Isolation of Eukaryotic Ribosomal Proteins

THE JOIJRNAL 01 B~.CGICAL CHEMISTW Vol. 253, No. 3, Issue of February 10, pp 946955, 1978

Printed m Cl S A

Isolation of Eukaryotic Ribosomal Proteins PURIFICATION AND CHARACTERIZATION OF THE 60 S RIBOSOMAL SUBUNIT PROTEINS La, Lb, Lf, Pl, P2, L13’, L14, L18’, L20, AND L38*

(Received for publication, August 22, 1977)

KUNIO TSURUGI, EKKEHARD COLLATZ, KAZUO TODOKORO, NORBERT ULBRICH, HAIDEH N. LIGHTFOOT, AND IRA G. Wool

From the Department of Biochemistry, University of Chicago, Chicago, Illinois 60637

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Ten proteins (La, Lb, Lf, Pl, P2, L13’, L14, L18’, L20, and L38) were isolated from three groups (A60, B60, and D60) by ion exchange chromatography on carboxymethylcellulose and DEAE-cellulose, and by filtration through Sephadex. The amount of protein obtained varied from 0.3 to 3.8 mg. Two of the proteins (La and L18’) had no detectable contamination; the impurities in the others were not greater than 8%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined. Several additional acidic proteins were identified: Pla and Plb are phosphorylated derivatives of PI; P2a, P2b, and P2c are phosphorylated derivatives of P2. Pl and P2 are distinct proteins but both have large amounts of alanine (20.4 and 17.5 mol %).

The purification of the proteins is necessary for an analysis of the structure and function of eukaryotic ribosomes. Knowl- edge of the structure of ribosomes may in turn provide the foundation for an understanding of how regulation of protein synthesis is achieved. With those purposes in mind, we have undertaken to isolate and characterize rat liver ribosomal proteins. We reported before on the purification and properties of 32 proteins of the 40 S subparticle (1, 16) and 38 of the large subunit (2, 3). We describe now the isolation and characteris- tics of several additional 60 S proteins including the resolution of some of the relatively acidic proteins.

EXPERIMENTAL PROCEDURES

Preparation of Ribosomes, Ribosomal Subunits and Ribosomal Proteins - Subunits were prepared from rat liver ribosomes (4) on a large scale by centrifugation in a zonal rotor (5) and the proteins extracted from the 60 S subparticles with 67% acetic acid in 10 rn~ TrislHCl containing 33 mM magnesium acetate (6, 7). After extrac- tion, the proteins were precipitated with acetone, collected by

_____

* This work was supported by Grants GM-21769, CA-19265, AM- 04842, and AM-17046 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘hduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

centrifugation (45 min at 1000 x g), dissolved in 10% acetic acid, dialyzed overnight against 10 volumes of 2% acetic acid, and stored at -20”.

Group Fractionation of 60 S Ribosomal Subunit Proteins-The proteins of the large subunit were separated into seven groups (A60, B60, C60, D60, E60, F60, and G60) by stepwise elution from carboxymethylcellulose (Whatman CM32) at 12-15” with LiCl at pH 6.5 (8).

Isolation of 60 S Ribosomal Subunit Proteins-Three groups of proteins (A60, B60, and D60) were resolved by chromatography on carboxymethylcellulose. The proteins in Group A60 (94 mg) in Buffer E (6 M urea, 0.02 M sodium acetate, 0.05% P-mercaptoethanol, 0.1% methylamine, adjusted to pH 4.2 with glacial acetic acid’) were applied to a column of carboxymethylcellulose (2.6 x 30 cm) and eluted with an B-liter linear gradient of 0 to 0.1 M LiCl in Buffer E. Group B60 proteins (200 mg) in Buffer A (6 M urea, 0.02 M H,PO,, 0.05% fi-mercaptoethanol, adjusted to pH 6.5 with methylamine) were applied to a column (2.6 x 40 cm) of carboxymethylcellulose and eluted with a lo-liter linear gradient of 0 to 0.2 M LiCl in Buffer A (2). Group D60 proteins (335 mg) in Buffer C (6 M urea, 0.05 M glycine, 0.05% P-mercaptoethanol, adjusted to pH 10.5 with methylamine) containing 0.02 M LiCl, were applied to a column of carboxymethylcellulose (2.6 x 50 cm) and eluted with a IO-liter linear gradient of 0.02 to 0.1 M LiCl in Buffer C. The flow rate was 42 ml/h and 15-ml samples were collected.

The proteins in subfraction A60-I from A60 were resolved by chromatography on DEAE-cellulose (Whatmann DE321 at pH 4.6. The proteins (21 mg) in Buffer F (6 M urea, 0.01 M sodium acetate, 0.05% /3-mercaptoethanol, 0.1% methylamine, adjusted to pH 4.6 with glacial acetic acid) were applied to a column of DEAE-cellulose (1.6 x 25 cm) and eluted with 1.2-liter linear gradient of 0 to 0.06 M LiCl in Buffer F. The proteins (15 mg) in fraction AGO-I-P1 were separated by chromatography on DEAE-cellulose in a similar way but were eluted with a 2.6-liter linear gradient of 0 to 0.04 M LiCl in Buffer F. The proteins in subfraction BGO-IV, from chromatography of Group B60, were resolved by rechromatography on carboxymethylcellulose at pH 9.0. The proteins (9 mg) in Buffer G (6 M urea, 0.05 M glycine, 0.05% P-mercaptoethanol, adjusted to pH 9.0 with methylamine) were applied to a column (1.6 x 20 cm) of carboxymethylcellulose and eluted with a P-liter linear gradient of 0 to 0.15 M LiCl in Buffer G. The proteins in subfraction DGO-III from Group D60 were resolved by rechromatography on carboxymethylcellulose at pH 9.0. The proteins (30 mg) in Buffer G containing 0.05 M LiCl were applied to a column of carboxymethylcellulose (1.6 x 30 cm) and eluted with a 2-liter linear gradient of 0.05 to 0.28 M LiCl in Buffer G. The flow rate was 28 ml/h and 12-ml samples were collected.

