Ribosomal Protein S6 from Xenopus Zaevis Ovaries

Transcription

1 Eur J Blohem. 122, (1982) i FEBS 1982 Ribosomal Protein S6 from Xenopus Zaevis Ovaries Isolation, Phosphorylation in vivo and Cross-Reation with Heterologous Anti-S6 Antibodies Holger KALTHOFF. Dorothea DAKMER. Harry TOWBIN, Julian GORDON. Reinout AMONS. Wim MGLI.FR. ant1 Dittll'lr RICHTER Institut fur Pliqsiologislie Chemie. Abteilulig Zellbiohemie. Hamhurg; Friedrili-Miesher Institut. Baal: and Sylvius Laboratoria, Leiden (Reeived November ) Ribosomal protein S6 from Xriopus lurvi.7 ovaries was prepared by ion-exhange hromatography on phosphoellulose and gel filtration on Sephadex G-75. The protein was identified as S6 from its position on twodimensional polyarylamide gels and from its immunologial ross-reation with monolonal antibody raised against hiken liver S6, and from the fat that it is the major phosphorylated protein of the small subunit. When ooytes were inubated with ["P]orthophosphate in the presene of progesterone. 3zP inorporation of 40-S ribosoniul proteins was stimulated about 1 0-fold over ontrols without hormone. The bulk of the 32P radioativity was inorporated into protein S6. In reent years, the importane of protein phosphorylation as a devie to regulate enzyme ativity has beome inreasingly lear [I]. As a result, the restrition of phosphorylation to a few of the many ribosomal proteins led investigators to searh for a speial funtion in ribosome ativity [2]. In spite of onsiderable work. espeially on phosphorylation of the ribosomal protein designated S6 [3], the major phosphorylated protein of the small subunit [4], no lear answer has emerged onerning the effet of this modifiation on translation (reviewed in [5.6]). Part of the problem is that no speifi funtion has been established for S6. Evidene has been presented for its role in initiation omplex binding, and for the involvement of the phosphorylation in regulating mrna interation. but none of the evidene has been onlusive (reviewed in [6]). In order to help larify this situation, we have studied the protein in the Xeriopirs ooyte system, where translational ativities an be monitored in vivo. This system has the additional advantage that the breakdown of germinal vesiles [7] and the ativity of endogenous protein synthesis [8] are under the ontrol of the steroid progesterone. This breakdown, whih is stimulated by the hormone, is aompanied by inreased protein synthesis and phosphorylation [h, 91. This artile desribes the isolation and haraterization of the protein S6 from the IYpnopus ooyte and the influene of progesterone treatment on the phosphorylation of S6. MATERIALS AND METHODS Pwpirntiorz of Ribosotiies. Rihosonznl SirOirnits rrizrl Ribosonzul Proteitis Ribosomes from XP/?OpllS kurvis ovaries were prepared as desribed for Arteniia sulimi ribosomes by Zassloff and ~ Ahhriviutioris. PhMeSO?F, plienylmetliylsulphonyl fluoride; TP40 and TP60, total protein mixture from the small (40-S) and large (604) ribosomal subunits, respetivelv: 'Gf-Sb'. group fration 'S6'. the set of proteins separated by hromatography on phosphoellulose highly enrihed with protein S6. Ohoa [lo]. In a typial preparation 90 g of ovary tissue were washed in low-salt buffer (70m.M KCI, 9mM MgCI mm EDTA, 35 mm Tris/HCl ph mm surose, 2 mm dithiothreitol) and homogenized in 270 ml of the same buffer by ;I Sorvall oninimixer. To inrease the yield of ribosomes. Triton X-100 and sodium deoxyholate were eah added to? ",, final onentration. Ribosomal pellets were stored at - 80 C. From 1350 g of ovary tissue 10.4 g of ribosomes were isolated. One A26t~ unit nxs taken to he equivalent to 90 pg 80-S ribosomes [l 11. Subunits were prepared from thse rude ribosomes ( A2h0/,4280 ratio of 1.76) on a large sale by zonal entrifugation [I?]