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Yeast ribosomal protein L26 is located at the ribosomal subunit interface as determined by chemical cross-linking
Biochbnie (1998) 80. 321-324 © Soci6td frangaise de bh~chimie e~ biologic m~lOcukm-c / Elsevier. Paris
Yeast ribosoma| protein L26 is located at the ribosoma| subunR interface as determined by chemical crossdinking J Villarreal Jr, JC Lee* Department of Biochemisto; The University q]"Texas Health Science Centeg 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA (Received 10 June 1997; accepted 18 Decernber 1997) Summary ~ Previous studies suggested that yeast ribosomal protein L26 was a candidate for the ribosomal subunit interfitce region. The present study used protein-pro|ehi cross°linking to identify neighboring proteins in intact 80S ribosomes of Saccharomvces ccrevisiae. To facilitate identification of cross-linked pairs involving L26, 80S ribosomes were first treated with 5-iodoacetamidofluorescein to selectively label L26. Protein cross-links were produced with dithiobis[succinimidyl] propionate or N-succinflnidyl-3-[2-pyridyldithio] propionate and analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing SDS. L26 was detected under UV and its crosslinked partner, detectable after staining with Coomassie blue, was located below the diagonal and was coincident with L26 on a single vertical axis. The identity of the partner was determined by its co-migration with 60S and ,l()S ribosomal protein markers on two-dimensional gels. Two protein pairs involving L26 and 40S sub,nit proteins were identified. The finding provided experimental evidence to support that L26 is located at the ribosomal subunit interface. A model that incorporates the present findings and the published data on intra-subunit protein pairs is proposed for the yeast 80S ribosome, (© Soci6t6 fran~aise de biochhme el bit~logie moldculaire I Elsevier, Paris).
ribosome / ribosome subunit interface I chemical cross-linking / yeast Introduction Current k n o w l e d g e on protein synthesis indicates that numerous time(tonal centers of the ribosome are located near or at the subunit interlace. Proteins located at the subunit interface of the E colt ribosomes have been identified Ii, 21, but little is known about the interface of eukaryotic ribosomes 131. Differential labeling experiments with 5-iodoacetamido-fluorescein (5-1AF) showed that L7 and L26 were the only two labeled proteins in the 80S ribosomes. They were only marginally labeled in the 60S subunits. The finding agreed with the idea that protein L26 might be located at or near the yeast ribosomal subunit interface 141. in the present study, protein cross-linking with two bifunc(tonal reagents was used to examine this supposition. Two protein pairs involving L26 and S 11 and S i 3 were detected. The evidence strongly suggests that L26, S II and S ! 3 are located at the interface region of the yeast 80S ribosome.
OR, USA) for 2 h at 0°C in the dark as previously described [41. Labeled ribosomes were purified by Sephadex G-25 chromato° graphy and centrifugation through a sucrose cushion ( 10% sucrose, 5% ammoniu.n sulfate in 50 mM Tris~acetate, pH 7.4, 12 mM MgCI~, and 51) mM ammonium chhu'ide). Under these conditions, only L7 and L2(~ are labeled 141. Chemical cross-linking of SOS rii~osmm'.~' with dithiol~i.q succinimidvl ] proiJiomttc t I)SI~ .Vmc'er h,ngth Of 12 A ) .ml N-.~'m't'inimidyl~3q2ol~yridyhtilhio) pr,lmmatc ISPI)P. spat'er length of 6.8 A )
Materials and methods
Ribosomes were treated as described 15, 61. Briefly, purified 5o!AFo labeled 80S particles in 8 mM MgCI~, 150 mM KCh and 50 mM triethanolamine HCI (pH 8), were treated with 20 mM DSP f~r 4 h or 4 mM SPDS for 2.5 h at O°C in the dark. The reaction was stopped with 100 mM glycylglycine or 20 mM glycinamide (pH 8.0). The SPDP-modified ribosomes were l'urther incubated in 40 mM H202 for 30 min at 0°C to promote disulfide bond formation. Excess H202 was removed by catalase. In both cases, ribosomes were subsequently incubated with 40 mM iodoacetamide for I h at (i°C in the dark to prevent disulfide interchange. Proteins were isolated I"1"o!11the cross-linked ribosomes using glacial acetic acid.