Fractions were resolved further by filtration through columns (1.6 x 200 cm) of Sephadex G-75 (superfine). Prior to gel filtration, the samples were treated with dithiothreitol and their volumes were reduced (11. The flow rate was about 4 ml/h and 1.5-ml samples were collected.

i The pH of solutions and buffers was determined at 20”.

946

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Purification of Eukaryotic Ribosomal Proteins

Plbo Pla m

La 0

Lb o

P2 *

v (a) -E

FIG. 1. Two-dimensional polyacrylamide gel electrophoresis of group A60 proteins: effect of treatment with intestinal alkaline phosphatase. a, schematic representation of the proteins in Group A60. b, electrophoretogram of Group A60 proteins (20 pg). c, Group A60 proteins (200 pg) were incubated at 37” for 16 h with 200 units

Ion exchange chromatography was done at 4” and gel filtration was at 12-15”. The elution of the proteins was monitored by determining the absorption at 280 nm. In some instances, the turbidity in 15% trichloroacetic acid was determined at 400 nm (2).

Two-dimensional Polyacrylamide Gel Electrophoresis - The ribosomal proteins were identified by micro-two-dimensional polyacrylamide gel electrophoresis (9) using a modification (10) of the usual procedure (11, 12). The proteins in fractions from ion exchange chromatography were precipitated with 15% trichloroacetic acid, washed with acetone, and dissolved in sample buffer (10) before electrophoresis; the proteins in fractions from Sephadex filtration were lyophilized and then dissolved in the same buffer.

Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate- The molecular weight and the purity of the isolated ribosomal proteins were estimated by gel electrophoresis in sodium dodecyl sulfate (1) using the Laemmli procedure (13), but with 15% polyacrylamide.

Determination of Amino Acid Composition of Ribosomal Proteins - The isolated ribosomal proteins (1 to 2 nmol) were hydro- lyzed in 6 M HCl for 24 h at 110”. The concentration of the amino acids in the hydrolysate was determined with a Durrum D500 analyzer. No corrections for incomplete hydrolysis or for decomposi- tion were made; tryptophan and cysteine were not determined.

Treatment of Ribosomal Proteins with Alkaline Phosphatase- Group A60 proteins (200 pg) in 0.5 ml of buffer (0.1 M glycine, adjusted to pH 9.5 with 1 M sodium hydroxide) were incubated with 200 units of intestinal alkaline phosphatase (EC 3.1.3.1, Sigma) at 37” for 16 h. The reaction was stopped by addition of 15% trichloroacetic acid.

RESULTS AND DISCUSSION

Isolation of 60 S Ribosomal Subunit Proteins in Group A60 - The proteins of the large ribosomal subunit were separated into seven groups (designated A60 through G60) by elution from carboxymethylcellulose with increasing concentrations of LiCl (added in steps) at pH 6.5 (see Fig. 1 in Ref. 2).

Group A60 contains about 14 proteins, i.e. the number of spots that can be identified on two-dimensional polyacrylamide gel plates (Fig. 1). The proteins are relatively acidic: they migrate to the anode on electrophoresis at pH 8.6 and they do not bind to carboxymethylcellulose at pH 6.5. Very few of the A60 proteins stain intensely even when large amounts are analyzed by gel electrophoresis; indeed, poor staining and variability are their salient properties and have

of intestinal alkaline phosphatase and a sample was analyzed. The enzyme is designated e on the electrophoretogram. Electrophoresis was from right to left in the first dimension and from top to bottom in the second. The origin (at top right) is marked 0.

made identification and characterization difficult (8). It is not certain which, if any, are ribosomal structural proteins; which

are factors transiently associated with the ribosomes during protein synthesis and hence present in TP60* only in trace amounts; and which are merely contaminants having no function in protein synthesis. An attempt was made to purify and characterize the proteins as a first step in resolving the several possibilities.

Group A60 has La, Lb, Lc, Ld, Le, Lf, S21, and two complexes of three (Pl, Pla, Plb) and four (P2, P2a, P2b, P2c) proteins each. In the original identification of the proteins of the large subunit of rat liver ribosomal proteins by two- dimensional polyacrylamide electrophoresis (12), two acidic proteins, Ll and L2, were designated although they were only seen when large amounts of TP60 were analyzed. We are not certain if Ll and L2 correspond to any of the acidic proteins we now find in Group A60. S21 is a relatively acidic

protein of the small subparticle that contaminates TP60. We presume 521 to be a small, rather than a large, subunit protein since greater amounts are found in A40 than in A60. It is possible it is an interface protein that partitions between the two ribosomal subunits when couples are dissociated (141. The Pl and P2 proteins are phosphorylated in vivo by a kinase that has not been identified and in vitro by a cyclic

AMP-independent protein kinase (15) that transfers the y- phosphor-y1 group of GTP3. The observation suggested that the seven spots (Pl, Pla, Plb, P2, P2a, P2b, P2cl are phosphorylated derivatives of one or more polypeptides. To test the possibility the A60 proteins were incubated with intestinal alkaline phosphatase for 16 h at 37”. Two proteins, Pl and P2, remained after incubation (Fig. lc). It seems likely that Pla and Plb are phosphorylated derivatives of Pl and that P2a, P2b, and P2c are phosphorylated derivatives of P2 (see also

below). An attempt was made to isolate and characterize the Group

A60 proteins: 94 mg were applied to a column of carboxymeth-

2 The abbreviation used is: TP60, the total protein of the 60 S ribosomal subunit.

3 A. Lin and I. G. Wool, unpublished data.

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948 Purification of Eukaryotic Ribosomal Proteins

ylcellulose and eluted with a lo-liter linear gradient of 0 to 0.1 M LiCl at pH 4.2 (Fig. 2). The identity of proteins in the fractions was determined by a combination of one-dimensional (in sodium dodecyl sulfate) and two-dimensional (in urea) polyacrylamide gel electrophoresis.

