113
Biochimica et Biophysica Acta, 563 (1979) 113--128 © Elsevier/North-Holland Biomedical Press
BBA 99454
KINETIC STUDIES ON RIBOSOMAL PROTEINS ASSEMBLY IN PRERIBOSOMAL PARTICLES AND RIBOSOMAL sUBUNITS OF MAMMALIAN CELLS
MARIE-ANNICK AUGER-BUENDIA *, MICHI~LE LONGUET and ARMAND TAVITIAN
Laboratoire d'Hdmatologie Expdrimentale, Institut de Recherches sur les Leuc~mies et les Maladies du Sang, H6pital Saint-Louis, 2 place du Dr. Alfred-Fournier, 754 75 Paris Cddex 10 (France) (Received October 5th, 1978)
Key words: Ribosomal protein assembly; Ribosomal subunit; (Kinetics, Mammalian)
Summary Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar preribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process. * To whom correspondence should b e
addressed.
114
Introduction In mammalian cells, the first steps of ribosome assembly take place in the nucleolus, where the newly transcribed large pre-rRNA is associated with a set of specific proteins [1--4]. Further processing of preribosomal particles involves endonucleolytic cleavages of the pre-rRNA and concomitant modifications in the protein content of the processed particles [ 5,6 ]. Recent reports have demonstrated the presence of some proteins which are added to the cytoplasmic ribosomal subunits at the later stages of the maturation process [7--11] and also in the absence of 'de novo' synthesis of 28-S and 18-S RNA [1,7,12,13]. However, little is known about the individual metabolism of the about 70 different protein components of eucaryotic ribosomes, especially at the earlier stages of ribosome assembly. The amount of individual ribosomal proteins in isolated 80-S preribosomes (the most nascent precursor particles) was measured under pulse and steadystate labeling conditions. Comparative experiments on the labeling kinetics of the proteins of cytoplasmic ribosomal subunits are also described. By using a modification of the two-dimensional electrophoretic method of Kaltschmidt and Wittmann [14], we obtained evidence that a significant number of ribosomal and nucleolar proteins present particular metabolic characteristics during the ribosome formation.
Materials and Methods
Cell culture and labeling L5178Y, a murine leukemic cell line, was cultivated in suspension as previously described [4,15]. For analysis of labeled ribosomal proteins, cells were grown for the indicated periods of time in Fischer medium lacking valine and leucine, and supplemented with 5% dialysed calf serum, 1% normal Fischer medium and 20 mCi/1 of a mixture of [3H]valine and [3H]leucine (30---35 Ci/mmol, from the CEA, Saclay, France).
Isolation of proteins from ribosomal and preribosomal particles The method used for the preparation of polyribosomes and the separation of ribosomal subunits has been described in our previous reports [4,16]. Nucleolar preribosomes were obtained by a modification [4] of the method developed by Warner and Soeiro [1], and highly purified by sucrose gradient centrifugation. Extraction of proteins from ribosomal and preribosomal particles was achieved by treatment with LiC1/urea, as detailed previously [4]. The specific activity of the proteins varied from 4 • 104 to 20 • 106 cpm • mg-l, depending on the duration of labeling.
Protein electrophoresis and radioactivity measurements Labeled proteins were mixed with carrier unlabeled proteins of polyribosomes, and analysed by two-dimensional polyacrylamide gel electrophoresis, using a modification [4] of the procedure of Howard and Traut [17]. Get slabs were stained by Coomassie Brilliant Blue, destained and either fluorographed and exposed to X-ray films [18], or used for labeling ratios measurements. In
115
this last case, stained spots were cut out from the slab gel with a scalpel, crushed in a 1 ml plastic syringe and solubilized at 45°C overnight in 0.5 ml
H202. 5 ml Picofluor (Packard Instrument Company) were added to each vial. Radioactive values were corrected for background by including appropriate standards during scintillation counting.
Results Incorporation of tritiated ribosomal proteins into 80-8 nucleolar preribosomes 80-S preribosomal particles were prepared from the nucleoli of routine L5178Y cells after incubation with tritiated valine and leucine for different periods of time. A period of 30 min was chosen for 'pulse-labeling' and 4 h for 'steady-state labeling' experiments, according to our previous results on the rates of ribosomal precursor RNA maturation as well as on the accumulation of labeled nucleolar ribonucleoproteins under these conditions [4,19]. Proteins were extracted from purified 80-S particles and analysed by two-dimensional gel electrophoresis. Comigration of tritiated preribosomal proteins with a large excess of unlabeled proteins from cytoplasmic polysomes ensures a precise identification of ribosomal and non-ribosomal proteins by superposition of the exposed X-ray film to the original stained gel plate. Fluorographic patterns of 80-S particle proteins are shown in Fig. 1A (after 30 rain labeling) and Fig. 1B (after 4 h labeling). Proteins were numbered and classified according to a code already described [4] with minor modifications. 40 major protein spots were identified and 20 additional minor spots, indiscernible on the photographs, appeared when the exposure of the dried gel plate to the X-ray film was prolonged. Comparison of the fluorograms shown for 'pulse' and 'steady-state' labeling experiments (Fig. 1A and B), revealed some differences in the relative intensities of several proteins. Spots L33, L34, $22-$24 and P9 appeared more intense in Fig. 1A than in Fig. lB. In contrast, proteins L5, L6-L7, L12, L13-L15, L14 and L21-L23 were more heavily labeled in Fig. 1B than in Fig. 1A. In some cases, two spots were observed at the position of protein L5; the intensity of labeling was increased for both spots in Fig. lB. Proteins Px and Py were only observed when the incubation period was decreased to 30 rain. As previously described [4], a set of three nucleolar protein spots appeared around P3 when the period of labeling was prolonged. Quantitative measurements of preribosomal proteins were carried out on parallel gel plates. The radioactivity contained in each ribosomal protein was determined as described in Materials and Methods. Non-ribosomal proteins were absent from the staining patterns of carrier polysomal proteins and thus were not counted, excepted for Pg, which has previously been shown to associate with polyribosomes [4]. The total radioactivity recovered from ribosomal protein spots was calculated for each gel plate, and fractions of the total radioactivity corresponding to each spot were listed in Table I. In experiments where triplicate gels from the same preparation or different preparations of the same set of proteins were compared, an average error of 15--20% was estimated for these percentages. Several regions of the two-dimensional pattern, such as L13-L15-S10, S14-$15, $26-L35, L33-$23, $22-$24-P9, were rather poorly
li6
0 P3
L3 $3
j~ ml_
L4
L5
241
Fig. 1.A.