. Ribosomes ( g) were layerd on the surose gradient in high-salt buffer (500 mm KCI. 10 mm magnesium aetate. 20 mm Tris/HCI ph 7.6, 20 mm 2-meraptoethanol. The 40-S and 60-S subunits were poold from the peak frations only and pelleted, whereas the material of the peak shoulders was reentrifuged. The aumulated yield of pure 403 sub- units (A2hO/A2X0 ratio of 1.91) was 906 nig. Proteins were extrated from the 40-S subunits (about 18 mg/ml in 100 mm magnesium aetate, 10 mm Tris,'HCl ph 7.6) with 670,(: aeti aid [13]. 'The resulting RNA pllki were reextrated twie and the total proteins of the 40-S ribo- somal subunits (TP40) were reovered by lyophilization after dialysis against a large exess of 1 I);, aeti aid. Group Frrtinnntion qj' TP40 The proteins of the small subunit were separated by phosphoellulose hromatography essentially aording to Hardy et al. [13]. About 180 mg of TP40 (determined aording to Lowry et al. [14]) were dissolved in 18 nil standard buker (6 M urea, 0.05 M NaH2P04, 14 mm methyl:imine, 0.7 mm 2-meraptoethanol. ph 5.8 at 20 'C) and applied to the olumn. The aidi proteins were eluted with of a linear gradient (0-0.6 M NaCI), followed by steps of of 0.8 M NaCI, and of 1 M NaCI. eah in standard buffer with a flow rate of 40 mlh. The absorbane of the 10-mI frations was measured at 230 nni (Fig. 1). The data of Du Vernay

2 440 and Traugh [15] on the elution of rabbit retiuloyte S6 from phosphoellulose has been used as a guide for the loation of the position of the Xenopus ooyte S6 in the frations from the phosphoellulose olumn. Every fration in the range M NaCl was subjeted to sodium dodeylsulfate/ polyarylamide gel eletrophoresis (data not shown). Eletrophoresis was arried out in 15 polyarylamide gels aording to Laemmli [16]. Purifiation of Group Fration Gf-S6 Those frations with the highest ontent of a M, protein were pooled (Fig. 1) yielding a group fration Gf-S6 (Fig. 2A) and further purified by Sephadex (3-75 superfine gel filtration. The lyophilized Gf-S6 (6.2 mg) was dissolved in 1 ml of standard buffer ontaining 25 pg/ml PhMeS02F and eluted from the olumn (1.5 x 130 m) with a flow rate of 2.1 ml/li. Absorbane was monitored at 280 nm. 1.8-ml Fration number Fig. 1. Group frationation oftp40. TP40 Was separated by phosphoehlose hromatography as desribed in Materials and Methods. The protein frations from the 0.5 M NaCl eluate (hathed peak area) were pooled yielding Gf-S6 (see Fig. 2A) frations were pooled aording to the protein pattern on polyarylamide gels, dialyzed against 1 aeti aid and l yophilized. Protein Transfer and Immunologial Assay Proteins were labeled with tritium [17], subjeted to sodium dodeylsulfate/polyarylamide gel eletrophoresis, then transferred to nitroellulose sheets and inubated with monolonal anti-sb antibody. The speifially bound antibody was visualized with a peroxidase-oupled seond antibody as desribed [18], exept that bloking and dilutions of antibody reagents were in 10 % (v/v) horse serum. Monolonal anti-s6 sereting hybridoma was derived from a mouse immunized with hiken liver S6 (H. Towbin, H.