Chapter 4
The Ribosomal Proteins of Saccharomyces cerevisiae JONATHAN R. WARNER AND CHARLES GORENSTEIN Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, New York. New York
. . . . A. One-Dimensional Gels . . . . . B. Two-Dimensional Gels . . IV. Conclusion . . . . . . . A. What is a Ribosomal Protein? . . . B. Uniformity of Eukaryotic Ribosomal Proteins . . . . . References I. Preparation of Ribosomes .
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11. Preparation of Ribosomal Proteins 111. Analysis of Ribosomal Proteins .
Note Added in Proof
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While studies on the ribosomal proteins of Escherichia coli have made great strides in the past few years (Stoffler and Wittman, 1977; Nomura, 1976), eukaryotic ribosomal proteins have been relatively neglected. Now that methods for purifying ribosomal proteins from rat liver have been developed (Tsurugi et al., 1976), further work in this area should proceed more rapidly. We have been studying the ribosomal proteins of Succharomyces cerevisiae for several years because we are interested in the coordinated regulation of their synthesis (Gorenstein and Warner, 1976; Warner and Gorenstein, 1977) and because it is possible to obtain mutants which are likely to involve ribosomal proteins (Mortimer and Hawthorne, 1966; Skogerson et al., 1973; Grant et al., 1976). This chapter describes methods of preparing and analyzingthe ribosomal proteins of S. cerevisiae and points out certain properties that may be shared by the ribosomal proteins of all eukaryotes. 45
46
JONATHAN R. WARNER AND CHARLES GORENSTEIN
I. Preparation of Ribosomes A 500-ml culture of S. cerevisiue strain A364A (Hartwell, 1967; Warner, 1971) growing in synthetic medium (Warner, 1971; Sripati and Warner, this volume) at a concentration of 2 to 4 x 107/mlis harvested by centrifugation and washed twice with water. The cells are suspended in 10 ml of TMN (50 mM Tris-acetate, pH 7.0, 50 mMNH,Cl, 12 mMMgCl,, 1 mMdithiothreitol), added to 5 ml of glass beads (0.1 mm, V W R ScientificNo. 34007-088), and shaken in Bronwill homogenizer (VWR Scientific No. 34006-008) for two 30-second bursts. The glass beads are allowed to settle, the supernate removed, and the beads rinsed with 4 ml of TMN. The combined supernatants are centrifuged at 10,OOO g for 10 minutes. The supernatant is r e moved, and the centrifugation repeated one or two times until there is no gross turbidity. Five milliliters of extract is then layered over 4 ml of 10% sucrose in HKB (500 mMKCl,5 mMMgCl,, 20 mMTris-HC1, pH 7.4) in a polycarbonate screw-cap tube (Beckman No. 339574) and centrifuged for 120 minutes at 60,OOO rpm in a Beckman 75 Ti or 65 rotor. The high concentration of KC1 used in the sucrose cushion strips loosely bound proteins from the ribosomes (Sherton and Wool, 1974). The supernatant is carefully sucked off, and the ribosomes are resuspended by stirring in 1.5 ml of TMN. If it is desired to separate subunits, the pellet is suspended in 1.5 ml of HKB and incubated at 30°C for 15 minutes with 1 mMpuromycin (Sherton and Wool, 1974). The sample is layered on 10-25% (w/w) sucrose gradients in HKB and centrifuged at 15°C for 20 hours at 18,000 rpm in the large bucket of a SW 27 rotor. Each tube accommodates about 50-75 A,,, units of ribosomes. Centrifugation at 15" C reduces the dimerization of 40s subunits. The gradients are collected through a recording spectrophotometer, and the 60 and 40s fractions are pooled, diluted with an equal volume of HKB, and centrifuged for 16 hours at 4°C at 50,000 rpm. The pellet is suspended in 1-1.5 ml of TMN. To obtain subunits free of contamination at least two cycles of sucrose gradients are necessary (see Fig. 2).
11. Preparation of Ribosomal Proteins The preparation of ribosomal proteins is essentiallyas developed by Hardy et ul. (1969) for Escherichiu coli. To the ribosome suspension stirring in ice is added 0.1 volume of 1 M MgC1, ,2.5 volumes of glacial acetic acid, and 0.1 volume of 0.1 M dithiothreitol. Eukaryotic ribosomal proteins appear to be
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
47
more susceptible to disulfide-induced aggregation than prokaryotic ribosomal proteins. The acetic acid solubilizes the protein and causes the RNA to precipitate. After 30-60 minutes, the sample is centrifuged at 20,000 g for 10 minutes, and the supernatant carefully removed into Spectrapor No. 3 membrane tubing (Spectrum Medical Industries, Los Angeles, Calif.). This is a low-molecular-weight cutoff tubing. Ribosomal proteins are small, and high osmotic pressure builds up during the dialysis. Using conventional tubing we often suffered appreciable losses. After extensive dialysis against 1% acetic acid, the proteins can be stored at -20°C indefinitely. In some cases it is necessary to prepare ribosomal proteins directly from a whole-cell lysate (Gorenstein and Warner, 1976). If so, the lysate is treated with MgCl,, dithiothreitol, and acetic acid as described above for the ribosome suspension and, after dialysis, subjected to two-dimensional gel electrophoresis (see Section III,B,2).
111. Analysis of Ribosomal Proteins A. One-Dimensional Gels Ribosomal proteins can be analyzed directly on one-dimensional SDS gels without removing RNA. Figure 1 shows such a pattern of 60 and 40s subunits. Cells were labeled for several generations with 15 amino acids uniformly labeled with I4C. Subunits were prepared as described in Section 1. Samples from a sucrose gradient were treated with 10% trichloroacetic acid for 3Ominutes at 0 ° C and collected by centrifugation at 20,000 gfor 15 minutes. A sample was suspended in 0.2 ml of 0.01 M NaPO,, pH 7, 1% SDS, and 1% mercaptoethanol, heated in boiling water for l,minute, and analyzed on a cylindrical gel as described by Maize1 (1969). The gel was fractionated and counted (Warner, 1971). Although a few of the larger proteins of the 60s subunit are resolved, most peaks represent a mixture of proteins (Warner, 1971). To obtain clear a resolution of ribosomal proteins, two-dimensional methods are necessary.
B. Two-Dimensional Gels We have employed two systems of two-dimensional polyacrylamide gel analysis to separate yeast ribosomal proteins. The first is based on the Kaltschmidt-Wittman (1970; Wittman, 1974) system and employs 6 Murea at pH 8.6 in the first dimension and at pH 4.5 in the second dimension. The second is a modified version of the Mets and Bogorad (1974) system and
48
JONATHAN R. WARNER AND CHARLES GORENSTEIN
(u
I
M.W. x IC? I
I
SWLL
I
I
I
SUBUNIT
1
I
1 l
a
J
50
40
30
20
15
10
M.W a I O - ~
FIG. I . One-dimensional analysis of yeast ribosomal proteins. Proteins from ribosomal subunits were analyzed on SDS gels as described in the text. Protein markers in parallel gels provided the molecular-weight scale. Reprinted from Warner (1971) by permission.
