Molee. gen. Genet. 142, 299--316 (1975) © by Springer-Verlag 1975
Charaeterisation of Eukaryotie Ribosomal Proteins O. H. W. Martini and H. J. Gould Department of Biophysics, King's College, London Received April 30, 1975 Summary. A simple method of two-dimensional polyacrylamide gel electrophoresis is described which affords: (1) high resolution of eukaryotic ribosomal proteins; (2) good recovery of protein in the transfer from first to second dimension; and (3) characterisation of tile separated proteins in terms of molecular weights and other electrophoretie properties. Using this method, we have characterised 70 proteins in rabbit reticulocyte ribosomes, 30 from the small subunit and 40 from the large subunit. The molecular weight distribution is compared with those obtained by other authors after fractionation of the proteins in two dimensions. The fractionation of ribosomal proteins is a formidable problem on account of their complexity. The simplest prokaryotic ribosomes, e.g. those of E. coli, contain 55 different proteins, most of which have now been fractionated on a preparative scale by column chromatography (Hardy et al., 1969; Mora et al., 1971 ; I-Iindennach et al., 1971 a, b) and extensively characterised. The complete set of 55 can be separated analytically by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt and Wittman, 1970a, b) which has proved useful as a rapid and simple method of characterisation. Eukaryotie ribosomal proteins present an even greater problem than those of prokaryotcs. Two-dimensional polyacrylamide gel electrophoresis reveals a minimum of about 70 different proteins (Welfle etal., 1971 ; Martini and Gould, 1971 ; Sherton and Wool, 1972; and later publications). On average, the eukaryotie proteins appear to be considerably larger, as well as more numerous, than E. coli proteins (Bickle and Traut, 1971) : Lin and Wool (1974) report number average molecular weights of 25,400 and 28,000 for the small and large subunit proteins, respectively, of rat liver ribosomes, whereas those of E. coli (both subnnits) are 19,000 and 16,300, respectively (Dzionara et al., 1970). Number average molecular weights reported by other authors (summarised by Howard et al., 1975) are in close agreement. These factors contribute to the fact t h a t eukaryotic ribosomes contain 2-3 times as much protein as E. coli ribosomes (reviewed by Martini et al., 1973). The extra mass is perplexing, since the mechanism of protein synthesis, as far as it is understood, is basically the same for prokaryotic and eukaryotie ribosomes. Only about half of the 70 proteins of eukaryotic ribosomes have been fractionated on a preparative scale (Westermann et al., 1971 ; Westermann and Bielka, 1973), and the procedures are neither routine, nor readily reproducible. As much information as possible must therefore be extracted from the patterns obtained in analytical scale separations, the most effective being two-dimensional polyacrylamide gel electrophoresis. 4
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Two methods of two-dimensional polyacrylamide gel electrophoresis of eukaryotic ribosomal proteins are in routine use. The first is identical, in principle, to that originally applied by Kaltschmidt and Wittmann (1970a, b) to E. coli ribosomal proteins: in the first dimension the proteins are separated, mainly on the basis of net charge, at pH 8.6, at relatively low polyaerylamide gel concentration (4 or 8 % ) which gives minimal molecular sieving. In the second dimension, the proteins are separated, mainly on the basis of size, at a high gel concentration (18%). This system was first used by Welfle et al. (1971), Huynh et al. (1971), and Sherton and Wool (1972), to enumerate the proteins in rat liver ribosomes. The second type of two-dimensional system involves the separation of the proteins on the basis of net charge at pH 4.5, instead of pH 8.6, in the first dimension system, and introduces sodium dodecyl sulfate (SDS) to obtain an essentially completely molecular weight-based separation in the second dimension (Shapiro et al., 1967). This system was originally used by Hultin and Sj6qvist (1971) and ourselves (1971) to examine proteins in eukaryotic ribosomes. It is claimed that about 70 different proteins are resolved, whichever method of two-dimensional separation is used. The relative advantages of the two types of separations may be summarised as follows : the Kaltsehmidt-Wittmann technique, designated by the characteristic features of the two coordinates as t h e " pH 8.6/pH 4.5" system, may give marginally better resolution both for prokaryotic and for eukaryotic ribosomal proteins. In the ease of E. coli proteins, this higher resolution seems to be critical for the separation of certain protein pairs (Subramanian, 1974). But in the case of eukaryotic proteins, the same number (70) of proteins seem to be resolved in both systems, although this may signify merely that the same number of, not necessarily the same individual, proteins are unresolved in both systems. The alternative " p H 4.5/SDS" system has a number of distinct advantages over the " p H 8.6/SDS" system. In the first place, the lower pH, used in the first dimension, favours the solubility of the proteins. Combined with other precautionary measures (see below), this leads to highly efficient transfer of protein into the gel, which is important, particularly for determinations of the stoiehiometry (Martini ct al., 1975). At pH 4.5, all the ribosomal proteins are cationic; at pH 8.6, several arc anionic, necessitating more complicated, timeconsuming, procedures for setting up the run (Howard and Traut, 1974). Also the absolute mobilities are lower at pH 8.6 than at pI-I 4.5, and longer runs are therefore required for a comparable separation. As to the second dimension, the use of SDS gives the clear advantage that, with little extra effort, the molecular weights of all the separated proteins can be directly determined (Martini and Gould, 1975). Further work is required, using both systems, to determine whether all zones observed represent single homogeneous proteins. In the absence of complementary data from preparative fraetionations, it is still necessary to rely on the two-dimensional gel separations for characterising eukaryotic ribosomal proteins. Sherton and Wool (1974) explored modifications of the original " p H 8.6/pH 4.5" system to improve resolution. They found that no single set of conditions was optimal for the resolution of all the proteins from rat liver ribosomes.
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W e h a v e c a r r i e d o u t c o m p l e m e n t a r y studies, u s i n g t h e " p H 4 . 5 / S D S " s y s t e m to e n u m e r a t e a n d c h a r a e t e r i s e r a b b i t r e t i e u l o c y t e r i b o s o m a l p r o t e i n s , a n d i~ s e e m s a p p r o p r i a t e a t t h i s s t a g e t o c o m p a r e t h e r e s u l t s of t h e s e a n d o t h e r similar studies.