Preparation and labeling of 80S ribosomes
Analysis and ident~lh'ation of crossolinked I,vteins
Ribosomes were prepared from Saccharonayces cerevisiae Iollowh~g the published procedure 141 and were labeled with ! m M 5-iodoaeetamidofluarescein (5-1AE Molecular Probes lnc, Eugene.
Cross-linked proteins were analyzed by the two-dimensional diag o onai gel electrophoretic system 17 I. Electrophoresis in the first and second dimension were carried out in 10% and 17.5% (w/v) acrylamide under non-reducing and reducing conditions, respectively. Under reducing conditions, cross-linked pairs were cleaved to generate the monomeric components which would migrate under
* Correspondence and reprints
322 the diag~mal.After electrophoresis, the fluorescent L2Owas viewed under an UV light and the other ribosomal proteins were detected by Coomassie blue R-250 staining. The cross-linked partner of L26 was identified as described 171, excised, and eluted from the gel with 0.1% SDS and 50 mM ammonium bicarbonate (pH 7.8). The eluted lnotein was radiolabeled with t251using chloramine-T, and analyzed on Mets,Bogorad 2-D polyacrylamide gels [8] in the pre~nce of yeast 60S and 40S subunits ribosomal proteins 15.61. I~'otein spois were detected by staining with Coomassie blue and were exci~sedfrom the gel. The amount of radioactivity in each spot was determined by gamma spectrometry. The nomenclature of the ribosomal pin, ins followed that proposed by Michel et ai 19].
All
Results and discussion Treatment of yeast 80S ribosomes labeled with 5-1AF with DSP or SPDP generated several cross-link pairs which were analyzed by diagonal gel electrophoresis. Figure I A shows a photograph of a typical diagonal gel displaying the fluorescence of 5-1AF-labeled 80S ribosomal proteins after treatment with DSP. Fluorescent L7 and L26 migrated on the diagonal containing non-cross-linked proteins. No cross4inked pairs involving L7 were detected. Cross-linked pairs involving L26 migrated as a dimer in the first dimension. After reduction of the cross-links by incubation of the first dimension gel with I]-mercaptoethanol, L26 and its p~tner migrated below the diagonal in the second dimension. The protein partner was detectable only after staining with Coomassie blue (fig I B). and was selected according Io tile criteria formerly established 171, ie L26 and its monomeric partner were matched by their coincidence on a single vertical axis below tile diagonal and tile additivity of their apparent molecular masses to produce tile upparent molecular mass of tile parent diluer. Tile apparent molecular mass of the dimer was determined to be 36 600 (fig IB). Protein constituents of the dimer were located on a vcrtica! line underneath the dimer on tile diagonal, The apparent molecular mass of the non-fluo~scent cross4inked partner of L26 was 22 60(t :t: 25(X) (n = 5), The identity of tile parther was determined by radioiodination of the protein and analysis l'or the distribution of radioactivity among the 60S and 40S ribosomal proteins on two-dimensional gels, 'the majority of the radioactivity was detected in a gel spot correSl~mding to S II whose apparent molecular mass was 22 ~ , No significant radioactivity was detected in any gel spots corresponding to 60S subunit proteins. Tile crosslinked pair must be L26-S I I, Only one cross-linked protein pair was generated by treatment of 80S ribosomes with SPDP and it was analyzed in a ,~imilar manner, The appa~nt molecular mass of the dimer was 36 000, "l~o-dimensional diagonal gel analysis of the protein Sl~ts on a vertical line underneath the dimer conlaining the fluorescent L26 revealed one protein spot ~,hose mole~:ular mass was 21 6i~) ~ 1300 O~ = 4), The idehfify of the protein was analyzed as described above and it was S 13. Therefore, the cross-linked pair must be L26-
B.