resolved, and quantitative data must be regarded with caution. The percentage of total counts was determined only for the basic proteins (right part of the gel) with the exception of proteins $12, $21 and L22. For the 30-min labeling experiments, the a m o u n t of radioactivity of proteins L10 and S19 could not be determined, due to the contamination by Px and Py, respectively. No spot corresponding to L10 and $19 could be seen on the fluorograms, even when the duration of X-ray film exposure was prolonged. In the upper left region of the gel plate, the tailing of major protein spots, as well as the presence of many faintly labeled spots, did n o t allow precise determinations, and was n o t taken into account. Fractions o f the total radioactivity in each protein spot were compared for 'pulse-labeled' and longer incubation
117
Fig. I . T w o - d i m e n s i o n a l f l u o r o g r a p h 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 80-S p r e r i b o s o m a l p a r t i c l e s a f t e r 3 0 r a i n ( A ) a n d 4 h (B) o f l a b e l i n g w i t h t r i t i a t e d v a l i n e a n d l e u c i n e . 1 0 0 # g o f 8 0 - S p r o t e i n s (1 • 1 0 6 c p m ) w e r e a n a l y s e d . M i g r a t i o n in t h e first d i m e n s i o n w a s f r o m t h e o r i g i n ( O ) t o t h e r i g h t ( c a t h o d e ) a n d to the left (anode) at pH 8.6. Migration in the 2nd dimension was toward the bottom (cathode) at pH 4.6. T h e gel p l a t e s w e r e s u b j e c t e d t o f l u o r o g r a p h y a n d e x p o s e d t o X - r a y f i l m s f o r 3 w e e k s a t - - 8 0 ° C . S e v e r a l s p o t s w h i c h w e r e a p p a r e n t o n t h e f i l m s a r e n o t visible o n t h e p h o t o g r a p h s .
experiments. Values thus obtained for these ratios (Table I, 3rd column) reflect the relative rate of newly synthesized protein incorporation into preribosomes and were compared to the average value (0.91). Whereas the bulk of preribosomai proteins presented a 30 min/4 h ratio of 0.3:1, corresponding to a gradual incorporation of radioactivity, its appears that a few proteins had a higher ratio than the average, and took up label more rapidly than the others: these were $6-$8, $22-S24, L9, L l l , L17, L31, L33, L34, L38. Non-ribosomal
118 TABLE I RELATIVE
LABELING
RATIOS OF PROTEINS
FROM 80-S PRERIBOSOMES
The major stained spots were cut out and their radioactivity measured. The 3H activity in individual spots ranged between 35 and 7000 cpm. The ratios of total counts were corrected by subtracting background counts. The reproducibility of the results was estimated by calculating the standard errors from the values of two similar experiments not shown here. An average error of 16% was estimated for ratios of total radioactivity of individual spots. 30 rain/4 h ratios were determined only for spots containing more than 0.3% of total counts. The average value of the ratios was 0.91, and proteins exhibiting a 30 rain/4 h ratio o f g r e a t e r t h a n 1 are i n d i c a t e d b y a s t e r i s k s . Protein
Percent total radioactivity 30 rain
4 h
30 min/4 h
L3 L4 L5 L6°L7 L8 L9 * LIO Lll * L12 L13-15 L14 L16 L17 * L18 L19 L20 L21-23 L22 L24 L26 L27 L28 L29 L30 L31 * L33 * L34 * L35 L36 L37 L38 * L39
1.1 1.9 1.7 1.9 2.6 3.4 -0.6 3.3 3.9 1.6 1.9 4.1 0.2 -0.5 3.1 0 0.1 1.8 1.6 0.5 0.3 0.2 2.3 6.1 5.5 0.4 0.6 0.2 4.0
3.2 2.8 3.4 5.8 3.0 2.5 -0.4 5.3 7.1 2.8 2.1 2.9 0.5 1.1 1.5 7.8 0 0 2.5 2.1 1.1 0.1 0.6 1.6 3.4 3.1 1.1 0.5 0.8 3.4
0.3 0.7 0.5 0.3 0.9 1.3 -1.7 0.6 0.5 0.6 0.9 1.4 0.4 -0.3 0.4 --0.7 0.8 0.5 -0.3 1.4 1.8 1.8 0.4 1.0 0.3 1.2
0
0
--
L40
0
0.I
--
Protein
$2 S3-Sa $4 $5 $6-$8 * $7 $9 SIO Sll $12 $13 $14 $15 S16-$18 $17 S19 $20 $21 $22-$24 * $23 $25 $26 $27 $28 P9 *
Percent total radioactivity 30 rain
4 h
30 rain/4 h
0 0.4 2.2 0.5 5.0 0.6 0 0.7 0.2 0.1 0.1 3.2 1.2 1.6 0.1 -0.2 0 6.7 0.8 0.2 0.2 0.1 0.2
0 0.7 2.8 0.3 3.6 0.6 0 1.3 0.4 0.3 0.6 3.2 1.1 2.2 0.4 -0.1 0 3.7 1.3 0.5 0.5 0 0.1
-0.7 0.8 -1.4 1.0 -0.6 0.5 0.3 0.2 1.0 1.1 0.7 ----1.8 0.6 -0.4 ---
17.2
3.2
5.4
protein P9 exhibited a particularly high ratio. The distribution o f preribosomal proteins among rapidly and gradually labeled classes is illustrated in Fig. 2.