employs 8 M urea at pH 5.0 in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension. The two systems are described separately, and the results then compared. For both gel systems the importance of using proteins free of RNA cannot be overemphasized. RNA binds to ribosomal proteins even in urea solutions and causes poor resolution and decreased yields. Parenthetically, several attempts to adapt to ribosomal proteins the O’Farrell ( 1975) two-dimensional system, based on isoelectric focusing,
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
49
have failed because the isoelectric points of most of the ribosomal proteins are higher than the working range of presently available ampholytes. 1. METHOD 1: p H 8.6
pH 4.5
6 M UREA This method is a slightly modified version of the method described by Wittman (1974). The solutions needed are: AND
IN
Solution A: Sample buffer 100 m18 M urea 0.085 gm Na,EDTA 0.32 gm boric acid Store frozen in small aliquots. Add 1% mercaptoethanol just before use. Solution B: First-dimension gel 6.0 gm acrylamide, recrystallized 0.225 gm bisacrylamide, recrystallized 0.8 gm Na,EDTA 3.2 gm boric acid 4.85 gm Tris 10 ml 3% linear polyacrylamide (BDH 29788) 75 m18 M urea Water to 100 ml For polymerization of 10 ml, use 30 pl 10% ammonium persulfate and 15 pl TEMED. A 5 x 100 mm gel requires - 2 ml. Solution C: First-dimension running buffer 2.4 gm Na,EDTA 9.6 gm boric acid 14.55 gm Tris Water to 1000 ml Degas Solution D: Soaking gel after first dimension (1) 2.5 ml concentrated HCl (first soak for 30 minutes) 100 ml 8 M urea (Avital and Elson, 1974) (2) 0.074 ml acetic acid 0.24 ml 5 N KOH (second soak for 30 minutes) 100 m18 M urea
50
JONATHAN R. WARNER AND CHARLES GORENSTEIN
Solution E: Second-dimension gel 180 gm acrylamide 5 gm bisacrylamide 52.3 ml acetic acid 9.6 m15 N KOH 750 m18 M urea Water to 1000 ml Use 80 ml per slab; add 1.32 ml 10% ammonium persulfate and 0.45 ml TEMED. Solution F: Second-dimension running buffer 14 gm glycine 1.5 ml acetic acid Water to 1000 ml Note: All 8 M urea is deionized, filtered, and stored in a cold room. a. First-Dimension Gel. A 5 x 140 mm glass tube is sealed at oneend with Parafilm and taped to the side of a bench. It is filled to 100 mm with degassed solution B to which TEMED and ammonium sulfate have been added, and overlaid with water. After polymerization, which occurs in 30-60 minutes, the Parafilm is removed, and the tube is placed in the apparatus and filled with running buffer containing 300 mg/ml urea. The urea prevents the precipitation of any proteins which migrate upward. The sample (100-300 p g of ribosomal protein) is dissolved in 100 p1 of solution A and layered under the running buffer using microsyringe. For the separation of basic proteins run toward the cathode at 110 V for 20 hours. For the separation of acidic proteins run toward the anode at 60 V for 20 hours. b. Equilibration for the Second Dimension. Remove gels with a syringe containing glycerol. Mark the bottom by inserting a No. 27, f-inch needle. Equilibrate for 30 minutes in solution D 1 with gentle shaking and then for 30 minutes in solution D2. Notes: (1) We found that polymerizing the sample in the middle of the first-dimension gel, as described by Kaltschmidt and Wittmann (1970), gave poor and irreproducible yields of yeast ribosomal protein. Therefore we analyze acidic and basic ribosomal proteins on separate gels. The improved yield and pattern more than compensate for the duplicate sample required. (2) The linear polyacrylamide in the first dimension provides strength and dimensional stability to the gel during the several steps in which it must be handled. (3) Touch the first-dimensional gel only with gloves during equilibration. An electrophoresed fingerprint can complicate the analysis of the gel.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
51
c. Second-Dimension Gel. The apparatus for the second-dimension gel, shown in Studier (1973), has been modified as described in Stewart and Crouch (1977). The separation gel is 140 mm wide, 130 mm high, and 3 mm thick. One glass plate is beveled, and the first-dimension gel is placed in the bevel between the two plates. One hundred milliliters of solution E, containing TEh4ED and ammonium persulfate, is degassed and poured around and finally over the first-dimension gel. The apparatus can be tilted to avoid the formation of bubbles at the seal between the two gels. A good seal between the gels is essential to keep the spots from spreading. The gel is overlaid with water and allowed to polymerize. The second-dimension running buffer, solution F, is placed in both wells, and electrophoresis toward the cathode carried out for 20 hours at 85 V. The gels are stained in 0.2% coomassie brilliant blue in 50%methanol for 4-6 hours and destained in 30% methanol containing a mesh bag ofwashed charcoal (Sherton and Wool, 1974). The gels are stored in plastic bags (No. F-13178, Be1 Art Products, Pequannock, N.J.), sealed with a T-bar plastic sealer (Harwill Company, Santa Monica, Calif.), and keep indefinitely. d. Results ofMethod 1. The results of the Wittman gel analysisof yeast ribosomal proteins are described in Zinker and Warner (1976), from which Figs. 2-4 have been extracted. Similar but not identical patterns have been presented by Kruiswijk and Planta (1974) and by Ishiguro (1976). There are
FIG.2. Separation of basic yeast ribosomal proteins by method 1 (pH 8.6 and pH 4.5). Proteins of 60 and 40s subunits were subjected to two-dimensional electrophoresis as described for basic proteins, and the gels stained. A small amount of cross-contamination permits alignment of the two gels; e.g., L1 and L2 can be seen faintly above S1; S3 and S4 can be seen faintly above L8. Reprinted from Zinker and Warner (1976) by permission.
H
n6-e
BASIC PROTEINS
I"
60s
y
-6 8
8
BASIC PROTEINS
40s
Yy
yi
FIG. 3. Numbering system of yeast basic ribosomal proteins separated by method 1 . Compare with Fig. 2. Reprinted from Zinker and Warner (1976) by permission.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
53
FIG.4. Separation of acidic yeast ribosomal proteins by method 1 (pH 8.6 and pH 4.5). Total ribosomal proteins were subjected to two-dimensional electrophoresis as described for acidic proteins, and the gels stained. The attribution of proteins to individual subunits was determined from the analysis of separated subunits. Proteins L34 and S25 are sometimes seen in the upper right corner which, however, is where most contaminating proteins run. Reprinted from Zinker and Warner (1976) by permission.
sufficient differences that a compatible numbering system must await further comparisons. As is the case with bacteria (Wittman, 1974)andmammalian cells(Sherton and Wool, 1972), nearly all yeast ribosomal proteins are very basic, migrating toward the cathode at pH 8.6. A comparison between yeast and mammalian ribosomal proteins reveals that: 1. The 60s subunits of all eukaryotes have two proteins larger than all the rest. This is particularly clear in one-dimensional SDS gels, e.g., Fig. 1. It is likely therefore that proteins L1 and L2 in Fig. 1 correspond to proteins L3 and LA of rat liver (Sherton and Wool, 1972). 2. Proteins S5 and S6 are two isomers of aphosphorylated protein whose unphosphorylated form is probably S7 (Zinker and Warner, 1976). Because of their comigration in a Wittman gel and in a gel containing SDS (see Section III.B.2) these proteins are undoubtedly the functional counterparts of the phosphorylated protein, S6, of rat liver (Gressner and Wool, 1974). 3. Proteins L35 and L36 are very acidic proteins, which are also phosphorylated (Zinker and Warner, 1976). They appear to be analagous to LAO and L41 of rat liver, which may have a functional relationship with proteins L7 and L12 of E. coli (Wool and Stoffler, 1974). It remains to be seen whether or not L35 and L36 have the same amino acid sequence as L7 and L12.
