In vivo isotope incorporation patterns into HeLa ribosomal proteins

104

Biochimica et Biophysica Acta, 432 (1976) 1 0 4 - - 1 1 2 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

BBA 98571

IN VIVO ISOTOPE INCORPORATION PATTERNS INTO HELA RIBOSOMAL PROTEINS

J O B S T P. V A N D R E Y , C A R L O S J. G O L D E N B E R G and G E O R G E L. E L I C E I R I

Department of Pathology, St. Louis University School of Medicine, St. Louis, Mo. 63104 (U.S.A.) (Received S e p t e m b e r 22nd, 1975 )

Summary When cells were incubated with L-[3ss] methionine plus L-[Me-3H] methionine, it was found that a t least four ribosomal proteins had 3H/3SS ratios higher than the rest of the ribosomal proteins, suggesting that they were methylated. The rate of apparent methylation paralleled the rate of amino acid incorporation. Amino acid incorporation into ribosomal proteins revealed several rapidly labeled components. When 3H-labeled amino acid incorporation was chased for 10 min with an excess of non-radioactive amino acids, several proteins reached at least 60% of the specific activity they showed after 150 min of chase. The time lapse between the onset of 3H-labeled amino acid incorporation and arrival at its plateau appeared to differ among various ribosomal proteins of a subunit, suggesting a heterogeneity in the pools of ribosomal proteins.

Introduction

The cytoplasmic ribosome is a useful system to study the regulation of assembly of macromolecules into a mammalian cell organelle. Although much has been learned about the synthesis and processing of its RNA components, very little is known about the synthesis, assembly, and maturation of its 70-odd proteins [1]. By one-dimensional gel electrophoresis of HeLa large subunit ribosomal proteins, Warner [2] found that some proteins were maximally labeled quickly in pulse chase experiments, and were also labeled even if preincubated with actinomycin D. With the availability of the high resolution of two-dimensional gel electrophoresis [3] and active mammalian ribosomal subunits with a very low protein content [4], we tried to pursue further some of Warner's [2] pulse chase studies of radioactive amino acid incorporation into HeLa ribosomal proteins. Abbreviations: 60 S, large ribosomal subunit; 40 S, small ribosomal subunit.

105 Two types of in vivo modifications of mammalian ribosomal proteins have been described: phosphorylation [5--7] and acetylation [8]. In this report we present data suggesting the existence of methylation as a third t y p e of in vivo modification of mammalian ribosomal proteins.