Proteins Pl and P2 did not bind to carboxymethylcellulose at pH 4.2 (Fig. 2, Peak I). The single protein in Peak II was identified as S21 from its position on two-dimensional gel plates and from its molecular weight and amino acid composition, which accorded with that of S21 isolated from TP40 (16). Peak III contained only La. Lb was in Peak IV and was purified by filtration through Sephadex G-75 (Fig. 3) since it had a small amount of contamination. Protein Lf eluted in two peaks, V and VI (Fig. 2); it was resolved by gel filtration (Fig. 3). Lf from Peak V and from Peak VI had the same molecular weight and very similar amino acid composition. Thus, Lf appears to be a single protein despite its elution at different LiCl concentrations during chromatography (Fig. 2).

Proteins Lc, Ld, and Le, which eluted in Peaks V and VI (Fig. 21, were not resolved (Fig. 3).

:f Lf LS

Lb Lb Ld t 1 1

FRACTION

FIG. 2. Chromatography on carboxymethylcellulose of 60 S ribosomal subunit proteins in Group A60. The proteins (94 mg) were eluted from a column (2.6 x 30 cm) of carboxymethylcellulose with an 8-liter linear gradient of 0 to 0.1 M LiCl at pH 4.2. The proteins in the fractions (15 ml) were identified by micro-two-dimensional polyacrylamide gel electrophoresis and electrophoresis in gels containing sodium dodecyl sulfate. The proteins are listed from top to bottom. in order of decreasing amounts.

460-P

20 40 60 FRACTION

20 40 60

FIG. 3. Separation of Group A60 proteins by filtration through Sephadex G-75. The protein mixtures (2 to 6 mg in 10% acetic acid) were filtered through a column (1.6 x 200 cm) of Sephadex G-75 and 1.5-ml fractions were collected. Dextran blue was added to the samples to designate the void volume; fractions containing the dye (and its concentration) are indicated by - - -. The ribosomal proteins contained in the fractions were identified by gel electrophoresis and the results are given on the chromatogram. The length of the line under the designation of the protein indicates the range of fractions that contained that protein; the brackets indicate fractions containing purified proteins that were pooled.

Proteins Pl and P2 (21 mg) were applied to a column of DEAE-cellulose and eluted with a 1.2-liter linear gradient of 0 to 0.06 M LiCl at pH 4.6 (Fig. 4u). The proteins eluted in separate peaks, but each had a trailing shoulder. The first peak contained mainly P2; the shoulder had P2a, P2b, and P2c (as judged from two-dimensional gel electrophoresis). The proteins in the peak and the shoulder showed only a single band, with* an apparent molecular weight of 15,200, when analyzed by electrophoresis in gels containing sodium dodecyl sulfate.

The second peak and shoulder (Fig. 4o) contained three main proteins, Pl, Pla, and Plb, and small amounts of P2b and P2c. This fraction gave a single prominent band with a molecular weight of 16,100 when electrophoresis was in gels containing sodium dodecyl sulfate. Pl and its putative derivatives were resolved from the peak material (excluding the shoulder-Fig. 4a) by chromatography of 15 mg on DEAE- cellulose using a similar but shallower gradient (2.6 liters of 0 to 0.04 M LiCl) than in the original separation (Fig. 4b). Pl had the same molecular weight (16,100) and a very similar amino acid composition as a mixture of Pla and Plb (Tables I and II).

The identity of the proteins isolated from A60 (La, Lb, Lf, Pl, and P2) was determined and their purity was assessed (Figs. 5 and 6): P2 appeared pure when electrophoresis was in gels containing sodium dodecyl sulfate (Fig. 61, but analysis of a large excess by two-dimensional electrophoresis in urea gels (Fig. 5) indicated small amounts of P2a, P2b, and P2c were present.

Isolation of L20 from Group B60-During the original separation of the proteins of Group B60, L20 had not been purified (2). A new attempt was made to prepare L20. About 200 mg of B60 proteins were applied to a column of carboxymethylcellulose and eluted with a lo-liter linear gradient of 0 to 0.2 M LiCl (Fig. 7). The elution profile was, with minor exceptions, the same as that reported before (2). More proteins from 40 S ribosomal subunits were, however, identified on this occasion. It may be that the TP60 used to make B60 for this experiment suffered greater contamination with 40 S ribosomal subunit proteins. The protein in Peak III (Fig. 7) was determined to be S5, not L9 (as indicated in Fig. 2, Ref. 2). L9 had been isolated before from Peaks V and VI so the

I t&O-I-PI b

FFUTION

1

FIG. 4. Separation of 60 S ribosomal subunit proteins in Peak A60-1 by chromatography on DEAE-cellulose. a, the proteins (21 mg) were eluted from a column (1.6 x 25 cm) of DEAE-cellulose with a 1.2-liter linear gradient of 0 to 0.06 M LiCl at pH 4.6. b, the proteins (15 mg) were eluted with a 2.6-liter linear gradient of 0 to 0.04 M LiCl at pH 4.6. The proteins contained in the fractions (12 ml) were identified and the results are indicated on the chromatogram.

