Molecular Morphology of Ribosomes

Transcription

1 Eur. J. Biohem. 33, (1973) Moleular Morphology of Ribosomes Loalization of Ribosomal Proteins in 54 Subunits Chester J. MCHALSX, Brue H. SELLS, and Martin MORRSON Laboratories of Biohemistry, St. Jude Children's Researh Hospital, Memphis, Tennessee (Reeived Otober 31, 1972) Using the latoperoxidaseatalyzed iodination system as a probe for exposed proteins, the loation of ribosomal proteins on 5S subunits of Esherihia oli was examined. These studies demonstrated that at least three of the proteins in the 5S ribosome are iodinated and may be termed exposed proteins. Most of the proteins, on the other hand, are not iodinated and an be lassified as buried or partially buried proteins. Unfolding of the 55 ribosome by the removal of Mg2+ resulted in a dereased organization of the ribosome struture and allowed all the proteins exept one to be iodinated. The Esherihia oli 55 subunit is omposed of approximately 34 distint proteins [l]. The studies of Traut et al. [] have suggested that, exept for two of these proteins, the remaining exist in one opy per ribosome. A question whih remains unresolved is the arrangement of these proteins within the subunit struture. Speifially, in the present study the topography of the 5S subunit has been investigated to establish whih proteins are buried and whih are exposed or on the surfae of the ribosome. To answer this question, advantage has been taken of the latoperoxidaseatalyzed halogenation system whih, under the onditions employed, is a maromoleular probe for exposed proteins in organized maromoleular systems [Z]. MATERALS AND METHODS The methionine and argininerequired mutant of E. oli strain K711 (RCrel) was used throughout these studies. The bateria were grown at 37 "C with shaking in minimal media [3] supplemented with 1 mm glyerol.33 mm Lmethionine and.24mm Larginine. Bateria were grown to an absorbane at 575 nm of.1 and inubated for three generations in the presene of tritiated tyrosine to label all proteins uniformly with tritium. The 3S and 5S ribosomal subunits were isolated as previously desribed [4]. The ells, olleted by entrifugation, were washed with 1 mm TrisHC1,6 mm 2meraptoethanol, 3nM ammonium hloride buffer ph 7.4, supplemented with 1 nm magnesium aetate and resuspended in 3 ml of TrismeraptoethanolNH4Cl Enzymes. Latoperoxidase (EC ). 32 Eur. J. Uiohem., Vo1.33 buffer plus 1OmM Mg2+. Cells were broken in a Frenh pressure ell at 6 to 8 lb/in2 and the debris removed as previously desribed [4]. The ribosomes were pursed as outlined earlier [4]. To frationate monosomes into subunits, approximately 125 Azao units (one A,,, unit is equal to an absorbane of at 26 nm in a uvette of light path 1 m) were layered on 27 ml of a linear 5 to ZOO/, surose gradient in TrismeraptoethanolNH4Cl buffer plus 1 mm Mg2+ and entrifuged at 215 rev./& for 1 h in an SW 25.1 rotor. Gradients were olleted through an SCO ultraviolet Monitor (nstruments Speialties Company) and the peaks orresponding to 3S and 5S ribosomal subunits olleted. The 5S fration was plaed in a length of dialysis tubing and onentrated by paking the tubing in fine granular surose. The onentrated sample was dialyzed against 5 mm Trisaetate, 1 mm magnesium aetate for 4 h and isolated from a linear density gradient (52/,). 2Meraptoethanol was removed from all ribosome preparations prior to iodination by dialysis against 1mM Tris ph 7.2, mm magnesium aetate and 3 mm NH4C1. Unfolding of 58 Ribosomes Unfolded 5S ribosome preparations were obtained by the method of Gesteland [5]. Approximately 57 mg of ribosomes in 35 ml Tris buffer were dialyzed against 3 mm EDTA, 1 mm Tris and.3 M NH4Cl for 6, 9 and 12min. mmediately after dialysis eah ribosome preparation was iodinated, layered on 27 ml linear 52 /, surose gradient,s and entrifuged at 17 rev./min for 16 h in an