MWt
9$kD 68 kD 39 kD 29 kD 20,4 kD 14 kD Fill I. Amdysis of DSP or SPDS cross-linked proleins by dk,gonal g¢! electrophoresis. A. A photogr~ph of the fluorescent-labeled yeast 8(IS rthosomal pmtein.~. Spots A and B t~re located on the dit~gontd and ~lre13 ~md L2f~,respectively. Spot C is located below Ills diagonal and is a illelllber o!' a DSP cros.~olinked p~fir. B. Cot~massie blue slail~ed im~ge, h~dicaled with arrows are A (L7, 20,5 kO,), 8 (utlerossotinked L26, 14 kD~t), C (previously crosslinked L26, 14 kDa) ~md D (the previously cross-linked protein with an apparent molecular mass of 22,6 kDa that was excised), e and D were components of a dimer. SI3, No significant amounts of radioactivity co-migrated with any of the 60S ribosomal subunit proteins. That L26 t~an be cross-linked to two proteins located on the small ribosome subunit strongly suggests that S I I, S 13, and L26 are in the ribosome subunit interface region. Hence, the present finding supports the previous supposition that protein L26 might be located near the ribosomal subunit interface 141, Furtlrermore, since tile length of the cl~ss-linking agents used in the present study was 12 to 13.6 A, the CUtTentfinding indicates that the reactive amino groups on proteins S I I or S 13 and L26 must lie within this distance. Determination on the cross-linked sites of these proteins will be informative, The length of the cross-linking agents and their reactivity must also be taken into account when assessing the failure to detect additional cros.~-iinked pairs.
$
--.., "',,
:
proteins plus ~wo pairs involving 40S aad (~OS ribosomal proteins. The modet assumes ~ha~ ~hc cros.,,-linkcd dam obtained wi~h purified ribosomal subtmits are applicable ~o 80S ribosomes. Tha~ ~he E colt 7{}S ribosome and the free subunits yielded identical protein pairs I l l ] subsmmiate,, the supposition. There is an excellem correlation between the interface proteins identified by protein cross-linking and those implicated by other experimental approaches. For example, S 11 and S 13 are sensitive to trypsin digestion of intact ribosomal subunits [12]. The rat liver analog of yeast S13 is $9 [131 which can be cross-linked to elF-3 suggesting that it may be located near the ribosomal subunit interface region 1141. Furthermore, in our model, S13 is cross-linked to $26 which in turn is cross-linked to S I0 [6J, The m a m m a l i a n an:dog of S l0 is $6 which can be cross-linked to 60S ribosomal proteins suggesting that ttle mammalian $6 is located at the ribosomal subunit interfi~ce [3 I. Taken together, these data support the hypothesis that yeast S 13 protein is present in the ribosomal subunit interface and may be involved in several ribosomal functions.
60S Acknowledgments The authors thank tile Robert A Welch Foundation for the financial support {AQ-1360~ and Dr Gerald Merrill for performfl~g the radioiodination tff proteins. The work is in parlial fulfilhnent of the requirements for tile Degree of Master of Science at the University of Texan Health Science Center at San Antonh~ by J Viilarreai.
