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652 Biochimica et Biophysica Acta, 475 (1977) 652--658 © Elsevier/North-Holland Biomedical Press BBA 98904 IN VIVO INCORPORATION OF RIBOSOMAL PROTEINS INTO HeLa CELL RIBOSOMAL PARTICLES LEONARDO O. AIELLO, CARLOS J. GOLDENBERG and GEORGE L. ELICEIRI Department of Pathology, St. Louis University School of Medicine, St. Louis, Mo. 63104 (U.S.A.) (Received July 27th, 1976) Summary Using high salt-washed ribosomal subunits from HeLa cells we detect three ribosomal proteins from the small subunit and five ribosomal proteins from the large subunit that enter ribosomal particles in the absence of ribosome forma- tion (actinomycin D-treated cells); in untreated cells, they enter the ribosomal particles quickly, while the rest of the ribosomal proteins are incorporated gradually. At least two of the large subunit actinomycin D-resistant ribosomal proteins seem to be absent in the 55 S nucleolar ribosomal precursor. Introduction Warner [ 1] originally found about three protein bands in 0.2 M NaCl-washed large subunits of HeLa cell ribosomes which were labeled quickly and were also labeled in the absence of ribosome formation (cells treated with actinomycin D), and which he interpreted to be exchangeable ribosomal proteins. Kumar and Subramanian found six major protein spots in 0.2 M NaCl-washed 60 S subunits from HeLa cells, which were quickly labeled comparing cells labeled for 15 min vs. 24 h [2]. In Saccharomyces cerevisiae Warner and Udem [3] have found three 60 S ribosomal proteins which were incorporated into subunits when ribosome formation was shut off in a temperature sensitive mutant incubated at its non- permissive temperature, and they were also considered to be exchangeable ribosomal proteins. The presence of quickly labeled HeLa cell ribosomal proteins (as opposed to most of the ribosomal proteins, which are gradually labeled) in high salt-washed large and small ribosomal subunits has been recently reported [4]. In the present report, we were interested in the effect of actinomycin D on the in vivo Abbreviations: 60 S, large ribosomal subunit; 40 S, small ribosomal subunit

In vivo incorporation of ribosomal proteins into HeLa cell ribosomal particles

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652

Biochimica et Biophysica Acta, 475 (1977) 652--658 © Elsevier/North-Holland Biomedical Press

BBA 98904

IN VIVO INCORPORATION OF RIBOSOMAL PROTEINS INTO HeLa CELL RIBOSOMAL PARTICLES

LEONARDO O. AIELLO, CARLOS J. GOLDENBERG and GEORGE L. ELICEIRI

Department of Pathology, St. Louis University School of Medicine, St. Louis, Mo. 63104 (U.S.A.)

(Received July 27th, 1976)

Summary

Using high salt-washed ribosomal subunits from HeLa cells we detect three ribosomal proteins from the small subunit and five ribosomal proteins from the large subunit that enter ribosomal particles in the absence of r ibosome forma- tion (actinomycin D-treated cells); in untreated cells, they enter the ribosomal particles quickly, while the rest of the ribosomal proteins are incorporated gradually. At least two of the large subunit act inomycin D-resistant ribosomal proteins seem to be absent in the 55 S nucleolar ribosomal precursor.

Introduction

Warner [ 1] originally found about three protein bands in 0 .2 M NaCl-washed large subunits of HeLa cell r ibosomes which were labeled quickly and were also labeled in the absence of r ibosome formation (cells treated with actinomycin D), and which he interpreted to be exchangeable ribosomal proteins. Kumar and Subramanian found six major protein spots in 0.2 M NaCl-washed 60 S subunits from HeLa cells, which were quickly labeled comparing cells labeled for 15 min vs. 24 h [2] .

In Saccharomyces cerevisiae Warner and Udem [3] have found three 60 S ribosomal proteins which were incorporated into subunits when r ibosome formation was shut off in a temperature sensitive mutant incubated at its non- permissive temperature, and they were also considered to be exchangeable ribosomal proteins.

The presence of quickly labeled HeLa cell ribosomal proteins (as opposed to most of the ribosomal proteins, which are gradually labeled) in high salt-washed large and small ribosomal subunits has been recently reported [4] . In the present report, we were interested in the effect of actinomycin D on the in vivo

Abbreviations: 60 S, large ribosomal subunit; 40 S, small ribosomal subunit

653

incorporation of ribosomal proteins into high salt-washed ribosomal subunits from HeLa cells. The method of preparation we have used has been shown to produce active HeLa cell ribosomal subunits with a low protein content [5].

