXCESS PRESENCE OF CYCLOI-IEXIMIDE

OF

NON-RADIOACTIVE

RIBOSOMAL PLUS [35S]-

METHIONINE,

IN T H E

T h i s e x p e r i m e n t is l i k e t h a t i n T a b l e V I I , e x c e p t t h a t w h e n L - [ M e - 3 H ] m e t h i o n i n e was a d d e d , it was m i x e d w i t h [ 35 S ] m e t h i o n i n e i n t h e s a m e p r o p o r t i o n as i n T a b l e I. P r o t e i n s f r o m d i s s o c i a t e d s u b u n i t s p e l l e t e d t h r o u g h a c u s h i o n w e r e a n a l y z e d . C o l u m n s a, b a n d c are as i n t h e l e g e n d t o T a b l e V I I . Spot No.

a

b

3H 6 9 18 53 60

35 S

3H

35 S

3H

35 S

20

0

163 0 9

7 0 0

59 35 253 3 31

10 6 12 2 4

45 31 540 1 7

3 2 8 1 0

was made in experiments like the one in Table VII. When protein synthesis was allowed to recover, and although the 3H pulse had been stopped 150 min earlier with a chase of non-radioactive methionine, 3H counts increased in protein 18 (from a ~H/14C ratio of 1.4 to 8.4). This p h e n o m e n o n could also be seen with isolated ribosomal subunits (Expt. 3 in Table VII). The same t y p e of exper i m ent can be seen in Table VIII, but this time the

a 4

3f

5 2~

I -1- (3

o~

I 0

2 0 0 50

42

3O 150

,/

2700 50

I0

,i

o~./, O5O

~SO

I 150

27~

~l 2

I

5

27O 0 SO

m i n u t e s o f chose

~SO

270

0

LJll

5

I

I0

L

1.5

I

20

hours Qfter cycloheximide removQI

F i g . 3. T i m e c o u r s e o f 3 H i n c o r p o r a t i o n i n t o i n d i v i d u a l r i b o s o m a l p r o t e i n s i n H e L a c e l l s i n c u b a t e d w i t h c y c l o h e x i m i d e a n d L - [ M e - 3 H ] m e t h i o n i n e . T h i s e x p e r i m e n t is l i k e a a n d b i n T a b l e V I I , e x c e p t t h a t s o m e extra time points were haxvested earlier, a and b in this figure denote two similar experiments. The numb e r inside each s e c t i o n i d e n t i f i e s t h e i n v i d u a l p r o t e i n s p o t a n a l y z e d . T h e o p e n circle in the g r a p h for prot e i n s p o t 1 8 r e p r e s e n t s a s a m p l e 2 h a f t e r c y e l o h e x i m i d e r e m o v a l , as i n d i c a t e d i n t h e l e g e n d o f T a b l e V I I . Fig. 4. T i m e course of 3H i n c o r p o r a t i o n i n t o r i b o s o m a l p r o t e i n 18 a f t e r r e m o v a l of c y c l o h e x i m i d e , in HeLa cells which had been pulsed with L-[Me-3H]methionine and chased with an excess of non-radioact i v e m e t h i o n i n e i n t h e p r e s e n c e o f c y c l o h e x i m i d e . T h i s e x p e r i m e n t is l i k e a a n d c i n T a b l e V I I , e x c e p t that some extra time points were collected.

232 radioactive pulse was with a mixture of [Me-3H]methionine and [3SS]methionine. The 3H counts in protein 18 increased after removal of cycloheximide, b u t the 3sS counts did not. Fig. 3 shows the kinectics of 3H incorporation into several m e t h y l a t e d proteins in a pulse with [Me-3H]methionine followed by a chase with an excess of non-radioactive methionine, in the presence of cycloheximide. Proteins like 1 and 30 reached a plateau at about 150 min of chase, while 42 had not reached a plateau after 270 min. The kinetics of 3H incorporation into protein 18 after removal of cycloheximide, in the presence of an excess of non-radioactive methionine, are shown in Fig. 4. Discussion A summary of our findings and proposals is presented in Table XI. A minim u m of seven ribosomal proteins appear to be m e t h y l a t e d in HeLa cell ribosomes, three of them belong to the small subunit (spots 1, 18 and 38) and two to the large subunit (spots 30 and 42); the subunit assignment of two others (spots 62 and 63) is still uncertain. These proteins fall under the current definition o f ribosomal proteins because t hey are associated with subunits washed with 0.5 M KC1, and are present in significant concent rat i on with respect to o t h e r ribosomal proteins (see last 14C column in Table V for a comparison of ribosomal proteins labeled for 24 h with a mixture of 15 14C-labeled amino acids). Most of the me t hyl a t i on in proteins 62 and 63 appears to occur in nascent ribosomes, while at least some of the m e t h y l a t i o n in protein 38 seems to take place in previously made ribosomes. Protein 18 is m e t hyl a t e d in untreated cells, where most, if n o t all, of its m e t h y latio n seems to occur in nascent ribosomes. After a pulse with [Me-3H] methionine and a chase with non-radioactive methionine, both in the presence

T A B L E IX SUMMARY

OF METHYLATION

OF RIBOSOMAL

Subunit assignment

Ribosomal protein

Maturing ribosome

40 S

1 18 38

+

30

+

42

+

62 63

+ +

60 S

Undetermined

Mature ribosome

PROTEINS Methyllysine

Methylarginine

+

+ + +

+ + +

In absence of protein synthesis

Apparently enhanced Apparently enhanced Unaffected

+ +

* 3H labeling under chase conditions, beginning 150 rain after chase had started.