References Fig 2. Protein topography of tile yeast 80S ribosome. Yeast ril~osoma_! proteins are depicled by circles and nulllbcred according to Michel et al 1191,The size of each circle is approximately proporo tional to the apparent molecular mass of the protein. Crossolinked proteins are joined by connecting lines and were generated by chemical cross-linking with DTBP (solid lint:), 2-IT (solid line), DSP and SPDP (dashed line)o The length of the connecting lines is arbitrary. The 40S cross-link pairs are shown on the top, and the 60S cross-link pairs below. Arrangements of proteins within the 40S and 60S subunits were established previously 15, 6, 101. The present finding forms a bridge between the two sets of published
information. Previous cross-linking studies have established models showing the arrangement of numerous proteins within the 40S and the 60S subunits from Sacchmrm~yces c e w v i s i a e [5, 6, 10l. The present finding provides a bridge linking the two yeast ribosomal subunits and forms the basis for the schematic representation of the cross-linked proteins of the yeast 80S ribosome (fig 2). The current model includes a total of 42 pairs involving 25 of the 32 40S ribosomal proteins and 26 pairs involving 20 of the 45 60S ribosomal
I Cove1 .IA, |.anll~¢rl .IM, Nt~FiIla {.'M, Ttau| RR, I~J~l) hh,!l|lili~:alt,~ll t~l plOl¢lll~, ill the ~,|lhtlnil il|lt'rl'a~'t' ~I ~thc l u h,,ri, l,,i , o h i d ~ m w
hy crns,~olinking with dinl~thyl 3,Y.dilhit~bi~lpr~plontmldalel. Bto* ,.hemisll 3' 20, 2843- 2852
2 Latnbcrt ,IM, Tratll RR ( Ig81 ) The suhunit interface ~1: file E~'dwri~ chia colt i"ihnst~!ne,hlenlificalitm of pmteimi at the interface between the 3()S and 50S s|lbullils by cro~,~.linking with 2-inmlt~lhiohtn¢, ,I Mol Biol 149, 451 --476 3 Nygard O. Nika H {1982) ltlcntificatmn by RNA.pmt¢in crt~ssolinking of ribosomal proteins located at the interface between the ~,mall and the hu'ge suhunits of mammalian rihosomes. EMBO J I. 357~3t~2 4 Lee JC, Horowitz PM (19921 Sullhydryl groups t,n yeasl ribtv~omal proteins L7 and L26 are significantly more rcaclive in the 8()S partides than in the 60S subunits. J Biol (7wm 207, 25(12~25(10 5 Lee JC, Traut RR (19841 Proximity of 5,8S l{NA-bindm/:~,pr~tcin~ and A-site proteins hi ycasl ribosonles inlerrcd I'rtnn ¢rw,s.linkillg, J Bioi Chem 259. 9t)71-g974 6 Yell YC, Trlull RR. I.ee JC 1198fi) Pratein mpt~graphy ~t' the 411S ribosomal subunit fron| Sacc'haromvc'es cereri.~ia~' a.~ ~ht~w'll by chemical cross-linking. J Biol Chem 26 I. 14148-14153 7 Kenny JW. l,aml~ert JM. Trau! RR ( 19791Cro~-Iinking of rib~stnn¢~ using 2.iminothiolane {|nethyl 4-mercaplobulyrimidale)and idenlifio ,'atio|| of cross-linking proteins by diagonal polyacrylamide/.,it~dium dodccyl ~If;tle gel eleclmphore:,is. Methods En:vmol l.IX. 534 55(I 8 Mets LJ, Bogorad 1. (1974) Two dimensional Imlyacrylamide elcclrophoresis: An improved method tiu"riimsOl|lal proleins. Anal Itiovhem 57, 2{10-210
324 9 Michel S. Traat RR, Lee JC (1983) Yeast ribosomal proteins: Electrophoretic analysis in four two-dimensional gel systems..Correlation of nomenclatures. Mol Gen Genet 191.251~256 10 Xiang RH. Lee JC (1989) Identification of neighboring protein pairs in the 60S ribo~mal subunits from e , . ::::.'.amyces cer~ ,isi,ae "t,~ chemical cro~-!~nki~.$. ,, iiwt C'hem 264. 10542-10546 II Lambert JM. Boile,au 13, Cover JA. Traut RR (19:83) Crosslinks be~tween ribosomal proteim of 30S subunits in 70S tight couples and in 30S subunits. Biochemistry 22, 3913-3920
12 ~ JC, Haley WT (1983) Reactivity of proteins in ribosomes from Saccharomyces cerevisiae with trypsin. Arch Bit~'hem Biophys 224, 69-76 13 Wood I, Chan YL, Gluck A (1996) Mammalian ribosomes: Structure and the evolution of the proteins, b~: Translational Control (Hershey .iWB, Mathews MB, Sonenburg N, eds) Cold Spring Harbor Laboratory Press, 685-732 j 14 Westermann P, Nygard O (1983) The spatial arrangement of the col,plex between eukaryotic initiation factor elF-3 and 40S ribosomal subunit. Biochim Biophys Acta 741,103-108