Exl~erimental Procedures Preparation o / R a b b i t Reticulocyte Ribosomal Proteins The method of Schweet et at. (1958), essentially unmodified, was used to prepare rabbit reticulocyte ribosomes (Martini, 1974). After pH 5.4 precipitation the ribosome pellets were resuspended in Medium A4 (25 mM KC1, 1.5 mM MgC12, 10 mM fi-mereaptoethanol, 50 mM Tris buffer, p i t 7.8 at 38 ° C), containing 5% (g/v) sucrose (ca. 5 ml/rabbit), and stored frozen for up to two weeks at --20 ° C. " R u n - o f f " ribosomes (stripped of messenger RNA) were prodnced by incubating the ribosomes in the conditions described by Nair and Arnstein (1965). Ribosomes were purified by discontinuous centrifugation through a layer of 3.5 ml 2M sucrose in Medium A4 (Wettstein et at., 1963). To obtain subunits, "run-off" ribosomes were dissociated according to Martin and Wool (1968) in Medium W (12.5 mM MgCl~, 500 mM KC1, 10 mM fl-mercaptoethanol, 50 mM Tris, pH 8.1 at 25 ° C) and the subunits separated by zonal centrifugation through a sucrose gradient in the same Medium. Protein was extracted from pelleted subunits and ribosomes, using the method of Spitnik-Elson (1965), with 4M LiC1, 6 M urea, 20 mM fi-mercapteethanol. After pelleting the RNA, the protein supernatants were dialysed against Sample Buffer for electrophoresis in the first dimension (0.1 volume of a stock solution of 120 mM K acetate, pH 5.4, added to freshly deionised 6.7 ~ urea).
Electrophoresis : F i r s t D i m e n s i o n The method essentially follows Reisfeld et al. (1964), as modified by Leboy et al. (1964) for ribosomal proteins. Immediately before use, the Sample Buffer was made 20 mM in /3-mercaptoethanol. Solutions (A)-(D) were used to prepare the gel, Solution (E) for overlayering and storing gels and Solution (F) in the buffer reservoirs. Solution A (Acrylamidebisacrylamide Gel Solution): 20g recrystallised aerylamide and 0.66 g recrystallised bisacrylamide (Loening, 1967), 36 g deionised urea and distilled water up to 100 ml. Solution B (Urea Solution): 36 g deionised urea and water up to 100 ml. Solution C (Buffer): 8.6 ml glacial acetic acid, 4.8 ml 5 N KOH, 18 g deionised urea, 3 ml N,N,N',N'-tetramethylenediamine (TEMED) and water up to 50 ml. Solution D (Persulfate Solution): 1.1 g ammonium persulfate, 18 g deionised urea and water up to 50 ml. Solution E (Overlay) : 1.2 ml 5 N KOH, 2.15 ml glacial acetic acid, 36 g deionised urea and water up to 100 ml. Solution F (Reservoir Buffer) : 31.2 g fl-alanine, 8 ml glacial acetic acid and water up to one 1. Before use, aliquots were diluted with 4 volumes of water. We have varied the gel concentration in the first dimension between 3.2 and 10%. At lower concentrations, a number of relatively small basic proteins are poorly resolved at the front, although larger and/or relatively acidic proteins are well separated. At the higher concentration, the separation is more predominantly size- than charge-based, and the resulting two-dimensional spots lie close to a diagonal line and are poorly resolved, except for certain low molecular weight species near the front. The most satisfactory gel concentration for most purposes was found to be 4.l%. 4.1% gels were made by mixing 2.4 ml of Solution A, 7.35 ml of Solution B, 1.5 ml of Solution C and 0.75 ml of Solution D. The solution was deaerated, poured into glass tubes of (usually) 4.5 mm i.d. × 10 cm long, to a height of 8.5 cm, and overlayered with water. In these conditions, polymerisation ensues after about 12-20 rain at room temperature. If the gels were not used immediately, the water layer was replaced with Solution E, and the tubes were capped. Gels could be stored for up to one month without apparent deterioration. Depending on the purpose of electrophoresis, the dimensions of the first dimensional rods were changed. Superior resolution was obtained with 12 cm, rather than 8.5 cm long gels, but the standard length was used when two gel rods were run simultaneously in a single second-dimensional slab to provide mirror symmetrical patterns for comparison. Decreasing
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the rod thickness, at constant protein load, from 4.5 to 3.5 mm, led to an improvement in resolution, but a higher proportion of the sample was trapped at the origin of the second dimensional separation, i.e. in the gel rods. The converse effects were observed on increasing the diameter of the gel rods. The use of relatively thick gels is recommended for quantitation of the separated proteins, accepting some sacrifice in resolution (Martini et al., 1975). Miniature separations were obtained using the GroAipore apparatus, with dimensions similar to those described by Mets and Bogorad (1974) and Howard and Traut (1974). Before use, the tops of the gels were dried with tissue and the tubes positioned symmetrically in the apparatus, e.g. the one of circular design made by Shandon or that of rectangular design made by Joyce Loebl. The reservoirs were then filled with Buffer F. A 10 ~l aliquot of positively charged dye, e.g. 0.05 w/v Pyronine Y in 4 M urea, is layered on top of the gel. This is underlayered by the protein sample, 50-250 ~g protein, in 50-150 ~l of sample buffer. Mixing, to be avoided, is revealed by disturbance of the above dye layer. Electrophoresis is carried out for 4 hours at 70-80 V (1.5-2 mA/gel) at room temperature.