-P. R. Ramjoue, J. Staehehn, G. Thomas, and J. Gordon; unpublished data). When blots from dodeylsulfate/polyarylamide gels are made, the eletrophoreti transfer is not always quantitative. Therefore, the proteins remaining in the gel after transfer to nitroellulose sheets were stained with Coomassie brilliant blue. Two-Dimensional Polyurylumide Gel Eletrophoresis Ribosomal proteins were identified aording to the original Kaltshmidt-Wittmann proedure [19] exept that the proteins were dissolved in 9.5 M urea buffer instead of the sample gel in order to ahieve a better penetration into the first-dimension gel. Eletrophoresis was arried out for 20 h, 11 5 V (first dimension) and 90 V (seond dimension). 32p Inorporation in viva into Ooyte Ribosomal Proteins Inubation and handling of ooytes was as desribed previously [17]. Bathes of 40 defolliulated ooytes (stage Fig. 2. Dod~~lsu!farr gel antr/~:~is of TP60, TP40, Gf-S6 and S6. (A) The moleular weight of the major protein from GFS6 was estimated to be Lane 1 ontains TP40, lane 2 TP60; lanes 3-5 ontain iiireiisiiig amounts of Gf-S6. (B) Purifiation of Gf-S6 by Sephadex (3-75 superfine gel filtration. The ontaminating proteins (lanes 1 and 2) of GfX6 (see A) ould be separated from protein S6 (lane 4) by gel filtration. Lme 3 ontailis (h letrophoresis alibration kit (Plidrmaia: pliospliorylase b, 94000; bovine serum albumin, 67000; ovalbumin, 43000; arboni aiiliydrnse, 30000: soybean trypsin inhibitor, ; a-lataibumin, 14400)

3 44 1 Fig. 3. The two-dimensional gel eletrophoresis pattern qf TP60 (lft) and TP40 (right) of X. laevis ovary ribosomes aording to the Kaltslmzidt- Wirtmunn system. Between 38 and 40 of the 60-S subunit proteins and of the 40-S subunit proteins were learly distinguishable in the gels, eah loaded with approximately 1 mg of protein. The position of protein S6 is marked by the arrow Coomassie blue Blots /S6-Antibodies Fluorogramm Fig.4. Immunologial ross-reation of protein S6 from X. laevis ovury ribosomes with monolonal antibodies raised against protein S6 from hiken liver. Protein S6 was subjeted to sodium dodeylsulfate/polyarylamide gel eletrophoresis after purifiation by gel filtration (Fig. 2) yielding two slightly different preparations of S6 (slots 3 and 4) and a ontaminating protein (slot 2). These three protein solutions were labeled by tritium [17] for the miroinjetion studies with frog ooytes [25]. Slot 5 ontains TP40, slot 1 the eletrophoresis alibration kit (see legend to Fig.2B) and slot 6 I4C eletrophoresis alibration kit (NEN: myosin, ; piiosphorylase b, ; bovine serum albumin, 69000; ovalbumin, 46000; arboni anhydrase, 30000; lysoryme, ) V-VI) were inubated with or without 1 pm progesterone [9] in 0.4 ml modified Bart11 medium ontaining 200 pci (32P)orthophosphate (arrier-free) for the indiated periods. Ribosomes from washed ooytes were isolated as desribed [I71 and ribosomal subunits were purified by two suessive surose density gradient entrifugations in high-salt buffer. Proteins of 40-S and 60-S subunits were subjeted to dodeylsulfate/polyarylamide gel eletrophoresis with or without RNase inubation. 32P inorporated into proteins was deteted by autoradiography or by nieasuring the trihloroaeti-aid-insoluble 32P-labeled material of the surose gradient frations. RESULTS Isolation and Purifiation of Protein S6 from Xenopus Ovary Ribosomes In order to obtain suffiient ribosomal protein for haraterization and identifiation, a large-sale purifiation proedure was devised as outlined in Materials and Methods. The key feature of the purifiation was the use of detergent in the initial homogenate to improve the yield. In this way, 10 g of rude ribosomes were obtained from 1.3 kg of ovaries, obtained from 82 fully grown Xenopus. From these ribosomes, 900mg of purified small subunits and 310mg of extrated