Yeast ribosoma| protein L26 is located at the ribosoma| subunR interface as determined by chemical crossdinking J Villarreal Jr, JC Lee* Department of Biochemisto; The University q]"Texas Health Science Centeg 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA (Received 10 June 1997; accepted 18 Decernber 1997) Summary ~ Previous studies suggested that yeast ribosomal protein L26 was a candidate for the ribosomal subunit interfitce region. The present study used protein-pro|ehi cross°linking to identify neighboring proteins in intact 80S ribosomes of Saccharomvces ccrevisiae. To facilitate identification of cross-linked pairs involving L26, 80S ribosomes were first treated with 5-iodoacetamidofluorescein to selectively label L26. Protein cross-links were produced with dithiobis[succinimidyl] propionate or N-succinflnidyl-3-[2-pyridyldithio] propionate and analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing SDS. L26 was detected under UV and its crosslinked partner, detectable after staining with Coomassie blue, was located below the diagonal and was coincident with L26 on a single vertical axis. The identity of the partner was determined by its co-migration with 60S and ,l()S ribosomal protein markers on two-dimensional gels. Two protein pairs involving L26 and 40S sub,nit proteins were identified. The finding provided experimental evidence to support that L26 is located at the ribosomal subunit interface. A model that incorporates the present findings and the published data on intra-subunit protein pairs is proposed for the yeast 80S ribosome, (© Soci6t6 fran~aise de biochhme el bit~logie moldculaire I Elsevier, Paris).
ribosome / ribosome subunit interface I chemical cross-linking / yeast Introduction Current k n o w l e d g e on protein synthesis indicates that numerous time(tonal centers of the ribosome are located near or at the subunit interlace. Proteins located at the subunit interface of the E colt ribosomes have been identified Ii, 21, but little is known about the interface of eukaryotic ribosomes 131. Differential labeling experiments with 5-iodoacetamido-fluorescein (5-1AF) showed that L7 and L26 were the only two labeled proteins in the 80S ribosomes. They were only marginally labeled in the 60S subunits. The finding agreed with the idea that protein L26 might be located at or near the yeast ribosomal subunit interface 141. in the present study, protein cross-linking with two bifunc(tonal reagents was used to examine this supposition. Two protein pairs involving L26 and S 11 and S i 3 were detected. The evidence strongly suggests that L26, S II and S ! 3 are located at the interface region of the yeast 80S ribosome.
OR, USA) for 2 h at 0°C in the dark as previously described [41. Labeled ribosomes were purified by Sephadex G-25 chromato° graphy and centrifugation through a sucrose cushion ( 10% sucrose, 5% ammoniu.n sulfate in 50 mM Tris~acetate, pH 7.4, 12 mM MgCI~, and 51) mM ammonium chhu'ide). Under these conditions, only L7 and L2(~ are labeled 141. Chemical cross-linking of SOS rii~osmm'.~' with dithiol~i.q succinimidvl ] proiJiomttc t I)SI~ .Vmc'er h,ngth Of 12 A ) .ml N-.~'m't'inimidyl~3q2ol~yridyhtilhio) pr,lmmatc ISPI)P. spat'er length of 6.8 A )
Materials and methods
Ribosomes were treated as described 15, 61. Briefly, purified 5o!AFo labeled 80S particles in 8 mM MgCI~, 150 mM KCh and 50 mM triethanolamine HCI (pH 8), were treated with 20 mM DSP f~r 4 h or 4 mM SPDS for 2.5 h at O°C in the dark. The reaction was stopped with 100 mM glycylglycine or 20 mM glycinamide (pH 8.0). The SPDP-modified ribosomes were l'urther incubated in 40 mM H202 for 30 min at 0°C to promote disulfide bond formation. Excess H202 was removed by catalase. In both cases, ribosomes were subsequently incubated with 40 mM iodoacetamide for I h at (i°C in the dark to prevent disulfide interchange. Proteins were isolated I"1"o!11the cross-linked ribosomes using glacial acetic acid.
