J. Mol. BioZ. (1976) 93,391-404

Ribosomal Proteins from Rabbit Reticulocytes : Number and Molecular Weights of Proteins from Ribosomal Subunits GUY A. HowmDt,

A. T~AUQH$, ELIZABETH AND ROBERT R. TRAUT

JOLJXDA

A. CROSER

University of California Department of Biological Ch,emistry Xchool of Medicine

Davis, Calif. 95616, U.S.A. (Received 27’ August 1974) The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulooytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomaesie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot, was mixed with proteins of known molecular weight end then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of ewzh of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits ; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomaesie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.

1. Introduction Ribosomes, whether procaryotic or eucaryotic, perform the same function of translating the genetic code into specific proteins. Initially, this was thought to be accomplished by the action of the ribosome as an inert matrix upon which the soluble components for protein synthesis interacted. However, beginning with the demonstration that the enzyme catalyzing formation of the peptide bond was an integral part of the larger ribosomal subunit both in procaryotes (Maden et al., 1968) and eucaryotes (Vasquez et al., 1969), it has become increasingly clear that the ribosome participates actively in protein synthesis. &1oreover, recent reports indicate that t Present sddress: Friedrioh Miescher-Institut, P.O. Box 273, CH-4002 Bssel, Switzerland.. $ Present address: Department of Biochemistry, University of CalifornL, Riverside, Calif. 92602, U.S.A.

392

G. A. HOWARD

ET

AL

ribosomes may also have effects on regulation of DNA and RNA synthesis (Ove & Coetzee, 1971; Morris $ Gould, 1971). These observations exemplify the need for a detailed understanding of ribosome structure in order to elucidate t’he various structure-function relationships that constitute the mechanism of protein synthesis and its regulation. Much work has been done on the isolation and characterization of Escherichia. coli ribosomal proteins, their spatial arrangements and on the relationship between specific proteins and specific functions. By contrast, relatively little is known about the structure of eucaryotic ribosomes, apart from the fact tha,t they are larger and have a higher protein content than procaryotic ribosomes. The protein moiety consists of a heterogeneous population of approximately 70 polypeptides (reviewed by Traugh & Traut, 1973; see references cited below), the average molecular weight of which is substantially greater than that of procaryotic proteins (Bickle & Traut, 1971). Studies on eucaryotic riboaomal proteins have included attempts to define the amino acid composition of the total protein (Mathais & Williamson, 1964), separation of the ribosomal proteins by various two-dimensional acrylamide gel electrophoretic procedures (Huhn-Van-Tan et al., 1971; Martini & Gould, 1971; Welfle, 1971; Welfle et al., 1971,1972; Hultin & Sjoqvist, 1972; Lambertsson, 1972; Sherton & Wool, 1972: Chatterjee et al., 1973; Peeters et al., 1973; Pratt & Cox, 1973) and determinations of molecular weights (Gould, 1970; Bickle & Traut, 1971; King et al., 1971; Westermann et al., 1971; Bielka et aZ., 1972; Teroa & Ogata, 1972; Ochiai et al., 1973; Pratt & Cox, 1973; Thomas, 1973; Peeters et al., 1973). Most of the molecular weights reported in the latter studies were not determined for individual proteins, but represented averages for pairs or larger groups of proteins. Thus, while there is general agreement on the number of eucaryotic ribosomal proteins (approx. 70), questions about the molecular weights and molar ratios of those 70 proteins remain unanswered. We report here the separation of all the ribosomal proteins from the large and small subunits of rabbit reticulocytes using a miniature two-dimensional polyacrylamide gel electrophoresis system (Howard & Traut, 1973). Similar studies have been reported by Chatterjee et al. (1973). Each protein has been numbered in relation to its position on the gel slabs, much as was done with E. coli (Wittmann et al., 1971) and rat liver ribosomes (Sherton & Wool, 1972). In addition, we have determined the molecular weight of each protein by analyzing the components extracted from each spot on the two-dimensional gel slabs by electrophoresis in polyacrylamide-sodium dodecyl sulfate disc gels (Weber & Osborn, 1969; Howard & Traut, 1974). The agreement between the sum of these individual molecular weights and values reported for the total protein in each eucaryotic ribosomal subunit is consistent with the possibility that each type of subunit has equimolar amounts of the constituent polypeptides, i.e. that the ribosome population is relatively homogeneous. Studies on the stoichiometry of ribosomal proteins from rabbit reticulocytes will be reported in a subsequent paper.

2. Materials and Methods (a) Preparation

of reticulocytes, lysates, ribosomes and su,bunits

lteticulocytes were obtained from New Zealand White (female) rabbits given 7 daily injections of 2.5% (v/v) phenylhydrazino . HCl neutralized with NaOH to pH 7 ; the amount administered was determined by the weight of the animal (Adamson et al., 1968). The animals were bled by cardiac puncture on the 8th day. The reticulocytes were washed twice in an isotonic saline solution (Godchaux et al., 1967) and lysed by addition of 1 vol.