Labeling pattern of 60-,9 ribosomal subunit proteins Cytoplasmic 60-S ribosomal subunits were isolated from L5178Y cells labeled with tritiated valine and leucine for 30 min and 4 h. The preparation o f ribosomal subunits was carried out from the cell cultures which were used for the isolation o f nucleolar 80-S preribosomes (as reported above). It must be pointed out that only traces of newly synthesized 28-S rRNA appeared in the
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cytoplasm of L5178Y cells labeled with [3H]uridine for 30 min, whereas small amounts of labeled 18-S rRNA were detected in 'nascent' 40-S subunits (unpublished data). Moreover, 4 h is not generally considered as a 'steady-state' labeling period for cytoplasmic ribosomes. However, these conditions were chosen in order to compare the metabolic properties of ribosomal proteins within both nucleolar and cytoplasmic particles, after identical periods of labeling. The two-dimensional fluorographic patterns of the large ribosomal subunit proteins of L5178Y cells are shown in Fig. 3A (30 rain label) and 3B (4 h label). It can be seen that the two patterns were considerably different. Particularly striking were the intensities of the three major spots at the earlier labeling time, i.e. L10, L24 and L29, and to a lesser extent, L9, L13, L38 and L40. The occurrence of two components of L10, frequently observed also for L14, L17, L24 and $4, could be due to a chemical alteration of the molecule [21]. Many protein spots were not visible on the fluorogram. In contrast, the labeling intensity of 60-S proteins after a longer incubation (Fig. 3B) looked more
120
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38
40
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uniform. These observations were confirmed by the quantitative determination of radioactivity in individual protein spots, expressed in Table II as a fraction of total radioactivity incorporated during 30 min and 4 h in 60-S subunits. A comparative study of the fractions of total counts in spots of pulse- and longlabeled proteins (Table II, 3rd column) showed a higher than average ratio for proteins L10, L22, L24, L29 and L40. Rapidly labeled proteins of 60-S subunit are represented as filled spots in Fig. 5. Another group of proteins, including L6-L7, L8, L9, L21wL23 and L28, did not reveal a particularly high 30 min/ 4 h ratio, but yet were present in noticeable amounts in pulse-labeled patterns of 60-S subunit proteins.
121
Fig. 3. T w o - d i m e n s i o n a l f l u o r o g r a p h 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 6 0 - S r i b o s o m a l s u b u n i t s a f t e r 3 0 r a i n ( A ) a n d 4 h (B) o f l a b e l i n g w i t h t r i t i a t e d a m i n o - a c i d s . 6 0 - S p r o t e i n s (3 • 1 0 5 c p m ) w e r e a n a l y s e d a s in Fig. 1. T h e e x p o s u r e o f t h e f i l m s w a s f o r 2 w e e k s .
Labeling pattern of 40-S ribosomal subunit proteins Fig. 4 shows the two-dimensional fiuorographic patterns of the small ribosomal subunit proteins from cells labeled for 30 rain (A) and 4 h (B) with tritiated valine and leucine. The 30 rain labeled major 40-S ribosomal proteins were identified as $9, S3-Sa, $7, S15, S19, S20 and $26 in Fig. 4A. Several minor spots as $2, $4, $6-S8, S16-S18, S17 and $27 were barely discemable on the photograph. In contrast, the radioactive level of 40-S subunit proteins looked more uniform after a longer incubation (Fig. 4B). Table III shows that about one quarter of
122
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total radioactivity contained in 40-8 subunit after 30 min labeling was localized in $9, and that the seven major proteins revealed on the fluorogram (Fig. 4A) represented 77% of the total counts. Comparative anaiysis of t h e fractions o f total radioactivity in individual spots under pulse- or long-labeled c o ~ i t i o n s indicated that proteins 87, 89, $20 and $29 had higher 30 ~ n / 4 h ratios than the average (1.45). These proteins which picked up label more rapidly that the r e m a i ~ e r are represented as filled spotsin Fig. 5. Proteins $2, $4, ~ 8 8 , 8 1 5 a n d $22-$24 were significantly labeled in short, term labeling patterns, but their 30 rain/4 h ratios were not so high.
123
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13
16
26
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Fig. 4. F l u o r o g r a p h 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 40-S r i b o s o m a l s u b u n i t s . A. Cells w e r e i n c u b a t e d f o r 3 0 r a i n w i t h 3 H - l a b e l e d a m i n o acids. B. 4 h i n c u b a t i o n w i t h 3 H - l a b e l e d a m i n o acids. 40-S p r o t e i n s ( 1 . 5 • 105 c p m ) w e r e a n a l y s e d as in Fig. 1. F l u o r o g r a p h i c e x p o s u r e was f o r 4 w e e k s .
Discussion
During the course of our previous characterization of isolated 80-S preribosomal particles, we observed in the fluorograms of short.labeled cells important variations in the intensity of the tritiated proteins spots [4]. We have attempted to gain further insight into this study by quantitative measurements of the radioactivity which is incorporated into individual preribosomal particles after short and longer periods of incubation. We intended to identify the sequence of ribosomal protein assembly during the process of ribosome matura-
124
TABLE II QUANTITATIVE
DETERMINATION
O F T H E L A B E L I N G P A T T E R N S O F 60-S S U B U N I T P R O T E I N S
Fractions of total radioactivity in the well defined spots from 30-rain and 4-h patterns were determined a n d c o m p a r e d as in T a b l e I. A v e r a g e v a l u e s a n d s t a n d a r d errors w e r e c a l c u l a t e d w h e n t h r e e o r m o r e d e t e r m i n a t i o n s w e r e available. T h e a v e r a g e 3 0 m i n / 4 h r a t i o w a s 0 . 9 2 . Protein
L3 L4 L5 L6-L7 L8 L9 L10 * Lll L12 L13-15 L14 L16 L17 L18 L19 L20 L21-23 L22 * L24 * L26 L27 L2S L29 * L30 L31 L33 L34 L35 L36 L37 L38 L39 L40 *
Percent of total radioactivity 30 min
4 h (S.E.)
