Separation of the Proteins in Human Tonsillar Cytoplasmic Ribosomes by Two-Dimensional Polyacrylamide-Gel Electrophoresis

Transcription

1 Eur. J. Biochem. 50, (1974) Separation of the Proteins in Human Tonsillar Cytoplasmic Ribosomes by TwoDimensional PolyacrylamideGel Electrophoresis Ugur UCER and Engin BERMEK Abteilung Molekulare Genetik, MaxPlanckInstitut fur Experimentelle Medizin, Gottingen (Received June 10/August 27, 1974) Proteins of cytoplasmic ribosomes from human tonsillar lymphatic tissue were analyzed by twodimensional polyacrylamidegel electrophoresis. 34 proteins were found to be associated with the 404 subunit and 35 proteins with the 60S subunit. 65 proteins had different electrophoretic mobilities. Two proteins found in the small subunit (SIO and S20) comigrated with two large subunit proteins (3 and 0 respectively). A comparison of our results with those obtained by the same method for ribosomal proteins from human placenta, rat liver and rabbit reticulocytes suggested a possible identity of the electrophoretic mobilities of S subunit proteins and subunit proteins. Proteins of cytoplasmic ribosomes from various eucaryotic species and within the same species from various tissues have recently been analyzed by twodimensional gel electrophoresis [l 131. These studies revealed a considerable similarity between the eucaryotic ribosomal proteins of different species. However, some interspecific and also intraspecific differences have been observed [5 7,111. This communication will report the separation of ribosomal proteins from human tonsillar lymphatic tissue using the method of Kaltschmidt and Wittman [14]. A tentative comparison of the proteins found in human tonsillar and some other eucaryotic (mammalian) ribosomes [l 41 permits a correlation of the proteins with identical electrophoretic mobilities. MATERIALS AND METHODS Chemicals Acrylamide, methylenen,wbisacrylamide were obtained from Biomol (Ilvesheim) ; urea, trizma base from Baker (Deventer); N,N,N',N'tetramethylethylendiamine from Serva (Heidelberg). All the other reagentgrade chemicals were purchased from Merck (Darmstadt). Preparations of Ribosomes and Extraction of Ribosomal Proteins Ribosomes [15] and ribosomal subunits [16] from human tonsillar lymphatic tissue were prepared as previously described. Ribosomal proteins were extracted according to the method of SpitnikElson [17]. 80S ribosomes corresponding to 100,4260 units, 60S ribosomal subunits corresponding to 60 A,,, units or 40S ribosomal subunits corresponding to 40,4260 units were suspended in 75 pl of medium A minus sucrose (50 mm TrisHC1 ph 7.4,5 mm MgC12, 25 mm KCl, and 7 mm 2mercaptoethanol) in 2ml tubes and mixed with an equal volume of 4 M LiCl in 8 M urea. After 24 h at 4 "C the RNA precipitate was pelleted by centrifugation for 10 min at x g in a Sorvall SS34 rotor. The pellet was resuspended in 50 pl of medium A minus sucrose and extracted again. The supernatant fractions were combined and dialyzed for 30 h against several changes of sample gel solution ~41. TwoDimensional Electrophoresis and Numbering of Ribosomal Proteins Twodimensional polyacrylamidegel electrophoresis was performed according to the method of Kalt

2 184 Human Tonsillar Ribosomal Proteins Fig. 1. Twodimensional electrophoretograms of human tonsil ribosomal proteins. (A) 2 mg of 40S ribosomal subunit proteins, (B) 2.4 mg of 603 ribosomal subunit proteins, (C) 2 mg of 406 and 2.4 mg 60S ribosomal subunit proteins, (D) 4 mg 80S ribosomal proteins Schmidt and Wittman [14]. Electrophoresis in the first dimension was carried out in 7.5% acrylamide gels, ph 8.6, for 12 h at 200 V with buffer circulation. Electrophoresis in the second dimension was performed in 18 % acrylamide gels, ph 4.5, for 26 h at 100 V. The electrophoresis was done at room temperature. The numbering of ribosomal proteins followed the method described [18]. The spots marked by open circles were only occasionally observed in the electrophoretograms of proteins from some ribosomal preparationsand were, therefore, not enumerated. Correlation of Ribosomal Proteins from Diflerent Eucaryotic Sources For the correlation of our results with those published for human placenta 141, rat liver [1,2] and rabbit reticulocyte [3] ribosomal proteins, the twodimensional mobility of each protein was considered relative to the whole pattern and relative to local neighbouring proteins. The staining intensity of the proteins were also taken into account. RESULTS Proteins of the Small Ribosomal Subunit Proteins extracted from the 40S subunit were separated into 34 distinct spots by twodimensional gel electrophoresis (Fig. 1 A,Fig. 2); The protein spots had varying sizes and stained with varying intensity. Three small subunit proteins (Sl, S22 and S28) migrated towards the anode at ph 8.6 (first dimension). Two of these acidic proteins (S1 and S22) were faintly