54
JONATHAN R. WARNER AND CHARLES GORENSTEIN
2. METHOD2: pH 5.0 IN 8 M UREAAND SDS This method is a modification (Gorenstein and Warner, 1976) of the method of Mets and Bogorad (1974). Briefly, in the first dimension the proteins are separated based on their charge at pH 5.0. In the second dimension they are separated on the basis of their molecular weight in the presence of SDS. a . First Dimension Solution A: First-dimension gel mix 4% (w/v) acrylamide 0.1% (w/v) bisacrylamide 8 M urea 0.057 M bis Tris adjusted to pH 5.0 with acetic acid Prior to polymerization this solution is degassed and 3 p1 of 10% ammonium persulfate and 1 p1 of TEMED are added per milliliter of solution A. Solution B: Upper electrode solution 0.01 M bis Tris adjusted to pH 4.0 with acetic acid Solution C: Lower electrode solution 0.179 M potassium acetate adjusted to pH 5.0 with acetic acid Solution D: First-dimension sample buffer 10% (v/ v) p -Mercaptoet hanol 10% (v/v) glycerol 1% (v/v) acetic acid 0.01% (w/v) basic fuschin 8 M urea This solution is stored in aliquots at -20°C. Solution E: equilibration buffer 0.5 M Tris, 1% SDS, adjusted to pH 6.8 with HCl b. Second Dimension Solution F: Resolving gel 17% (w/v) acrylamide 0.45% (w/v) bisacrylamide 0.1% (w/v) SDS 0.175 M Tris adjusted to pH 8.8 with HCl If the gel is to be dried for autoradiography, inclusion of 0.3%linear polyacrylamide in solution F reduces the tendency of the gels to crack during drying. Prior to polymerization this solution is degassed, and 6 pl of 10%ammonium persulfate and 0.5 pl of TEMED are added per milliliter of solution F.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
55
Solution G: Stacking gel 4% (w/v) acrylamide 0.11% (w/v) bisacrylamide 0.1% (w/v) SDS 0.0625 M Tris adjusted to pH 6.8 with HCl The gel is degassed and polymerized with 10 pl 10%ammonium persulfate and 0.5 pl TEMED per milliliter of solution G. Solution H: Electrode buffer 0.05 M Tris 0.38 M glycine 0.05% (w/v) SDS c. First Dimension. Six-millimeter (I.D.) 150-mm glass tubes, washed with chromic acid, methanolic KOH, and 1% Siliclad (Beckman Instruments), are sealed at the bottom with Parafilm, filled to 110 mm with gel mix solution A, and overlayered with water. The gels are allowed to polymerize for 1 hour. The gel tubes are placed in the apparatus, and the upperreservoir filled with electrode solution B and the lower reservoir with electrode solution C. The sample, consisting of up to 400 pg of lyophilized protein is dissolved in 100 pl of sample buffer D. Occasionally difficulties are encountered in solubilizing total yeast extracts. They can be overcome by increasing the acetic acid concentration in sample buffer D to 10%(w/v) without affecting the resolution of the gel. The sample is layered on the surface of the gel with a microsyringe. The gels are electrophoresed toward the cathode at constant voltage for 900 volt-hours or until the tracking dye, basic fuschin, has reached the bottom of the gel. d. Equilibration. The first-dimension gels are removed from the glass tubes by rimming them with a 0.1% SDS solution. Each gel is then placed in a 125-1111 Erlenmeyer flask containing 25 ml of solution E and shaken for 45 minutes. This procedure changes the pH of the first-dimensional gel to that of the second-dimension stacking gel and introduces the detergent
SDS.
e. Second Dimension. A 100 x 3 mm second-dimension slab gel is polymerized between two glass plates in the apparatus described in Section III,B,l. Solution F is added and overlayered with 0.1% SDS, using an atomizer. After the gel has polymerized, the excess unpolymerized material is removed and the equilibrated first-dimension gel is laid across the apparatus, resting between the beveled edge and the straight glass plate. The cylindrical gel is cemented into place by means of the stacking gel solution G. Care
56
JONATHAN R. WARNER AND CHARLES GORENSTEIN
must be taken to see that no bubbles are trapped under the cylindrical gel. If a reference sample or a tracking protein is to be used, a well can be made by placing a small piece of Plexiglas near one edge of the apparatus and allowing the stacking gel to polymerize around it. After polymerization, the reservoirs are filled with electrode buffer, and electrophoresis is conducted at constant voltage toward the anode for 1100 volt-hours or until a cytochrome-c marker is within 1 cm of the bottom of the gel. The gels are stained, destained, and stored as described in Section III,B, 1. f: Quantitation. The amount of radioactively labeled protein in a spot can be quantitated from an autoradiograph by comparing the intensity of the protein spot with a series of known standards. A more accurate means of measuring the amount of radioactively labeled protein is to excise the spot from the gel and count the radioactivity in ascintillation counter (Gorenstein and Warner, 1976). Capillary stainless-steel tubing (Small Parts, Inc.), sharpened at one end, make very convenient borers. The excised spots are dried in glass scintillation vials at 60°C for 2 hours; then 0.5 ml of freshly made 30% H,O, (made by diluting 50% H,O,) is added. The vials are tightly capped and incubated at 60°C overnight. After cooling, 10 ml of Aquasol (New England Nuclear) or Readisolv G P (Beckman Instruments) is added. Because of the large amount of peroxide present, problems with fluorescence may be encountered. We avoid this problem by storing the vials in a cool, dark place for several hours before counting in a refrigerated counter. g . Results of Method 2. The analysis of yeast ribosomal proteins by method 2 is shown in Fig. 5. Most ofthe proteins have been attributed to one of the two subunits (Table I). In only a few cases has it been possible to cross-identify proteins in the two systems: 1. Because of their size, proteins 1 and 2 are likely to L1 and C2. The identification of L2 with protein 2 is confirmed by the finding that both are methylated (Cannon et al., 1977; D. Barton, unpublished). 2. Protein 9 is the unphosphorylated form of the 40s protein S5, S6, S7 (P2 of Zinker and Warner, 1976). The phosphorylated form runs slightly to the left of protein 9. 3. Protein 14 is the phosphorylated protein S27 (P3 of Zinker and Warner, 1976). 4. Protein 16 is the exchangeable 60s protein L7 (E2 of Zinker and Warner, 1976). Although method 1 probably gives better resolution of more proteins, method 2 has three distinct advantages: 1. It is more reliable. 2. It gives better separation of marginally basic ribosomal proteins, e.g., protein 14.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
57
FIG.5. Separation of yeast ribosomal proteins by method 2 (pH 5.0 SDS). Total ribosomal proteins were subjected to two-dimensional electrophoresis as described, and the gels stained. Reprinted from Gorenstein and Warner (1976) by permission.
3. Proteins are more soluble in its sample buffer. Therefore method 2 is preferable when isolating ribosomal proteins from a total cell extract (Gorenstein and Warner, 1976).