0.9 0.6 0.1 3.7 3.5 5.8 35.9 0.1 0 6.4 0 0 0.2 0 0.6 0.5 2.9 1.4 11.6 0.7 0.8 3.5 11.0 0 0.3 1.7 0.2 0 0 0 6.0 0 2.8
5.0 4.3 0.7 10.7 3.3 5.6 5.3 1.6 2.3 7.0 2.5 0.6 2.5 1.6 1.4 1.5 9.0 0.7 2.3 2.9 2.9 4.1 2.4 0.8 1.6 1.6 0.7 1.9 3.1 2.4 4.9 0.3 0.9
(0.20) (1.00) (0.14) (0.44) (0.28) (0.62) (0.90) -(0.20) (1.20) (0.52) (0.20) (0.52) (0.44) (0.44) (0.28) (3.06) (0.20) (0.20) (0.94) (0.62) (0.56) (0.44) (0.20) (0.20) (1.03) (0.95) (0.44) (0.10) (0.20) (0.20) -(0.10)
30 min/4 h
0.2 0.1 0.2 0.3 1.0 1.0 6.8 0.1 -0.9 --0.1 -0.4 0.3 0.3 1.9 5.0 0.2 0.3 0.8 4.6 -0.2 1.1 0.3 ---1.2 -3.1
tion, by analysing the uptake of radioactivity within the proteins of cytoplasmic ribosomal subunits, under the same conditions of labeling. The two-dimensional electrophoretic method used for this purpose has been widely utilized for the analysis of eucaryotic ribosomal proteins and allows the correlation of the results obtained by several authors on the properties of individual proteins, such as molecular weight, phosphorylation, exchange, reactivity, differences during evolution or differentiation, and amino acid composition [ 17,20,21--29]. However, this method provides a better resolution for basic than for acidic proteins, and a large fraction o f the applied proteiv~ are retained at the origin, which could involve a differential loss o f individual proteins. For these reasons, other electrophoretic procedures are now developed for the analysis of ribosomal and preribosomal proteins [3,30], but
125
T A B L E III RELATIVE
LABELING RATIOS OF 40-S SUBUNIT PROTEINS
S e e t h e c a p t i o n s o f T a b l e s I and II. A n average value o f 1 . 4 5 w a s c a l c u l a t e d f o r t h e 3 0 m i n / 4 h ratios. Protein
S2 S3-Sa $4 $5 S6-S8
Percent total radioactivity
30 min
4 h (S.E.)
30 min/4 h
4.6 10.3 1.8 0.5 1.9
5.4 7.7 7.1 0.8 10.8
(0.75) (1.14) (0.81) (0.07) (2.00)
0.8 1.3 0.2 0.7 0.2
2.3 (0.83)
3.1
4.7 4.1 5.2 1.5
5.9 0.2 0.1 0.2
S7 *
7.1
$9 * S10 $11 S12 S13
27.9 0.7 0.6 0.3 0
$14
0.5
S15 S16-S18 S17 S19 $20 * S21 $22-$24 $23
8.0 4.1 3.4 5.7 9.3 0.2 2.8 0.7 0
$25 $26 * $27
$28
8.6 1.1 0
(2.36) (0.58) (0.99) (0.14)
2.8 (0.68) 9.6 (1.02)
-0
8.0 8.3 2.3 4.0 1.1 0.5 4.6 4.2
(0.81) (2.40) (0.70) (0.77) (0.20) -(0.70) (0.96)
1.0 0.5 1.5 1.4 8.1 0.5 0.6 0.2
0.4 (0.I0) 3.3 (0.38) 0.5 (0.20) 0 . 8 (0.06)
-2.6 2.1 --
the informations provided by these studies on the metabolic characteristics of individual proteins are still unreliable. Cells were incubated with a mixture of valine and leucine, which are shared with all ribosomal proteins at a rather uniform level [28,29]. As far as our study deals with the comparison of the radioactive level of the same protein at different times of labeling, discrepancies in the distribution of valine and leucine would not interfere with our results. By comparing the results obtained in Tables I, II and III and in Figs. 3 and 5, it is clear that a number of ribosomal proteins behaved differently from the remainder on the basis o f their accumulation o f labeled amino acids. Most of the rapidly labeled proteins of nucleolar 80-S preribosomes were distinct from the rapidly labeled proteins of ribosomal subunits. A m o n g the about 60 distinct major proteins found in 80-S preribosomes, 12 incorporated label more quickly than the remainder. Most of them are structural constituents of the large ribosomal subunit, and to a lesser extent of the small subunit. Three nucleolar non-ribosomal proteins (Px, Py and P9) were shown to be labeled quite rapidly, and particularly striking was the high proportion o f P9 in pulse-labeled samples. The presence of a few rapidly labeled nucleolar proteins has been reported in 55-S preribosomal particles o f HeLa cells [10], although most o f the non-ribosomal proteins were shown to incorporate the radioactivity at a slower rate, as should be expected [31].