2 482 Topography of Esherihia oli Ribosome Eur. J. Biohem. SW 25.1 rotor. The ribosome peaks were then pooled and onentrated before protein extration. odination of Ribosomes Latoperoxidase was isolated and purified as previously desribed [6,7] and the onentration was determined by absorbane at 412 run using an absorption oeffiient of 114 mm1 ml [8]. Hydrogen peroxide onentration was measured by absorbane at 23nm using an absorption oeffiient of 72.4 Ml m1 [9]. Enzymati iodination aording to the method of Morrison et al. [2] was performed with ribosome partiles. An iodidespeifi eletrode (Orion Researh, n., Cambridge, Massahusetts ; or National nstrument Laboratories, Cambridge, Massahusetts) onneted to a Metrohm ombination eletrometer and titrator (Brinkmann nstruments) was employed to monitor the reation. The reation mixture ontained 51 mg ribosome partiles, 5. pm iodine, and 2 nm latoperoxidase in.5 M Trisaetate buffer ph 7. at "C. Reation was initiated and ontrolled by small additions of.1 M H2,. The onentration of peroxide in the reation flask was kept below.1 mm. A maximum of 2 mmol iodine was inorporated per mg ribosome. To detet the presene of bound iodine in the isolated protein, the 1311 isotope waa employed in the iodination reation. Although the speifi ativity of the Na 1311 varied in the different experiments, beause of deay, the onentration of arrier Na used and the amount of iodine inorporated was the same in eah experiment. Extration and Separation of Ribosomal Protein After iodination the ribosomal subunits were olleted by entrifugation. The iodination proedure did not affet their sedimentation properties. The partiles then were onentrated to at least 2 A,,, unitsfml and dialyzed against Trismeraptoethanol NH,Cl buffer plus 12 M Mga+. The extration of ribosomal protein was arried out essentially as desribed by Waller and Harris [lo]. Two volumes of glaial aeti aid were added to the old ribosome solution and the RNA whih preipitated was kept in suspension with a Teflon homogenizer. After 45 min the RNA was pelleted by entrifugation at 2xg for 2min. The supernatant then was dialyzed against 5 ml old &tilled water and two 5ml hanges of.5m aeti aid ontaining 6 M urea. Samples were then onentrated to 23 mg/ ml ribosomal protein as determined by the method of Groves et al. [ll]. The ribosomal proteins were separated by polyarylamidegel eletrophoresis using a modifiation of the proedure of Reisfeld et al. [12] as previously desribed [4]. Following separation of the protein the gels were slied, dissolved and the isotope ontent of the various protein bands determined. RESULTS AND DSCUSSON Proteins odinated in the 5S Subunit To determine whih proteins were exposed on the outside of the 55 ribosomes the following experiments were performed. Ribosomal subunits were harvested from ells inubated for three generations with tritiated tyrosine to label all ribosomal proteins uniformly ; tritiumlabelled 55 subunits then were iodinated as desribed above. The doublelabelled ribosomal proteins were extrated and separated by polyarylamidegel eletrophoresis. The radioative profiles for 1311 and 3H were determined following proessing of the gels. 1._ E T. E T 3 x O L % >.;._ 1..._, Slie no. Fig. 1. Polyarylamidegel eletrophoresis of 58 ribosomal proteins. The 5S ribosomal subunits were obtained from els inubated for three generations with [3H]tyrosine and then were iodinated with 1311 in the presene of latoperoxidase. Proteins were extrated and gels proessed as desribed in Materials and Methods. () Radioativity of 3H, () radioativity of 1311

3 Vol.33, No.3,1873 C. J. MCALSK, B. H. SELLS, and &. MORRSON 483 Latoperoxidase has a moleular weight of 78 and does not dissoiate into subunits. Sine the iodination atalyzed by the enzyme takes plae via the usual enzyme * substrate omplex [13,14], only those proteins whih are exposed on the surfae of the partile should be labelled [15]. Those proteins buried in the interior of the partile would be inaessible to the enzyme. The results obtained in Fig.1 show onsiderable variation in the amount of iodine inorporated into the different ribosomal proteins. Preliminary studies revealed that the extent of iodination does not alter the pattern of labelling of the proteins. Thus, the variations in iodination annot be attributed to differenes in the rate of labelling. Furthermore, the present data indiate that the amount of iodine inorporated is not diretly related to the tyrosine ontent of the protein in the 5S subunit. The results suggest, therefore, that at least three proteins are on the exterior of the subunit. The highly iodinated proteins 15, 19 and 2 are situated on the surfae of the partile while all the other proteins are either ompletely or partially buried. No statement an be made onerning the lak of iodination of proteins 35, sine the tyrosine ontent of these proteins is extremely low. Ribosomal Proteins odimted in the Unfolded 58 Partile Sine most proteins in the intat 5S subunit were not