Eleetrophoresis : Second D i m e n s i o n The method which separates proteins according to molecular weight is adapted from Shapiro et al. (1967). After disc electrophoresis in the first dimension, the gel rods were expelled from the tubes and incubated in a series of solutions which varied according to the purpose of the experiment. If necessary the gels could be stored in 10% TCA overnight before subjecting them to the usual incubation steps. Longer incubations in TCA are not recommended owing to irreversible immobilization of protein in the gel rod. Two stock solutions were used to prepare the three incubation media (A, B, C) for the first dimensional gel rods: 0.4 M phosphate buffer, pH 7.1, made by mixing 1,425 ml of 0.4 MNa2HPO a with 575 ml 0.4: M NaH2PO 3, and 4% w/v sodium dodeeyl sulfate (SDS). Solution A consists of 250 ml of phosphate buffer, 250 ml of SDS solution, 360 g deionised urea and water to one 1. Solution B consists of 25 ml of phosphate buffer, 250 ml of SDS solution, 360 g deionised urea and water to one 1. Solution C consists of 25 ml of phosphate buffer, 25 ml of SDS solution, 360 g deionised urea and water to one I. Just before use Solutions A and B were made 1% (v/v), and Solution C 0.1% or 0.2%, in fl-mercaptoethanol. Gel rods were incubated in a shaking water bath at 50 ° C, successively, in 50-100 ml aliquots of Solution A, B and C, for about 20 rain apiece, and finally in fl-mercaptoethanol-free solution C for l rain. The final step prevents some slight disturbance of polymerisation of acrylamide in the gel slab in the region of embedding. It is important for the highest reproducibility and resolution. I-Iowever, it leads to some retention of the protein sample at the origin i.e. in the gel rods if they are thin, and was therefore omitted in our quantitative studies on the stoichiometry of ribosomal proteins (Martini et al., 1975). Gel Solution, one of two Catalyst Solutions and Reservoir Buffer were required prior to electrophoresis in the second dimension. Solution A (Gel Solution for a 10% gel): 100 g recrystallised acrylamide, 3.33g recrystallised bisacrylamide, 250ml phosphate buffer, 25 ml stock SDS solution, 360 g deionised urea and water up to one 1. Proportionately more or less of the monomers (at fixed ratio) were used to vary the gel concentration. Solution B (Persulfate Catalyst) : 1 g ammonium persulfate, 7.2 g deionised urea a~d water up to 20 ml. Solution C (Riboflavin Catalyst): 12.5 mg riboflavin, 9 g deionised urea and water up to 25 ml. Solution D (Reservoir Buffer) : 1,500 ml phosphate buffer, 150 ml SDS stock solution and water up to six 1. Improvements in resolution of proteins in some areas of the gel could be obtained by increasing the final gel coneentration in the second dimension from 10 to 15 %. A cell for casting the gel slab was made up of two chromatography plates (22.5 cm × 22.5 cm × 3-4 ram) separated by two greased ]?erspex slats [1 cm × 22.5 cm × ca. 0.35 (varied)] held in place by fold-back clamps. To assemble the parts of the cell, glass plates were greased with silicone high vacuum grease along two opposite edges and the perspex slats, first only along one edge. The greased surfaces of the two 1)erspex pieces and of the glass plates were put together, exerting a slight pressure and longitudinal movement to ensure a good seal. The upper surfaces of the Perspex slats were then greased along the outer edges and the second glass plate was put in place to complete the seal. The seal was maintained by the pressure of 6 fold-back paper clamps, usually one of 3 cm span near the bottom and
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two of 1.5 cm span half way up and near the top on each side. The cell was supported vertically in a Perspex box of appropriate dimensions by means of a removable Perspex solid block which also serves the function of filling the empty space in the box. The gel was formed in three sections, two of ca. 15 ml each and the third of ca. 100 ml, which should reach to 1.5 em below the top of the cell. Each section was allowed to set before the next was poured. T o initiate polymerisation, 0.1% v/v TEMED and l % v/v of either Solution B, freshly made up, or of Solution C was added to Solution A. The pouring of the last gel section was timed to coincide with the completion of the gel rod incubations described above or, when Persulfate Catalyst (Solution B) was used, to precede it by a few minutes. The gel rods were laid lengthwise on the upper opening of the cell and pressed down gently, using a piece of Perspex slightly thinner than the side slats, and manouvred into a horizontal position so that they lay ca. 1 cm under the upper smfface of the gel solution. The diameter of the gel rod was about 20 % larger than the thickness of the Perspex side walls so that, once it was moved into the correct position, it was stationary. After the rod was positioned, the upper surface of the slab was overlayered with water. The gel sets in about 20 min, but was not used until about 2 hrs or more, after pouring. Two gels were set into one slab with their bottom (or top) ends touching to give patterns easily compared for mirror symmetry. Both electrode compartments (e.g. plastic "cake container" with platinum wires held in place with sellotape) were filled with 3 1 each of Solution D and put into their working positions. 1.5-2 hrs after embedding, the cell was taken out of the sealing box and rested with its two larger clips on the edge of the lower electrode compartment, so that the bottom end dipped into the buffer. Four layers of Whatman 3 MM paper cut to 20.0 em x 7.0 cm were wrapped around with a 41.0 cm X 5.5 em thick strip of polythene. The free overlapping end of the polythene was sellotaped down to form a sleeve which should be sufficiently wide to allow for expansion of the paper when it is soaked in buffer. This forms a flexible bridge protected against evaporation. The small free space above the gel was filled with electrode buffer (filtered to remove undissolved material), and the bridge was inserted beginning at one side to avoid trapping air bubbles. The sleeve should reach into the buffer. The bridge was then bent over the edge of the upper electrode compartment. The height of the cell was adjusted by moving the supporting clamps so that the upper edge of the cell was raised a few mm above the surface of the buffer in the upper electrode compartment. Electrophoresis was carried out for 24 hrs at 60 V and ca. 105 mA or for 37.5 hrs at 40 V and ca. 64 mA at room temperature. Reservoir Buffer (Solution D) was circulated between the two compartments using a pump. The gel slabs were stained by soaking them overnight in a mixture of methanol, acetic acid, and water (50:10:40 by volume) containing 0.1% (g/v) Coomassie Brilliant Blue. The dye, supplied by Sigma or G.T. Gurr Ltd. gave a range of colours from magenta to blue. The gels were destained and stored in a solution of methanol, acetic acid and water, 10:10:80 by volume.