PROTEINS

FROM

RETICULOCYTE

RIBOSOMES

393

cold 1 mM-MgCl, according to the method of Collier (1967). The lysatte was claritled by centrifugation in a Sorvall SS-34 rotor at 20,000 revs/min for 20 min at 4°C. This supernatant fraction was then centrifuged for 15 h at 50,000 revs/min at 4% in a Beckman Ti60 rotor. The ribosomal pellets were resuspended in buffer A (25 mu-KCl, 10 mM-Tris. HCl (pH 7*4), 1.5 mM-MgCl,, 1 maa-dithiothreitol) and diluted to a final concentration of 100 to 200 &,, units/ml. They were either used immediately or stored at -7O’C. Ribosomes prepared in this manner have been shown to be highly active in a cell-free, protein synthesizing system (Howard et al., 1970). Ribosomal subunits were prepared from 80 S ribosomes by centrifugation of the latter on 10% to 38% (w/v) hyperbolic sucrose gradients in a buffer containing 20 mu-Tris (pH 7*6), 500 mM-Kcl, 2 mM-MgCl,, 1 mM-dithiothreitol at 47,000 revs/min for 180 min at 4°C in a Beckman Ti-XIV rotor as described previously (Traugh et al., 1973).

(b) Preparation

of ribosomal

proteins

Two alternative procedures were used for preparation of ribosomal proteins from the purified subunits. In the first method, the ribosomal RNA was precipitated by addition of acetic acid to a final concentration of 66% after first raising the MgCls concentration to 0.1 M (Hardy et al., 1969). The supernatant fraction containing the proteins was dialyzed 10 mM-NH4HC03, 1 mM-2-mercaptoethanol and concentrated by dialysis against 8 M-UI?%, against dry Sephadex G200. In the second method, the ribosomal RNA was precipitated by adding to the ribosomes an equal volume of a solution containing 8 M-urea and 6 M-Licl (Spitnik-Elson, 1965). The extracted proteins were then precipitated from the supernatant solution at 4°C by addition of an equal volume of 20% trichloroacetic acid. The precipitat0 was washed successively with 5 to 10 vol. each ethanol, ethanol/diethyl ether (1: l), and diethyl ether at 4°C. It was then solubilized in a small volume of a solution containing 8 M-urea, 10 mM-NH,HCO,, 1 mM-2-mercaptoet’hanol. The protein solutions obtained wit,11 either method were stored at - 70°C in small portions. Tho protein patterns obtained by oloctrophoresis in 2-dimensional polyacrylamide gals w0r0 the same for both methods of sample preparation. (c) Analysis

of ribosomal

RIVA

by polyacrylamide

gel electrophoresis

The purity of the preparations of ribosomal subunits was established by analysis of RNA by electrophoresis in polyacrylamide gels. The RNA obtained after extracting the total protein from the subunits by the methods described above was washed twice with 4 M-ur0a, 3 M-LiCl to remove any non-specifically bound proteins, resuspended in 10 mMTris (pH 7.4), 0.3 mM-Mgcl,, 30 mM-NH&l, 6 mM-2-mercaptoethanol and dialyzed until clear (Fahnestock et al., 1973). The RNA preparations were then submitted to electrophoresis on 2.8% polyacrylamide gels in the presencc~ of 0.2% sodium dodecyl sulfate for 45 to 60 min at 5 mA/gel at room temperature (Williamson et al., 197 1; Lockard & Lingrel, 1972). The gels were stained for 30 min in methylene blue (0.2% in 0.4 M-sodium acetate, 0.4 M-acetic acid). Excess stain was removed by soaking the gels overnight in water. (d) Two-dimensional

polyacrylamide

gel electrophoresis

The procedure for 2-dimensional gel electrophoresis has beon described by Howard & Traut (1973,1974) and represents a miniaturization of the method first described by Kaltschmidt & Wittmann (1970). The first-dimension disc gels contained 4% acrylamide. Tbo proteins were electrophoresed in 2 separate gels at pH 8.7: for 3 to 6 h at 4 mA/g01 toward the cathode and for 5 h at 4 mA/gel toward the anode. The second-dimension slab gels contained 18% acrylamide. The disc gels were embedded with their origins adjacent at tho top of the go1 slab and 0lectrophoresed at pH 4.5 Flither for 7 h at 150 V, or for 16 11 at 80 V. Electrophoresis in both dimensions was carried out at room temperature. The sample of ribosomal proteins contained 150 to 300 pg of protein/gel in a volume of 100 ~1. Protein concentration was determined by the method of Lowry et al. (1951). The gels were stained for 1 to 4 h in Coomassie brilliant blue R-250 (0.1% in 7.5% destained electrophoretically for 10 to 30 min in 7.5% acetic acid/500,& methanol/water), acetio acid/b% methanol/water and photographed (Howard & Traut, 1973,1974). The stained protein spots for each subunit were numbered along vertical lines starting at the

394

G. A. HOWARD

ET AL.

origin of the gels in the upper left-hand corner. The numbers assigned to the spots are prefixed with L or S for the large or small subunits, respectively. No attempt has been made at this time to correlate the proteins of rabbit retioulocyte ribosomes with those from other eucaryotic organisms or tissues.