Materials and Methods

Cell culture and labelingconditions. HeLa $3 cellswere maintained at a concentration of 2 • I0s--4 • 10 s cells/ml in spinner culture using Joklik modified m i n i m u m essential medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 7 % horse serum. To prepare uniformly labeled ribosomal proteins, cells were grown for 24 h with either 0.075/~Ci/ml of a ~4C-labeled amino acid mixture (NEC445, N e w England Nuclear, Boston, Mass.) or with [3H] leucine, [3H] lysine, [3H] alanine, and [3H] glycine at a final concentration of 1 /~Ci/ml of each of the four 3H-labeled amino acids. For short term labeling with methionine, cells were resuspended at 4 • 106 cells/ml in methionine-free Joklik m e d i u m supplemented with 7 % dialyzed horse serum, and incubated for 10 min at 37°C. A mixture of 1.25 mCi of I~[Me-3H] methionine (11 Ci/mmol) and 0.25 mCi of L-[3sS]methionine (166 Ci/mmol) was added per 100 ml of culture, followed by a 15 rain incubation at 37°C. At the end of this initial period of incubation, the culture was supplemented with one-tenth the normal level of methionine, and aliquots were withdrawn later at the indicated time intervals. For amino acid incorporation studies, the culture was initiallylabeled for 24 h with the ~4C-labeled amino acid mixture mentioned above, and then resuspended at 4 • 106 cells/mlin Joklik m e d i u m lacking lysine, leucine, alanine, and glycine, and supplemented with 7% dialyzed horse serum. After a 10 min incubation the cells were labeled with [3H]leucine, [3H]lysine, [3H]alanine, and [3H] glycine (5 #Ci of each of the four amino acids per ml of culture). At the end of 15 min of 3H pulse, the cells were diluted to 2" l 0 s cells/ml in normal Joklik medium, and aliquots of the chase were taken at the indicated time intervals. Isolation of ribosomes. HeLa ribosomes were isolated substantially as described by McConkey [4]. After the initial harvest, the cells were washed with cold phosphate-buffered saline at a concentration of 3 - 1 0 6 cells/ml and resuspended in hypotonic buffer (10 mM Tris • HC1, pH 7.4, 10 mM KC1, 1 mM MgC12, 1 mM dithiothreitol) at 2 • 107 cells/ml of buffer. The cells were allowed to swell in ice for 5 min and they were then broken with a Dounce glass homogenizer. One-tenth volume of 3 M KC1, 20 mM MgC12 was added and the preparation was centrifuged at 17 000 × g for 15 min to remove nuclei and mitochondria. The supernatant was then adjusted to 0.5% sodium deoxycholate and 0.5% Brij 58. The ribosomes were finally sedimented through a 2-ml cushion o f 1.75 M sucrose, 50 mM Tris • HC1, pH 7.4, 0.1 M KC1, 1 mM MgC12, and 1 mM dithiothreitol in a Beckman (Palo Alto, Calif.) 50 Ti rotor at 50 000 rev./min for 15 h. Isolation of ribosomal subunits. Ribosomal subunits were prepared essentially as described by Blobel and Sabatini [9]. The crude ribosomal pellets were resuspended in 50 mM Tris • HC1, pH 7.4, 100 mM KC1, 1 mM MgC12, and 1 mM dithiothreitol, and incubated at 37°C for 15 min with 0.5 mM puromycin. The resulting subunits were then separated in 5--20% sucrose linear gradients con-

106 taining 0.5 M KC1, 5 mM MgC12, 10 mM Tris • HC1, pH 7.4, and 1 mM dithiothreitol. Peak fractions were pooled and collected by centrifugation at 50 000 rev./min for 18 h in a Beckman 50 Ti rotor. The ribosomal proteins were extracted from crude ribosome preparations and from purified subunits by a modification of the m e t h o d of Hardy et al. [10]. The preparations were resuspended in water and extracted at 4°C for 1 h with a final concentration of 67% acetic acid, 3.3 mM Tris • HC1, and 33 mM magnesium acetate. The RNA precipitate was collected by centrifugation, and re-extracted as before. After the second centrifugation, the two supernatants were combined and precipitated with eight volumes of cold acetone at --20°C overnight. The precipitated ribosomal proteins were then collected by centrifugation and resuspended in a small volume of 8 M urea, 22 mM EDTA, 0.5 M H3BO3, 0.4 M Tris • HC1, pH 6.6, and 1 mM dithiothreitol, and stored at --20°C. Gel electrophoresis. The two-dimensional gel electrophoresis was a slightly modified version of the procedure described by Kaltschmidt and Wittmann [3], including also some modifications of Sherton and Wool [1] and Howard and Traut [11]. For the first dimension l l - c m long gel cylinders with a diameter of 5 mm were used, composed of 6% acrylamide, 0.3% methylenebisacrylamide, 0.5 M H3BO3, 0.4 M Tris • HC1, pH 8.7, 22 mM EDTA, 8 M urea, and 0.045% N,N,N',N'-tetramethylenediamine. The gel was polymerized with a final concentration of 0.05% (NHa)2S2Os. The anodic end of each gel was loaded with 200--300 pg of extracted protein in 30--50 pl, and overlayered with 0.2 ml of a mixture of three parts of protein resuspension buffer and one part of electrode buffer. The composition of the first dimension electrode buffer was as follows: 6.5 mM EDTA, 77 mM H3BO3 and 60 mM Tris • HC1, pH 8.2. The first dimension electrophoresis was carried out at constant voltage, at 130 V for 28 h. The gel cylinder was then dialyzed as described by Howard and Traut [11], and placed between the walls which were to hold the second dimension gel slab. The distance between the ~irst dimension gel and the cathodic edge of the gel slab was about 10 cm and the thickness of the second dimension gel slab was 4.5 mm. The second dimension gel solution consisted of 18% acrylamide, 0.83% ethylene diacrylate (w/v), 6 M urea, 5.2% acetic acid, 48 mM KOH, and 0.58% N,N,N',N'-tetramethylenediamine, adjusted to a final pH of 4.6. The gel was polymerized with a final concentration of 0.33% (NH4)2 $208. The second dimension electrode buffer was made of 0.37 M glycine in 0.3% acetic acid. Electrophoresis was carried out at constant voltage, at 120 V for 21 h. Gel slabs were stained with 0.05% Coomassie blue, and destained with 7.5% acetic acid, 10% methanol, pumping the destaining solution through a charcoal filter to remove the dye continuously [12]. Liquid scintillation counting of gel slabs. Destained gel slabs were photographed and individual spots were cut out with an 8-mm diameter cellulose nitrate centrifuge tube acting as a punch. These gel pieces were then completely digested in sealed glass scintillation vials, using 2.4 ml of 1.5% NH4OH per vial at 70°C overnight. To each digested gel piece 10 ml of the following mixture was added : five parts of xylene (0.6% Omnifluor, New England Nuclear), three parts of Triton X-114, and two parts of Triton X-100. The vials were mixed with the scintillation mixture while still warm and then allowed to stand for 12 h before counting in a liquid scintillation counter.