126
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Biochimica et Biophysica Acta, 563 (1979) 113--128 © Elsevier/North-Holland Biomedical Press
BBA 99454
KINETIC STUDIES ON RIBOSOMAL PROTEINS ASSEMBLY IN PRERIBOSOMAL PARTICLES AND RIBOSOMAL sUBUNITS OF MAMMALIAN CELLS
MARIE-ANNICK AUGER-BUENDIA *, MICHI~LE LONGUET and ARMAND TAVITIAN
Laboratoire d'Hdmatologie Expdrimentale, Institut de Recherches sur les Leuc~mies et les Maladies du Sang, H6pital Saint-Louis, 2 place du Dr. Alfred-Fournier, 754 75 Paris Cddex 10 (France) (Received October 5th, 1978)
Key words: Ribosomal protein assembly; Ribosomal subunit; (Kinetics, Mammalian)
Summary Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar preribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process. * To whom correspondence should b e
addressed.
114
Introduction In mammalian cells, the first steps of ribosome assembly take place in the nucleolus, where the newly transcribed large pre-rRNA is associated with a set of specific proteins [1--4]. Further processing of preribosomal particles involves endonucleolytic cleavages of the pre-rRNA and concomitant modifications in the protein content of the processed particles [ 5,6 ]. Recent reports have demonstrated the presence of some proteins which are added to the cytoplasmic ribosomal subunits at the later stages of the maturation process [7--11] and also in the absence of 'de novo' synthesis of 28-S and 18-S RNA [1,7,12,13]. However, little is known about the individual metabolism of the about 70 different protein components of eucaryotic ribosomes, especially at the earlier stages of ribosome assembly. The amount of individual ribosomal proteins in isolated 80-S preribosomes (the most nascent precursor particles) was measured under pulse and steadystate labeling conditions. Comparative experiments on the labeling kinetics of the proteins of cytoplasmic ribosomal subunits are also described. By using a modification of the two-dimensional electrophoretic method of Kaltschmidt and Wittmann [14], we obtained evidence that a significant number of ribosomal and nucleolar proteins present particular metabolic characteristics during the ribosome formation.
Materials and Methods
Cell culture and labeling L5178Y, a murine leukemic cell line, was cultivated in suspension as previously described [4,15]. For analysis of labeled ribosomal proteins, cells were grown for the indicated periods of time in Fischer medium lacking valine and leucine, and supplemented with 5% dialysed calf serum, 1% normal Fischer medium and 20 mCi/1 of a mixture of [3H]valine and [3H]leucine (30---35 Ci/mmol, from the CEA, Saclay, France).
Isolation of proteins from ribosomal and preribosomal particles The method used for the preparation of polyribosomes and the separation of ribosomal subunits has been described in our previous reports [4,16]. Nucleolar preribosomes were obtained by a modification [4] of the method developed by Warner and Soeiro [1], and highly purified by sucrose gradient centrifugation. Extraction of proteins from ribosomal and preribosomal particles was achieved by treatment with LiC1/urea, as detailed previously [4]. The specific activity of the proteins varied from 4 • 104 to 20 • 106 cpm • mg-l, depending on the duration of labeling.
Protein electrophoresis and radioactivity measurements Labeled proteins were mixed with carrier unlabeled proteins of polyribosomes, and analysed by two-dimensional polyacrylamide gel electrophoresis, using a modification [4] of the procedure of Howard and Traut [17]. Get slabs were stained by Coomassie Brilliant Blue, destained and either fluorographed and exposed to X-ray films [18], or used for labeling ratios measurements. In
115
this last case, stained spots were cut out from the slab gel with a scalpel, crushed in a 1 ml plastic syringe and solubilized at 45°C overnight in 0.5 ml
H202. 5 ml Picofluor (Packard Instrument Company) were added to each vial. Radioactive values were corrected for background by including appropriate standards during scintillation counting.
Results Incorporation of tritiated ribosomal proteins into 80-8 nucleolar preribosomes 80-S preribosomal particles were prepared from the nucleoli of routine L5178Y cells after incubation with tritiated valine and leucine for different periods of time. A period of 30 min was chosen for 'pulse-labeling' and 4 h for 'steady-state labeling' experiments, according to our previous results on the rates of ribosomal precursor RNA maturation as well as on the accumulation of labeled nucleolar ribonucleoproteins under these conditions [4,19]. Proteins were extracted from purified 80-S particles and analysed by two-dimensional gel electrophoresis. Comigration of tritiated preribosomal proteins with a large excess of unlabeled proteins from cytoplasmic polysomes ensures a precise identification of ribosomal and non-ribosomal proteins by superposition of the exposed X-ray film to the original stained gel plate. Fluorographic patterns of 80-S particle proteins are shown in Fig. 1A (after 30 rain labeling) and Fig. 1B (after 4 h labeling). Proteins were numbered and classified according to a code already described [4] with minor modifications. 