3 U. Uqer and E. Bermek S ribosomal proteins S22. S Fig. 2. Scheme of the twodimensional electrophoretogram of the proteins of the 40S ribosomal subunit from human tonsils I 60S ribosomal proteins B L m OW 4 * 135 Fig. 3. Scheme of the twodimensionul ekctrc~phoretogrum of the proteins of the 60S ribosomal subunit from human tonsils

4 186 Human Tonsillar Ribosomal Proteins I s1m S ribosomal proteins F Fig. 4. Scheme of the twodimensional electrophoretogram of the proteins of the recombined 403 and 603 ribosomal subunits from human tonsils s1 0 ga, as2 80S ribosomal proteins 0 S Fig. 5. Scheme of the twodimensional electrophoretogram of' the proteins of the 80S ribosome from human tonsils

5 U. UGer and E. Bermek 187 visible on the electrophoretograms of 80S proteins, but stained more distinctly and reproducibly on the electrophoretograms of 40S proteins. Several weakly stained satellite spots were seen below S31 in the electrophoretograms of the 40S proteins. Proteins of the Large Ribosomal Subunit Proteins extracted from the large ribosomal subunit were resolved into 35 protein spots of which one (1) migrated towards the anode in the first dimension (Fig. 1 B, Fig. 3). Spots corresponding to S11 and S12 were on occasion faintly detectable also in the electrophoretograms of 60S proteins. Proteins of 80S Ribosomes and Recombined 403 and 60S Subunits 67 protein spots were seen in the electrophoretograms of proteins extracted from 80S ribosomes or from recombined 40S and 60S subunits. Proteins S10 and 3, as well as S20 and 0 migrated as single spots (Fig. 1 C, D, Fig.4 and 5). Comparison of Proteins,from Human Tonsillar Cytoplasmic Ribosomes to Those.from Some Other Eucaryotic Sources Table 1 shows a tentative comparison of the proteins from human tonsillar 40S subunits to those from human placenta [4], rabbit reticulocyte [3] and rat liver ribosomes [l, 21. This comparison was possible because in all these studies the same method [14] was used to separate the proteins and because the separated proteins were numbered. 21 of the proteins seen in all the compared 40S subunits appear to have identical twodimensional electrophoretic mobilities (Table 1). The tonsillar proteins (S2, S3, S4, S5, S6 + S7, S8 + S9, S10, S13, S14,S15, S16, S17, S19, S20, S21,S24, S27, S29, S31, S32, S33) seem to have correlates with identical electrophoretic mobilities in all the compared 40S subunits. The tonsillar proteins S6 and S7 as well as S8 and S9, which occasionally migrated as doublets, correspond to single protein spots in the other ribosomes. Human tonsillar S11 seems to correspond to human placental 5, rat liver [I], rat liver 7 [2] and rabbit reticulocyte 8; tonsillar S12 to rabbit reticulocyte 7. As already mentioned above, spots corresponding to tonsillar S11 and S12 are occasionally faintly visible also in the electrophoretograms of tonsillar 60S proteins (Fig. 1 and 2). Proteins S23 and S26 are found only in the tonsillar ribosomes. Human tonsillar S1 apparently corresponds to reticulocyte S2; a corresponding spot was not found in the rat liver small subunit [l]. A comparison of the anionic Table 1. Correlation of 40S ribosomal proteins from human tonsil, human placenta [4], rat liver [l,zj and rabbit reticulocytes [3 J according to their electrophoretic mobilities All proteins with identical mobilities are given in the same line. indicates the absence of a corresponding protein spot among the separated proteins from the particular source. ( ) denotes that possibly corresponding proteins have not been found in ribosomes from human placenta and rat liver [2] because of the modified procedure employed. Protein numbers in parentheses denote that the respective proteins are likely but not certain correlates of the proteins from other sources given in the same line. As the correlates of tonsillar S11 and S12 were found among the 60S proteins from the sources compared, these particular proteins are in bold type tonsil placenta [I] PI reticulocyte s1 s2 s3 s5 S6 s7 S8 s9 s10 s11 s12 S13 S14 S15 S16 S17 S18 S19 s20 s21 s22 S23 S24 S25 S26 s27 S28 S29 S30 S31 S32 s33 s34 s2 s5 S6 s7 S8 s10 5 s9 s11 s12 S14 S13 s20 S15 S18 S19 s2 s3 s5 S6 s7 S8 s9 s10 s11 S14 S17 S13 S15 S18 S16 s12 S17 S19 S16 s21 s20 (1 s21 S23 S24 S24 S25 S25 S26 S26 S27 S28 S28 S proteins from tonsillar ribosomes to those from human placenta and rat liver [2] was not possible, owing to the slightly modified methods employed. Rat liver S2 [2], rabbit reticulocyte S4 and human placental S3 were not seen in human tonsillar nor in rat liver [l] ribosomes. Table 2 shows proteins found and numbered in the compared 40S subunits but not present in human tonsillar ribosomes. A protein spot b corresponding to rat liver S27 [l], rat liver S28 [2] and to the rabbit