Results E l e c t r o p h o r e t i c p a t t e r n s of p r o t e i n s f r o m r a b b i t r e t i c u l o c y t e r i b o s o m e s separated by the unmodified two-dimensional electrophoretic technique described e a r l i e r ( M a r t i n i a n d G o u l d , 1971), a r e s h o w n in Fig. 1 (small s u b u n i t ) , Fig. 2 (large s u b u n i t ) a n d Fig. 3 ( u n d i s s o c i a t e d ribosomes). T h e p r o t e i n s are h e a v i l y l o a d e d t o d e t e c t t h o s e p r e s e n t in s m a l l a m o u n t s a n d t h o s e of l o w m o l e c u l a r w e i g h t , w i t h c o n s i d e r a b l e sacrifice t o r e s o l u t i o n a n d s o l u b i l i t y of p r o t e i n a t t h e origin. O n e - d i m e n s i o n a l e l e c t r o p h o r e s i s in S I ) S was p e r f o r m e d in t h e s a m e slab a t t h e e d g e to g i v e a n i n d i c a t i o n of m o l e c u l a r w e i g h t d i s t r i b u t i o n . T h e separated proteins have been enumerated according to their electrophoretic m o b i l i t i e s in e a c h of t h e t w o d i m e n s i o n s . T h e p r o t e i n s are n u m b e r e d f r o m 1 . . . n, in o r d e r of d e c r e a s i n g m o l e c u l a r w e i g h t . I n t h e case of t w o or m o r e p r o t e i n s h a v i n g r o u g h l y t h e s a m e m o l e c u l a r w e i g h t s , suffixes b e g i n n i n g w i t h " a " for
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Fig. 1. Electrophoresis of rabbit reticulocyte small subunit ribosomal proteins. 160 txg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Protein sample aliquots of the same preparation as was subjected to the two-dimensional separation were dissolved in the second-dimensional buffer, which contains SDS, and electrophoresed one-dimensionally in the SDS containing second dimension, on either side of the centrally embedded gel rod, in order to obtain tile molecular weight scale on the right. A schematic diagram with the proteins enumerated according to the text is shown on the left
Fig. 2. Electrophoresis of rabbit reticulocyte large subunit ribosomal proteins. 220 tzg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Protein, in second-dimensional buffer, was run on the left margin of the slab to provide a reference for molecular weight scale (cf. Fig. 1). A schematic diagram with the proteins enumerated is shown on the right
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Fig. 3. Electrophoresis of proteins from unfractionated rabbit reticulocyte ribosomes purified as described in Experimental Procedures. 350 ~zg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Proteins were also run in the second dimension only (left), and a schematic diagram with code names derived by comparison with Figs. 1 and 2 is shown on the right
the most acidic proteins and proceeding through the alphabet, have been assigned. Some of the coded proteins are relatively faint, but they have been included in the enumeration, since they are invariably present in our standard preparations. By modifying the conditions of electrophoresis in various ways, as described in detail in the experimental section, and decreasing the protein load, higher resolution in different regions of the gel could be achieved. More complete enumeration was also made possible by taking into account characteristics other than coordinates of the zones, e.g. their size, shape, hue, density, and changes under selective conditions. The colour photograph of a polysomal protein pattern shown in Fig. 4 serves to illustrate several points concerning the use of various criteria to resolve additional proteins. With some batches of Coomassie Brilliant Blue, eolour variation from red to blue between individual zones m a y be observed, which m a y be used to characterise the separated ribosomal proteins. The quantitative extent of this variability is shown in Fig. 5, showing the spectra of the most extremely " r e d " (S12e) and the most extremely " b l u e " (89) proteins of the small subunit with ~max of 558 nm and 569 nm, respectively. The proteins which leaned most noticeably to the red end of the spectrum were L7c, L3b, L14, L15, L18b, S l l , S12c, S14, S12a, S12b and $5. The proteins which leaned towards the " b l u e " were L12b, L12d, L19, 89, S10 and 813c. In the case of the pair, L7e/L7d, which forms
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a single horizontally elongated spot, the reddish tinge of the zone at the right and the bluish tinge of the zone at the left serves to distinguish the two proteins (NB Fig. 45). Shape, as well as colour, of zones was also characteristic in the given conditions of eleetrophoresis. With reference to Fig. 4, it can be seen that some proteins, i.e. $6, L3a and L3b, exhibited vertically-elongated, cone-shaped zones, with the base of the cone para]lel to the bottom of the gel, and the concentration of protein increasing towards the point of the cone in the direction of the top of the gel. I t is possible that protein-protein interaction, which may be characteristic for these proteins, could account for the anomalous shape of the zones. Protein S15 was characterised by the unusual diffuseness of the zone generated, which was unaffected by changes in electrophoretic conditions. Cei~ain proteins displayed evidence of limited, specific, and selective proteolyric degradation. Such signs of degradation were characteristically manifested when the protein samples were exposed, inadvertently, to substandard treatment during preparation or storage. Protein L3b was particularly sensitive in this respect, and, to a lesser extent, so were proteins L3a and L10. The evidence for degradation was reduced density of the original zone, which could be correlated with increasing amounts of protein in one or more satellite zones of slight]y higher mobility in the second dimension. Quantities of protein in various zones have been estimated by densitometry (Martini et al., 1975) with results which confirm our visual impressions. There is a gradation of density roughly proportional to molecular weight, probably reflecting the fact that most of the proteins are equimolar in ribosomes. I t is characteristic of certain proteins, however, that they are unusually faint or unusually dark for their molecular weight. The list of proteins which give faint zones in normal preparations include S1, S2a, S15, S16b and $17c. In the case of S1, which is completely resolved from the remaining proteins of the
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Characterisation of Eukaryotie Ribosomal Proteins