4 I I 3-12 protein were obtained. The protein mixture was frationated using phospho~ellulose to yield 9.3 mg of pure M, protein and two ontaminants with moleular weights of about and 20000, whih ould be separated out by Sephadex G-75 gel filtration. Tlie polyarylamide gels of these proteins are shown in Fig. 2. Irlm t if Iwt ioii arid CIiuruttvYx t iori q f Pro teh Sh,/ivtn X. laevis The protein was provisionally identified as S6 during the purifiation by its kromatogmphi properties on pliosphoellulose (see Materials and Methods) and from its moleular weight of The two-dimensional gel pattern shows the same readily reognized onstellation of proteins in the region of S6, illustrated here for Xtviopiu (Fig. 3). as is well known for a variety of other vertebrates [3.20]. The position of S6 an thus be reognized and is indiated in Fig.3. As ii further riterion for the identity of this protein we, made use of the speies ross-reativity of a monolonal antibody originally raised against tlie hik protein S6 (H. Towbin. H.-P. R. Ratnjouk, J. Staehelin, G. Thomas, and J. Gordon; unpublished data). This was arried out by performing antibody binding on eletrophoreti blots from polyarylamide gel eletroplierograms [18]. Fig.4A shows tlie stained protein remaining in the gel after the eletrophoreti blotting (the transfer is not quantitative), Fig.4B the antibody stained blot and Fig. 4C the fluorogram of tlie tritium-labeled proteins. It an be seen that the major antibody-stained omponent orresponds to the M, protein in TP40 (Fig.4A and B, hnnnel 5) and the purified protein (Fig.4B and C, lanes 3 or 4). No ross-reation was obtained with an impurity separated out on the last purifiation step of the S6 (Fig. 4, hannel 2). Cross-reation was, however. obtained with some lower-moleular-weight peptides in TP40 (Fig. 4B, hannel 5). This is probably a proteolyti produt whih is removed on purifiation. The protein was further onfirmed as S6 by being tlie major protein omponent of the small ribosomal subunit apable of being pliosphorylated. In many systems tliis phosphorylation orrelates with tlie stimulation of protein synthesis [6]. Sine protein synthesis [8] and phospliorylation [9] are both stimulated by progesterone in Xm1pzr.r ooytes we tested these onditions for the stimulation of phosphorylation of S6. We inubated ooytes with ["2P]ortliopliosph~ite in the presene and absene of progesterone atid examined the inorporation into sinall ribosomal subunit proteins. The results in Fig. 5 show that the inorporation was stimulated about 10-fold prior to and during germiiial vesile breakdown. The distribution of radioativity in the ribosomal proteins is shown in tlie autoradiogram (inset to Fig. 5). More than 85 'I,, of the radioativity is inorporated into S6. The rest is distributed into proteins with moleular weights of approximately and indiated by arrows in the figure. The latter may be S16, known to be phosphorylated in virus-infeted ells [2 I]. The stimulation of the phosphorylation aoinpanies the stimulated translation reation, sine it orresponds with maximal inorporation of ["S]methionine into protein (not i 11 us t rii ted ). DISCUSSION A proedure is desribed for the bulk isolation ofribosomes from k'riopirs ooytes, and for the frationation of the pro C 3 D E ln 3 U z : U B -u 2 Q N 0 X S X ll Inubation time (h) Fig. 5. Pho.