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Purification of Eukaryotic Ribosomal Proteins 949

FIG. 5. Two-dimensional polyacrylamide gel electrophoresis of isolated 60 S ribosomal subunit proteins. The isolated acidic proteins La, Lb, Lf, Pl, and P2 (1 to 3 pg) were analyzed on the right half of the second dimension gel (origin at top right) to assess its purity; and on the left side (origin at top center) with a small amount (about 10 pg) of A60 to provide a background to assist in the identification of the protein. The isolated basic proteins L13’, L14, L18’, L20, and L38 (1 to 3 pg) were analyzed alone on the left half (origin at top left); and on the right side with a small amount (about 10 pg) of TP60. The origin is marked 0.

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published (2) amino acid composition and molecular weight are correct. In the present experiments, Peak IV had L20 and S20 (not L22 as in Ref. 2). About 9 mg of Peak IV proteins were applied to a column of carboxymethylcellulose and eluted with a 2.6-liter linear gradient of 0 to 0.15 M LiCl at pH 9.0 (Fig. 8). L20 was separated in that way from S20 and its identity and purity were determined (Figs. 5 and 6).

Isolation of L13’, L14, L18’, and L38 from Group D60- L14 and L38 had not been purified when the proteins in Group D60 were first resolved (2). An attempt was made now to isolate the proteins. During the preparation of L14 and L38 from D60, two additional proteins, L13’ and LB’, not previ- ously recognized were identified and they too were isolated. About 335 mg of Group D60 proteins were applied to a column of carboxymethylcellulose and eluted with a lo-liter linear gradient of 0.02 to 0.1 M LiCl at pH 10.5 (Fig. 9). The elution profile was different from that obtained before (Fig. 6, Ref. 2) since a glycinelmethylamine, rather than a T&/borate, buffer was used, the glycine buffer is more efficient in the main- tenance of a high pH. In addition, this preparation contained several proteins (L3, L4, L6, L8, LlO, L17, and L38) which are not ordinarily in Group D60.

In retrospect it would appear that in the earlier separation (21, L13’ and L18’ eluted mainly between Peaks VI and VII

TWO A60 Lo Lb Lf PI P2 L13’ Ll4 Ll8’ ~20 L38

FIG. 6. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of purified 60 S ribosomal subunit proteins. Electrophoresis was as described by Laemmli (12) except that the concentration of acrylamide was 15%. The analysis was of 3 to 5 gg of isolated proteins and 40 pg of TP60 or A60.

(Fig. 6, Ref. 2). Proteins L13’ and L18’ had not been distin- guished originally from L13 and L18 which occupy the same position on two-dimensional gel plates. Their existence was suggested by the appearance of bands on sodium dodecyl sulfate gels that could not be correlated with any proteins identified before.

Protein L38, which had not been isolated before, was in Peak I with several other proteins. L38 was resolved by filtration through Sephadex G-75 (Fig. 10). The preparation of L38 contained a minor contaminant, either S27 or S27’ from its position on two-dimensional gel electrophoretograms (Fig. 5); the amount of contamination could not be determined

TABLE II

Yield and purity of isolated 60 S ribosomal subunit proteins

Electrophoresis of the isolated proteins (3 to 5 pg) was in polyacrylamide gels containing sodium dodecyl sulfate (Fig. 5). The extent of the contamination was estimated after scanning the gels at 540 nm; the part of the total absorption that did not derive from the main band (in a sample shown to contain one, or predominantly one, spot after two-dimensional polyacrylamide gel electrophoresis - Fig. 6) was taken to give the percentage contamination.

Protein Yield Contamination

w %

La 1.8 0 Lb 1.4 8 Lf 0.8 2 Pl 3.8 4 P2 3.0 0” L13’ 1.9 2 L14 1.0 4 L18’ 0.9 0 L20 1.5 2 L38 0.3 0*

n P2 was demonstrated by two-dimensional gel electrophoresis of a large excess to contain small amounts of P2a, P2b, and P2c (Fig. 5); since the latter have the same molecular weight as P2 they are not resolved by electrophoresis in gels containing sodium dodecyl sulfate.

* L38 was shown by two-dimensional gel electrophoresis to contain a small amount of S27 (Fig. 5); L38 and S27 have the same molecular weight and are not resolved by electrophoresis in gels containing sodium dodecyl sulfate.