IV. Conclusion A. What is a Ribosomal Protein? There are no covalent bonds between the components of a ribosome. Since ribosomes are well known to bind many proteins non- or quasispecifically, e.g., RNase of E . coli (Waller, 1964) and initiation factors in both prokaryotes and eukaryotes (Weissbach and Ochoa, 1976), it has been a
58
JONATHAN R. WARNER A N D CHARLES GORENSTEIN
TABLE I ASSIGNMENT TO SUBUNIT OF PROTEINS BY METHODFIG. Sa SEPARATED
1 2 3 6 8
10 11 15 16 18
22 23 24 25 27 28 29 31 32 33
38 39 44 45 47 48 49 51 57 58
61 62 64 65
5 9 12 13 14 19
4
37 0 41 42 50 52
20
55
21 30 36
60 61 63
uprotein 61 is found in both subunits. Whether this represents two unresolved proteins or a single protein that can be purified with either subunit is unclear at present.
continuous problem to determine which proteins should be considered truly ribosomal. The question is complicated by the fact that the salt washes necessary to remove contaminating proteins often remove some ribosomal proteins (Hardy, 1975). Clearly certain proteins in Figs. 2 and 5 aremuch less distinct than others; it is unlikely that the variation is due only to different affinities for the coomassie stain. Furthermore, since only a small portion of the ribosomes is active in most in vitro systems, a functional assay is seldom feasible. Thus, while it is likely that most of the spots in Figs. 2-5 are ribosomal proteins, a few may not be. To distinguish between the two it may be useful to develop criteria based on the assembly of ribosomal particles or on the regulation of the synthesis of these components, as we have proposed for mammalian cells (Warner, 1966) and for S . cerevisiae (Gorenstein and Warner, 1976).
B. Uniformity of Eukaryotic Ribosomal Proteins From an evolutionary point of view ribosomal proteins appear to be very stable, as first demonstrated by Nomuraet af. (1968) when they reconstituted active 30s subunits from 16s RNA of E . cofi and ribosomal proteins of Baciffisstearothermophifus. It is not surprising therefore that the ribosomal proteins of all mammals appear to be very similar (Delaunay et af., 1973; S. Morgan and J. R. Warner, unpublished).
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
59
Nevertheless, it is interesting to note that the two phosphorylated yeast proteins revealed on a pH 5 SDS gel, proteins 9 and 14, migrate in positions essentially identical to those of the two phosphorylated proteins of hamster 40s ribosomes (Schubart et al., 1977). Furthermore, the three exchangeable proteins of yeast (Warner and Udem, 1972) migrate in positions essentially identical to those of the three exchangeable proteins of HeLa (Warner, 1966) on a pH 5 SDS gel. We suggest that for most of the ribosomal proteins there is likely to be a 1:1 correlation between eukaryotes as distant as yeast and humans. ACKNOWLEDGMENTS Work from this laboratory was supported by grants from the American Cancer Society No. NP 72 G, the National Science Foundation No. PCM 7503938 and the National Institutes of Health No. P 30 CA 13330. J.R.W. was a Faculty Career Awardee of the American Cancer Society.
REFERENCES Avital, S., and Elson, D. (1974). Anal. Biochem. 57, 287-292. Cannon, M., Schindler, D., and Davies, J. (1977). FEBS Lett. 75, 187-191. Delaunay, J., Creusot, F., and Schapira, G. (1973). Eur. J. Biochem. 39, 305-312. Gorenstein, C., and Warner, J. R. (1976). Proc. Nutl. Acad. Sci. U.S.A. 73, 1547-1551. Grant, P., Schindler, D., and Davies, J. (1976). Geneticr 83, 667-673. Gressner, A. M., and Wool, I. G. (1974). J. Biol. Chem. 249, 6917-6925. Hardy, S. J. (1975). Mol. Gen. Genet. 140, 253-274. Hardy, S. J., Kurland, C. G., Voynow, P., and Mora, G. (1969). Biochemistry 8, 2897-2905. Hartwell, L. (1967). J . Bucteriol. 93, 1662-1670. Ishiguro, J. (1976). Mol. Gen. Genet. 145, 73-79. Kaltschmidt, E., and Wittman, H. G. (1970). Anal. Biochem. 36,401407. Kruiswijk, T., and Planta, R. J. (1974). Mol. Biol. Rep. 1,409414. Maizel, J. V., Jr. (1969). In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 334-357. Academic Press, New York. Mets, L., and Bogorad, L. (1974). Anal. Biochem. 57, 200-207. Mortimer, R., and Hawthorne, D. (1966). Genetics 53, 165-173. Nomura, M.;Traub, P., and Bechmann, H. (1968). Nuture (London) 219, 793-795. OFarrell, P. H. (1975). J. Biol. Chem. 250,4007-4013. Schubart, U.K., Shapiro, S., Fleischer, N., and Rosen, 0. M. (1977). J . Biol. Chem. 252, 92-101. Sherton, C., and Wool, I. G. (1972). J. Biol. Chem. 4460-4467. Sherton, C., and Wool, I. G. (1974). In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 30, pp. 506-526. Academic Press, New York. Skogerson, L., McLaughlin, C., and Wakatama, E. (1973). J . Bucteriol. 116, 818-822. Stewart, M., and Crouch, R. (1977). Anal. Biochem. (in press). Stoffer, G., and Wittman, H. G. (1977). I n “Molecular Mechanisms of Protein Biosynthesis” H. Weissbod and S. Pestka, eds.), pp. 117-202. Academic Press, New York. Studier, F. W. (1973). J. Mol. Biol. 79, 237-244. Tsurugi, K., Collatz, E., Wool, I. G., and Lin, A. (1976). J. Biol. Chem. 251, 7940-7946. Waller, J. P. (1964). J. Mol. Biol. 10, 319-336.
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Warner, J. R. (1966). J. Mol. Biol. 19, 383-398. Warner, J. R. (1971). J. Biol. Chem. 246,447454. Warner, J. R., and Gorenstein, C. (1977). Cell 11 (201-212). Weissbach, H., and Ochoa, S. (1976). Annu. Rev. Biochem. 45, 191-216. Wittman, H. G. (1974). In “Methods in Enzymology” (L. Grossman and R. Moldave, eds.), Vol. 30, pp. 497-518. Academic Press, New York. Wool, I . G., and Stofller, G. (1974). In “Ribosomes” (M. Nomura, A. Tissieres, and P. Lengyel, eds.), p. 417. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Zinker, S., and Warner, J. R. (1976). J. Biol. G e m . 251, 1799-1807.
NOTEADDEDIN PROOF Using as a third dimension the analysis of proteolytic digests of proteins separated on 2D gels (Cleveland, D., Fischer, S., Kirschner, M., and Laemmli, U. (1977). J. Biol. Chem. 252, 1102-1 106), P. J. Wejksnora has cross-identified a number of ribosomal proteins as separated by the two 2D gel methods (see Table 11). These results demonstrate the identity of the phosphorylated proteins S5 and S7 with No. 9. Also No. 61 is shown to consist of two proteins, one from each subunit. TABLE I1 CROSSIDENTIFICATION OF PROTEINS AS SEPARATED BY METHODSI AND 2
System 1 SI
s2
s3 s4 s5 s7 S8 s9
SIO
s12 S13 S14 S16
s20
s21 s22 S27 S28
System 2 12 5 30 40 9 9 21
19 55 41 42 52 50 61?a 37 45 14 13
System I LI L2 L3 L4 L6 L8 L11 L13 L16 L17 L18 L20 L2 1 L24
System 2 2 1
6 II 8 25 16 18?“ 15?”
22 33 62? “ 31?” 29? “
“Protein pairs marked (?) are likely matches, but not confirmed unquestionably by either method.