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Charaeterisation of Eukaryotie Ribosomal Proteins O. H. W. Martini and H. J. Gould Department of Biophysics, King's College, London Received April 30, 1975 Summary. A simple method of two-dimensional polyacrylamide gel electrophoresis is described which affords: (1) high resolution of eukaryotic ribosomal proteins; (2) good recovery of protein in the transfer from first to second dimension; and (3) characterisation of tile separated proteins in terms of molecular weights and other electrophoretie properties. Using this method, we have characterised 70 proteins in rabbit reticulocyte ribosomes, 30 from the small subunit and 40 from the large subunit. The molecular weight distribution is compared with those obtained by other authors after fractionation of the proteins in two dimensions. The fractionation of ribosomal proteins is a formidable problem on account of their complexity. The simplest prokaryotic ribosomes, e.g. those of E. coli, contain 55 different proteins, most of which have now been fractionated on a preparative scale by column chromatography (Hardy et al., 1969; Mora et al., 1971 ; I-Iindennach et al., 1971 a, b) and extensively characterised. The complete set of 55 can be separated analytically by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt and Wittman, 1970a, b) which has proved useful as a rapid and simple method of characterisation. Eukaryotie ribosomal proteins present an even greater problem than those of prokaryotcs. Two-dimensional polyacrylamide gel electrophoresis reveals a minimum of about 70 different proteins (Welfle etal., 1971 ; Martini and Gould, 1971 ; Sherton and Wool, 1972; and later publications). On average, the eukaryotie proteins appear to be considerably larger, as well as more numerous, than E. coli proteins (Bickle and Traut, 1971) : Lin and Wool (1974) report number average molecular weights of 25,400 and 28,000 for the small and large subunit proteins, respectively, of rat liver ribosomes, whereas those of E. coli (both subnnits) are 19,000 and 16,300, respectively (Dzionara et al., 1970). Number average molecular weights reported by other authors (summarised by Howard et al., 1975) are in close agreement. These factors contribute to the fact t h a t eukaryotic ribosomes contain 2-3 times as much protein as E. coli ribosomes (reviewed by Martini et al., 1973). The extra mass is perplexing, since the mechanism of protein synthesis, as far as it is understood, is basically the same for prokaryotic and eukaryotie ribosomes. Only about half of the 70 proteins of eukaryotic ribosomes have been fractionated on a preparative scale (Westermann et al., 1971 ; Westermann and Bielka, 1973), and the procedures are neither routine, nor readily reproducible. As much information as possible must therefore be extracted from the patterns obtained in analytical scale separations, the most effective being two-dimensional polyacrylamide gel electrophoresis. 4
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Two methods of two-dimensional polyacrylamide gel electrophoresis of eukaryotic ribosomal proteins are in routine use. The first is identical, in principle, to that originally applied by Kaltschmidt and Wittmann (1970a, b) to E. coli ribosomal proteins: in the first dimension the proteins are separated, mainly on the basis of net charge, at pH 8.6, at relatively low polyaerylamide gel concentration (4 or 8 % ) which gives minimal molecular sieving. In the second dimension, the proteins are separated, mainly on the basis of size, at a high gel concentration (18%). This system was first used by Welfle et al. (1971), Huynh et al. (1971), and Sherton and Wool (1972), to enumerate the proteins in rat liver ribosomes. The second type of two-dimensional system involves the separation of the proteins on the basis of net charge at pH 4.5, instead of pH 8.6, in the first dimension system, and introduces sodium dodecyl sulfate (SDS) to obtain an essentially completely molecular weight-based separation in the second dimension (Shapiro et al., 1967). This system was originally used by Hultin and Sj6qvist (1971) and ourselves (1971) to examine proteins in eukaryotic ribosomes. It is claimed that about 70 different proteins are resolved, whichever method of two-dimensional separation is used. The relative advantages of the two types of separations may be summarised as follows : the Kaltsehmidt-Wittmann technique, designated by the characteristic features of the two coordinates as t h e " pH 8.6/pH 4.5" system, may give marginally better resolution both for prokaryotic and for eukaryotic ribosomal proteins. In the ease of E. coli proteins, this higher resolution seems to be critical for the separation of certain protein pairs (Subramanian, 1974). But in the case of eukaryotic proteins, the same number (70) of proteins seem to be resolved in both systems, although this may signify merely that the same number of, not necessarily the same individual, proteins are unresolved in both systems. The alternative " p H 4.5/SDS" system has a number of distinct advantages over the " p H 8.6/SDS" system. In the first place, the lower pH, used in the first dimension, favours the solubility of the proteins. Combined with other precautionary measures (see below), this leads to highly efficient transfer of protein into the gel, which is important, particularly for determinations of the stoiehiometry (Martini ct al., 1975). At pH 4.5, all the ribosomal proteins are cationic; at pH 8.6, several arc anionic, necessitating more complicated, timeconsuming, procedures for setting up the run (Howard and Traut, 1974). Also the absolute mobilities are lower at pH 8.6 than at pI-I 4.5, and longer runs are therefore required for a comparable separation. As to the second dimension, the use of SDS gives the clear advantage that, with little extra effort, the molecular weights of all the separated proteins can be directly determined (Martini and Gould, 1975). Further work is required, using both systems, to determine whether all zones observed represent single homogeneous proteins. In the absence of complementary data from preparative fraetionations, it is still necessary to rely on the two-dimensional gel separations for characterising eukaryotic ribosomal proteins. Sherton and Wool (1974) explored modifications of the original " p H 8.6/pH 4.5" system to improve resolution. They found that no single set of conditions was optimal for the resolution of all the proteins from rat liver ribosomes.
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W e h a v e c a r r i e d o u t c o m p l e m e n t a r y studies, u s i n g t h e " p H 4 . 5 / S D S " s y s t e m to e n u m e r a t e a n d c h a r a e t e r i s e r a b b i t r e t i e u l o c y t e r i b o s o m a l p r o t e i n s , a n d i~ s e e m s a p p r o p r i a t e a t t h i s s t a g e t o c o m p a r e t h e r e s u l t s of t h e s e a n d o t h e r similar studies.