(e) Polyawylamide-sodium

dodecyl sulfate gel electrophorerria of ribosomal

proteins

The total ribosomal protein from each subunit or the isolated ribosomal proteins that had been eluted as single stained spots from the 2-dimensional slab gels (Howard & Traut, 1974) were electrophoresed on 10 y. and 15% polyacrylamide gels containing 0.1 y. sodium dodecyl sulfate. Single stained spots were cut out of the slab and gel segments were dialyzed against 4 to 5 ml distilled water for 30 min at 4°C to remove excess acetic acid from the gel. They were then macerated in small tubes containing 0.2 to 0.3 ml 8 M-Urea, 1% sodium dodecyl sulfate and left in this solution overnight at room temperature. The contents of each tube were then adjusted to pH 7.0 with 10 N-NaOH, heated at 65°C for 10 min, and both gel fragments and supernatant solution were transferred to the top of the polyacrylamide-sodium dodecyl sulfate gel tubes. To determine accurately the molecular weight of each protein spot resolved on the slab gel, protein standards of known molecular weight, chosen so as not to coincide with the unknown ribosomal proteins, were added either directly to the macerated gel before the heating step, or electrophoresed on separate gels run at the same time. Both procedures gave similar results, but the use of internal standards gave better precision. Electrophoresis was for 20 to 30 min at 3 mA to allow the relatively large sample to form a compact zone at the top of the gel, then the current was raised to 10 mA and electrophoresis was continued for 3 to 4 h at room temperature. The gels were stained with Coomassie brilliant blue (0.1%) overnight, and then excess stain was removed by transverse electrophoresis (Bickle & Traut, 1971). (f) Spectrophotometriic scanning of gels The stained polyacrylamide-sodium dodecyl sulfate disc gels of the total protein from each subunit were scanned at 550 nm in a Gilford 2400-S recording spectrophotometer. The height of the absorbance curve was measured by hand every O-1 in from the origin was processed in a Wang 700 computer (programmed to the tracking dye. This information by Dr T. A. Bickle) to determine weight average @I,) and number average (H,) molecular weights (Bickle & Traut, 1971,1974). (g) Materials Acrylamide (technical grade), N,N-methylene bisacrylamide and N,N,N’,N’-tetraethylmethylenediamine were obtained from Eastman Chemicals; sodium dodecyl sulfate was from BDH Chemicals Ltd. ; EDTA(Ng) and Tris *HCl were from Sigma ; the remaining reagents were from Mallinckrodt. Recrystallization of the aorylamide was unnecessary, aa results were the same with the crude or purified reagent. The 8 M-urea solutions were passed through a column of AG 501-X8 (D) Dowex resin (BioRad Laboratories) before use.

3. Results (a)

Preparation and characterization of ribosmnd subunits

Retioulooyte ribosomes were separated into subunits by zonal oentrifugation in sucrose density gradients as described in Materials and Methods. Fractions containing well-resolved subunits, designated by the hatched areas in Figure 1, were pooled, diluted with buffer A and centrifuged at 40,000 revs/min in a Beckman Ti60 rotor for 16 hours. The pellets containing the subunits were resuspended in buffer A to a final concentration of 100 to 200 A 26,,units/ml, and stored in small portions at -70°C. The purity of each preparation of subunits was determined by oentrifugation on small sucrose gradients in buffer A. The inset in Figure 1 shows that the subunits are free

PROTEINS

FROM

RETICULOCYTE

Fraction

no

RIBOSOMES

396

TOP

Fra. 1. Preparative separation of 40 S and 60 S ribosomal subunits of reticulocytes. Approximately 2500 Azao units of ribosomes were treated with O-6 M-KCl and then separated on a sucrose gradient formed in a Beckman Ti-XIV zonal rotor as described in Materials and Methods. A tracing of the separation obtained is shown in (a), with the fractions pooled from each subunit peak indicated by the hatched areas. Small sucrose gradients (4.5 ml; 6% to 20%) in buffer A were centrifuged at 65,000 revs/min in a Beckman SW56 rotor at 4°C to analyze the purity of the pooled zone1 fractions from (b) the 60 S region, and (c) the 40 S region.

of cross-contamination. The designations 60 S and 40 S are somewhat arbitrary, although consistent with the sedimentation velocity as compared to subunits from E. coli run at the same time. To confirm the purity of each subunit preparation, ribosomal RNA was analyzed by polyacrylamide gel electrophoresis. As shown in Plate I, only 18 S RNA was present in the 40 S subunit fraction, and only 28 S RNA in the 60 S subunit fraction. The experiments shown in Figure 1 and Plate I establish the purity of the subunits used in subsequent experiments. Total protein was extracted from the purified ribosomal subunits as described. Since it has been reported in other systems that certain proteins remain bound to the rRNA under the conditions of extraction (Ford, 1971; Fahnestock et al., 1973), the resuspended washed RNA from each of the subunits (after protein extraction) was submitted to electrophoresis on 10% and 15% acrylamide gels containing O*lo/o sodium dodecyl sulfate. These gels did not reveal any significant protein bands, even when a tenfold excess of sample over the amount used routinely was analyzed. The results were the same with either extraction procedure. It was concluded that the extraction of the proteins from the subunits was complete. (b) Tulo-dimensional gel dectrophoresis

of 40 S and 60 S ribosomal proteins

The total proteins from each subunit were separated by electrophoresis on twodimensional polyacrylamide gels (see Materials and Methods). Plate II shows typical 26

396

G. A. HOWARD

ET

AL.

separation patterns of the proteins extracted from (a) the small subunit and (b) large subunit. Each of the stained spots has been assigned a number. (c) Polyacrylamide-sodium dodecyl suljate gel electrophoretic alzalysis of components separated by two-chbensional electrophoresis A further analysis of the components resolved by two-dimensional electrophoresis was carried out, both in order to determine the molecular weight of the material in each spot and to investigate the possibility that a single spot contained more than one polypeptide. The individual stained spots from the gel slabs were cut out, extracted and submitted to electrophoresis on 10% and 15% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (see Materials and Methods). Typical examples of the results of this analysis are shown in Plate III. The polyacrylamide-sodium dodecyl sulfate gels show the position of proteins eluted from two-dimensional gels together with protein standards of known molecular weight, which were coelectrophoresed in order to calibrate the gels. The molecular weight values obtained from standard semilogarithmic graphs are reproducible and the same with gels of either polyacrylamide concentration; however, the 15% gels gave greater precision for proteins of low molecular weight. TABLE