Preparation and labeling of 80S ribosomes
Analysis and ident~lh'ation of crossolinked I,vteins
Ribosomes were prepared from Saccharonayces cerevisiae Iollowh~g the published procedure 141 and were labeled with ! m M 5-iodoaeetamidofluarescein (5-1AE Molecular Probes lnc, Eugene.
Cross-linked proteins were analyzed by the two-dimensional diag o onai gel electrophoretic system 17 I. Electrophoresis in the first and second dimension were carried out in 10% and 17.5% (w/v) acrylamide under non-reducing and reducing conditions, respectively. Under reducing conditions, cross-linked pairs were cleaved to generate the monomeric components which would migrate under
* Correspondence and reprints
322 the diag~mal.After electrophoresis, the fluorescent L2Owas viewed under an UV light and the other ribosomal proteins were detected by Coomassie blue R-250 staining. The cross-linked partner of L26 was identified as described 171, excised, and eluted from the gel with 0.1% SDS and 50 mM ammonium bicarbonate (pH 7.8). The eluted lnotein was radiolabeled with t251using chloramine-T, and analyzed on Mets,Bogorad 2-D polyacrylamide gels [8] in the pre~nce of yeast 60S and 40S subunits ribosomal proteins 15.61. I~'otein spois were detected by staining with Coomassie blue and were exci~sedfrom the gel. The amount of radioactivity in each spot was determined by gamma spectrometry. The nomenclature of the ribosomal pin, ins followed that proposed by Michel et ai 19].
All
Results and discussion Treatment of yeast 80S ribosomes labeled with 5-1AF with DSP or SPDP generated several cross-link pairs which were analyzed by diagonal gel electrophoresis. Figure I A shows a photograph of a typical diagonal gel displaying the fluorescence of 5-1AF-labeled 80S ribosomal proteins after treatment with DSP. Fluorescent L7 and L26 migrated on the diagonal containing non-cross-linked proteins. No cross4inked pairs involving L7 were detected. Cross-linked pairs involving L26 migrated as a dimer in the first dimension. After reduction of the cross-links by incubation of the first dimension gel with I]-mercaptoethanol, L26 and its p~tner migrated below the diagonal in the second dimension. The protein partner was detectable only after staining with Coomassie blue (fig I B). and was selected according Io tile criteria formerly established 171, ie L26 and its monomeric partner were matched by their coincidence on a single vertical axis below tile diagonal and tile additivity of their apparent molecular masses to produce tile upparent molecular mass of tile parent diluer. Tile apparent molecular mass of the dimer was determined to be 36 600 (fig IB). Protein constituents of the dimer were located on a vcrtica! line underneath the dimer on tile diagonal, The apparent molecular mass of the non-fluo~scent cross4inked partner of L26 was 22 60(t :t: 25(X) (n = 5), The identity of tile parther was determined by radioiodination of the protein and analysis l'or the distribution of radioactivity among the 60S and 40S ribosomal proteins on two-dimensional gels, 'the majority of the radioactivity was detected in a gel spot correSl~mding to S II whose apparent molecular mass was 22 ~ , No significant radioactivity was detected in any gel spots corresponding to 60S subunit proteins. Tile crosslinked pair must be L26-S I I, Only one cross-linked protein pair was generated by treatment of 80S ribosomes with SPDP and it was analyzed in a ,~imilar manner, The appa~nt molecular mass of the dimer was 36 000, "l~o-dimensional diagonal gel analysis of the protein Sl~ts on a vertical line underneath the dimer conlaining the fluorescent L26 revealed one protein spot ~,hose mole~:ular mass was 21 6i~) ~ 1300 O~ = 4), The idehfify of the protein was analyzed as described above and it was S 13. Therefore, the cross-linked pair must be L26-
B.