Exl~erimental Procedures Preparation o / R a b b i t Reticulocyte Ribosomal Proteins The method of Schweet et at. (1958), essentially unmodified, was used to prepare rabbit reticulocyte ribosomes (Martini, 1974). After pH 5.4 precipitation the ribosome pellets were resuspended in Medium A4 (25 mM KC1, 1.5 mM MgC12, 10 mM fi-mereaptoethanol, 50 mM Tris buffer, p i t 7.8 at 38 ° C), containing 5% (g/v) sucrose (ca. 5 ml/rabbit), and stored frozen for up to two weeks at --20 ° C. " R u n - o f f " ribosomes (stripped of messenger RNA) were prodnced by incubating the ribosomes in the conditions described by Nair and Arnstein (1965). Ribosomes were purified by discontinuous centrifugation through a layer of 3.5 ml 2M sucrose in Medium A4 (Wettstein et at., 1963). To obtain subunits, "run-off" ribosomes were dissociated according to Martin and Wool (1968) in Medium W (12.5 mM MgCl~, 500 mM KC1, 10 mM fl-mercaptoethanol, 50 mM Tris, pH 8.1 at 25 ° C) and the subunits separated by zonal centrifugation through a sucrose gradient in the same Medium. Protein was extracted from pelleted subunits and ribosomes, using the method of Spitnik-Elson (1965), with 4M LiC1, 6 M urea, 20 mM fi-mercapteethanol. After pelleting the RNA, the protein supernatants were dialysed against Sample Buffer for electrophoresis in the first dimension (0.1 volume of a stock solution of 120 mM K acetate, pH 5.4, added to freshly deionised 6.7 ~ urea).
Electrophoresis : F i r s t D i m e n s i o n The method essentially follows Reisfeld et al. (1964), as modified by Leboy et al. (1964) for ribosomal proteins. Immediately before use, the Sample Buffer was made 20 mM in /3-mercaptoethanol. Solutions (A)-(D) were used to prepare the gel, Solution (E) for overlayering and storing gels and Solution (F) in the buffer reservoirs. Solution A (Acrylamidebisacrylamide Gel Solution): 20g recrystallised aerylamide and 0.66 g recrystallised bisacrylamide (Loening, 1967), 36 g deionised urea and distilled water up to 100 ml. Solution B (Urea Solution): 36 g deionised urea and water up to 100 ml. Solution C (Buffer): 8.6 ml glacial acetic acid, 4.8 ml 5 N KOH, 18 g deionised urea, 3 ml N,N,N',N'-tetramethylenediamine (TEMED) and water up to 50 ml. Solution D (Persulfate Solution): 1.1 g ammonium persulfate, 18 g deionised urea and water up to 50 ml. Solution E (Overlay) : 1.2 ml 5 N KOH, 2.15 ml glacial acetic acid, 36 g deionised urea and water up to 100 ml. Solution F (Reservoir Buffer) : 31.2 g fl-alanine, 8 ml glacial acetic acid and water up to one 1. Before use, aliquots were diluted with 4 volumes of water. We have varied the gel concentration in the first dimension between 3.2 and 10%. At lower concentrations, a number of relatively small basic proteins are poorly resolved at the front, although larger and/or relatively acidic proteins are well separated. At the higher concentration, the separation is more predominantly size- than charge-based, and the resulting two-dimensional spots lie close to a diagonal line and are poorly resolved, except for certain low molecular weight species near the front. The most satisfactory gel concentration for most purposes was found to be 4.l%. 4.1% gels were made by mixing 2.4 ml of Solution A, 7.35 ml of Solution B, 1.5 ml of Solution C and 0.75 ml of Solution D. The solution was deaerated, poured into glass tubes of (usually) 4.5 mm i.d. × 10 cm long, to a height of 8.5 cm, and overlayered with water. In these conditions, polymerisation ensues after about 12-20 rain at room temperature. If the gels were not used immediately, the water layer was replaced with Solution E, and the tubes were capped. Gels could be stored for up to one month without apparent deterioration. Depending on the purpose of electrophoresis, the dimensions of the first dimensional rods were changed. Superior resolution was obtained with 12 cm, rather than 8.5 cm long gels, but the standard length was used when two gel rods were run simultaneously in a single second-dimensional slab to provide mirror symmetrical patterns for comparison. Decreasing
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O . H . W . Martini and H. J. Gould
the rod thickness, at constant protein load, from 4.5 to 3.5 mm, led to an improvement in resolution, but a higher proportion of the sample was trapped at the origin of the second dimensional separation, i.e. in the gel rods. The converse effects were observed on increasing the diameter of the gel rods. The use of relatively thick gels is recommended for quantitation of the separated proteins, accepting some sacrifice in resolution (Martini et al., 1975). Miniature separations were obtained using the GroAipore apparatus, with dimensions similar to those described by Mets and Bogorad (1974) and Howard and Traut (1974). Before use, the tops of the gels were dried with tissue and the tubes positioned symmetrically in the apparatus, e.g. the one of circular design made by Shandon or that of rectangular design made by Joyce Loebl. The reservoirs were then filled with Buffer F. A 10 ~l aliquot of positively charged dye, e.g. 0.05 w/v Pyronine Y in 4 M urea, is layered on top of the gel. This is underlayered by the protein sample, 50-250 ~g protein, in 50-150 ~l of sample buffer. Mixing, to be avoided, is revealed by disturbance of the above dye layer. Electrophoresis is carried out for 4 hours at 70-80 V (1.5-2 mA/gel) at room temperature.