iodinated, experiments were performed to determine whether this lak of reativity was a funtion of the organization of the proteins in the partile. A series of studies was undertaken to assess the effet that unfolding of the 55 partile had upon the suseptibility of various proteins to iodination. The 55 subunits were dialyzed against EDTA to produe unfolding of the ribosome by the removal of Mg2f ions. Fig. 2 illustrates the sedimentation profiles of untreated and EDTAtreated 55 subunits. After 9 min approximately 5 /, of the ribosome partiles exist in the 345 to 365 form as desribed by Gesteland [5]. Dialysis against 3 mm EDTA for 12 min renders an almost ompletely unfolded 53 preparation. Following various stages of dialysis the unfolded partiles were iodinated. From the labelling patterns of Fig. 3 and 4, it is observed that, as the 55 partile unfolds, proteins 1, 2 and 613 beome aessible to iodination. The inrease in the normalized ratios, as presented in Table 1, also indiates that these proteins are most readily iodinated as the degree of unfolding inreases. Unfolded 55 subunits appear to be struturally omplete, sine 235 RNA and normal protein patterns an be reovered. Sine relatively few of the 55 ribosomal proteins in the intat subunit are exposed for iodination, the 32' ' 1 2 Fration no Fration no. C n Fration no. Fig. 2. Sedimentation patterns of (A) untreated 5S ribosomes, (B) and (C) 5S ribosomes dialyzed against EDTA for 9 min and 12 rnin respetively. Ribosomes (2 mg/ml) were dialyzed against 3 mm EDTA, mm Tris and.3 M NH,C. Sedimentation analyses of all the above were arried out immediately after iodination at 24 OC, 17 rev./min for 16 h in an SW 25.1 rotor on 27 ml linear 52 /, surose gradients above data suggest that the 55 subunit has a fairly ompat struture and/or muh of the surfae struture may onsist of RNA. The apparent lak of rosslinking among 55 proteins in the presene of bifuntional imidoesters also led Slobin [61 to a similar onlusion onerning the ompatness of the 5S subunit. t is interesting to note that protein 17, whih apparently is one of the last proteins added in the 5S subunit assembly [li'], is not exposed. This observation suggests that the subunit may undergo onformational hanges upon addition of the final

4 484 Topography of Esherihia wli Ribosome Eur. J. Biohem. Slie no. Fig.3. Polyarylamidegel eletrophoresis of unfolded 5S ribosomal proteins. The unfolded 53 ribosomal subunits were obtained from ells inubated for three generations with [ah]tyrosine, dialyzed against EDTA for 9 min and then were iodinated with 1311 in the presene of latoperoxidase. Proteins were extrated and gels proessed as desribed in Materials and Methods. () Radioativity of 3H, () radioativity of ls1._ E l. Y) f 3 N A Slie no Fig. 4. Polyarylamidetgel eletrophoresis of unfolded 58 ribosomal proteins. The unfolded 55 ribosomal subunits were obtained from ells inubated for three generations with [3H]tyrosine, dialyzed against EDTA for 12 min and then were iodinated with 1311 in the presene of latoperoxidase. Proteins were extrated and gels proessed as desribed in Materials and Methods. Symbols as in Fig.3 protein resulting in the burial of protein 17. An alternative possibility is that the tyrosine residues are masked for other reasons. Chang and Flasks [is] have reported a study of the topography of the 3S subunits in whih trypsin was used as a probe for position of ribosomal protein. On the basis of their results, they onluded that the loation of proteins orrelated with the sequene in whih they were assembled. Those proteins assembled last were the ones most exposed. With partiular referene to protein 17, this orrelation does not appear to hold for 5S subunits. The present approah to an examination of the morphology of ribosomal partiles has ertain advantages over other methods urrently employed. The iodination proedure an be performed at room temperature and does not employ onditions whih require extremes in ph. Other methods whih have been employed to investigate the struture of ribosomal subunits have inluded the use not only of trypsin but also of protein reagents. Sine the protein reagents used are of small moleular weight [19], they might be expeted to penetrate the ribosomal subunit and reat with protein groups inside as well as on the surfae of the partile. Although trypsin ats under mild onditions, hanges may result in the ribosome sine the digested proteins leave the struture. This effet may result in an alteration of the ribosome topography, thus giving an inaurate piture of the arrangement of proteins. SpitnikElson and Breiman [2] reently have observed that when the 55 subunit was unfolded, trypti digestion