The Ribosomal Proteins of Saccharomyces cerevisiae JONATHAN R. WARNER AND CHARLES GORENSTEIN Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, New York. New York
. . . . A. One-Dimensional Gels . . . . . B. Two-Dimensional Gels . . IV. Conclusion . . . . . . . A. What is a Ribosomal Protein? . . . B. Uniformity of Eukaryotic Ribosomal Proteins . . . . . References I. Preparation of Ribosomes .
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11. Preparation of Ribosomal Proteins 111. Analysis of Ribosomal Proteins .
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47 47 47 57 57 58 59
60
While studies on the ribosomal proteins of Escherichia coli have made great strides in the past few years (Stoffler and Wittman, 1977; Nomura, 1976), eukaryotic ribosomal proteins have been relatively neglected. Now that methods for purifying ribosomal proteins from rat liver have been developed (Tsurugi et al., 1976), further work in this area should proceed more rapidly. We have been studying the ribosomal proteins of Succharomyces cerevisiae for several years because we are interested in the coordinated regulation of their synthesis (Gorenstein and Warner, 1976; Warner and Gorenstein, 1977) and because it is possible to obtain mutants which are likely to involve ribosomal proteins (Mortimer and Hawthorne, 1966; Skogerson et al., 1973; Grant et al., 1976). This chapter describes methods of preparing and analyzingthe ribosomal proteins of S. cerevisiae and points out certain properties that may be shared by the ribosomal proteins of all eukaryotes. 45
46
JONATHAN R. WARNER AND CHARLES GORENSTEIN
I. Preparation of Ribosomes A 500-ml culture of S. cerevisiue strain A364A (Hartwell, 1967; Warner, 1971) growing in synthetic medium (Warner, 1971; Sripati and Warner, this volume) at a concentration of 2 to 4 x 107/mlis harvested by centrifugation and washed twice with water. The cells are suspended in 10 ml of TMN (50 mM Tris-acetate, pH 7.0, 50 mMNH,Cl, 12 mMMgCl,, 1 mMdithiothreitol), added to 5 ml of glass beads (0.1 mm, V W R ScientificNo. 34007-088), and shaken in Bronwill homogenizer (VWR Scientific No. 34006-008) for two 30-second bursts. The glass beads are allowed to settle, the supernate removed, and the beads rinsed with 4 ml of TMN. The combined supernatants are centrifuged at 10,OOO g for 10 minutes. The supernatant is r e moved, and the centrifugation repeated one or two times until there is no gross turbidity. Five milliliters of extract is then layered over 4 ml of 10% sucrose in HKB (500 mMKCl,5 mMMgCl,, 20 mMTris-HC1, pH 7.4) in a polycarbonate screw-cap tube (Beckman No. 339574) and centrifuged for 120 minutes at 60,OOO rpm in a Beckman 75 Ti or 65 rotor. The high concentration of KC1 used in the sucrose cushion strips loosely bound proteins from the ribosomes (Sherton and Wool, 1974). The supernatant is carefully sucked off, and the ribosomes are resuspended by stirring in 1.5 ml of TMN. If it is desired to separate subunits, the pellet is suspended in 1.5 ml of HKB and incubated at 30°C for 15 minutes with 1 mMpuromycin (Sherton and Wool, 1974). The sample is layered on 10-25% (w/w) sucrose gradients in HKB and centrifuged at 15°C for 20 hours at 18,000 rpm in the large bucket of a SW 27 rotor. Each tube accommodates about 50-75 A,,, units of ribosomes. Centrifugation at 15" C reduces the dimerization of 40s subunits. The gradients are collected through a recording spectrophotometer, and the 60 and 40s fractions are pooled, diluted with an equal volume of HKB, and centrifuged for 16 hours at 4°C at 50,000 rpm. The pellet is suspended in 1-1.5 ml of TMN. To obtain subunits free of contamination at least two cycles of sucrose gradients are necessary (see Fig. 2).
11. Preparation of Ribosomal Proteins The preparation of ribosomal proteins is essentiallyas developed by Hardy et ul. (1969) for Escherichiu coli. To the ribosome suspension stirring in ice is added 0.1 volume of 1 M MgC1, ,2.5 volumes of glacial acetic acid, and 0.1 volume of 0.1 M dithiothreitol. Eukaryotic ribosomal proteins appear to be
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
47
more susceptible to disulfide-induced aggregation than prokaryotic ribosomal proteins. The acetic acid solubilizes the protein and causes the RNA to precipitate. After 30-60 minutes, the sample is centrifuged at 20,000 g for 10 minutes, and the supernatant carefully removed into Spectrapor No. 3 membrane tubing (Spectrum Medical Industries, Los Angeles, Calif.). This is a low-molecular-weight cutoff tubing. Ribosomal proteins are small, and high osmotic pressure builds up during the dialysis. Using conventional tubing we often suffered appreciable losses. After extensive dialysis against 1% acetic acid, the proteins can be stored at -20°C indefinitely. In some cases it is necessary to prepare ribosomal proteins directly from a whole-cell lysate (Gorenstein and Warner, 1976). If so, the lysate is treated with MgCl,, dithiothreitol, and acetic acid as described above for the ribosome suspension and, after dialysis, subjected to two-dimensional gel electrophoresis (see Section III,B,2).
111. Analysis of Ribosomal Proteins A. One-Dimensional Gels Ribosomal proteins can be analyzed directly on one-dimensional SDS gels without removing RNA. Figure 1 shows such a pattern of 60 and 40s subunits. Cells were labeled for several generations with 15 amino acids uniformly labeled with I4C. Subunits were prepared as described in Section 1. Samples from a sucrose gradient were treated with 10% trichloroacetic acid for 3Ominutes at 0 ° C and collected by centrifugation at 20,000 gfor 15 minutes. A sample was suspended in 0.2 ml of 0.01 M NaPO,, pH 7, 1% SDS, and 1% mercaptoethanol, heated in boiling water for l,minute, and analyzed on a cylindrical gel as described by Maize1 (1969). The gel was fractionated and counted (Warner, 1971). Although a few of the larger proteins of the 60s subunit are resolved, most peaks represent a mixture of proteins (Warner, 1971). To obtain clear a resolution of ribosomal proteins, two-dimensional methods are necessary.
B. Two-Dimensional Gels We have employed two systems of two-dimensional polyacrylamide gel analysis to separate yeast ribosomal proteins. The first is based on the Kaltschmidt-Wittman (1970; Wittman, 1974) system and employs 6 Murea at pH 8.6 in the first dimension and at pH 4.5 in the second dimension. The second is a modified version of the Mets and Bogorad (1974) system and
48
JONATHAN R. WARNER AND CHARLES GORENSTEIN
(u
I
M.W. x IC? I
I
SWLL
I
I
I
SUBUNIT
1
I
1 l
a
J
50
40
30
20
15
10
M.W a I O - ~
FIG. I . One-dimensional analysis of yeast ribosomal proteins. Proteins from ribosomal subunits were analyzed on SDS gels as described in the text. Protein markers in parallel gels provided the molecular-weight scale. Reprinted from Warner (1971) by permission.