TABLE I

Molecular weight and amino acid composition of isolated 60 S subunit proteins

La Lb Lf PI Pl8.b P2. P2a.b.c L13’ I.14 I.,* I.20 I.38

M. (x 10-3) 37. 9 29. 8 14.6 16.1 16.1 15. 2 15. 2 24. 6 25. 8 21. 3 16. 2 11. 5

ASP 10. 2 9. 6 10. 2 10. 5 10. 5 11 7 12. 9 56 5. 5 5 3 9. 4 82

Thr 6. 8 5. 5 4. 3 1. 8 2. 1 1. 2 04 4. 0 3. 7 5 8 50 5. 4

Ser 5. 7 4. 6 5. 6 5 9 87 8. 4 8. 8 1 8 2. 3 5 8 10 6 5. 8

GlU 14. 4 13 4 12. 5 11.6 12. 4 10. 5 11.0 8. 5 8. 5 7. 9 12 3 10. 1

Pro 6. 8 6. 5 3. 7 7. 7 6. 5 5. 0 4. 9 6 2 6. 0 6 9 4. 9 6. 0

GlY 8. 4 8. 7 10 9 10 5 12. 9 10. 6 11. 5 7. 7 6. 9 6. 2 13 8 4 7

Al.3 14.6 12. 5 IO. 1 20. 4 19. 1 17. 5 18 5 9. 0 19. 8 6. 7 6 4 6 3

va1 5. 5 4. 8 7. 5 6. 8 5. 9 6 9 6. 5 8. 6 6 8 6.6 5. 9 8 0

Met 1.6 1. 7 0. 1 0. 7 0. 3 1 3 1. 3 1 2 1 9 2. 7 1. 3 0. 5

ue 4. 2 4. 6 50 4. 6 4. 3 46 4. 5 3. 6 4. 2 3 8 5. 0 4. 3

Leu 7. 0 8. 8 96 7. 1 6. 4 7. 2 6. 8 11. 2 4. 3 5. 2 5. 6 11 2

TV 1. 8 1 7 1. 7 0. 5 0. 5 1 7 1. 5 3. 1 1. 2 3 7 1. 9 2. 6

Phe 3. 1 2.6 i 6 3. 3 2 7 1. 8 1. 8 3 4 3. 7 5. 9 2. 0 3 1

HIS 1.6 2. 1 2. 2 1 0 1 2 0. 4 0. 4 2. 3 1. 0 5. 2 2. 7 2. 1

LYS 4. 2 6. 0 10. 0 6. 5 6. 0 8 5 8. 3 13. 2 15. 7 11. 0 6. 7 13 8

At9 4. 2 5. 3 5. 1 0. 0 0. 5 3. 2 1. 4 10. 7 8. 2 12. 2 6.6 7 8

The values for am,“0 aads are in moles Per cent.

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accurately. Peak III proteins (30 mg) containing L18’ were separated by rechromatography on carboxymethylcellulose at pH 9.0 (Fig. 11). L18’ eluted in Peak “c”, with L27. The proteins (18’ and L27) were resolved by filtration through Sephadex G-75 (Fig. 10). L13’ was purified from Peak IV (which also had L7 - Fig. 9) by gel filtration. Purification of L14 required chromatography on Sephadex G-75 (Fig. 10) of Peak DGO-VI (Fig. 9) and then refiltration of a subfraction designated “a” (Fig. 10).

The identity of the newly purified proteins (L13’, L14, L18’, and L38) was confirmed by two-dimensional gel electrophoresis (Fig. 5) and the purity was assessed by sodium dodecyl sulfate gel electrophoresis (Fig. 6).

L13’ and L18’ occupied the same position on two-dimensional electrophoretograms as L13 and L18, respectively, however, the molecular weight and amino acid composition of L13’ and L18’ were different than those of L13 and L18 (Table I and Refs. 2 and 3).

Yield and Purity of Isolated Proteins-The amount of the

FRACTION

FIG. 7. Chromatography on carboxymethylcellulose of 60 S ribosomal subunit proteins in Group B60. The proteins (200 mg) were eluted from a cblumn (2.6 x 40 cm) of carbokymethylcellulose with a lo-liter linear gradient of 0 to 0.2 M LiCl at pH 6.5. The proteins contained in the fractions (15 ml) were identified and the results are indicated on the chromatogram. The proteins are listed, from top to bottom, in order of decreasing amounts.

individual purified ribosomal proteins obtained varied from 0.3 to 3.8 mg (Table II); the yield was determined, as in the previous studies (l-3), by the quantity of starting material (i.e. the amount in the group fractions); the quality of the chromatography; and the losses tolerated for the sake of purity.

The purity of the isolated proteins was assessed (11, after electrophoresis in gels containing sodium dodecyl sulfate, by scanning at 540 nm (Fig. 5 and Table II). Proteins La and L18’ had no detectable contamination; the impurities in the others were no more than 8%.

Molecular Weight and Amino Acid Composition-The molecular weight of each purified protein was estimated after electrophoresis in sodium dodecyl sulfate gels by comparison with standards (Fig. 5 and Table I).

The amino acid content of the isolated proteins was determined (Table I). There are several features of the composition of proteins Pl and P2 that are worth remarking on. First, it needs emphasizing that the amino acid compositions of Pl and P2 were distinct providing additional evidence that the proteins are unique; on the other hand the amino acid composition (and the molecular weight as well) of Pl very closely approximated that of a mixture of Pla and Plb, just as the composition of P2 was very similar to a mixture of P2a, P2b, and P2c. If the amino acid content and the molecular weight are considered together with the results of the treatment of the proteins with phosphatase (Fig. l), it seems certain that Pla and Plb are phosphorylated derivatives of Pl and P2a, P2b and P2c are phosphorylated forms of P2. From their positions on two-dimensional gel plates we assume Plb contains more phosphate residues than Pla and P2c is more phosphorylated than P2b which in turn has more phosphate than P2a. Pl and P2 are unique polypeptides; it is not known whether they too are phosphoproteins. However, circumstan- tial evidence suggests they are not. Treatment of Group A60 proteins with alkaline phosphatase (Fig. 1) converts Pla and Plb to Pl, and P2a, P2b, and P2c to P2, but does not lead to the formation of a new species (i.e. a new protein spot on two-dimensional gels) as would be expected if Pl and P2 were

phosphoproteins. Moreover, while Pla, P2a, P2b, and P2c are phosphorylated when 60 S subunits are incubated in vitro

1

FRACTION FRACTION

FIG. 8 clefi). Separation of 60 S ribosomal subunit proteins in FIG. 9 (right). Chromatography on carboxymethylcellulose of 60 S Peak BGO-VI by chromatography on carboxymethylcellulose. The ribosomal subunit proteins in Group D60. The proteins (335 mg) proteins (9 mg) were eluted from a column (1.6 x 20 cm) of were eluted from a column (2.6 x 50 cm) of carboxymethylcellulose carboxymethylcellulose with a a-liter linear gradient of 0 to 0.15 M with a lo-liter linear gradient of 0.02 to 0.1 M LiCl at pH 10.5. The LiCl at pH 9.0. The proteins in fractions (12 ml) were identified and proteins contained in the fractions (15 ml) were identified and the the results are given on the chromatogram. results are indicated on the chromatogram.