Eleetrophoresis : Second D i m e n s i o n The method which separates proteins according to molecular weight is adapted from Shapiro et al. (1967). After disc electrophoresis in the first dimension, the gel rods were expelled from the tubes and incubated in a series of solutions which varied according to the purpose of the experiment. If necessary the gels could be stored in 10% TCA overnight before subjecting them to the usual incubation steps. Longer incubations in TCA are not recommended owing to irreversible immobilization of protein in the gel rod. Two stock solutions were used to prepare the three incubation media (A, B, C) for the first dimensional gel rods: 0.4 M phosphate buffer, pH 7.1, made by mixing 1,425 ml of 0.4 MNa2HPO a with 575 ml 0.4: M NaH2PO 3, and 4% w/v sodium dodeeyl sulfate (SDS). Solution A consists of 250 ml of phosphate buffer, 250 ml of SDS solution, 360 g deionised urea and water to one 1. Solution B consists of 25 ml of phosphate buffer, 250 ml of SDS solution, 360 g deionised urea and water to one 1. Solution C consists of 25 ml of phosphate buffer, 25 ml of SDS solution, 360 g deionised urea and water to one I. Just before use Solutions A and B were made 1% (v/v), and Solution C 0.1% or 0.2%, in fl-mercaptoethanol. Gel rods were incubated in a shaking water bath at 50 ° C, successively, in 50-100 ml aliquots of Solution A, B and C, for about 20 rain apiece, and finally in fl-mercaptoethanol-free solution C for l rain. The final step prevents some slight disturbance of polymerisation of acrylamide in the gel slab in the region of embedding. It is important for the highest reproducibility and resolution. I-Iowever, it leads to some retention of the protein sample at the origin i.e. in the gel rods if they are thin, and was therefore omitted in our quantitative studies on the stoichiometry of ribosomal proteins (Martini et al., 1975). Gel Solution, one of two Catalyst Solutions and Reservoir Buffer were required prior to electrophoresis in the second dimension. Solution A (Gel Solution for a 10% gel): 100 g recrystallised acrylamide, 3.33g recrystallised bisacrylamide, 250ml phosphate buffer, 25 ml stock SDS solution, 360 g deionised urea and water up to one 1. Proportionately more or less of the monomers (at fixed ratio) were used to vary the gel concentration. Solution B (Persulfate Catalyst) : 1 g ammonium persulfate, 7.2 g deionised urea a~d water up to 20 ml. Solution C (Riboflavin Catalyst): 12.5 mg riboflavin, 9 g deionised urea and water up to 25 ml. Solution D (Reservoir Buffer) : 1,500 ml phosphate buffer, 150 ml SDS stock solution and water up to six 1. Improvements in resolution of proteins in some areas of the gel could be obtained by increasing the final gel coneentration in the second dimension from 10 to 15 %. A cell for casting the gel slab was made up of two chromatography plates (22.5 cm × 22.5 cm × 3-4 ram) separated by two greased ]?erspex slats [1 cm × 22.5 cm × ca. 0.35 (varied)] held in place by fold-back clamps. To assemble the parts of the cell, glass plates were greased with silicone high vacuum grease along two opposite edges and the perspex slats, first only along one edge. The greased surfaces of the two 1)erspex pieces and of the glass plates were put together, exerting a slight pressure and longitudinal movement to ensure a good seal. The upper surfaces of the Perspex slats were then greased along the outer edges and the second glass plate was put in place to complete the seal. The seal was maintained by the pressure of 6 fold-back paper clamps, usually one of 3 cm span near the bottom and
Characterisation of Eukaryotic Ribosomal Proteins
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two of 1.5 cm span half way up and near the top on each side. The cell was supported vertically in a Perspex box of appropriate dimensions by means of a removable Perspex solid block which also serves the function of filling the empty space in the box. The gel was formed in three sections, two of ca. 15 ml each and the third of ca. 100 ml, which should reach to 1.5 em below the top of the cell. Each section was allowed to set before the next was poured. T o initiate polymerisation, 0.1% v/v TEMED and l % v/v of either Solution B, freshly made up, or of Solution C was added to Solution A. The pouring of the last gel section was timed to coincide with the completion of the gel rod incubations described above or, when Persulfate Catalyst (Solution B) was used, to precede it by a few minutes. The gel rods were laid lengthwise on the upper opening of the cell and pressed down gently, using a piece of Perspex slightly thinner than the side slats, and manouvred into a horizontal position so that they lay ca. 1 cm under the upper smfface of the gel solution. The diameter of the gel rod was about 20 % larger than the thickness of the Perspex side walls so that, once it was moved into the correct position, it was stationary. After the rod was positioned, the upper surface of the slab was overlayered with water. The gel sets in about 20 min, but was not used until about 2 hrs or more, after pouring. Two gels were set into one slab with their bottom (or top) ends touching to give patterns easily compared for mirror symmetry. Both electrode compartments (e.g. plastic "cake container" with platinum wires held in place with sellotape) were filled with 3 1 each of Solution D and put into their working positions. 1.5-2 hrs after embedding, the cell was taken out of the sealing box and rested with its two larger clips on the edge of the lower electrode compartment, so that the bottom end dipped into the buffer. Four layers of Whatman 3 MM paper cut to 20.0 em x 7.0 cm were wrapped around with a 41.0 cm X 5.5 em thick strip of polythene. The free overlapping end of the polythene was sellotaped down to form a sleeve which should be sufficiently wide to allow for expansion of the paper when it is soaked in buffer. This forms a flexible bridge protected against evaporation. The small free space above the gel was filled with electrode buffer (filtered to remove undissolved material), and the bridge was inserted beginning at one side to avoid trapping air bubbles. The sleeve should reach into the buffer. The bridge was then bent over the edge of the upper electrode compartment. The height of the cell was adjusted by moving the supporting clamps so that the upper edge of the cell was raised a few mm above the surface of the buffer in the upper electrode compartment. Electrophoresis was carried out for 24 hrs at 60 V and ca. 105 mA or for 37.5 hrs at 40 V and ca. 64 mA at room temperature. Reservoir Buffer (Solution D) was circulated between the two compartments using a pump. The gel slabs were stained by soaking them overnight in a mixture of methanol, acetic acid, and water (50:10:40 by volume) containing 0.1% (g/v) Coomassie Brilliant Blue. The dye, supplied by Sigma or G.T. Gurr Ltd. gave a range of colours from magenta to blue. The gels were destained and stored in a solution of methanol, acetic acid and water, 10:10:80 by volume.