5 ~ Vol.33, N.3, 1973 C. J. MCHALSK, B. H. SELLS, and M. MORRSON 485 Table 1. sotope ontent {normalized ratios of l3ll3h) of 58 ribosomal protein from exponentially growing ell8 inubated for three generations with [ 3H]tyrosine and iodinated with NalS1 in the presene of latoperoxidase The data presented in this table represent information derived from Fig. 1, 3, and 4. To loate the slies ontaining eah band, the distane (obtained from the photograph of eah gel) and the number of gel slies separating bands 2 and 17 were determined. From these values a ratio (number of gel slies/distane) was alulated. Using simple proportionality, the value alulated from this ratio an be multiplied by the distane of a given band from band 17 to yield the slie numbers in whih the band was loated. The (1s11/3H) ratio for eah band was alulated from the profiles of radioativity illustrated in Pig.1, 3, and 4. The mean (1311/3H) ratio was alulated and the normalized ratios determined by dividing the appropriate mean (1311/3H) ratio into the individual (l"'13) ratio Normalized ratios of lal/*h for Band no. 55 protein 55 protein 59 protein unfolded unfolded for 9 min for 12 min was faster, but there was little indiation of signifiant hanges in the relative vulnerabilities of the different proteins [ZO]. Thus it would appear that trypti digestion does not seletively attak surfae proteins and is not an effetive method for determining the topography of the ribosome. One limitation of the latoperoxidaseatalyzed iodination of ribosomal proteins is that it is dependent upon the availability of the exposed tyrosine residues of surfae proteins. Consequently, proteins at or near the surfae of the ribosome would not be iodinated if their tyrosine residues were not exposed. However, the use of other probes in onjuntion with the iodination tehniques should be effetive in providing a more omplete understanding of the topography of the ribosome. Sine the present disgeleletrophoresis proedure resolves only 2 of the 34 proteins of the 5S subunit, we are urrently employing a more sophistiated system allowing for more omplete resolution of aidi as well as basi proteins. The tehnial assistane of Mrs Diana Jones and Mrs Aurelia Mihaels is gratefully aknowledged. This study was supported by U.S. Publi Health Servie Grants AM7375, GM15913, and the Damon Runyon Memorial Fund. C.M. is a postdotoral fellow of the National Caner nstitute. Speial thanks are also due to Dr G. Bayse for assistane in early studies. REFERENCES 1. Traut, R. R., Delius, H., AhmadZadeh, C., Bikle, T. A. Pearson, P. & Tissiere, A. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, Morrison, M., Bayse, G. S. & Webster, R. G. (1971) mmunohemistry, 8, Cohen, S. S. & Arbogast, R. J. (195) J. Ezp. Med. 91, Davis, F. C. & Sells, B. H. (1969) J. MoZ. Biol. 39, Gesteland, R. F. (1966) J. Mol. Biol. 18, Morrison, M. & Hultquist, D. E. (1963) J. Biol. Chem. 238, Rombauts, W. A., Shroeder, W. A. & Morrison, Af. (1967) Biohemistry, 6, Morrison, M., Hamilton, H. B. & Stotz, E. (1957) J. Biol. Chem. 228, George, P. (1953) Biohem. J. 54, Waller. J. P. & Harris. J. L. (1961) Pro. Nut. Aad.. Si. b. S. A. 47, Groves, W. E., Davis, F. C. & Sells, B. H. (1968)., Anal. Biohem. 22; Reisfeld, R. A., Lewis, E. J. & Williams, D. E. (1962) Nature (London) 195, Morrison, M. & Bayse,. E. (197) Biohemistry, 9, Morrison, M., Bayse, G. S. & Danner, D. J. (197) in Biohemistry of the Phagoyti Proess (Shultz, J., ed.) p. 51, North Holland Publishing Company. 15. Phillips, D. R. & Morrison, M. (197) Biohern. Biophys. Res. Commun. 4, Slobin, L.. (1972) J. Mol. Biol. 64, Sells, B. H. & Davis, F. C. (197) J. Mol. Biol. 47, Chang, F. N. & Flasks, J. G. (197) Pro. Nat. Ad. Si. U. S. A. 67, Craven, G. R. & Gupta, V. (197) Pro. Nat. Ad. Xi. U. S. A. 67, SpitnikEison, P. & Breiman, A. (1971) Biohim. Biophys. Ata, 254, 457. Present address of C. J. Mihalski and B. H. Sells1 Laboratories of Moleular Biology, Faulty of Mediine Memorial University of Newfoundland, St. Johns, Newfoundland, Canada M. Morrison Laboratories of Biohemistry St. Jude Children's Researh Hospital, Memphis, Tennessee 3811, U.S.A. 1 To whom orrespondene should be addressed.