40 major protein spots were identified and 20 additional minor spots, indiscernible on the photographs, appeared when the exposure of the dried gel plate to the X-ray film was prolonged. Comparison of the fluorograms shown for 'pulse' and 'steady-state' labeling experiments (Fig. 1A and B), revealed some differences in the relative intensities of several proteins. Spots L33, L34, $22-$24 and P9 appeared more intense in Fig. 1A than in Fig. lB. In contrast, proteins L5, L6-L7, L12, L13-L15, L14 and L21-L23 were more heavily labeled in Fig. 1B than in Fig. 1A. In some cases, two spots were observed at the position of protein L5; the intensity of labeling was increased for both spots in Fig. lB. Proteins Px and Py were only observed when the incubation period was decreased to 30 rain. As previously described [4], a set of three nucleolar protein spots appeared around P3 when the period of labeling was prolonged. Quantitative measurements of preribosomal proteins were carried out on parallel gel plates. The radioactivity contained in each ribosomal protein was determined as described in Materials and Methods. Non-ribosomal proteins were absent from the staining patterns of carrier polysomal proteins and thus were not counted, excepted for Pg, which has previously been shown to associate with polyribosomes [4]. The total radioactivity recovered from ribosomal protein spots was calculated for each gel plate, and fractions of the total radioactivity corresponding to each spot were listed in Table I. In experiments where triplicate gels from the same preparation or different preparations of the same set of proteins were compared, an average error of 15--20% was estimated for these percentages. Several regions of the two-dimensional pattern, such as L13-L15-S10, S14-$15, $26-L35, L33-$23, $22-$24-P9, were rather poorly
li6
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L3 $3
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resolved, and quantitative data must be regarded with caution. The percentage of total counts was determined only for the basic proteins (right part of the gel) with the exception of proteins $12, $21 and L22. For the 30-min labeling experiments, the a m o u n t of radioactivity of proteins L10 and S19 could not be determined, due to the contamination by Px and Py, respectively. No spot corresponding to L10 and $19 could be seen on the fluorograms, even when the duration of X-ray film exposure was prolonged. In the upper left region of the gel plate, the tailing of major protein spots, as well as the presence of many faintly labeled spots, did n o t allow precise determinations, and was n o t taken into account. Fractions o f the total radioactivity in each protein spot were compared for 'pulse-labeled' and longer incubation
117
Fig. I . T w o - d i m e n s i o n a l f l u o r o g r a p h 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 80-S p r e r i b o s o m a l p a r t i c l e s a f t e r 3 0 r a i n ( A ) a n d 4 h (B) o f l a b e l i n g w i t h t r i t i a t e d v a l i n e a n d l e u c i n e . 1 0 0 # g o f 8 0 - S p r o t e i n s (1 • 1 0 6 c p m ) w e r e a n a l y s e d . M i g r a t i o n in t h e first d i m e n s i o n w a s f r o m t h e o r i g i n ( O ) t o t h e r i g h t ( c a t h o d e ) a n d to the left (anode) at pH 8.6. Migration in the 2nd dimension was toward the bottom (cathode) at pH 4.6. T h e gel p l a t e s w e r e s u b j e c t e d t o f l u o r o g r a p h y a n d e x p o s e d t o X - r a y f i l m s f o r 3 w e e k s a t - - 8 0 ° C . S e v e r a l s p o t s w h i c h w e r e a p p a r e n t o n t h e f i l m s a r e n o t visible o n t h e p h o t o g r a p h s .
experiments. Values thus obtained for these ratios (Table I, 3rd column) reflect the relative rate of newly synthesized protein incorporation into preribosomes and were compared to the average value (0.91). Whereas the bulk of preribosomai proteins presented a 30 min/4 h ratio of 0.3:1, corresponding to a gradual incorporation of radioactivity, its appears that a few proteins had a higher ratio than the average, and took up label more rapidly than the others: these were $6-$8, $22-S24, L9, L l l , L17, L31, L33, L34, L38. Non-ribosomal
118 TABLE I RELATIVE
LABELING
RATIOS OF PROTEINS
FROM 80-S PRERIBOSOMES
The major stained spots were cut out and their radioactivity measured. The 3H activity in individual spots ranged between 35 and 7000 cpm. The ratios of total counts were corrected by subtracting background counts. The reproducibility of the results was estimated by calculating the standard errors from the values of two similar experiments not shown here. An average error of 16% was estimated for ratios of total radioactivity of individual spots. 30 rain/4 h ratios were determined only for spots containing more than 0.3% of total counts. The average value of the ratios was 0.91, and proteins exhibiting a 30 rain/4 h ratio o f g r e a t e r t h a n 1 are i n d i c a t e d b y a s t e r i s k s . Protein
Percent total radioactivity 30 rain
4 h
30 min/4 h
L3 L4 L5 L6°L7 L8 L9 * LIO Lll * L12 L13-15 L14 L16 L17 * L18 L19 L20 L21-23 L22 L24 L26 L27 L28 L29 L30 L31 * L33 * L34 * L35 L36 L37 L38 * L39
1.1 1.9 1.7 1.9 2.6 3.4 -0.6 3.3 3.9 1.6 1.9 4.1 0.2 -0.5 3.1 0 0.1 1.8 1.6 0.5 0.3 0.2 2.3 6.1 5.5 0.4 0.6 0.2 4.0
3.2 2.8 3.4 5.8 3.0 2.5 -0.4 5.3 7.1 2.8 2.1 2.9 0.5 1.1 1.5 7.8 0 0 2.5 2.1 1.1 0.1 0.6 1.6 3.4 3.1 1.1 0.5 0.8 3.4
0.3 0.7 0.5 0.3 0.9 1.3 -1.7 0.6 0.5 0.6 0.9 1.4 0.4 -0.3 0.4 --0.7 0.8 0.5 -0.3 1.4 1.8 1.8 0.4 1.0 0.3 1.2
0
0
--
L40
0
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Protein
$2 S3-Sa $4 $5 $6-$8 * $7 $9 SIO Sll $12 $13 $14 $15 S16-$18 $17 S19 $20 $21 $22-$24 * $23 $25 $26 $27 $28 P9 *
Percent total radioactivity 30 rain
4 h
30 rain/4 h
0 0.4 2.2 0.5 5.0 0.6 0 0.7 0.2 0.1 0.1 3.2 1.2 1.6 0.1 -0.2 0 6.7 0.8 0.2 0.2 0.1 0.2
0 0.7 2.8 0.3 3.6 0.6 0 1.3 0.4 0.3 0.6 3.2 1.1 2.2 0.4 -0.1 0 3.7 1.3 0.5 0.5 0 0.1
-0.7 0.8 -1.4 1.0 -0.6 0.5 0.3 0.2 1.0 1.1 0.7 ----1.8 0.6 -0.4 ---
17.2
3.2
5.4
protein P9 exhibited a particularly high ratio. The distribution o f preribosomal proteins among rapidly and gradually labeled classes is illustrated in Fig. 2.