employs 8 M urea at pH 5.0 in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension. The two systems are described separately, and the results then compared. For both gel systems the importance of using proteins free of RNA cannot be overemphasized. RNA binds to ribosomal proteins even in urea solutions and causes poor resolution and decreased yields. Parenthetically, several attempts to adapt to ribosomal proteins the O’Farrell ( 1975) two-dimensional system, based on isoelectric focusing,
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
49
have failed because the isoelectric points of most of the ribosomal proteins are higher than the working range of presently available ampholytes. 1. METHOD 1: p H 8.6
pH 4.5
6 M UREA This method is a slightly modified version of the method described by Wittman (1974). The solutions needed are: AND
IN
Solution A: Sample buffer 100 m18 M urea 0.085 gm Na,EDTA 0.32 gm boric acid Store frozen in small aliquots. Add 1% mercaptoethanol just before use. Solution B: First-dimension gel 6.0 gm acrylamide, recrystallized 0.225 gm bisacrylamide, recrystallized 0.8 gm Na,EDTA 3.2 gm boric acid 4.85 gm Tris 10 ml 3% linear polyacrylamide (BDH 29788) 75 m18 M urea Water to 100 ml For polymerization of 10 ml, use 30 pl 10% ammonium persulfate and 15 pl TEMED. A 5 x 100 mm gel requires - 2 ml. Solution C: First-dimension running buffer 2.4 gm Na,EDTA 9.6 gm boric acid 14.55 gm Tris Water to 1000 ml Degas Solution D: Soaking gel after first dimension (1) 2.5 ml concentrated HCl (first soak for 30 minutes) 100 ml 8 M urea (Avital and Elson, 1974) (2) 0.074 ml acetic acid 0.24 ml 5 N KOH (second soak for 30 minutes) 100 m18 M urea
50
JONATHAN R. WARNER AND CHARLES GORENSTEIN
Solution E: Second-dimension gel 180 gm acrylamide 5 gm bisacrylamide 52.3 ml acetic acid 9.6 m15 N KOH 750 m18 M urea Water to 1000 ml Use 80 ml per slab; add 1.32 ml 10% ammonium persulfate and 0.45 ml TEMED. Solution F: Second-dimension running buffer 14 gm glycine 1.5 ml acetic acid Water to 1000 ml Note: All 8 M urea is deionized, filtered, and stored in a cold room. a. First-Dimension Gel. A 5 x 140 mm glass tube is sealed at oneend with Parafilm and taped to the side of a bench. It is filled to 100 mm with degassed solution B to which TEMED and ammonium sulfate have been added, and overlaid with water. After polymerization, which occurs in 30-60 minutes, the Parafilm is removed, and the tube is placed in the apparatus and filled with running buffer containing 300 mg/ml urea. The urea prevents the precipitation of any proteins which migrate upward. The sample (100-300 p g of ribosomal protein) is dissolved in 100 p1 of solution A and layered under the running buffer using microsyringe. For the separation of basic proteins run toward the cathode at 110 V for 20 hours. For the separation of acidic proteins run toward the anode at 60 V for 20 hours. b. Equilibration for the Second Dimension. Remove gels with a syringe containing glycerol. Mark the bottom by inserting a No. 27, f-inch needle. Equilibrate for 30 minutes in solution D 1 with gentle shaking and then for 30 minutes in solution D2. Notes: (1) We found that polymerizing the sample in the middle of the first-dimension gel, as described by Kaltschmidt and Wittmann (1970), gave poor and irreproducible yields of yeast ribosomal protein. Therefore we analyze acidic and basic ribosomal proteins on separate gels. The improved yield and pattern more than compensate for the duplicate sample required. (2) The linear polyacrylamide in the first dimension provides strength and dimensional stability to the gel during the several steps in which it must be handled. (3) Touch the first-dimensional gel only with gloves during equilibration. An electrophoresed fingerprint can complicate the analysis of the gel.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
51
c. Second-Dimension Gel. The apparatus for the second-dimension gel, shown in Studier (1973), has been modified as described in Stewart and Crouch (1977). The separation gel is 140 mm wide, 130 mm high, and 3 mm thick. One glass plate is beveled, and the first-dimension gel is placed in the bevel between the two plates. One hundred milliliters of solution E, containing TEh4ED and ammonium persulfate, is degassed and poured around and finally over the first-dimension gel. The apparatus can be tilted to avoid the formation of bubbles at the seal between the two gels. A good seal between the gels is essential to keep the spots from spreading. The gel is overlaid with water and allowed to polymerize. The second-dimension running buffer, solution F, is placed in both wells, and electrophoresis toward the cathode carried out for 20 hours at 85 V. The gels are stained in 0.2% coomassie brilliant blue in 50%methanol for 4-6 hours and destained in 30% methanol containing a mesh bag ofwashed charcoal (Sherton and Wool, 1974). The gels are stored in plastic bags (No. F-13178, Be1 Art Products, Pequannock, N.J.), sealed with a T-bar plastic sealer (Harwill Company, Santa Monica, Calif.), and keep indefinitely. d. Results ofMethod 1. The results of the Wittman gel analysisof yeast ribosomal proteins are described in Zinker and Warner (1976), from which Figs. 2-4 have been extracted. Similar but not identical patterns have been presented by Kruiswijk and Planta (1974) and by Ishiguro (1976). There are
FIG.2. Separation of basic yeast ribosomal proteins by method 1 (pH 8.6 and pH 4.5). Proteins of 60 and 40s subunits were subjected to two-dimensional electrophoresis as described for basic proteins, and the gels stained. A small amount of cross-contamination permits alignment of the two gels; e.g., L1 and L2 can be seen faintly above S1; S3 and S4 can be seen faintly above L8. Reprinted from Zinker and Warner (1976) by permission.
H
n6-e
BASIC PROTEINS
I"
60s
y
-6 8
8
BASIC PROTEINS
40s
Yy
yi
FIG. 3. Numbering system of yeast basic ribosomal proteins separated by method 1 . Compare with Fig. 2. Reprinted from Zinker and Warner (1976) by permission.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
53
FIG.4. Separation of acidic yeast ribosomal proteins by method 1 (pH 8.6 and pH 4.5). Total ribosomal proteins were subjected to two-dimensional electrophoresis as described for acidic proteins, and the gels stained. The attribution of proteins to individual subunits was determined from the analysis of separated subunits. Proteins L34 and S25 are sometimes seen in the upper right corner which, however, is where most contaminating proteins run. Reprinted from Zinker and Warner (1976) by permission.
sufficient differences that a compatible numbering system must await further comparisons. As is the case with bacteria (Wittman, 1974)andmammalian cells(Sherton and Wool, 1972), nearly all yeast ribosomal proteins are very basic, migrating toward the cathode at pH 8.6. A comparison between yeast and mammalian ribosomal proteins reveals that: 1. The 60s subunits of all eukaryotes have two proteins larger than all the rest. This is particularly clear in one-dimensional SDS gels, e.g., Fig. 1. It is likely therefore that proteins L1 and L2 in Fig. 1 correspond to proteins L3 and LA of rat liver (Sherton and Wool, 1972). 2. Proteins S5 and S6 are two isomers of aphosphorylated protein whose unphosphorylated form is probably S7 (Zinker and Warner, 1976). Because of their comigration in a Wittman gel and in a gel containing SDS (see Section III.B.2) these proteins are undoubtedly the functional counterparts of the phosphorylated protein, S6, of rat liver (Gressner and Wool, 1974). 3. Proteins L35 and L36 are very acidic proteins, which are also phosphorylated (Zinker and Warner, 1976). They appear to be analagous to LAO and L41 of rat liver, which may have a functional relationship with proteins L7 and L12 of E. coli (Wool and Stoffler, 1974). It remains to be seen whether or not L35 and L36 have the same amino acid sequence as L7 and L12.