Materials and Methods

The conditions of cell culture and labeling, isolation of ribosomes and ribosomal subunits, and extraction of ribosomal proteins were as described before [4]. When indicated, the ribosomal subunits were not separated by sucrose gradient centrifugation, but were pelleted through a cushion of 0.5 M sucrose, 0.5 M KC1, and 5 mM MgC12, as described previously [6].

The ribosomal proteins were separated by the two-dimensional gel electro- phoresis system of Mets and Bogorad [7]. After electrophoresis, gel slabs were stained and destained as indicated by Subramanian [8], 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 [9]. Each digested gel piece was counted in a liquid scintillation counter after addition of 10 ml of a scintilla- tion mixture containing a 3/2 ratio of toluene and ethylene glycol monomethyl ether, with a final concentration of 0.4% Omnifluor (New England Nuclear, Boston, Mass.).

Results and Discussion

Fig. 1. shows the numbering system used in this report to identify the various ribosomal proteins found in HeLa ribosomal subunits. Only the

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proteins which are more reproducible, associated with subunits, and present in significant concentrations are shown. We tentatively identify proteins 1--4, 18--21, and 33--41 as belonging to 40 S, and 5--13, 15--17, 22--30, 32, and 42--55 as belonging to 60 S. We cannot exclude the possibility of two proteins, from the same or different subunits, comigrating in this gel electrophoresis system. It appears that most mammalian ribosomal proteins known today should have entered the first dimension gels used in this report (Mets and Bogorad [ 7] ), because L40 and L41, among the most acidic ribosomal proteins known in rat liver ribosomes migrate like Escherichia coli acidic ribosomal proteins L6/L12 in Kaltschmidt and Wittmann gels [10,11] , and L7/L12 have been shown to enter Mets and Bogorad gels [8].

With 0.2 M NaCl-washed ribosomal subunits Warner [1] had shown that about three bands from the large subunit of HeLa cell ribosomes could be separated in a one~limensional gel electrophoresis system, which entered ribosomes when ribosome formation was shutt off with actinomycin D, and considered them to be exchange proteins. When we repeated this experiment with high salt-washed ribosomal subunits and two<limensional gel electro- phoresis, Table I shows that three ribosomal proteins from the small subunit (1, 3, and 4) and five ribosomal proteins from the large subunit (5, 32, 46, 47,

T A B L E I

E F F E C T OF A C T I N O M Y C I N D ON I N C O R P O R A T I O N OF R I B O S O M A L P R O T E I N S I N T O R I B O S O M A L P A R T I C L E S

Cells w e r e labe led w i t h 14C-labeled a m i n o acids for 24 h, pulsed wi th 3H-labeled a m i n o acids, and chased w i t h an e x c e s s o f non-rad ioac t ive a m i n o acids as descr ibed in ref . 4. In these e x p e r i m e n t s the durat ion of the 3H pulse was 15 rain, chased for 120 rain in Exp t . 1 and for 150 rain in Exp t . 2. The cells w e r e e x p o s e d to a c t i n o m y c i n D (5 ~g/ ra l in c o l u m n a and 0.1 ~g/ra l in c o l u m n b ) for 120 m i n pr io r to the pulse, as well as during the pulse and the chase. In the case of the c o n t r o l preparat ion (no a c t i n o m y c i n D t r e a t m e n t ) and co lu ran a of the a c t i n o m y c i n D samples the r i b o s o m a l subuni t s w e r e pe l l e ted through a 0.5 M sucrose , 0 .5 M KCI, 5 raM MgCl 2 cush ion , whi l e the ac t i no rayc in D p r e p a r a t i o n in c o l u m n b had b e e n separated in to subuni t s by sucrose gradient centr i fugat ion in the presence of 0.5 M KC1 and 5 m M MgC12. F o r f u r t he r e x p e r i m e n t a l detai ls , see Materials and Methods . The da ta are expressed as 3H/14C rat ios to cor rec t for possible r e c o v e r y variat ions during the i so la t ion o f the r i b o s o m a l prote ins and cut t ing of the gel slabs. The miss ing f igures are due to loss o f prote ins during handl ing or l o w 14C recover ies .

Spot 3 H / 1 4 C ratio n u m b e r

Contro l Act ino ray cin D

a b

Expt . 1 6 5.9 0.3 8 4.6 0.5 9 7.5 O.5 3 5.8 4.7 4 4.5 2.3

46 14.7 5.7 5 6.8 6.6

47 11.1 6.8 1 5.7 3.8

32 6.5 53 6.9

Expt . 2 53 17.7 16 .0

0.5 0.4 0.5 4.7 2.8 5.0

6.5

655

and 53) enter ribosomal particles in the absence of ribosome formation (cells exposed to actinomycin D). By pulse chase experiments with [3H] uridine and actinomycin D we have found that in less than 15 min (the shortest time tested) after addition of 0.1 or 5 pg of actinomycin D/ml no new 18 S or 28 S ribosomal RNA exits the nucleus into the cytoplasm of HeLa cells (data not shown). Therefore, as in the experiments in this report the actinomycin D preincubation lasted 2 h, all incorporation of 3H-labeled amino acids should have occurred in previously made ribosomes, as there were no remaining new ribosomal subunits going from nucleus to cytoplasm when the pulse with 3H- labeled amino acids started.