~j~ho~~/~itrriorr u/' 40-S atid 60-S riho.wtml srthirriii protrirrs in vivo. Defolliiilated ooytes were pre-inubated with ("P]orthopIios- pliate (or in order to ahieve high labeling in the nuleotide pool (still inreasing afki ; riot shown). Ribosonial subunits were prpared after the indiated times :is desribed in Materials and Methods. The 32P radioalivitj preipitable by trihloroaeti aid from the surose density gradient frations ( S ribosomal hubunit: H. 0, 60-S ribosomal subunit) wi5 determined and alulated per,4260 unit. The inubatiui medium was adjusted to 1 pm progesterone at zero time (0, a), the ontrol values being without progesterone (0. H); after the addition of the hormone the phosphorylation of 40-S ribosomal subunit proteins wiis stimulated 8.8-fold and 10.2-fold. respetively, whereas ilie phosphorylntiori of the 604 ribosomal subunit proteins remained nearly onstant. The autoradiogram (inset) after dodeyl- sul~~te/polyuryl~imid gel eletrophoresis of tlit ribosomal subunit proteins shows that SO is the major phosphorylated protein. The 15 SOO-M, protein phosphorylnted in the large ribosomal subunit may be one of Ihe aidi proteins also known to be pliosphorylated in other systems 12.51: tli M, baiid observed in the 603 subunit onstitutes a minor ontamination of S6. The appearane of the germinal vesile breakdown is indiatd by the tno iirrows: mor than 90",, of the ooytes shoa.ed illis typial phenomenon after 6 11 of' progesterone treatment. Although some IRNA was labeled after this period of inubation. tlie urves reflet mainly the progesieron-iiidued stimulation of S6 pliosp1ior)lntion: this has beeii sliowii in pni-allel experiments by do~leylsulf~~teipol).ar).lamide gel eletrophoreti analysis of TP40. Hiitoradiograpliy and ounting of gel slies [25] teins of the small subunit. Tlie.Ytwopiis ooyte system has a number of lear advantages over other systems, sine it is amenable to tlie injetion of defined or labeled omponents into an effiient translating system iii vivo. For example, one of our laboratories has extensively studied the exhangeability of exogenous ribosomal proteins with pre-existing ribosomes it1 this system [I 71. The ability to purify suh proteins in bulk from this system is therefore important and obviates the need to use proteins from other speies with possible attendant artefats.

5 443 The isolation proedure iiivolves an initial treatment with detergent, whih minimizes the losses attributable to tlie high ontent of yolk protein, fat, glyogen, ferritin, et. The use of detergent preludes the initial separation of intat mitohondria, but the amount of mitohoiidrial ribosomes should be so low ( of the ytoplasmi ribosome ontent) [22,23] that they do not onstitute a serious ontamination. Further, sine the majority of ooyte ribosomes are in a storage form, ativated only following fertilization [24], tlie subunit separation an be ahieved with high-salt treatment only and no run-off step; a lean subunit separation is obtained. No rossontamination of the subunit proteins is apparent from Fig. 3 nor from analytial surose gradients of the isolated 40-S and 60-S ribosomal subunits (not shown). The proteins of the small subunit of the Xenopus ribosome are suffiiently onserved that the position of the protein S6 an be reognized by inspetion of tlie two-dimensional polyarylamide gel fingerprint and omparison with published gel patterns [3,20]. The homology of the protein S6 with that of vertebrates is further supported by omparison of the moleular weight with published values for rabbit retiuloytes [5], by tlie overlap of S6 from hik liver with that of Xenopus in two-dimensional polyarylamide gels (G. Thomas, personal ommuniation) and the similar moleular weights of the ribosomal proteins from rabbit, hik, human and trout having determinants reognized by tlie monolonal antibody against S6 (H. Towbin, H.