1

Comparison of the molecular weights of E. coli 30 S ribosomal proteins determined by various procedures

Protein

Sl s2 53 54 Sb S6 s7 58 s9 SlO 514 S16 S16 518 s19 s20 521

Proteins isoletedt from 2.dimensions1 gels M, x 10-s 63.6 27.9 29.0 26.9 20.6 14.8 19.3 14.9 17.8 9.8 14.2 11.3 8.8 12.4 12.4 12.9 19.6

Proteins isolated byi CM-cellulose chromatography M, x 1O-3

Sedimentation$ equilibrium M, x 10-s

68.0 29.8 29.9 26.6 20.2 13.5 19.6 14.1 16.2 10.6 10.7 9.6 11.0 11.4 10.8 19.7

65.0 28.3 28.2 26.7 19.6 16.6 22.7 16.6 16.2 12.4 14.0 12.6 11.7 12.2 13.1 120 12.2

E. wli ribosomes and ribosomal subunits were prepared as desoribed by Bickle & Traut (1971). The ribosomal proteins were prepared by the method of Spitnik-Elson (1966) and separated on 2-dimensional polyacrylamide gels ss previously described (Howard t Traut, 1973). The number of each of the proteins is es defined by Wittmann et al. (1971). t This study. $ Traut et al. (1969). Protein fractions isolated either from 2dimensional gels or from CM-cellulose analyzed on polyaorylemide-sodium dodecyl sulfate gels. 5 Taken from Dzionara et al. (1970).

columns

were

PLATES

I-III

I’LATE I. Polyacrylamide gel electropherogrsms of RN.4 from rabbit raticulocyte ribosomen. The bands shown represent RNA fram (a) 40 S subunits. (b) 80 S ribosornos and (c) 60 R suhunit,s c~lw3rophoresrtl on 2.S”,& acrylamitln prls as described in Mntcrials and Methods.

PLATE II. Two-dimensional gel clectrophoretograms of rlbouomal proteins from rabbit reticulocytr 40 S (a) and 60 S (b) subunit,s. Preparation of the prot,eins for eleatrophoresis, and the elwt.rophoresi~ met,hod are dercribed in Materials and Met.hods. Electrophoresis in thv first tlimrnsion was for 5 h at 4 mA/gel, and for 7 h at, 150 V/gel m the second dimension. The 0 indicates the origin in the first dimension, and the arums sh ow the migration direction in both tlimmsions. The protein patterns shown here were obtained wit,h samples of approximatel> 6 .-I Lb0 units of 40 R subunit* and I2 A,,, units of 60 S snhlmits. I’LATE 111. Polyacrylamid~.sotii~lm tlutlt:cyl ;iulfatr gt’l clwt,rophoresis of individual rabbit reticulocyte ribosomal proteins. Stained protein spots were excised from %dimensional slab gels like those in Plate II(a) and (b), then treated as described in the text, and applied to the gels. The arrows show the position of the ribosomal protein; the* other bands are the molecular weight st,nndards. (a) 15% and (b) 10% acrylamidc gels. The ahbreviatjions for the standard proteins clectrophoresed and their molecular wcight,s are: HXA, bovine serum albumin, 67,500; OV. ovalbumin, 46,000; CHY, chymotryp+mgen, 15,700; RN&se A, ribonucleare A, 13,600. Elecbrophorenis was from (-) to (+) itt 10 mA/grl. Lmear srmilog plots of migration dist,ences of standard proteins DWSUS molecular wright wew made, and the, mcrlccular weights of the mataerial in the% l:ands indicatlvl by the arrows were cst,imattvl

d E n”

E

1st D -o-

(-)

s22

1st

D t-3

-o-

i27

L34 t 3c L38 L38 a”L39

L28

L19

L19

h-4

Acrylrmido

L29

-*

et3

-.

PLATE

RNAase

III.

L32

s2

L19

Li4

s24

RUAase

PROTEINS

FROM

RETICULOCYTE

RIBOSOMES

397

The accuracy of the method was established by a similar experiment with the proteins of E. coEi 30 S ribosomes. Table 1 shows that the molecular weights obtained by the method described here are in agreement with those obtained for the bacterial ribosomal proteins by other methods. The results of the analysis of each spot resolved by two-dimensional electrophoresis of the proteins from 40 S and 60 S subunits of rabbit reticulocytes are shown in Tables 2 and 3. TABLE 2

Molecular weight of reticulocyte 40 8 ribosomal proteins

Protein

Sl 52 53 54 56 S6 57 58 s9 SIO Sll s12 513 s14 s15 Sl6a S16b 517 518 Sl!l 520 521 522 s23 524 525 S26 527 528 529 s30 531

Average M, x 10-s

33.4 39.3 31.7 30.9 22.8 19.8 16.5 16.3 14.4 33.3 29.1 29.0 31.7 22.0 17.2 18.6 16.4 16.1 12.4 14.6 20.6 18.8 8.6 26.7 18.4 20.8 18.4 15.5 14.6 18.4 16.8 20.6