Without drug treatment, Warner [1] had also shown that the 60 S actino- mycin D-resistant protein bands were labeled very quickly, while the rest of the ribosomal proteins were labeled gradually. We had shown previously [4] that several quickly labeled ribosomal proteins could be detected in high salt- washed ribosomes, analyzed by the t~vo<limensional gel electrophoresis method of Kaltschmidt and Wittmann [10]. In the absence of actinomycin D, the actinomycin D-resistant ribosomal proteins shown in Table I were found to incorporate isotope quickly, instead of gradually like the rest of the ribosomal proteins. Some examples are shown in Fig. 2, which also shows that fast incorporation of isotope also occurred in the presence of actinomycin D (after only 15 min of 3H pulse).

The content of individual actinomycin D-resistant ribosomal proteins per ribosomal subunit is similar to that of other ribosomal proteins, as shown in the steady state-labeled subunits in Table II. We do not propose that the varia- tions in levels of various ribosomal proteins in Table II may reflect an in vivo phenomenon; they may very well be due to a differential recovery of various ribosomal proteins during the isolation procedure.

Nucleolar 55 S ribosomal precursor proteins from cells incubated with 3H- labeled amino acids for 30 min were mixed with cytoplasmic mature 60 S proteins from cells incubated for 24 h with 14C-labeled amino acids. In this experiment the 14C-labeled 60 S preparation provided both 14C counts to monitor recovery in each spot and also provided the mass of 60 S proteins which could be stained to guide us in cutting the gel slab. When the 60 S stained spots were cut out, high 3H counts were found in most of the 60 S pro- teins, and Table III shows that at least two of the actinomycin D-resistant ribosomal proteins (32 and 47) were virtually unlabeled, while the 60 S cyto- plasmic mature particles from the same 3H-labeled cells showed counts in spots 32 and 47, but not in other ribosomal proteins. The possibility of not detecting a ribosomal protein in the 55 S precursor if it were present as a precursor pro- tein form with different electrophoretic mobility, cannot be excluded.

It is difficult to compare our results with those of Kumar and Subramanian [2], because although we both used HeLa cells and the gel electrophoresis method of Mets and Bogorad [7], our ribosomal protein gel patterns are different. Perhaps this might be partly related to the fact that their ribosomes were dissociated with ethylenediaminetetraacetate in the presence of 0.2 M NaC1, while our ribosomes were dissociated in the presence of puromycin, 0.5 M KC1, and 5 mM MgC12 [13].

While this manuscript was in preparation, McConkey and coworkers, using

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Fig. 2. Time course of r ibosomal protein incorporat ion into ribosomal particles. The condit ions of I4C labeling, act inomycin D t rea tment (at 0.1 pg/ml in this ease), and 3H pulse-labeling were as in Table I. The numbers identify various ribosomal proteins. A and B are two similar experiments. • m control (no act inomycin D); o, act inomyein D.

high salt-washed ribosomes from HeLa cells, reported finding three 60 S actino- mycin D-resistant proteins (none in 40 S) [14] ; in untreated cells two of them appeared to be metabolically less stable than most of the ribosomal proteins [14] , while one of these two was also present in nucleoli [15] . They used a gel electrophoresis method similar to that of Kaltschmidt and Wittmann [9] , and analyzed only the ribosomal proteins whose isoelectric points were above 8.6.

Our data indicate that eight ribosomal proteins from the large and small subunits of HeLa cells seem to enter ribosomal particles in the absence of r ibosome formation. By current criteria these proteins would be considered ribosomal proteins [16] as they are found in subunits washed with 0.5 M KC1 [17] , and their concentrations in ribosomal subunits are similar to those of other ribosomal proteins. Considering that no ribosomal subunits exit the nucleus within a few minutes after addition of actinomycin D and that in these experi- ments 2 h preincubations with actinomycin D were used, it seems very unlikely that the entry of these proteins into ribosomes is related to nuclear (or cyto- plasmic) processing of new ribosomes.