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Purification of Eukaryotic Ribosomal Proteins

0.2

I

8

a?

0. I

FRACTION

060-III L35’ L21 L21 L35’ L26

1 I 0 b

I-

150 200 250 FRACTION

-0

-0

FIG. 11. Chromatography on carboxymethylcellulose of 60 S ribosomal subunit proteins in Peak DGO-III. The proteins (about 30 mg) were eluted from a column (1.6 x 30 cm) of carboxymethylcellulose with a Z-liter linear gradient of 0.05 to 0.28 M LiCl at pH 9.0. The proteins contained in the fractions (12 ml) were identified and the results are indicated on the chromatogram. Proteins are listed, from top to bottom, in order of decreasing amounts.

with protein kinase and [-,J2PlGTP, Pl, and P2 are not; nor are they phosphorylated in vivo when ascites cells are incubated with [32P]orthophosphate.3

A second distinctive aspect of the structure of Pl and P2 is the large content of alanine (20.4 and 17.5 mol %, respectively). Only one other rat liver ribosomal protein, L14, has so much alanine - 19.8 mol % (Table I). It is perhaps notable that L14 has been reported to be phosphorylated too (17). However, there is unlikely to be a direct correlation between a large content of alanine and the phosphorylation of ribosomal proteins, since the main phosphoprotein in mammalian ribosomes is S6 (18) and it has only 7 mol % alanine (1).

There are two acidic proteins, L7 and L12, in E. coli ribosomes that have relatively large amounts (24 mol %) of alanine (19). They are by no means the only E. coli ribosomal proteins that contain a great deal of the amino acid: S20 has 19.2 mol %; L8 or L9, 16.3; LlO, 17.6; L20, 18.5 (19). However, the comparison with E. coli L7/L12 is especially pertinent since rat liver ribosomes have a pair of acidic proteins that are structurally and functionally homologous (20, 21). More- over, E. coli L7/L12 are selectively and exclusively phosphorylated if 50 S subparticles are incubated in vitro with a kinase that transfers the y-phosphoryl group of GTP to protein (22). It may be that L7/L12 are not ordinarily phosphorylated only because the organism lacks protein kinase (23). The

FIG. 10. Separation of 60 S ribosomal subunit proteins in Group D60 by gel filtration through Sephadex G-75. The protein mixtures (5 to 12 mg in 10% acetic acid) were filtered through a column (1.6 x 200 cm) of Sephadex G-75 and I.&ml fractions were collected. The fractions were analyzed and the results are designated as described in the legend to Fig. 3. The fraction marked +, in D60-VI was rechromatographed (DGO-VIa).

question that arises then is whether rat liver Pl or P2 or both are homologous with E. coli ribosomal proteins L7 and L12 (L7 differs from L12 only in the acetylation of the NH,- terminal se&e). The homologous rat liver ribosomal proteins had been identified before, using immunological methods, and designated L40 and L41 (20, 21). L40 and L41 have not yet been isolated and until they are there can be no certainty-despite the similarities in amino acid composition, isoelectric point, and molecular weight-about the difference or identity in the structure of rat liver Pl, P2, L40, and L41 on the one hand, and E. coli L7/L12 on the other.

A useful method for determining the possible relationship of two proteins, if no sequence data are available, is to compare their amino acid composition (19, 24). For the comparison, the sum of the differences in the content of individual amino acids is divided by two: the calculation gives a value that is referred to as the difference index (24). The smaller the number, the more likely it is that the proteins are structurally related. It is understood that the method must be interpreted judiciously, and that it is best applied in conjunc- tion with other criteria of relatedness of proteins, such as molecular size, behavior during electrophoresis, peptide fingerprints, and immunological analyses.

A correlation of the amino acid composition of Pl with L7/ L12 gives a value (difference index) of 20.5; the value for the comparison of P2 with L7/L12 is 21.3 (Table III). The values are sufficiently large as to make it unlikely the proteins are related, although it must be conceded the decision is somewhat arbitrary. Preliminary immunological experiments (ra- dioimmunoassay and Ouchterlony immunodiffusion) have failed to reveal cross-reaction between the proteins. As anti- serum against E. coli L7 did not react with rat liver Pl or P2, and antisera against Pl and against P2 did not react with L71 L12. Inasmuch as only positive results in immunological experiments carry weight these findings must be considered tentative.

Two acidic proteins have been isolated from the large subunit of Artemia salina ribosomes and characterized (26). The Artemia proteins have structural and functional properties that resemble those of E. coli L7/L12, and hence were designated EL7 and EL12 (E, for eukaryotic). EL7 and EL12 have the same amino acid composition and molecular weight (13,000), and similar peptide fingerprints, and are presumed, therefore, to be closely related. They form a pair of spots on two-dimensional electrophoretograms that are comparable in position to E. coli L7/L12. The Artemia proteins are required for EF-1 dependent binding of aminoacyl-tRNA to the ribosome (26). However, there is no direct evidence that Artemia EL7/EL12 are homologous with E. coli L7/L12; nor for that matter that EL7/EL12 are related to the rat liver acidic ribosomal proteins.