Range of M, x 1O-3

33.2-33.6 39-l-39.6 31-2-32.3 30.7-31.2 21.8-23.8 19.7-19.9 16.k16.7 15.3-15.4 14.2-14.6 32.9-33.8 28-2-29.5 286-31.0 31.2-32.3 21-b-22.6 16.9-17.6 18.6-18.7 16.2-16.6 14.8-16.6 12.2-12.7 14.6-14.8 19.7-21.5 18.7-19.0 8.1- 9.0 26.8-27.7 18.2-18.7 20.7-21.0 18.2-18.7 14.8-16~2 13.916.1 l&1-18*7 15.7-16.0 206-21.3

Number of independent preparations 3 3 3 4 3 3 3 3 3 3 3 3 3 3 3 4 4 3 3 4 3 3 3 3 3 3 4 3 4 3 3 3

Number of mol. wt determinations 4 4 4 6 4 4 4 4 4 4 4 5 5 4 4 6 6 4 4 6 4 4 4 6 4 6 6 6 6 5 4 4

The protein number refers to its position on the 2dimensional gel slebs as shown in Plate II. The number of mol. wt determinations, shown for eeoh protein in the last column, included at leaet one run on a 10% aorylamide gel and a 16% acrylamide gel. Selected proteins of known mol. wt were chosen to braaket the weight of the protein being examined and mixed with each ribosomal protein before electrophoresis.

G. A. HOWARD

398

ET

AL.

TABLE 3

Molecular weight of reticulocyte 60 X ribosomal proteins

Protein

Ll L2 L3 L4 Lb L6 L7 Lfl L9 LlO Lll L12 L13 L14 Lib L16 L17 L18 L19 L20 L21 L22 L23 L24 L26 L26 L27 L28 L29 L30 L31 L32 L33 L34 L36 L36 L37 L38 L39

M, x 1O-3

Range of M, x 10-a

Number of independent preparations

26.4 21,2 21.1 21.7 44.7 58.5 65.0 22.3 16.7 39.2 40.0 27.0 26.8 27.3 26.3 26.1 46.1 38.0 31.6 30.0 27.1 29.6 22.8 31.1 21.0 20.9 23.1 8.9 19.7 27.0 28.8 40.3 20.7 13.6 12.8 12.5 12.3 11.6 10.6

24.3-26.6 20.1-22.4 21.1-21.2 20622.9 44.6-44.9 68.2-68.9 54+-66.0 22.2-22.5 16.6-16.9 39.2-39.3 39.7-40.3 26.9-27.2 26.3-26.4 26.6-28.8 25.7-26.9 26.0-26.2 444-46.9 37.6-38.5 30.7-32.3 29.9-30.1 26.8-27.4 29.3-29.7 226-23.1 29.8-31.4 19.1-22.6 20.8-21.0 22.6-23.7 8.8- 9.0 184-20.9 26.9-27.2 27.7-29.8 40.2-40.5 194-22.1 13.2-14-l 12.7-12.9 12~3-12.7 12.1-12.6 114-11.9 10~1-11~1

3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 3 3 4 4 3 3 3 4 4 3 3 3 3 3 3 3 3 3 3 3 3

Average

Number of mol. wt determinations 4 4 4 4 4 4 4 4 4 4 4 4 4 6 6 6 5 6 6 b 6 6 b 4 5 6 6 4 4 4 4 4 4 4 4 4 4 4 4

Refer to the legend of Table 2 for explanation.

(d) Number of proteins in 40 ii’ and 60 S subunits of rabbit reticdocytes All the protein spots on the two-dimensional gels except S16 from 40 S subunits had only one protein band when each was electrophoresed on gels containing sodium dodecyl sulfate. In some instances stained spots overlapped slightly. They could be separated into discrete spots on the two-dimensional gel slabs by varying the electrophoresis time to increase separation of the overlapping proteins. It was concluded from the combined analysis that there are 32 proteins in the 40 S subunit and 39 proteins in the 60 5 subunit.

PROTEINS

FROM

RETICULOCYTE

RIBOSOMES

399

(e) Comparison of molecular weight averages with single proteins and unfractionated total protein Molecular weight averages were calculated from the data of Tables 2 and 3 and compared to those obtained with unfractionated protein analyzed by polyacrylamidesodium dodecyl sulfate gel electrophoresis (Bickle & Traut, 1974), and with results from other laboratories (Gould, 1970; King et al., 1971). For the latter experiments the intact 40 S and 60 S subunits were electrophoresed directly on 10% polyacrylamide gels containing 0.1 o/osodium dodecyl sulfate to determine the molecular weight distribution of the total unfractionated protein. The use of intact subunits for these analyses precluded any possible loss of protein during extraction procedures. Scans of the stained protein bands are shown in Figure 2 for (a) 40 S subunits and (b) 60 S subunits.

iI((

b)

L o ETMolecularB weight ’ ----x IO-’

40-S subunit

60 S subunit

IO

W

FIG. 2. Spectrophotometric soans of polyacrylamide-sodium dodecyl sulfate gels of unfractionated ribosomal proteins from each subunit stained with Coomassie brilliant blue. Stained 10% aorylamide gels for retioulocyte (a) 40 S and (b) 60 S ribosomal subunits were scanned at 600 nm as described in Material8 and Methods. The top of the gels is to the left.