MWt
9$kD 68 kD 39 kD 29 kD 20,4 kD 14 kD Fill I. Amdysis of DSP or SPDS cross-linked proleins by dk,gonal g¢! electrophoresis. A. A photogr~ph of the fluorescent-labeled yeast 8(IS rthosomal pmtein.~. Spots A and B t~re located on the dit~gontd and ~lre13 ~md L2f~,respectively. Spot C is located below Ills diagonal and is a illelllber o!' a DSP cros.~olinked p~fir. B. Cot~massie blue slail~ed im~ge, h~dicaled with arrows are A (L7, 20,5 kO,), 8 (utlerossotinked L26, 14 kD~t), C (previously crosslinked L26, 14 kDa) ~md D (the previously cross-linked protein with an apparent molecular mass of 22,6 kDa that was excised), e and D were components of a dimer. SI3, No significant amounts of radioactivity co-migrated with any of the 60S ribosomal subunit proteins. That L26 t~an be cross-linked to two proteins located on the small ribosome subunit strongly suggests that S I I, S 13, and L26 are in the ribosome subunit interface region. Hence, the present finding supports the previous supposition that protein L26 might be located near the ribosomal subunit interface 141, Furtlrermore, since tile length of the cl~ss-linking agents used in the present study was 12 to 13.6 A, the CUtTentfinding indicates that the reactive amino groups on proteins S I I or S 13 and L26 must lie within this distance. Determination on the cross-linked sites of these proteins will be informative, The length of the cross-linking agents and their reactivity must also be taken into account when assessing the failure to detect additional cros.~-iinked pairs.
$
--.., "',,
:
proteins plus ~wo pairs involving 40S aad (~OS ribosomal proteins. The modet assumes ~ha~ ~hc cros.,,-linkcd dam obtained wi~h purified ribosomal subtmits are applicable ~o 80S ribosomes. Tha~ ~he E colt 7{}S ribosome and the free subunits yielded identical protein pairs I l l ] subsmmiate,, the supposition. There is an excellem correlation between the interface proteins identified by protein cross-linking and those implicated by other experimental approaches. For example, S 11 and S 13 are sensitive to trypsin digestion of intact ribosomal subunits [12]. The rat liver analog of yeast S13 is $9 [131 which can be cross-linked to elF-3 suggesting that it may be located near the ribosomal subunit interface region 1141. Furthermore, in our model, S13 is cross-linked to $26 which in turn is cross-linked to S I0 [6J, The m a m m a l i a n an:dog of S l0 is $6 which can be cross-linked to 60S ribosomal proteins suggesting that ttle mammalian $6 is located at the ribosomal subunit interfi~ce [3 I. Taken together, these data support the hypothesis that yeast S 13 protein is present in the ribosomal subunit interface and may be involved in several ribosomal functions.
60S Acknowledgments The authors thank tile Robert A Welch Foundation for the financial support {AQ-1360~ and Dr Gerald Merrill for performfl~g the radioiodination tff proteins. The work is in parlial fulfilhnent of the requirements for tile Degree of Master of Science at the University of Texan Health Science Center at San Antonh~ by J Viilarreai.