The apparent molecular weight of rat liver Pl (16,100) and

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TABLE III

Comparison of molecular weight and amino acid composition of acidic ribosomal proteins

P1 Ph. b P2 P2.% b, c EL7 EL12 AI A2 L7 LIZ

Rat Liver ArteWa YeaSta c. colia

M, (x 1o-3) 16. 1 16.1 15. 2 15. 2 13. 0 13. 0 13. 0 13. 0 11. 5 11.5

10. 5 10. 5 11. 7 12. 9 1.8 2. 1 1. 2 0. 4 5. 9 8. 7 8. 4 8. 8

11.6 12. 4 10. 5 11. 0 7. 7 6. 5 5. 0 4. 9

10. 5 12. 9 10.6 11. 5 20.4 19. 1 17. 5 18. 5

6. 8 5. 9 6. 9 6. 5 0. 7 0. 3 1. 3 1. 3 4. 8 4. 3 4.6 4. 5 7. 1 6. 4 7. 2 6. 8 0. 5 0. 5 1. 7 1. 5 3. 3 2. 7 1. 8 1. 8 1.0 1. 2 0. 4 0. 4 6. 5 6.0 8. 5 8. 3 0. 0 0. 5 3. 2 1. 4

7. 4 7. 8 9. 3 10. 8 6. 3 6. 2 2. 9 3. 1 2. 1 2. 6 2. 6 2. 6 6.4 6.4 7. 9 7. 9 5. 6 5. 3

17. 3 16.9 16.6 15. 5 14. 8 15. 0 4. 0 4. 2 2. 2 3. 1 1. 6 1. 7

14. 0 10.6 12. 5 11. 1 7. 6 7. 6 18. 5 19. 1 20. 3 18. 8 24. 0 23. 4 2. 4 2. 7 5. 1 4. 8 13. 8 14. 0 3. 7 4. 1 1.5 1. 6 2. 6 2. 5 3. 8 4. 2 3. 8 4. 5 5. 0 5. 0 8. 1 8. 8 8. 9 9. 5 6. 9 7. 0 1. 7 1. 3 1. 4 1.6 0. 0 0. 0 1. 4 1.6 2. 5 2. 3 0. 0 0. 0 0. 2 0. 0 0. 2 0. 3 0. 0 0. 0 8. 0 8. 2 6.2 66 10. 7 IO. 5 0. 7 0. 8 0. 7 0. 0 0. 8 0. 9

-c3- -4. O-J L4.O-J L5,O-J -1. o- -10.3-

17. s- 12. 3 19. 0

L-16.3' -Il.0 , L-22.2

-9.li- -19.1

-19.3-

* The amino acid composition for Artemia salina EL7 and EL12 is from Moller et al. (26); for Saccharomyces cereuisiae Al and A2 from Itoh and Osawa”; for E. coli L7 and L12 from Moller et al. (25).

b The values for amino acids are in moles per cent. c The value is derived by summing the differences in content of individual amino acids and dividing by 2 (19, 24).

P2 (15,200) is greater than that of EL7/EL12 (13,000), and the amino acid compositions are quite distinct (Table III). None- theless, further structural studies are necessary before a final decision can be reached on the relation, if any, of rat liver Pl and P2 to Artemia salina EL7 and EL12.

Two acidic proteins have been isolated from yeast (Saccha- romyces cerevisiae) ribosomes and designated Al and A24. Since the molecular weight (13,000) and the amino acid composition of the two proteins are very similar it is likely they are chemical variants of a single polypeptide. The NH,- terminal sequence of the yeast proteins has been determined (27) and there appears to be some homology with an acidic protein of Halobacterium cutirubrum but none with E. coli L7/L12. An evaluation of the exact relationship to bacterial and animal ribosomal proteins awaits publication of the details of the characterization of yeast Al and A2. Still, the amino acid composition of yeast Al and A2 does suggest some (albeit not a close) relation to Pl and P2 (Table III).

As was pointed out before, there is no certainty that the acidic proteins found in Group A60 are structural components of the ribosome. The uncertainty is compounded because they are found in TP60 in amounts much less than those of the other proteins. One possibility is that the acidic proteins are initiation, elongation, or termination factors, ordinarily only transiently associated with the ribosome, but found in Group A60 because of the large amount of TP60 (approximately 8 g) that was used as starting material. We have considered the possibility. The first thing that is pertinent is that none of the acidic proteins we have isolated has a molecular weight that exceeds 38,000 (La, Table II). Estimates of the molecular weight of EF-1 range from 47,000 to 60,000 (28, 32). The factor is reported, however, to be composed of subunits (30, 33): EF- la acts like prokaryotic EF-Tu and has a molecular weight of

4 T. Itoh and S. Osawa, personal communication.

53,000; EF-l/3 acts like EF-Ts and is composed of two polypeptides of 30,000 (p) and 55,000 (yl molecular weight. The amino acid composition of the 30,000 molecular weight species differs significantly from that of Lb (M, = 29,800Y. The amino acid composition of La (M, = 37,900) and Lb (M, = 29,800) differ from that of two species of EF-1 (M, = 52,000 and 47,000; Ref. 29). The molecular weight of EF-2 is approximately 100,000 (34, 35) and thus is unlikely to correspond to any of the proteins we have isolated.

The eukaryotic release factor has a molecular weight of about 56,000 (36) so it too can be left out of consideration. Of the initiation factors (which, incidentally, are ordinarily associated with the small rather than the large subunit and with native rather than derived subunits), only eIF-1 (M, = 15,000), eIF-4C (M, = 17,600), eIF-4D (M, = 15,000), and the small subunit of eIF-2 (M, = 35,000) need be evaluated (cf. Ref. 37 for the nomenclature used with eukaryotic initiation factors). The amino acid composition of eIF-1 has not yet been reported so we cannot compare it with the acidic proteins we have isolated; that of eIF-4C and eIF-4D have been determined (38); neither eIF-4C nor eIF-4D has an amino acid composition that resembles that of Lf, Pl, or P2, the acidic proteins in their molecular weight range. The content of amino acids in the small subunit of eIF-2 (molecular weight 32,000 to 38,000) has also been analyzed6; it was compared with La and Lb (and S6 as well, see below) and no similarity was found. There is then no evidence that any of the acidic proteins that were isolated are protein synthesis factors, although not all can be excluded with certainty.