The intensity of staining with Coomassie brilliant blue is proportional to the amount of material present (Fazekm de St. Groth et al., 1963; Bickle & Traut, 1971). The data shown in Figure 2 were used to calculate weight average (gW) and number average (18,) molecular weights for the total protein from each subunit. Values for a, and

G. A. HOWARD

400

TABLE

ET

AL.

4

Average molecdar weights a?adtotal proteilt content of eucuyotic subunits 40s Total protein (mol. wt/partiole X 10-e) a. SDS gel *c&n8 A B C D E F

24,000 18,000

b. Pure proteins 22,000

c. SDS gel 8cans 26,000 21,000

d. Pure proteins 24,600

0. c Pure proteins

f. Physioochemical data

0.68 0.64

20,400

0.66 28,000

26,400

28,400

0.68 0.69

30,600

l-06

60s A B c D E

27,000 22,000

26,900

31,000 26,000

l-09

24,300

o-94 30,000

28,000

31,600

1.07

The data, in line A are from the studies described here. Those in line B from King et al. (1971); line C, H. Gould (personal communication); line D, Westermarm et al. (1971); line E, Lin & Wool (1974) ; line F, Teroe & Ogata (1972). See references cited for experimental details. Molecular weights for pure proteins were obtained either as described here (A and E), or from Z-dimensional gels that oonteined sodium dodecyl sulfate in the seoond dimension (D), or by separation of proteti on CM-oellulose followed by electrophoresis in gels oontaining sodium dodeoyl sulfate (F). The date in lines A to C (~TBfor rabbit reticulocyte ribosomes; in lines D to F for rat liver ribosomes.

Bn were also calculated using the molecular weights found for the individual proteins (Tables 2 and 3). The results are given in Table 4, line A. There is quite good agreement between the average molecular weights obtained by the two methods. These results strongly suggest that the proteins identified and recovered from the two-dimensional gels account for all the protein found on the particles. Table 4 (lines B to D) also shows the average molecular weights found in other laboratories by methods both similar to and different from those used here (see legend to TabIe 4 and references therein for experimental details). The variations in the values found in different laboratories are most likely due to the different methods used both to prepare ribosomes and to process data obtained from gel scans. However, all the results confirm the fact that the eucaryotic ribosomal proteins have an average molecular weight substantially greater than that for procaryotic ribosomal proteins (Bickle & Traut, 1971). (f) Total protein content of eucaryotic ribosomal subunits Earlier estimates for the total protein content of eucaryotic ribosomal subunits were derived from physico-chemical studies on the intact particles (Table 4, column f ) .

PROTEINS

FROM

RETICULOCYTE

RIBOSOMES

401

This parameter can also be calculated from the present results by assuming that each protein species is present in one copy and summing all individual molecular weights. These results, given in Table 4, column e, are in close agreement with the former values. (g) Relative molar ratios of proteins The data in Tables 2 and 3 along with data derived from the scans in Figure 2 were used to make comparative estimates of the relative total and molar amounts of protein present in defined ranges of molecular weight. The amount of material present in molecular weight classes of 5000 derived from the scans (Fig. 2) of gels (containing sodium dodecyl sulfate) of total proteins was plotted as the weight or mole fraction of the total material present. This plot makes no assumptions concerning the absolute molar amounts of the different protein species. Similar plots were made from the data a)

30

20

10 c a3 ‘0 k -. 0 0 s 20

IO

Mnlecular

weight x ICI-j

FIG. 3. Moleoular weight distribution of ribosomal proteins derived from molecular weights of individual proteins separated by 2-dimensional gel electrophoresis, and from soans of gels of total protein. The amount of protein in each molecular weight class of 6000 was determined by analyzing the gel soans of Fig. 2. The moleoular weights of individual proteins listed in Tables 2 and 3 were similarly divided into groups differing in mol. wt by 6000. The data are plotted as the weight percentage of total material present in each class ((b) and (d)) and as the mole pementage in each molecular weight olass ((a) and (a)). The hatched bars represent values obtained from the scans of total protein (Fig. 2); the open bars represent values from the data on individual proteins (Tables 2 and 3). The standard proteins used to calibrate the molecular weight soale were bovine serum albumin, 67,500; ovalbmnin, 46,000; carboxypeptidase a, 34,000; chymotrypsinogen, 25,700; myoglobin, 17,200; and ribonuclease A, 13,600.