107

Results Fig. 1. shows the pattern of HeLa 60 S and 40 S subunit ribosomal proteins, prepared and analyzed by two-dimensional gel electrophoresis, as described in Materials and Methods. Several of these spots represent multiple proteins, including superimposed large and small subunit proteins, that are only partially separated or not separated at all under the standard electrophoresis conditions. With different conditions of electrophoresis we have found that some apparently homogeneous spots separated into two or three components. The routine electrophoretic analysis described herein would fail to detect two groups of proteins shown to exist in low amounts in rat liver ribosomes [1]. Since the sample is loaded on top of the first dimension gel rather than between two gel columns, any protein that is negatively charged at pH 8.7 (the pH of the separation gel) would be lost. Furthermore, several low molecular weight proteins that travel very rapidly in the second dimension would also be lost in this analysis. To analyze this latter group of ribosomal proteins, samples were subjected to electrophoresis for reduced times (10 h for the first dimension and 8 h for the second, see Materials and Methods for other details). This permitted the visualization of three additional spots. Fig. 1 also presents the numbering identification used throughout this report.

Incorporation of L-[358]methionine and L-[Me-3H] methionine Methylation of some ribosomal proteins has been shown in bacteria [13-16]. To test if any HeLa ribosomal protein might be methylated in vivo, the cells were exposed to two forms of labeled methionine simultaneously (L-[Me3H]methionine and L-[3SS]methionine). Using L-[Me-3H]methionine, 3H can be incorporated into proteins as part of methionine, and also as part of 3H(~

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Fig. 2. T i m e c o u r s e o f r e l a t i v e i n c o r p o r a t i o n o f t h e m e t h y l g r o u p s o r i g i n a l l y d o n a t e d b y m e t h i o n i n e p l u s i n c o r p o r a t i o n o f [ 3 H ] m e t h i o n i n e vs. i n c o r p o r a t i o n o f [ 3 S S] m e t h i o n i n e i n t o v a r i o u s r i b o s o m a l p r o t e i n s . The e x p e r i m e n t s , u s i n g w h o l e r i b o s o m e s , w e r e as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . oo, s p o t n u m b e r 31 ; o . . . . . . ©, s p o t n u m b e r 15; X - X, s p o t n u m b e r 33. Fig. 3. T i m e c o u r s e o f 3 H - l a b e l e d a m i n o a c i d i n c o r p o r a t i o n i n t o p r o t e i n n u m b e r 6 (o o) a n d p r o t e i n n u m b e r 10 (© . . . . . . o) d u r i n g a p u l s e c h a s e . As d e t a i l e d in M a t e r i a l s a n d M e t h o d s , cells w e r e i n c u b a t e d f o r 24 h w i t h a 1 4 C - l a b e l e d a m i n o a c i d m i x t u r e , t h e n i n c u b a t e d f o r 15 m i n w i t h 3 H - l a b e l e d a m i n o acids, a n d t h e n (0 r a i n in t h e f i g u r e ) c h a s e s w i t h an e x c e s s o f n o n - r a d i o a c t i v e a m i n o a c i d s f o r t h e v a r i o u s t i m e s i n d i c a t e d in t h i s f i g u r e . W h o l e r i b o s o m e s w e r e u s e d .