657

TABLE II

RECOVERY OF RADIOACTIVITY IN SOME OF THE RIBOSOMAL PROTEINS FROM RIBOSOMAL SUBUNITS OF HELA CELLS INCUBATED WITH 14C-LABELED AMINO ACIDS FOR 24 h

Cells were incubated for 15 min at 4.106 cells/ml with a 14C-labeled amino acid mixture (NEC-445, New

England Nuclear) at 3 pCi/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, after 105 min the cells were diluted to 2 . lo5 cells/ml with complete medium, and 24 h later the cells were harvested, the rlbosomal subunits isolated. and their proteins extracted and analyzed as indicated in Materials and Methods.

60s 40s

spot number

cpm spot

number cpm Spot

number CPm

5 225 28 125 1 165 6 326 29 206 3 143 8 212 30 103 4 396

10 292 18 354 12 253 32 179 20 192 13 313 45 131 21 288

46 207 34 91 15 169 47 102 35 204

16 151 48 255 36 468 17 184 49 207 37 462 25 233 50 139 21 100 51 117

TABLE III

INCORPORATION OF RIBOSOMAL PROTEINS INTO NUCLEOLAR PRECURSOR RIBOSOMAL PARTICLES

Cells were resuspended at 4 * 106 cells/ml in Jokllk modified minimum essential medium (Grand Island Biological Co., Grand Island, N.Y.) lacking leucine and lyslne, supplemented with 7% dialyzed home serum, and labeled for 30 mln with [3Hllyslne, [3H31euclne and 13Hlglycine (10 pCl of each per ml of medium). The cells were then harvested, 55 S nucleolar precursors were isolated as described by Warner and Soeiro [121, and 60 S mature cytoplasmic subunits were isolated as indicated in Materials and Methods. 60 S mature cytoplasmic subunits from cells incubated for 24 h with 14C-labeled amino acids were prepared as indicated in Materials and Methods. The 3H-labeled 55 S preparation was mixed with the 14C-labeled 60 S preparation. the ribosomal proteins were extracted and analyzed by two-dimensional gel electrophoresis as indicated in Materials and Methods, Columns a and b refer to the 3H and 14C counts from the stained spots cut out from that gel slab. The 3H-labeled 60 S ribosomsl protein preparation was analyzed in a second gel slab (last column).

spot number

32 41 16 28 50 -

cpm cpm SH-labeled 55 S 1 4C-labeled 60 S

(a) (b)

31 179 38 102

517 151 384 125 481 139

a/b

-

0.17 0.37 3.4 3.1 3.5

cpm 3H-labeled 60 S

414 196

6 0

26

Acknowledgments

The technical assistance of Miss Rebecca A. Vuch during part of this work is gratefully acknowledged. This work was supported in part by a grant from the

658

National Institute o f General Medical Sciences. One of the authors (G.L.E.) holds a Research Career Development Award from the National Institute ol General Medical Sciences.

References

1 Warner, J.R. (1966) J. Mol. Biol. 19 ,383 - -398 2 Kurnar, A. and Subramanian, A.R. (1975) J. Mol. Biol. 94,409---423 3 Warner, J.R. and Udern, S.A. (1972) J. Mol. Biol. 65, 243--257 4 Vandrey, J.P., Goldenberg, C.J. and Elicelri, G.L. (1976) Biochirn. Biophys. Acta 432 ,104 - -112 5 McConkey, E.H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1379--1383 6 Fujisawa, T. and Eliceiri, G.L. (1975) Biochirn. BioPhys. Acta 402 , 238 - -243 7 Mets, L. and Bogorad, L. (1974) Anal. Biochern. 57, 200--210 8 Subramanian, A.R. (1974) Eur. J. Biochern. 45, 541--546 9 Goodman, D. and Matzura, H. (1971) Anal. Biochern. 42 ,481 - -486

10 Kaltschrnidt, E. and Wittrnann, H.G. (1970) Anal. Biochern. 36 ,401 - -412 11 Sherton, C.C. and Wool, I.G. (1974) J. Biol. Chem. 249, 2258--2267 12 Warner, J.R. and Soeiro, R. (1967) Proc. Natl. Acad. Sei. U.S. 58, 1984--1990 13 Blobel, G. and Sabatini, D. (1971) Proc. Natl. Acad. Sci. U.S. 68 , 390 - -394 14 Lastiek, S.M. and McConkey, E.H. (1976) J. Biol. Chem. 251, 2667--2875 15 PhiLlips, W.F. and McConkey, E.H. (1976) J. Biol. Chem. 251, 2876--2881 16 Zinker, S. and Warner, J.R. (1976) J. Biol. Chem. 251, 1799--1807 17 Sherton, C. and Wool, I.G. (1974) Methods Enzymol. 30, 506--526