Labeling pattern of 60-,9 ribosomal subunit proteins Cytoplasmic 60-S ribosomal subunits were isolated from L5178Y cells labeled with tritiated valine and leucine for 30 min and 4 h. The preparation o f ribosomal subunits was carried out from the cell cultures which were used for the isolation o f nucleolar 80-S preribosomes (as reported above). It must be pointed out that only traces of newly synthesized 28-S rRNA appeared in the
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® Fig. 2. D i s t r i b u t i o n o f r a p i d a n d s l o w l a b e l i n g p r o t e i n s i n 8 0 - S p r e r i b o s o m a l p a r t i c l e s . F r o m t h e d a t a p r e s e n t e d i n Fig. 1 a n d i n T a b l e I, p r o t e i n s w e r e c]A__~_ified as r a p i d l y l a b e l e d ( r e p r e s e n t e d as filled s p o t s ) a n d g r a d u a l l y l a b e l e d p r o t e i n s ( h a t c h e d s p o t s ) . R i b o s o m a l s t r u c t u r a l p o l y p e p t i d e s t h a t are a b s e n t f r o m t h e p a t t e r n s o f 8 0 - S p a r t i c l e s p r o t e i n s a r e r e p r e s e n t e d as o p e n circles.
cytoplasm of L5178Y cells labeled with [3H]uridine for 30 min, whereas small amounts of labeled 18-S rRNA were detected in 'nascent' 40-S subunits (unpublished data). Moreover, 4 h is not generally considered as a 'steady-state' labeling period for cytoplasmic ribosomes. However, these conditions were chosen in order to compare the metabolic properties of ribosomal proteins within both nucleolar and cytoplasmic particles, after identical periods of labeling. The two-dimensional fluorographic patterns of the large ribosomal subunit proteins of L5178Y cells are shown in Fig. 3A (30 rain label) and 3B (4 h label). It can be seen that the two patterns were considerably different. Particularly striking were the intensities of the three major spots at the earlier labeling time, i.e. L10, L24 and L29, and to a lesser extent, L9, L13, L38 and L40. The occurrence of two components of L10, frequently observed also for L14, L17, L24 and $4, could be due to a chemical alteration of the molecule [21]. Many protein spots were not visible on the fluorogram. In contrast, the labeling intensity of 60-S proteins after a longer incubation (Fig. 3B) looked more
120
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uniform. These observations were confirmed by the quantitative determination of radioactivity in individual protein spots, expressed in Table II as a fraction of total radioactivity incorporated during 30 min and 4 h in 60-S subunits. A comparative study of the fractions of total counts in spots of pulse- and longlabeled proteins (Table II, 3rd column) showed a higher than average ratio for proteins L10, L22, L24, L29 and L40. Rapidly labeled proteins of 60-S subunit are represented as filled spots in Fig. 5. Another group of proteins, including L6-L7, L8, L9, L21wL23 and L28, did not reveal a particularly high 30 min/ 4 h ratio, but yet were present in noticeable amounts in pulse-labeled patterns of 60-S subunit proteins.
121
Fig. 3. T w o - d i m e n s i o n a l f l u o r o g r a p h 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 6 0 - S r i b o s o m a l s u b u n i t s a f t e r 3 0 r a i n ( A ) a n d 4 h (B) o f l a b e l i n g w i t h t r i t i a t e d a m i n o - a c i d s . 6 0 - S p r o t e i n s (3 • 1 0 5 c p m ) w e r e a n a l y s e d a s in Fig. 1. T h e e x p o s u r e o f t h e f i l m s w a s f o r 2 w e e k s .
Labeling pattern of 40-S ribosomal subunit proteins Fig. 4 shows the two-dimensional fiuorographic patterns of the small ribosomal subunit proteins from cells labeled for 30 rain (A) and 4 h (B) with tritiated valine and leucine. The 30 rain labeled major 40-S ribosomal proteins were identified as $9, S3-Sa, $7, S15, S19, S20 and $26 in Fig. 4A. Several minor spots as $2, $4, $6-S8, S16-S18, S17 and $27 were barely discemable on the photograph. In contrast, the radioactive level of 40-S subunit proteins looked more uniform after a longer incubation (Fig. 4B). Table III shows that about one quarter of
122
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total radioactivity contained in 40-8 subunit after 30 min labeling was localized in $9, and that the seven major proteins revealed on the fluorogram (Fig. 4A) represented 77% of the total counts. Comparative anaiysis of t h e fractions o f total radioactivity in individual spots under pulse- or long-labeled c o ~ i t i o n s indicated that proteins 87, 89, $20 and $29 had higher 30 ~ n / 4 h ratios than the average (1.45). These proteins which picked up label more rapidly that the r e m a i ~ e r are represented as filled spotsin Fig. 5. Proteins $2, $4, ~ 8 8 , 8 1 5 a n d $22-$24 were significantly labeled in short, term labeling patterns, but their 30 rain/4 h ratios were not so high.
123
b
13
16
26
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Fig. 4. F l u o r o g r a p h 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 40-S r i b o s o m a l s u b u n i t s . A. Cells w e r e i n c u b a t e d f o r 3 0 r a i n w i t h 3 H - l a b e l e d a m i n o acids. B. 4 h i n c u b a t i o n w i t h 3 H - l a b e l e d a m i n o acids. 40-S p r o t e i n s ( 1 . 5 • 105 c p m ) w e r e a n a l y s e d as in Fig. 1. F l u o r o g r a p h i c e x p o s u r e was f o r 4 w e e k s .