6 Human Tonsillar Ribosomal Proteins Table 2. Compilation of 40S ribosomal proteins.from human placenta, rat liver and rabbit reticulocytes apparently not present in human tonsillar ribosomes Letters a and b denote the protein spots which were observed but not included in OUT enumeration; these do appear, however, to have correlates in some of the systems compared. The proteins in the lower section of the table are apparently unique for the particular source tonsil placenta [I] 121 reticulocyte S5, S6, S7 s15 (S25) S30 s33 ' S1, (S32) reticulocyte S30 was seen and marked by an open circle in our gels but was not numbered (see above). Rabbit reticulocyte S15 appears to be unique for these ribosomes, although a corresponding but only slightly stained spot a was also observed in the human tonsillar 40S subunit. 25 out of 35 human tonsillar 60S subunit proteins are apparently present also in the other ribosomes compared (Table 3). These 25 tonsillar proteins are,,,, L7,, 1, 2, 3, 4,, 7, 8,0, 2,5, 6, 7,8,0,1, 2,, 4 and 5. Spots corresponding to the tonsillar ribosomal proteins,, 9, 3, 4 and 9 were, however, not seen in the other systems. Tonsillar 60S subunit protein 1 corresponds to rabbit reticulocyte protein 6. Proteins found and numbered in human placental, rat liver or rabbit reticulocyte, but not in human tonsillar 603 subunits are compiled in Table4. Human placental L6, rat liver L7 [l] and rat liver [2] appear to have their correlate in the protein spot c. Spot c was reproducibly seen only in the electrophoretograms of our 604 or recombined 40S and 60S subunit proteins, but not on those of 80S proteins. It was, therefore, not included in our enumeration. The human tonsillar protein spot d corresponds to human placental 4 and to rat liver 4 [l]; and the human tonsillar protein spot r to human placental 0, rat liver 2 [l], rat liver 3 [2] and to rabbit reticulocyte 5. DISCUSSION 34 proteins from 40S subunits and 35 proteins from 603 subunits of human tonsillar ribosomes Table 3. Correlation of 60S ribosomal proteins jrom human tonsil, human placenta (41, rat liver [1,2] and rabbit reticulocytes [3] according to their electrophoretic mobilities All proteins with identical mobilities are given in the same line. indicates the absence of a corresponding protein spot among the separated proteins from the particular source. in the line corresponding to the tonsillar anionic protein 1 indicates that possibly corresponding proteins in human placenta or rat liver [2] ribosomes are not known, owing to the modified procedure employed in these two cases. Protein numbers in parentheses denote that the respective proteins are likely but not certain correlates of the proteins from other ribosomes given in the same line tonsil placenta [I] [21 reticulocyte L6 L &4 L (2) 5 L (3) () (L6) L7 4 3 LlO&ll 2 (L 18) &32 4 5(6) 7 8 L &16 Lll& (2) have been separated by twodimensional gel electrophoresis. This is in reasonable agreement with the number of ribosomal proteins from other eucaryotic ribosomes. The total number of eucaryotic ribosomal proteins being around 70 5 is, therefore, significantly higher than that found in Eschrrichiu coli ribosomes. A large number of ribosomal proteins in all the species thus far analyzed appear to have identical twodimensional polyacrylamide gel electrophoretic mobilities. Our comparison implies that the cytoplasmic ribo Eur. J. Biochern. 50 (1974)