labeled methyl groups originally donated by methionine (methylation). This double label would effectively point out the existence of possibly methylated proteins, since the 3H/3SS ratio should be higher for a methylated protein than for a non-methylated protein. While this manuscript was in preparation, a report by Chang and Chang [17] appeared, in which a mixture of L-[1-14C] methionine and L-[Me-3H]methionine was similarly used to study the methylated ribosomal proteins of Escherichia coli. Fig. 2 shows the 3H (incorporation of methionine plus incorporation of methyl groups originally donated by methionine) to 3sS (incorporation of methionine) ratio in three spots from whole ribosomes throughout the time course of the experiment. Most proteins have a certain 3H/3SS ratio in the first time point, which remains fairly constant later (protein number 33 is shown as an example). A few proteins have a higher ratio, which also stays rather constant (protein number 15 is shown in Fig. 3); these are the apparently methylated proteins. The stability of the ratio indicates that the rate of apparent methylation parallels the rate of amino acid incorporation. One spot (number 31), which is a mixture of a 60 S protein and a 40 S protein, starts with a high ratio which decreases later in the experiment. Experiment 1 in Table I illustrates the 3H/3SS ratio of the various spots from whole ribosomes after 165 min of incubation with the isotope mixture. It is apparent that a minimum of three proteins have high 3H/aSS ratios. Spot number 17 also has a relatively high ratio, however, its close proximity to spot number 15, which has a higher ratio, makes its significance doubtful. When the ribosomes from a methylation experiment were separated into subunits and their proteins analyzed, it was found that the large subunit protein in spot 31 was the one with the high SH/3SS ratio (Experiment 2 in Table I). The observed decrease in ~H/3SS ratio with incubation time was due to the masking effect of isotope incorporation into the apparently more slowly labeled, non-methylated 40 S ribosomal protein component in spot 31. Therfore, at least four ribosomal

109 TABLE I R E L A T I V E I N C O R P O R A T I O N O F T H E M E T H Y L G R O U P S O R I G I N A L L Y D O N A T E D BY M E T H I O N I N E P L U S I N C O R P O R A T I O N O F [ 3 H I M E T H I O N I N E vs. I N C O R P O R A T I O N O F [35 S] M E T H I O N I N E INTO VARIOUS RIBOSOMAL PROPERTIES E x p e r i m e n t 1 is the one from Fig. 2, proteins, after 1 6 5 m i n o f i n c u b a t i o n s h o w s the 3 H / 3 5 S ratio in the 6 0 S after separation o f the r i b o s o m e into in a n o t h e r e x p e r i m e n t .

s h o w i n g the 3 H / 3 5 S ratios o f i s o t o p e i n c o r p o r a t i o n into r i b o s o m a l w i t h L - [ 3 5 S ] m e t h i o n i n e and L-[Me-3H] m e t h i o n i n e . E x p e r i m e n t 2 s u b u n i t (L) a n d 4 0 S s u b u n i t (S) c o m p o n e n t s of s p o t n u m b e r 3 1 , subunits, from cells incubated for 6 0 rain w i t h the i s o t o p e m i x t u r e ,