After cycloheximide removal *

233

of cycloheximide, protein 18 becomes labeled upon removal of cycloheximide during the chase. The labeling of 40-S protein 18 after cycloheximide removal during a chase could be related to the restoration of cytoplasmic appearance of new 40-S subunits or to the restoration of protein synthesis. This labeling could be due to methyl groups, labeled during the pulse, either entering protein 18 after removal of cycloheximide, or entering previously made protein 18 in the presence of cycloheximide, and then protein 18 assembling with nascent ribosomes after removal of cycloheximide. The latter possibility seems unlikely because most ribosomal proteins synthesized during suppression of cytoplasmic appearance of new ribosomes are not utilized when ribosome formation is restored [21--23]. Moreover, more recent results (Goldenberg and Eliceiri, in preparation) directly support the first alternative. We propose then that protein 18 could be specifically methylated by a hypothetical methylating intermediate, whose demethylation is inhibited by cycloheximide, but whose methylation is not. Protein spot 30 is methylated in untreated cells, mostly on nascent ribosomes. This methylation appears to increase during suppression of protein synthesis, and these methyl groups seem to be metabolically stable when protein synthesis is restored. We did not detect methylation in protein spot 42 from untreated cells. In the case of 3H/3SS ratios (Table I) this could be due to low levels of methylation of protein 42 in untreated cells. If protein 42 in untreated cells were methylated on free carboxyl groups [24], its methylation would not have been detected in amino acid analyses like those in Fig. 1, due to volatilization during acid hydrolysis [25,26]. The results in the actinomycin D and cycloheximide plus actinomycin D columns of Table V support the existence of a methylation site in protein 42 in previously finished ribosomes that is not affected by inhibition of protein synthesis. The figures in the cycloheximide and the cycloheximide plus actinomycin D columns of Table V indicate the existence of a methylation site in nascent ribosomes in protein 42. A comparison between the values in the control and cycloheximide columns in Table V suggests an increase in methylation in the absence of protein synthesis. As the methylation site in protein 42 for previously made ribosomes appears to be unaffected by suppression of protein synthesis, it seems then that the methylation site for nascent ribosomes is the one apparently enhanced by inhibition of protein synthesis. The methyl groups incorporated into protein 42 in the presence of cycloheximide seem to be metabolically stable after removal of cycloheximide. If there were heterogeneity in the population of mammalian ribosomes, one could entertain the possibility that the cytoplasmic appearance of a certain population of nascent ribosomes was not affected by cycloheximide and that they were also the ones methylated in proteins 30 and 42. In that case the apparent increase in methylation levels of proteins 30 and 42 would not be as high as it seems. Gressner and Wool [27] have shown that cycloheximide or puromycin stimulate another modification of mammalian ribosomal proteins, phosphorylation of 40-S protein $6 in rat liver ribosomes. As the apparent stimulation of methylation during inhibition of protein synthesis seems to occur on the proposed "processing site" of both protein spots 30 and 42, this stimulation should not

234

be related to the functions of the ribosome in protein synthesis, but to an effect of protein synthesis inhibition on ribosome maturation. The apparent methylation of protein 42 in previously completed ribosomes, being unaffected by suppression of protein synthesis, would suggest a relationship with some activity of the mature ribosome other than protein synthesis. It is interesting to note that, in general, the ribosomal proteins that according to actinomycin D experiments appeared to be methylated in nascent ribosomes (most likely, nuclear methylations), contained methyllysine, while those methylated in mature ribosomes (cytoplasmic methylations) were methylated on arginine. This is in accordance with the known observations that the enzymatic activity for methylation of various cellular proteins in lysine residues is exclusively nuclear [24], while that for arginine is predominantly cytoplasmic [28,29].

Acknowledgments We thank Dr. Karl M. Dus for his advice on the techniques of amino acid analysis, Dr. David Schlessinger for critical reading of the manuscript and Mr. Richard Pinkston for hydrolyzing the protein samples. This work was supported in part by grant GM-19835 from the U.S. Public Health Service. One of the authors (G.L.E.) is a recipient of a Research Career Development Award from the U.S. Public Health Service.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

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Methylation of ribosomal proteins in HeLa cells.

220 Biochimica et Biophysica Acta, 4 7 9 ( 1 9 7 7 ) 2 2 0 - - 2 3 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press BBA 990...
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