Results E l e c t r o p h o r e t i c p a t t e r n s of p r o t e i n s f r o m r a b b i t r e t i c u l o c y t e r i b o s o m e s separated by the unmodified two-dimensional electrophoretic technique described e a r l i e r ( M a r t i n i a n d G o u l d , 1971), a r e s h o w n in Fig. 1 (small s u b u n i t ) , Fig. 2 (large s u b u n i t ) a n d Fig. 3 ( u n d i s s o c i a t e d ribosomes). T h e p r o t e i n s are h e a v i l y l o a d e d t o d e t e c t t h o s e p r e s e n t in s m a l l a m o u n t s a n d t h o s e of l o w m o l e c u l a r w e i g h t , w i t h c o n s i d e r a b l e sacrifice t o r e s o l u t i o n a n d s o l u b i l i t y of p r o t e i n a t t h e origin. O n e - d i m e n s i o n a l e l e c t r o p h o r e s i s in S I ) S was p e r f o r m e d in t h e s a m e slab a t t h e e d g e to g i v e a n i n d i c a t i o n of m o l e c u l a r w e i g h t d i s t r i b u t i o n . T h e separated proteins have been enumerated according to their electrophoretic m o b i l i t i e s in e a c h of t h e t w o d i m e n s i o n s . T h e p r o t e i n s are n u m b e r e d f r o m 1 . . . n, in o r d e r of d e c r e a s i n g m o l e c u l a r w e i g h t . I n t h e case of t w o or m o r e p r o t e i n s h a v i n g r o u g h l y t h e s a m e m o l e c u l a r w e i g h t s , suffixes b e g i n n i n g w i t h " a " for
304
O . H . W . Martini and H. J. Gould
Fig. 1. Electrophoresis of rabbit reticulocyte small subunit ribosomal proteins. 160 txg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Protein sample aliquots of the same preparation as was subjected to the two-dimensional separation were dissolved in the second-dimensional buffer, which contains SDS, and electrophoresed one-dimensionally in the SDS containing second dimension, on either side of the centrally embedded gel rod, in order to obtain tile molecular weight scale on the right. A schematic diagram with the proteins enumerated according to the text is shown on the left
Fig. 2. Electrophoresis of rabbit reticulocyte large subunit ribosomal proteins. 220 tzg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Protein, in second-dimensional buffer, was run on the left margin of the slab to provide a reference for molecular weight scale (cf. Fig. 1). A schematic diagram with the proteins enumerated is shown on the right
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Fig. 3. Electrophoresis of proteins from unfractionated rabbit reticulocyte ribosomes purified as described in Experimental Procedures. 350 ~zg protein was subjected to electrophoresis in standard conditions (Martini and Gould, 1971). Proteins were also run in the second dimension only (left), and a schematic diagram with code names derived by comparison with Figs. 1 and 2 is shown on the right
the most acidic proteins and proceeding through the alphabet, have been assigned. Some of the coded proteins are relatively faint, but they have been included in the enumeration, since they are invariably present in our standard preparations. By modifying the conditions of electrophoresis in various ways, as described in detail in the experimental section, and decreasing the protein load, higher resolution in different regions of the gel could be achieved. More complete enumeration was also made possible by taking into account characteristics other than coordinates of the zones, e.g. their size, shape, hue, density, and changes under selective conditions. The colour photograph of a polysomal protein pattern shown in Fig. 4 serves to illustrate several points concerning the use of various criteria to resolve additional proteins. With some batches of Coomassie Brilliant Blue, eolour variation from red to blue between individual zones m a y be observed, which m a y be used to characterise the separated ribosomal proteins. The quantitative extent of this variability is shown in Fig. 5, showing the spectra of the most extremely " r e d " (S12e) and the most extremely " b l u e " (89) proteins of the small subunit with ~max of 558 nm and 569 nm, respectively. The proteins which leaned most noticeably to the red end of the spectrum were L7c, L3b, L14, L15, L18b, S l l , S12c, S14, S12a, S12b and $5. The proteins which leaned towards the " b l u e " were L12b, L12d, L19, 89, S10 and 813c. In the case of the pair, L7e/L7d, which forms
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Fig. 4A and B. Colour photograph of gel sIabs showing separated rabbit reticulocyte ribosomal proteins stained with Coomassie Brilliant Blue. Shades of purple ranging from red to blue, which contribute to "resolution" and identification of proteins, are clearly evident: (A) The whole pattern; (B) enlarged section from the middle of (A) demonstrating the use of colonr criteria for the enumeration of LTd (left, blue) and LTc (right, red), which form a horizontally elongated spot near the centre of the photograph
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a single horizontally elongated spot, the reddish tinge of the zone at the right and the bluish tinge of the zone at the left serves to distinguish the two proteins (NB Fig. 45). Shape, as well as colour, of zones was also characteristic in the given conditions of eleetrophoresis. With reference to Fig. 4, it can be seen that some proteins, i.e. $6, L3a and L3b, exhibited vertically-elongated, cone-shaped zones, with the base of the cone para]lel to the bottom of the gel, and the concentration of protein increasing towards the point of the cone in the direction of the top of the gel. I t is possible that protein-protein interaction, which may be characteristic for these proteins, could account for the anomalous shape of the zones. Protein S15 was characterised by the unusual diffuseness of the zone generated, which was unaffected by changes in electrophoretic conditions. Cei~ain proteins displayed evidence of limited, specific, and selective proteolyric degradation. Such signs of degradation were characteristically manifested when the protein samples were exposed, inadvertently, to substandard treatment during preparation or storage. Protein L3b was particularly sensitive in this respect, and, to a lesser extent, so were proteins L3a and L10. The evidence for degradation was reduced density of the original zone, which could be correlated with increasing amounts of protein in one or more satellite zones of slight]y higher mobility in the second dimension. Quantities of protein in various zones have been estimated by densitometry (Martini et al., 1975) with results which confirm our visual impressions. There is a gradation of density roughly proportional to molecular weight, probably reflecting the fact that most of the proteins are equimolar in ribosomes. I t is characteristic of certain proteins, however, that they are unusually faint or unusually dark for their molecular weight. The list of proteins which give faint zones in normal preparations include S1, S2a, S15, S16b and $17c. In the case of S1, which is completely resolved from the remaining proteins of the
308
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Characterisation of Eukaryotie Ribosomal Proteins
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