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proteins appear to be methylated: numbers 15 and 31 (60 S), and 2 and 22 (40 S). Their 3H/3SS ratios were also high in puromycin-dissociated subunits treated with 0.5 M KC1, suggesting that the apparently methylated proteins are structural ribosomal proteins. Amino acid incorporation When we carried out pulse chase experiments with 3H-labeled amino acids, essentially as described by Warner [2], most ribosomal proteins incorporated 3H gradually. After 10 min of chase, isotope incorporation had started in some ribosomal proteins, like number 10 of the large subunit, but for others a lag in isotope incorporation could be detected until 20 min of chase, like number 6 of the large subunit (Fig. 3). These results, similar to those of Warner [2], are consistent with a sequential model of ribosome assembly. Fig. 4 also shows that within one subunit, isotope incorporation into various ribosomal proteins reached a plateau at different times. The time lapse between onset o f isotope incorporation and arrival to a plateau varied among different ribosomal proteins o f a subunit {Fig. 3). At least five proteins incorporated isotope very quickly, instead of gradually, as most of the ribosomal proteins. Their time course of 3H-labeled amino acid incorporation is shown in Fig. 4. One was from the large subunit (No. 39), two from the small subunit (Nos. 28 and 34), one was mixture of 60 S and 40 S

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ribosomal proteins (No. 27), and one was tentatively assigned to the small subunit (No. 41). After 10 min of chase, these proteins had incorporated at least 60% of the isotope incorporated afte 150 min of chase. As the amino acid incorporation studies into rapidly labeled ribosomal proteins were done with whole ribosomes, it is possible that some of these proteins migh come o f f the subunit in 0.5 M KC1, b u t comigrate in two -dimensional gel electrophoresis with subunit-associated ribosomal proteins. When the electrophoretically faster moving ribosomal proteins were analyzed (by running the electrophoresis for fewer hours in b o t h dimensions, as indicated above) no proteins were found which had a high 3H/3SS ratio or which rapidly incorporated radioactive amino acids. Discussion A double label technique was used to assay for possibly methylated ribosomal proteins in HeLa mature ribosomes. Using a mixture of t w o isotopes labeling different moieties of methionine, it could be possible to detect methylation. The proteins to be methylated would incorporate selectively 3H from the methyl moiety of L-[Me-3H] methionine, while both methylated and non-methylated proteins would incorporate at random the 3H- and aSS-labeled forms of methionine. Thus, in the case of non-methylated proteins, the 3H/3SS ratio would be an expression of the relative ratio of the added isotopes. In the case of a methylated protein the elevated 3H/3SS ratio would b e c o m e a measure of the ratio of methyl groups to methionine residues in that protein. This assay for methylation might give misleading results in two cases. If a protein is only slightly methylated it might be difficult to detect a small increase in the 3H/aSS ratio over the ratios of non-methylated proteins. If a protein does n o t contain methionine or has a very low methionine content, the ass counts might be t o o low to give a significant 3H/3SS ratio. We have n o t tested if the few ribosomal proteins which are negatively charged at pH 8.7, have high aH/3SS ratios. In an electrophoretic system that separated the 60 S ribosomal proteins into a b o u t 11 bands, Warner [2] detected at least three bands which incorporated a maximum of radioactive amino acid within 10 min of its chase with non-radioactive amino acid, instead of being labeled gradually. These proteins were also