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in Tables 2 and 3. In this case the proteins were assumed to be present in equimolar ratios. It can be seen in Figure 3 that there is close agreement between the two procedures, both when mole fractions and weight fractions are compared. These results, together with those shown in Table 4, are consistent with the hypothesis that the ribosomal proteins of rabbit reticulocytes are present in approximately equimolar amounts. This has been suggested by other workers (Ford, 1971; Pratt & Cox, 1973; Westermann & Bielka, 1973). 4. Discussion The proteins of rabbit reticulocyte ribosomes have been separated into 71 individual polypeptide components: 32 in the 40 S subunit and 39 in the 60 S subunit. Only one acidic protein from the 40 S subunit was observed, and that protein always stained very lightly on the two-dimensional gels, even when the sample concentration was greatly increased. This raises the question of whether or not it is truly a ribosomal protein. Two very acidic 60 S proteins, which also stained faintly, have been observed in experiments not shown here. More reproducible methods for increasing the intensity or yield of these proteins have recently been found (0. Issinger & R. Traut, unpublished results). Their behavior is reminiscent of that of proteins L7 and L12 in E. wli 50 S subunits and of acidic proteins from eucaryotic ribosomes reported by other laboratories. There are indications that the recovery of these two proteins from reticulocyte ribosomes decreases when 80 S ribosomes are treated with 0.5 M-KC1 to produce subunits (W. Moller, personal communication). Two similar proteins have been observed on two-dimensional gels of rat liver ribosomal proteins (Sherton t Wool, 1974). Furthermore, the two proteins from eucaryotic 60 S ribosomes have been shown to have immunochemical and functional homologies to the procaryotic proteins L7 and L12 (I. Wool & G. StGf%ler,personal communication). All the ribosomal proteins separated by two-dimensional gel electrophoresis as shown in Plate II(a) and (b) were characterized further by extracting them from the stained spots on the two-dimensional acrylamide gel slabs and then electrophoresing the extracted proteins on polyacrylamide gels containing sodium dodecyl sulfate. For the first time this made possible determination of molecular weights for each of the 70 proteins in a eucaryotic ribosome. Previous workers have also determined the number of eucaryotic ribosomal proteins separated by a variety of two-dimensional acrylamide gel methods (see review by Traugh t Traut, 1973; Welfle, 1971; Welfle et al., 1971; Lambertsson, 1972; Sherton & Wool, 1972; Chatterjee et al., 1973; Pratt & Cox, 1973; Traut et al., 1974; Peeters et al., 1973). The number of proteins resolved is quite similar in all cases cited, i.e. approximately 70. However, since various types of gel systems yield different separation patterns for the same ribosomes (see Fig. 2 of Chatterjee et al., 1973; Plates I and II of Martini t Gould, 1971), it is not feasible at present to adopt a general numbering scheme for eucaryotic ribosomal proteins. Moreover, it may be difficult to devise a general numbering system, insofar as there exist specific differences between ribosomes from different tissues within the same species (Houssais, 1971) as well as between ribosomes from different eucaryotic species (Delaunay et al., 1972,1973; Wikman-Coffelt et al., 1972). The work described here on the separation, enumeration and quantification of reticulocyte ribosomal proteins w&8 undertaken in order to provide a framework for

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the continued investigation of structure-function relationships in eucaryotic ribosomes. Such studies will include determination of protein neighborhood relationships as implicated by crosslinking, determination of the ribosomal binding sites for soluble factors in protein synthesis and the sites of phosphorylation of ribosomes by protein kinases. Unambiguous methods for the identification of ribosomal proteins are a prerequisite for the successful completion of all such investigations. In addition, in this and a succeeding paper, the question of whether eucaryotic ribosomes are homogeneous or heterogeneous with respect to their protein composition is examined. The results reported here are suggestive that most eucaryotic ribosomal proteins (or at least those found in rabbit reticulocytes) may be present in amounts equimolar with each other and with the ribosomal particle. These preliminary conclusions concerning the stoichiometry of ribosomal proteins derive from the following results reported here. (1) The sum of the molecular weights of the individual proteins (Table 4, column e) is in agreement with the total molecular weight of protein previously reported for rabbit reticulocyte ribosomes from physicochemical data (Table 4, column f). (2) The weight average and number average molecular weights derived from the individual molecular weight values for each of 71 proteins (Table 4, columns b and d), which are based on the assumption of equimolar&y, are in agreement with results derived from analyses on total proteins (Table 4, columns a and c). (3) The number of protein species, for which individual molecular weights have been obtained, that fall in defined molecular weight classes (defined arbitrarily as domains differing by 5000 from 5000 to SO,OOO),is in agreement with the distribution predicted by analyses performed on the total unfractionated protein mixtures from each subunit. None of these relations is found in the case of the E. coli 30 S ribosome, where protein heterogeneity is best documented (Voynow & Kurland, 1971; Traut et al., 1969). The results are consistent with the hypothesis that each protein is present in one copy per subunit. This has been suggested previously (Ford, 1971; Pratt & Cox, 1973; Westermann et al., 1973). Clearly, however, a method that allows quantitation of the relative mass of individual proteins present in the ribosome is necessary to answer fully the question of whether or not the proteins are present in equimolar amounts. Further studies on this problem will be reported elsewhere. This work was supported by grants to one of us (It. R. T.) from the American Heart Association and the Damon Runyon Memorial Fund for Cancer Research. REFERENCES Adamson, S. D., Herbert, E. & Godchaux, W. (1968). Arch. Biochem. Biophya. 125, 671-683. Bickle, T. A. & Traut, R. R. (1971). J. Biol. Chem. 246, 6828-6834. Bickle, T. A. & Traut, R. R. (1974). Method.9 EnzymoZ. 30, 545-553. Bielka, H., We&, H., Westermann, P., Noll, F., Grummt, F. & Stahl, J. (1972). In Proc. 7th FEBS Meeting, Varnu 1971 (Cox, R. A. 8: Hadjiolov, A. A., eds), vol. 23, pp. 19-40, Academic Press, New York. Chettejee, S. H., Kazemie, M. & Matthaei, H. (1973). Noppe-Seyler’s 2. Phyad. Chem. 345,481-486. Collier, R. J. (1967). J. MOE. BioE. 25, 83-98. Delaunay, J., Mathieu, C. & Schapira, G. (1972). Eur. J. Biochem. 31, 561-564. Delaunay, J., Creuset, F. & Schapira, G. (1973). Eur. J. Biochem. 39, 305-311. Dzionara, M., Kaltschmidt, E. & Wittmann, H. G. (1970). Proc. Nat. Ad. Sci., U.S.A. 67, 1909-1913.