References Fig 2. Protein topography of tile yeast 80S ribosome. Yeast ril~osoma_! proteins are depicled by circles and nulllbcred according to Michel et al 1191,The size of each circle is approximately proporo tional to the apparent molecular mass of the protein. Crossolinked proteins are joined by connecting lines and were generated by chemical cross-linking with DTBP (solid lint:), 2-IT (solid line), DSP and SPDP (dashed line)o The length of the connecting lines is arbitrary. The 40S cross-link pairs are shown on the top, and the 60S cross-link pairs below. Arrangements of proteins within the 40S and 60S subunits were established previously 15, 6, 101. The present finding forms a bridge between the two sets of published
information. Previous cross-linking studies have established models showing the arrangement of numerous proteins within the 40S and the 60S subunits from Sacchmrm~yces c e w v i s i a e [5, 6, 10l. The present finding provides a bridge linking the two yeast ribosomal subunits and forms the basis for the schematic representation of the cross-linked proteins of the yeast 80S ribosome (fig 2). The current model includes a total of 42 pairs involving 25 of the 32 40S ribosomal proteins and 26 pairs involving 20 of the 45 60S ribosomal
I Cove1 .IA, |.anll~¢rl .IM, Nt~FiIla {.'M, Ttau| RR, I~J~l) hh,!l|lili~:alt,~ll t~l plOl¢lll~, ill the ~,|lhtlnil il|lt'rl'a~'t' ~I ~thc l u h,,ri, l,,i , o h i d ~ m w
hy crns,~olinking with dinl~thyl 3,Y.dilhit~bi~lpr~plontmldalel. Bto* ,.hemisll 3' 20, 2843- 2852
2 Latnbcrt ,IM, Tratll RR ( Ig81 ) The suhunit interface ~1: file E~'dwri~ chia colt i"ihnst~!ne,hlenlificalitm of pmteimi at the interface between the 3()S and 50S s|lbullils by cro~,~.linking with 2-inmlt~lhiohtn¢, ,I Mol Biol 149, 451 --476 3 Nygard O. Nika H {1982) ltlcntificatmn by RNA.pmt¢in crt~ssolinking of ribosomal proteins located at the interface between the ~,mall and the hu'ge suhunits of mammalian rihosomes. EMBO J I. 357~3t~2 4 Lee JC, Horowitz PM (19921 Sullhydryl groups t,n yeasl ribtv~omal proteins L7 and L26 are significantly more rcaclive in the 8()S partides than in the 60S subunits. J Biol (7wm 207, 25(12~25(10 5 Lee JC, Traut RR (19841 Proximity of 5,8S l{NA-bindm/:~,pr~tcin~ and A-site proteins hi ycasl ribosonles inlerrcd I'rtnn ¢rw,s.linkillg, J Bioi Chem 259. 9t)71-g974 6 Yell YC, Trlull RR. I.ee JC 1198fi) Pratein mpt~graphy ~t' the 411S ribosomal subunit fron| Sacc'haromvc'es cereri.~ia~' a.~ ~ht~w'll by chemical cross-linking. J Biol Chem 26 I. 14148-14153 7 Kenny JW. l,aml~ert JM. Trau! RR ( 19791Cro~-Iinking of rib~stnn¢~ using 2.iminothiolane {|nethyl 4-mercaplobulyrimidale)and idenlifio ,'atio|| of cross-linking proteins by diagonal polyacrylamide/.,it~dium dodccyl ~If;tle gel eleclmphore:,is. Methods En:vmol l.IX. 534 55(I 8 Mets LJ, Bogorad 1. (1974) Two dimensional Imlyacrylamide elcclrophoresis: An improved method tiu"riimsOl|lal proleins. Anal Itiovhem 57, 2{10-210
324 9 Michel S. Traat RR, Lee JC (1983) Yeast ribosomal proteins: Electrophoretic analysis in four two-dimensional gel systems..Correlation of nomenclatures. Mol Gen Genet 191.251~256 10 Xiang RH. Lee JC (1989) Identification of neighboring protein pairs in the 60S ribo~mal subunits from e , . ::::.'.amyces cer~ ,isi,ae "t,~ chemical cro~-!~nki~.$. ,, iiwt C'hem 264. 10542-10546 II Lambert JM. Boile,au 13, Cover JA. Traut RR (19:83) Crosslinks be~tween ribosomal proteim of 30S subunits in 70S tight couples and in 30S subunits. Biochemistry 22, 3913-3920
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