111

the only ones labeled when the cells were preincubated with actinomycin D before the pulse chase. Kumar and Subramanian [18] also found several rapidly labeled HeLa 60 S ribosomal proteins. Fig. 4 shows the 60 S and 40 S ribosomal proteins which after 10 min of chase reached at least 60% of the specific activity they had after 150 min of chase. Other ribosomal proteins were labeled slightly less rapidly and are not shown. Some of these proteins could be cell sap proteins that exchange with proteins present in completed ribosomes [2], or could be structural proteins added int he cytoplasm independently of the nucleolar processing [18], or could be ribosomal proteins which have small pools and join the ribosomal subunits late in their processing, either in the nucleus or in the cytoplasm. The last interpretation is consistent with a rapid uptake of radioactive amino acid (late step in subunit maturation), and a rapid termination of this uptake in a pulse chase (small pool of such ribosomal protein}. Heterogeneity of ribosomal protein pool sizes for the same subunit type is suggested by our finding that the time lapse between beginning and end of radioactive amino acid incorporation in a pulse chase, varied within the ribosomal proteins of a subunit (Fig. 3). The existence of a massive exchange between soluble proteins in the cytoplasm and proteins in the ribosome reported by Dice and Schimke [19] would seriously affect our experiments as well as those of others in this field. Our attempts (unpublished data) as well as the attempts of Delaunay and Schapira [20] and Terao et al. [21], to reproduce the in vitro protein exchange experiments of Dice and Schimke [19], failed to indicate any large exchange. More over, immunological studies of Wool and StSffler [22] did not detect any appreciable amounts of ribosomal proteins in the cell sap. Acknowledgements We wish to thank Drs. E.H. McConkey, A. Kumar and A.R. Subramanian for communication of their results prior to publication. The technical assistance of Mrs. l~osemarie L. Reimert during part of this work is gratefully acknowledged. This work was supported in part by a grant from the U.S. National Institute of General Medical Sciences. One of the authors (G.L.E.) holds a Research Career Development Award from the U.S. National Institute of General Medical Sciences. References 1 2 3 4 5 6 7 8 9 10 11 12 13

Sherton, C.C. and Wool, I.G. (1972) J. Biol. Chem. 247, 4460---4467 Warner, J.R. (1966) J. Mol. Biol. 19, 383---398 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401--412 McConkey, E.H. (1970) Proc. Natl. Acad. Sci. U.S.A. 71, 1379--1383 Kabat, D. (1970) Biochemistry 9, 4 1 6 0 - - 4 1 7 5 Loeb, J.E. and Blat, C. (1970) FEBS Lett. 10, 105--108 Gressner, A.M. and Wool, LG. (1974) J. Biol. Chem. 249, 6 9 1 7 - - 6 9 2 5 Liew, C.C. and Gornall, A.G. (1973) J. Biol. Chem. 248, 977--983 Blobel, G. and Sabatini, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 390--394 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2 8 9 7 - - 2 9 0 5 Howard, G.A. and Traut, R.R. (1973) FEBS Lett. 29, 177--160 Sherton, C.C. and Wool, I.G. (1974) J. Biol. Chem. 249, 2 2 5 8 - - 2 2 6 2 Terhorst, C., Wittmann-Liebold, B. and M/Slier, W. (1972) Eur. J. Biochem. 25, 13--19

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T e r h o r s t , C., M611er, W., L a u r s e n , R. a n d W i t t m a n n - L i e b o l d , B. ( 1 9 7 3 ) Eur. J. B i o c h e m . 34, 1 3 8 1 5 2 Alix, J . H . a n d H a y e s , D. ( 1 9 7 4 ) J. Mol. Biol. 8 6 , 1 3 9 - - 1 5 9 Chang, F . N . , C h a n g , C.N. a n d Paik, W.K. ( 1 9 7 4 ) J. B a c t e r i o L 1 2 0 , 6 5 1 - - - 6 5 6 C h a n g , C.N. a n d C h a n g , F . N . ( 1 9 7 5 ) B i o c h e m i s t r y 14, 4 6 8 - - 4 7 7 K u m a r A. a n d S u b r a m a n i a n , A . R . ( 1 9 7 5 ) J. Mol. Biol. 9 4 , 4 0 9 - - 4 2 3 Dice, J . F . a n d S c h i m k e , R.T. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 9 8 - I I I D e l a u n a y , J. a n d S c h a p i r a , G. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 9 , 2 6 2 - - 2 6 8 T e r a o , K., T s u r u g i , K. a n d O g a t a , K. ( 1 9 7 4 ) J. B i o e h e m . T o k y o 76, 1 1 1 3 - - 1 1 2 2 W o o l I.G. a n d S t S f f l e r , G. ( 1 9 7 4 ) R i b o s o m e s ( N o m u r a , M., Tissi~res, A. a n d L e n g y e l , P., eds.), pp. 4 1 7 - 4 6 1 , C o l d S p r i n g H a r b o r Press