54
JONATHAN R. WARNER AND CHARLES GORENSTEIN
2. METHOD2: pH 5.0 IN 8 M UREAAND SDS This method is a modification (Gorenstein and Warner, 1976) of the method of Mets and Bogorad (1974). Briefly, in the first dimension the proteins are separated based on their charge at pH 5.0. In the second dimension they are separated on the basis of their molecular weight in the presence of SDS. a . First Dimension Solution A: First-dimension gel mix 4% (w/v) acrylamide 0.1% (w/v) bisacrylamide 8 M urea 0.057 M bis Tris adjusted to pH 5.0 with acetic acid Prior to polymerization this solution is degassed and 3 p1 of 10% ammonium persulfate and 1 p1 of TEMED are added per milliliter of solution A. Solution B: Upper electrode solution 0.01 M bis Tris adjusted to pH 4.0 with acetic acid Solution C: Lower electrode solution 0.179 M potassium acetate adjusted to pH 5.0 with acetic acid Solution D: First-dimension sample buffer 10% (v/ v) p -Mercaptoet hanol 10% (v/v) glycerol 1% (v/v) acetic acid 0.01% (w/v) basic fuschin 8 M urea This solution is stored in aliquots at -20°C. Solution E: equilibration buffer 0.5 M Tris, 1% SDS, adjusted to pH 6.8 with HCl b. Second Dimension Solution F: Resolving gel 17% (w/v) acrylamide 0.45% (w/v) bisacrylamide 0.1% (w/v) SDS 0.175 M Tris adjusted to pH 8.8 with HCl If the gel is to be dried for autoradiography, inclusion of 0.3%linear polyacrylamide in solution F reduces the tendency of the gels to crack during drying. Prior to polymerization this solution is degassed, and 6 pl of 10%ammonium persulfate and 0.5 pl of TEMED are added per milliliter of solution F.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
55
Solution G: Stacking gel 4% (w/v) acrylamide 0.11% (w/v) bisacrylamide 0.1% (w/v) SDS 0.0625 M Tris adjusted to pH 6.8 with HCl The gel is degassed and polymerized with 10 pl 10%ammonium persulfate and 0.5 pl TEMED per milliliter of solution G. Solution H: Electrode buffer 0.05 M Tris 0.38 M glycine 0.05% (w/v) SDS c. First Dimension. Six-millimeter (I.D.) 150-mm glass tubes, washed with chromic acid, methanolic KOH, and 1% Siliclad (Beckman Instruments), are sealed at the bottom with Parafilm, filled to 110 mm with gel mix solution A, and overlayered with water. The gels are allowed to polymerize for 1 hour. The gel tubes are placed in the apparatus, and the upperreservoir filled with electrode solution B and the lower reservoir with electrode solution C. The sample, consisting of up to 400 pg of lyophilized protein is dissolved in 100 pl of sample buffer D. Occasionally difficulties are encountered in solubilizing total yeast extracts. They can be overcome by increasing the acetic acid concentration in sample buffer D to 10%(w/v) without affecting the resolution of the gel. The sample is layered on the surface of the gel with a microsyringe. The gels are electrophoresed toward the cathode at constant voltage for 900 volt-hours or until the tracking dye, basic fuschin, has reached the bottom of the gel. d. Equilibration. The first-dimension gels are removed from the glass tubes by rimming them with a 0.1% SDS solution. Each gel is then placed in a 125-1111 Erlenmeyer flask containing 25 ml of solution E and shaken for 45 minutes. This procedure changes the pH of the first-dimensional gel to that of the second-dimension stacking gel and introduces the detergent
SDS.
e. Second Dimension. A 100 x 3 mm second-dimension slab gel is polymerized between two glass plates in the apparatus described in Section III,B,l. Solution F is added and overlayered with 0.1% SDS, using an atomizer. After the gel has polymerized, the excess unpolymerized material is removed and the equilibrated first-dimension gel is laid across the apparatus, resting between the beveled edge and the straight glass plate. The cylindrical gel is cemented into place by means of the stacking gel solution G. Care
56
JONATHAN R. WARNER AND CHARLES GORENSTEIN
must be taken to see that no bubbles are trapped under the cylindrical gel. If a reference sample or a tracking protein is to be used, a well can be made by placing a small piece of Plexiglas near one edge of the apparatus and allowing the stacking gel to polymerize around it. After polymerization, the reservoirs are filled with electrode buffer, and electrophoresis is conducted at constant voltage toward the anode for 1100 volt-hours or until a cytochrome-c marker is within 1 cm of the bottom of the gel. The gels are stained, destained, and stored as described in Section III,B, 1. f: Quantitation. The amount of radioactively labeled protein in a spot can be quantitated from an autoradiograph by comparing the intensity of the protein spot with a series of known standards. A more accurate means of measuring the amount of radioactively labeled protein is to excise the spot from the gel and count the radioactivity in ascintillation counter (Gorenstein and Warner, 1976). Capillary stainless-steel tubing (Small Parts, Inc.), sharpened at one end, make very convenient borers. The excised spots are dried in glass scintillation vials at 60°C for 2 hours; then 0.5 ml of freshly made 30% H,O, (made by diluting 50% H,O,) is added. The vials are tightly capped and incubated at 60°C overnight. After cooling, 10 ml of Aquasol (New England Nuclear) or Readisolv G P (Beckman Instruments) is added. Because of the large amount of peroxide present, problems with fluorescence may be encountered. We avoid this problem by storing the vials in a cool, dark place for several hours before counting in a refrigerated counter. g . Results of Method 2. The analysis of yeast ribosomal proteins by method 2 is shown in Fig. 5. Most ofthe proteins have been attributed to one of the two subunits (Table I). In only a few cases has it been possible to cross-identify proteins in the two systems: 1. Because of their size, proteins 1 and 2 are likely to L1 and C2. The identification of L2 with protein 2 is confirmed by the finding that both are methylated (Cannon et al., 1977; D. Barton, unpublished). 2. Protein 9 is the unphosphorylated form of the 40s protein S5, S6, S7 (P2 of Zinker and Warner, 1976). The phosphorylated form runs slightly to the left of protein 9. 3. Protein 14 is the phosphorylated protein S27 (P3 of Zinker and Warner, 1976). 4. Protein 16 is the exchangeable 60s protein L7 (E2 of Zinker and Warner, 1976). Although method 1 probably gives better resolution of more proteins, method 2 has three distinct advantages: 1. It is more reliable. 2. It gives better separation of marginally basic ribosomal proteins, e.g., protein 14.
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
57
FIG.5. Separation of yeast ribosomal proteins by method 2 (pH 5.0 SDS). Total ribosomal proteins were subjected to two-dimensional electrophoresis as described, and the gels stained. Reprinted from Gorenstein and Warner (1976) by permission.
3. Proteins are more soluble in its sample buffer. Therefore method 2 is preferable when isolating ribosomal proteins from a total cell extract (Gorenstein and Warner, 1976).