S6, which has a molecular weight of 31,000, is the main phosphoprotein of mammalian ribosomes (18); the small subunit of eIF-2 (M, = 32,000 to 38,000) can also be phosphoryl-

5 Y. Kaziro, personal communication. 6 B. Safer and W. C. Merrick, personal communication.

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ated (39). A comparison of the amino acid composition of S6 (1) and the small subunit of eIF-2fi would indicate they are distinct polypeptides.

Forty-eight proteins have been isolated from the 60 S subunit of rat liver ribosomes and characterized (Refs. 2 and 3 and the present study). The sum of the molecular weight of the 48 is approximately 1 x 10”. I f we take the molecular weight of 28 S rRNA to be 1.75 x 10” (40) and 5 S and 5.8 S to be about 1 x 105, then the mass of the rRNA in the 60 S subparticle of mammalian ribosomes is approximately 1.85 x

10” daltons. If we assume for the moment one copy of each protein, then the molecular weight of the large subunit should be about 2.85 x 10”. That is somewhat below the estimate of 3 x 10” (41) and hence there could be more than one copy of some proteins. However, the values for the molecular weight of eukaryotic ribosomes and subunits are not reliable and hence one must be cautious in making judgments about the stoichiometry of the proteins from that data.

The original identification and enumeration of the proteins of rat liver ribosomes was by two-dimensional polyacrylamide gel electrophoresis (12). The analysis led to a presumption that the 60 S subparticle contained 41 proteins, although there was uncertainty about some. Forty-eight proteins have been isolated from extracts prepared from the large subparticle (Refs. 2 and 3 and these experiments), and all have been characterized. Each of the purified proteins is unique, although there is no assurance all are structural components of the ribosome. There is doubt especially about the acidic proteins (Pl, P2, La, Lb, and Lf) since they seem to be present in extracts of ribosomal particles in smaller amounts than the other proteins.

Note Added in Proof-After submission of this manuscript, there appeared a number of reports describing the acidic proteins of eukaryotic ribosomes, their preparation, and their phosphorylation. Reyes et al. (42) has indicated that proteins resembling Pl, P2, and their derivatives can be extracted from rat liver 60 S ribosomal subunits with 1 M NH&l and 50% ethanol at 0”. Issinger (43) has found two phosphoproteins of 16,000 molecular weight in reticulocyte ribosomes that may correspond to Pla and Plb. A triplet of acidic proteins, most likely related to P2, P2a, and P2b have been seen in extracts of HeLa cell ribosomes (44, 45). Ascites cell ribosomes also contain an acidic ribosomal protein (46). The Artemia salina proteins EL7 and EL12 (2) have been redesignated eL12 and eLl2p (47) and shown to differ only in that the latter is phosphorylated; the immunological evidence is against a relationship between A. salina eL12 and E. coli L7lL12. There is limited data on the NH,-terminal sequences of A. salina eL12 and S. cereuisiae A protein (48). The two eukaryotic proteins may be structurally related; the eukaryotic sequences are also apparently homologous with the Halobacterium cutirubrum acidic ribosomal protein L20; there is no clear indica- tion of a homology of the eukaryotic acidic proteins with E. coli L7lL12.

If one sets aside the five acidic proteins, the number is 43, very close to the original estimate. However, they are not exactly the same group: the 43 include 8 (L7’, L13’, L18’, L23’, L27’, L35’, L36’, and L37’) that were not in the original classification.’ Moreover, several proteins (Ll, L2, L16, L24, L40, and L41) designated in the beginning have not been isolated. Ll, L2, L16, and L24 were not found in any of the group fractions (they had occurred only occasionally on two- dimensional gel electrophoretograms and then stained lightly) and there must now be considerable doubt that they are in fact structural ribosomal proteins. Finally, proteins L40 and L41 have not yet been prepared.

As has been pointed out (31, it is still difficult to state precisely the number of proteins in the 60 S subunit of rat liver ribosomes. The molecular weight, or the amino acid composition, or the behavior on electrophoresis (or most frequently all three parameters) of the 48 purified proteins is distinctive. Unless one (or more) was generated from another by some chemical modification (either physiological or during its preparation) we can take each to be unique. A problem is in establishing that all are structural constituents of the ribosome. Resolution of the issue will require immunological and functional analyses.

Acknowledgments -We are grateful to Robert L. Heinrik- son and Pamela Keim for aid with the amino acid analyses; and to Alan Lin and Walter MacKinlay for valuable assist- ance. The paper is dedicated to Joel Shapiro.

’ The proteins are numbered so as to indicate their nearest neighbor on two-dimensional gel plates; the prime distinguishes them from the neighbor and indicates that they were identified after the original nomenclature was established.

1.

2.

3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

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K Tsurugi, E Collatz, K Todokoro, N Ulbrich, H N Lightfoot and I G Wooland L38.

the 60 S ribosomal subunit proteins La, Lb, Lf, P1, P2, L13', L14, L18', L20, Isolation of eukaryotic ribosomal proteins. Purification and characterization of

1978, 253:946-955.J. Biol. Chem.

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Isolation of Eukaryotic Ribosomal Proteins