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G. A. HOWARD

ET

AL.

Fahnestock, S., Erdmann, V. & Nomura, M. (1973). Biochemistry, 12, 226-224. Fazekas de St. Groth, S., Webster, R. G. & Datyner, A. (1963). Biochim. Biophys. Acta, 71, 377-391. Ford, P. J. (1971). Biochm. J. 125, 1091-1107. Godchaux, W., Adamson, S. D. & Herbert, E. (1967). J. Mol. Biol. 27, 57-72. Gould, H. J. (1970). Nature (London), 227, 1145-1147. 8,2897-2905. Hardy, S. J. S., Kurland, C. G., Voynow, P. & Mora, G. (1969). Biochemistry, Houssais, J. F. (1971). Eur. J. Biochem. 24, 323-341. Howard, G. A. & Traut, R. R. (1973). FEBS Letters, 29, 177-180. Howard, G. A. & Traut, R. R. (1974). Method8 Enzymol. 30, 526-539. Howard, G. A., Adamson, S. D. & Herbert, E. (1970). J. Biol. Chem. 245, 6237-6239. H&n-Van-Tan, Delaunay, J. & Shapira, G. (1971). FEBS Letters, 17, 163-167. Hultin, T. & Sjoqvist, A. (1972). Anal. Biochem. 46, 342-346. Kaltschmidt, E. & Wittmann, H. G. (1970). Anal. Biochem. 36, 401-412. King, H. W. S., Gould, H. J. & Shearman, J. J. (1971). J. Mol. Biol. 61, 143-156. Lambertsson, A. G. (1972). Mol. Ben. Genet. 118, 215-222. Lin, A. t Wool, I. G. (1974). Mol. Ben. Genet. 134, l-6. Lockard, R. E. t Lingrel, J. B. (1972). J. BioZ. Chem. 247, 4174-4179. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265-275. Maden, B. E. H., Traut, R. R. & Munro, R. E. (1968). J. Mol. BioZ. 35, 333-345. Martini, 0. H. W. & Gould, H. J. (1971). J. Mol. Biol. 62, 403-405. Mathais, A. P. & Williamson, R. (1964). J. Mol. BioZ. 9, 498-502. Morris, M. E. t Gould, H. (1971). PTOC. Nat. Acad. Sci., U.S.A. 68, 481-485. Ochiai, H., Kanda, F. & Iwabuchi, M. (1973). J. B&hem. 73, 163-167. Ove, P. t Coetzee, M. L. (1971). Proc. Amer. Asa. Can. Res. 12, 10-17. Peeters, B., Vanduffel, L., Depuydt, A. L% Rombauts, W. (1973). FEBS Letters, 36, 217-221. Pratt, H. & Cox, R. A. (1973). Biochim. Biophys. Acta, 310, 188-204. Sherton, C. C. & Wool, I. G. (1972). J. BioZ. Chem. 247, 4460-4467. Sherton, C. C. & Wool, I. G. (1974). J. BioZ. Chem. 249, 2253-2267. Spitnik-Elson, P. (1965). Biochem. Biophye. Res. Commun. 18, 557-562. Teroa, K. & Ogata, K. (1972). Biochim. Biophys. Acta, 285, 473-482. Thomas, H. (1973). ExptZ Cell Res. 77, 298-302. Traugh, J. A. & Traut, R. R. (1973). In Methods in CeZZ Phy&oZogy (Prescott, D. M., ed.), vol. 7, pp. 67-103, Academic Press, New York. Traugh, J. A., Mumby, M. & Traut, R. R. (1973). Proc. Nat. Acad.Sci., U.S.A. 70,373-376. Traut, R. R., Delius, H., Ahmad-Zadeh, C., Bickle, T. A., Pearson, P. & Tissieres, A. (1969). Cold Spring

Traut,

Harbor

Symp.

Quant.

BioZ. 34, 25-38.

R. R., Howard, G. A. & Traugh, J. A. (1974). In MetuboZic Interconversion of New York. Enzymes, pp. 155-164, Springer-Verlag, Vasquez, D., Battner, E., Neth, R., Heller, G. & Munro, R. E. (1969). Cold Spring Harbor Symp. Quant. BioZ. 34, 369-375. Voynow, P. & Kurland, C. G. (1971). Biochemietry, 10, 517-524. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244, 44064412. Welfle, H. (1971). Acta BioZ. Med. Germ. 27, 547-551. Welfle, H., Stahl, J. & Bielka, H. (1971). B&him. Biophys. Actu, 243, 416-419. Welfle, H., Stahl, J. & Bielka, H. (1972). FEBS Letters, 26, 228-232. Westermann, P. & Bielka, H. (1973). MOE. Gen. Genet. 126, 349-356. Westermann, P., Bielka, H. & Bottger, M. (1971). Mol. Gen. Genet. 111, 224-234. Wikman-Coffelt, J., Howard, G. A. & Traut, R. R. (1972). B&him. Biophys. Actu, 277, 671-676. Williamson, R., Morrison, M., Lanyon, G., Eason, R. & Paul, J. (1971). Biochemistry, 10, 3014-3021. Wittmann, H. G., Stoffler, G., Hindennach, I., Kurland, C. G., Randall-Hazelbauer, L., Birge, E. A., Nomura, M., Kaltschmidt, E., Mizushima, S., Traut, R. R. &Bickle,T. A. (1971). Mol. Gen. Genet. 111, 327-333.