7 U. UGer and E. Bermek 189 Table 4. Compilation of 60S ribosomal proteins from human placenta, rat liver and rabbit reticulocytes apparently not present in human tonsillar ribosomes Letters c, dand e denote the protein spots which were observed but not included in our enumeration; these do appear, however, to have correlates in some of the systems compared. The proteins in the lower section of the table are apparently unique for the particular source tonsil placenta [l] PI reticulocyte c L6 L7 LS d 4 4 e (1) 6() LI,, 0, 3, 5 5, 2, 1 L7, somes of different eucaryotic (mammalian) species may have at least 46 (but probably as many as 50) proteins in common. If these proteins are in fact structurally (and thus functionally) homologous, this would strongly argue that in analogy to histone proteins many proteins of cytoplasmic ribosomes are highly conserved during evolution. Data published by Delauney ef al. [7] suggest the identity of a great number of ribosomal proteins throughout many different eucaryotic species. Nevertheless, some distinct inter and intraspecific differences do apparently exist. Human tonsillar ribosomes appear to contain seven proteins in the smaller and nine proteins in the larger subunit, not found in the human placental ribosomes. Conversely, nine human placental ribosomal proteins (three 40S and six 60S proteins) are apparently not present in the tonsillar ribosomes. This finding is in agreement with those of Delauney et al. [6], who have observed intraspecific differences involving around 20% of the ribosomal proteins separated from different organs of the rabbit. As cytoplasmic ribosomes of different eucaryotic species are widely interchangeable in cellfree proteinsynthesizing systems the implications and the possible biological significance of the observed inter and particularly intraspecific differences necessitate further elucidation. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. Sherton, C. C. & Wool, 1. G. (1972) J. Biol. Chem. 247, Welfle, H., Stahl, J. & Bielka, H. (1972) FEBS Leu. 26, Chatterjee, S. K., Kazemie, M. & Matthaei, H. (1973) HoppeSeyler s Z. Physiol. Chem. 354, Peeters, B., Vanduffel, L., Depuydt, A. & Rombauts, W. (1973) FEBSLett. 36, HuynhVanTan, Delaunay, J. & Schapira, G. (1971) FEBS Lett. 17, Delaunay, J., Mathieu, C. & Schapira, G. (1972) Eur. J. Biochem. 31, Delaunay, J., Creusot, F. & Schapira, G. (1973) Eur. J. Biochem. 39, Rodgers, A, (1973) Biochem. Biophys. Acta, 294, Hultin, T. & Sjoqvist, A. (1972) Anal. Biochem. 46, Hultin, T. (1972) Biochem. Biophys. Acta, 269, Huh, T. & Sjoqvist, A. (1971) Comp. Biochem. Physiol. 40B, Martini, 0. H. W. & Gould, H. J. (1971) J. Mol. Biol. 62, Hanna, N., Bellemare, G. & Godin, C. (1973) Biochem. Biophys. Acta, 331, Kaltschmidt, E. & Wittmann, H. G. (1970) Anal. Biochem Bermek, E., Kramer, W., Monkemeyer, H. & Matthaei, H. (1970) Biochem. Biophvs. Res. Commun. 40, Monkemeyer, H. & Bermek, E. (1973) HoppeSeyler s Z. Phvsiol. Chem Spgnik Elson, P. (1965) Biochem. Biophys. Res. Comrnun Kaltkhmidt, E. & Wittmann, H. G. (1970) Proc. Natl Acad Sci. U.S.A U. uqer and E. Bermek, MaxPlanckInstitut fur Experimentelle Medizin, Abteilung Molekulare Genetik, D3400 Gottingen, HermannReinStraBe 3. Federal Republic of Germany