IV. Conclusion A. What is a Ribosomal Protein? There are no covalent bonds between the components of a ribosome. Since ribosomes are well known to bind many proteins non- or quasispecifically, e.g., RNase of E . coli (Waller, 1964) and initiation factors in both prokaryotes and eukaryotes (Weissbach and Ochoa, 1976), it has been a
58
JONATHAN R. WARNER A N D CHARLES GORENSTEIN
TABLE I ASSIGNMENT TO SUBUNIT OF PROTEINS BY METHODFIG. Sa SEPARATED
1 2 3 6 8
10 11 15 16 18
22 23 24 25 27 28 29 31 32 33
38 39 44 45 47 48 49 51 57 58
61 62 64 65
5 9 12 13 14 19
4
37 0 41 42 50 52
20
55
21 30 36
60 61 63
uprotein 61 is found in both subunits. Whether this represents two unresolved proteins or a single protein that can be purified with either subunit is unclear at present.
continuous problem to determine which proteins should be considered truly ribosomal. The question is complicated by the fact that the salt washes necessary to remove contaminating proteins often remove some ribosomal proteins (Hardy, 1975). Clearly certain proteins in Figs. 2 and 5 aremuch less distinct than others; it is unlikely that the variation is due only to different affinities for the coomassie stain. Furthermore, since only a small portion of the ribosomes is active in most in vitro systems, a functional assay is seldom feasible. Thus, while it is likely that most of the spots in Figs. 2-5 are ribosomal proteins, a few may not be. To distinguish between the two it may be useful to develop criteria based on the assembly of ribosomal particles or on the regulation of the synthesis of these components, as we have proposed for mammalian cells (Warner, 1966) and for S . cerevisiae (Gorenstein and Warner, 1976).
B. Uniformity of Eukaryotic Ribosomal Proteins From an evolutionary point of view ribosomal proteins appear to be very stable, as first demonstrated by Nomuraet af. (1968) when they reconstituted active 30s subunits from 16s RNA of E . cofi and ribosomal proteins of Baciffisstearothermophifus. It is not surprising therefore that the ribosomal proteins of all mammals appear to be very similar (Delaunay et af., 1973; S. Morgan and J. R. Warner, unpublished).
4.
RIBOSOMAL PROTEINS OF SACCHAROMYCES
59
Nevertheless, it is interesting to note that the two phosphorylated yeast proteins revealed on a pH 5 SDS gel, proteins 9 and 14, migrate in positions essentially identical to those of the two phosphorylated proteins of hamster 40s ribosomes (Schubart et al., 1977). Furthermore, the three exchangeable proteins of yeast (Warner and Udem, 1972) migrate in positions essentially identical to those of the three exchangeable proteins of HeLa (Warner, 1966) on a pH 5 SDS gel. We suggest that for most of the ribosomal proteins there is likely to be a 1:1 correlation between eukaryotes as distant as yeast and humans. ACKNOWLEDGMENTS Work from this laboratory was supported by grants from the American Cancer Society No. NP 72 G, the National Science Foundation No. PCM 7503938 and the National Institutes of Health No. P 30 CA 13330. J.R.W. was a Faculty Career Awardee of the American Cancer Society.
REFERENCES Avital, S., and Elson, D. (1974). Anal. Biochem. 57, 287-292. Cannon, M., Schindler, D., and Davies, J. (1977). FEBS Lett. 75, 187-191. Delaunay, J., Creusot, F., and Schapira, G. (1973). Eur. J. Biochem. 39, 305-312. Gorenstein, C., and Warner, J. R. (1976). Proc. Nutl. Acad. Sci. U.S.A. 73, 1547-1551. Grant, P., Schindler, D., and Davies, J. (1976). Geneticr 83, 667-673. Gressner, A. M., and Wool, I. G. (1974). J. Biol. Chem. 249, 6917-6925. Hardy, S. J. (1975). Mol. Gen. Genet. 140, 253-274. Hardy, S. J., Kurland, C. G., Voynow, P., and Mora, G. (1969). Biochemistry 8, 2897-2905. Hartwell, L. (1967). J . Bucteriol. 93, 1662-1670. Ishiguro, J. (1976). Mol. Gen. Genet. 145, 73-79. Kaltschmidt, E., and Wittman, H. G. (1970). Anal. Biochem. 36,401407. Kruiswijk, T., and Planta, R. J. (1974). Mol. Biol. Rep. 1,409414. Maizel, J. V., Jr. (1969). In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 334-357. Academic Press, New York. Mets, L., and Bogorad, L. (1974). Anal. Biochem. 57, 200-207. Mortimer, R., and Hawthorne, D. (1966). Genetics 53, 165-173. Nomura, M.;Traub, P., and Bechmann, H. (1968). Nuture (London) 219, 793-795. OFarrell, P. H. (1975). J. Biol. Chem. 250,4007-4013. Schubart, U.K., Shapiro, S., Fleischer, N., and Rosen, 0. M. (1977). J . Biol. Chem. 252, 92-101. Sherton, C., and Wool, I. G. (1972). J. Biol. Chem. 4460-4467. Sherton, C., and Wool, I. G. (1974). In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 30, pp. 506-526. Academic Press, New York. Skogerson, L., McLaughlin, C., and Wakatama, E. (1973). J . Bucteriol. 116, 818-822. Stewart, M., and Crouch, R. (1977). Anal. Biochem. (in press). Stoffer, G., and Wittman, H. G. (1977). I n “Molecular Mechanisms of Protein Biosynthesis” H. Weissbod and S. Pestka, eds.), pp. 117-202. Academic Press, New York. Studier, F. W. (1973). J. Mol. Biol. 79, 237-244. Tsurugi, K., Collatz, E., Wool, I. G., and Lin, A. (1976). J. Biol. Chem. 251, 7940-7946. Waller, J. P. (1964). J. Mol. Biol. 10, 319-336.
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Warner, J. R. (1966). J. Mol. Biol. 19, 383-398. Warner, J. R. (1971). J. Biol. Chem. 246,447454. Warner, J. R., and Gorenstein, C. (1977). Cell 11 (201-212). Weissbach, H., and Ochoa, S. (1976). Annu. Rev. Biochem. 45, 191-216. Wittman, H. G. (1974). In “Methods in Enzymology” (L. Grossman and R. Moldave, eds.), Vol. 30, pp. 497-518. Academic Press, New York. Wool, I . G., and Stofller, G. (1974). In “Ribosomes” (M. Nomura, A. Tissieres, and P. Lengyel, eds.), p. 417. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Zinker, S., and Warner, J. R. (1976). J. Biol. G e m . 251, 1799-1807.
NOTEADDEDIN PROOF Using as a third dimension the analysis of proteolytic digests of proteins separated on 2D gels (Cleveland, D., Fischer, S., Kirschner, M., and Laemmli, U. (1977). J. Biol. Chem. 252, 1102-1 106), P. J. Wejksnora has cross-identified a number of ribosomal proteins as separated by the two 2D gel methods (see Table 11). These results demonstrate the identity of the phosphorylated proteins S5 and S7 with No. 9. Also No. 61 is shown to consist of two proteins, one from each subunit. TABLE I1 CROSSIDENTIFICATION OF PROTEINS AS SEPARATED BY METHODSI AND 2
System 1 SI
s2
s3 s4 s5 s7 S8 s9
SIO
s12 S13 S14 S16
s20
s21 s22 S27 S28
System 2 12 5 30 40 9 9 21
19 55 41 42 52 50 61?a 37 45 14 13
System I LI L2 L3 L4 L6 L8 L11 L13 L16 L17 L18 L20 L2 1 L24
System 2 2 1
6 II 8 25 16 18?“ 15?”
22 33 62? “ 31?” 29? “
“Protein pairs marked (?) are likely matches, but not confirmed unquestionably by either method.