-P. R. Ramjoue, J. Staehelin, G. Thomas, and J. Gordon; unpublished data). A further argument for tlie funtional as well as strutural homology of tlie Xenopus ooyte S6 with the mammalian S6 is its phosphorylation in response to a signal whih also stimulates protein synthesis (progesterone treatment) [S], as shown here and in the independent experiments of Nielsen, Maller and Thomas (unpublished results). Sine we have shown that tlie level of S6 phosphorylation an thus be manipulated in ooytes, and beause of the possibilities of miro-injetion offered by tlie ooyte system, this should provide an ideal experimental model for solving the still open question as to the real fuiitional role of the phosphorylation of tlie ribosomal protein S6. We would like to thank Dr R. Ivell (Hamburg) for stimulating disussions aiid Jan Kriek (Leiden) for exellent tehnial assistane. This work has been supported by the Deutshe Forshun~.s~emeinsliafi and by short-term fellowship to H.K. from the European Moleular Biology Organization. Some of the data presented here are part of the thesis of H. Kalthoff. REFERENCES 1. Coheii, L. (1980) in Moleulur Aspets of Cellulur Regulation (Cohen, P., ed.) vol. I, pp. 1-10, Elsevier/North-Holland Biomedial Press, Amsterdam. 2. Wool, 1. G. (1979) Annu. Rev. Biohem. 48, MConkey, E. H., Bielka, H., Gordon, J., Lastik, S. M., Lin, A., Ogata, K., Keboud, J.-P., Traugh, J. A,, Traut, R. R., Warner, J. R., Welfle, H. &Wool, I. G. (1979) Mol. Gen. Genet. 169, Gressner, A. M. & Wool, I. G. (1974) J. Biol. Chem. 249, Leader, P. (1980) in Moleular Aspets ofc,llulur Regulation (Cohen, P., ed.) vol. I, pp , Elsevier/North-Holland Biomedial Press, Amsterdam. 6. Gordon, J., Nielsen, P., Manhester, K. L., Towbin, H., Jimenez de Asua. L. & Thomas, G. (1982) Curr. Top. Cell. Regul., in the press. 7. Maller, J. L. & Krebs. E. G. (1977) J. Biol. Chem. 252, Younglai, E., Godeau, F. & Baulieu, E.-E. (1981) FEBS Lett. 127, Belle, R., Boyer. J. & Ozon, R. (1978) Bid. Cell Zassloff, M. & Ohoa, S. (1971) Pro. Nut1 Aad. Si. USA, 68, Stoffler, G., Wool, I. G., Lin, A. & Rak, K.-H. (1974) Pro. Nut1 Aad. Si. USA, 71, L. I Sherton, C. C., DiCamelli, R. F. & Wool, I. G. (1974) Methods Enzymol. 30F, Hardy, S. J. S., Kurland, C. G., Voynow, P. & Mora, G. (1969) Biohemistry, 8, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Du Vernay, V. H., Jr & Traugh, J. A. (1978) Biohemistry, 17, Laemmli, U. K. (1970) Nature (Lond.) 227, Kalthoff, H. & Rihter, D. (1979) Biohemistry, 18, Towbin, H., Staehelin, T. & Gordon, J. (1979) Pro. Natl Aud. Si. USA, 76, Kaltshmidt, E. & Wittmann, H. G. (1970) Anal. Bioliem. 36, Ramjoue, H. P. R. & Gordon, J. (1977) J. Bid. Chem. 252, Kennedy, I. M., Stevely. W. S. & Leader, D. P. (1981) J. Virol. 39, Sheer, U. (1972) Z. Zellforsh. 127, Sheer, U. (1973) DPV. Bid. 30, Woodland, H. R. (1974) Dev. Bid. 40, Kalthoff, H. & Rihter, D. (1982) Biohemi.stry, in the press. H. Kalthoff, D. Darmer, and D. Rihter, Iiistitut fiir Physiologishe Chemie, Abteilung Zellbiohemie, Physiologis~i-Chemishes liistitut der Universitat Hamburg. Uiiiversitatskraiikenhaus Eppendorf. MartinistraBe 52, D-2000 Hamburg 20; Federal Republi of Germany ti. Towbiii aiid J. Gordon, Friedrih Misher Institut, Postfab 273, CH-4002 Bnsl, Switzerland R. Amoiis and W. Mtiller, Laboratorium voor Fysiologishe Siieikunde, Sylvius Laboratoria, Rijksuniversiteit Leiden, Postbus 722, NL-2333-AL Leiden, The Netherlands