Discussion
During the course of our previous characterization of isolated 80-S preribosomal particles, we observed in the fluorograms of short.labeled cells important variations in the intensity of the tritiated proteins spots [4]. We have attempted to gain further insight into this study by quantitative measurements of the radioactivity which is incorporated into individual preribosomal particles after short and longer periods of incubation. We intended to identify the sequence of ribosomal protein assembly during the process of ribosome matura-
124
TABLE II QUANTITATIVE
DETERMINATION
O F T H E L A B E L I N G P A T T E R N S O F 60-S S U B U N I T P R O T E I N S
Fractions of total radioactivity in the well defined spots from 30-rain and 4-h patterns were determined a n d c o m p a r e d as in T a b l e I. A v e r a g e v a l u e s a n d s t a n d a r d errors w e r e c a l c u l a t e d w h e n t h r e e o r m o r e d e t e r m i n a t i o n s w e r e available. T h e a v e r a g e 3 0 m i n / 4 h r a t i o w a s 0 . 9 2 . Protein
L3 L4 L5 L6-L7 L8 L9 L10 * Lll L12 L13-15 L14 L16 L17 L18 L19 L20 L21-23 L22 * L24 * L26 L27 L2S L29 * L30 L31 L33 L34 L35 L36 L37 L38 L39 L40 *
Percent of total radioactivity 30 min
4 h (S.E.)
0.9 0.6 0.1 3.7 3.5 5.8 35.9 0.1 0 6.4 0 0 0.2 0 0.6 0.5 2.9 1.4 11.6 0.7 0.8 3.5 11.0 0 0.3 1.7 0.2 0 0 0 6.0 0 2.8
5.0 4.3 0.7 10.7 3.3 5.6 5.3 1.6 2.3 7.0 2.5 0.6 2.5 1.6 1.4 1.5 9.0 0.7 2.3 2.9 2.9 4.1 2.4 0.8 1.6 1.6 0.7 1.9 3.1 2.4 4.9 0.3 0.9
(0.20) (1.00) (0.14) (0.44) (0.28) (0.62) (0.90) -(0.20) (1.20) (0.52) (0.20) (0.52) (0.44) (0.44) (0.28) (3.06) (0.20) (0.20) (0.94) (0.62) (0.56) (0.44) (0.20) (0.20) (1.03) (0.95) (0.44) (0.10) (0.20) (0.20) -(0.10)
30 min/4 h
0.2 0.1 0.2 0.3 1.0 1.0 6.8 0.1 -0.9 --0.1 -0.4 0.3 0.3 1.9 5.0 0.2 0.3 0.8 4.6 -0.2 1.1 0.3 ---1.2 -3.1
tion, by analysing the uptake of radioactivity within the proteins of cytoplasmic ribosomal subunits, under the same conditions of labeling. The two-dimensional electrophoretic method used for this purpose has been widely utilized for the analysis of eucaryotic ribosomal proteins and allows the correlation of the results obtained by several authors on the properties of individual proteins, such as molecular weight, phosphorylation, exchange, reactivity, differences during evolution or differentiation, and amino acid composition [ 17,20,21--29]. However, this method provides a better resolution for basic than for acidic proteins, and a large fraction o f the applied proteiv~ are retained at the origin, which could involve a differential loss o f individual proteins. For these reasons, other electrophoretic procedures are now developed for the analysis of ribosomal and preribosomal proteins [3,30], but
125
T A B L E III RELATIVE
LABELING RATIOS OF 40-S SUBUNIT PROTEINS
S e e t h e c a p t i o n s o f T a b l e s I and II. A n average value o f 1 . 4 5 w a s c a l c u l a t e d f o r t h e 3 0 m i n / 4 h ratios. Protein
S2 S3-Sa $4 $5 S6-S8
Percent total radioactivity
30 min
4 h (S.E.)
30 min/4 h
4.6 10.3 1.8 0.5 1.9
5.4 7.7 7.1 0.8 10.8
(0.75) (1.14) (0.81) (0.07) (2.00)
0.8 1.3 0.2 0.7 0.2
2.3 (0.83)
3.1
4.7 4.1 5.2 1.5
5.9 0.2 0.1 0.2
S7 *
7.1
$9 * S10 $11 S12 S13
27.9 0.7 0.6 0.3 0
$14
0.5
S15 S16-S18 S17 S19 $20 * S21 $22-$24 $23
8.0 4.1 3.4 5.7 9.3 0.2 2.8 0.7 0
$25 $26 * $27
$28
8.6 1.1 0
(2.36) (0.58) (0.99) (0.14)
2.8 (0.68) 9.6 (1.02)
-0
8.0 8.3 2.3 4.0 1.1 0.5 4.6 4.2
(0.81) (2.40) (0.70) (0.77) (0.20) -(0.70) (0.96)
1.0 0.5 1.5 1.4 8.1 0.5 0.6 0.2
0.4 (0.I0) 3.3 (0.38) 0.5 (0.20) 0 . 8 (0.06)
-2.6 2.1 --
the informations provided by these studies on the metabolic characteristics of individual proteins are still unreliable. Cells were incubated with a mixture of valine and leucine, which are shared with all ribosomal proteins at a rather uniform level [28,29]. As far as our study deals with the comparison of the radioactive level of the same protein at different times of labeling, discrepancies in the distribution of valine and leucine would not interfere with our results. By comparing the results obtained in Tables I, II and III and in Figs. 3 and 5, it is clear that a number of ribosomal proteins behaved differently from the remainder on the basis o f their accumulation o f labeled amino acids. Most of the rapidly labeled proteins of nucleolar 80-S preribosomes were distinct from the rapidly labeled proteins of ribosomal subunits. A m o n g the about 60 distinct major proteins found in 80-S preribosomes, 12 incorporated label more quickly than the remainder. Most of them are structural constituents of the large ribosomal subunit, and to a lesser extent of the small subunit. Three nucleolar non-ribosomal proteins (Px, Py and P9) were shown to be labeled quite rapidly, and particularly striking was the high proportion o f P9 in pulse-labeled samples. The presence of a few rapidly labeled nucleolar proteins has been reported in 55-S preribosomal particles o f HeLa cells [10], although most o f the non-ribosomal proteins were shown to incorporate the radioactivity at a slower rate, as should be expected [31].
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