Bhwhimica el Bitq~hysi¢'u Acta. 1! 15 ( Iq¢)1) fi- 14
~e"19tJt ElsevierScience Publishers B.V. All rights reserved [13(14-4165/t~t/$[13.511
BBAGEN 236(12
Dictyostelium discoideum acidic ribosomal phosphoproteins: Identification and in vitro phosphorylation Jesfis Prieto, E i e n a C a n d e l , M a r g a r i t a F e r n f i n d e z - R e n a r t
and Antonio Coloma
Departaownto de Bioqufmica. l"acultad de Medici~'a de la Unit'er.~idad Aut{mcmta de Madrid and Institttto ~h' hwestigaciotws Biott~tlicas del CSIC, Madrid tSpain)
{Received 19 April 1991)
Key words: R;bt~somal:,cidicpht~spht~protcin;Casein kinase type ii: In vitro phosphorylation:( D. di.~cokh.um
Four acidic phosphoproteins from the ribosomes of the slime mold Dictyostelium discoideum have been identified and partially characterized. These proteins are selectively released from ribosomal particles by salt/ethanol washes, have low molecular weight and acidic p l, and tend to aggregate in solution to form homodimers. These features correspond to proteins of different origins that have been included in the conserved family of eukaryotic A-ribosomal proteins, and, therefore, we have named them Dictyostelium ribosomal proteins AI, A2, A3 and A4. We also demonstrate that Dictyostelium ribosomal A-proteins are specifically phosphorylated in vitro by a type !i casein kinase previously identified in Dictyostelium. lsoelectric focusing separation has permitted us to identify four proteins (or P-proteins) that may consist of the phosphorylated forms of A-proteins. A-proteins from Dictyostelium and yeast do not present immunological cross-reactivity. Dictyostelium A-proteins contain, therefore, some specific features in their amino acid sequence that distinguish them from other members of the conserved eukaryoti¢ A-protein family; this conclusion is coherent with data deduced from the nucleotide sequence of eDNA clones encoding two Dictyostelium A-proteins (PI and P2) which we have recently reported.
Introduction Acidic proteins have been identified in the large ribosomal subunit from diverse species [1,2]. These proleins, called A-proteins (acidic) or P-proteins (the phosphorylated eukaryotic A proteins), appear to be involved in the interaction of supernatant factors with the ribosome [3,4] in the translation of the genetic message, In E s c h e r i c h i a coli ribosomes, two acidic proteins (L7 and LI2) were identified which correspond to a unique polypeptide in which the amino terminus can appcar either in free (protein L12) or acetylated (L7) form. They form a complex in which two L 7 / L I 2 dimers arc mounted on a single L10 ribosomal protein, constituting a characteristic structure on the large ribosomal subunit [5]. In higher organisms, two proteins analogous to L 7 / L I 2 are generally tk~und. The A eukaryotic pro-
('orrespt~ndencc: A. Coloma, Dcpartamento de Bioquimica, FaculIr.td de Mcdicinade la UnivcrsidadAut6noma dc Madrid. Arzobispo Morcillo. 4. 2802~)Madrid, Spain.
teins are coded by independent genes, have a molecular weight of about 12000 and show important analogies in their amino acid sequence [6-8]. These two proteins interact with eukaryotic elongation factors EFI and EF2, and are required for aminoacyl-tRNA binding, EF2-dependent GTPase activity and polypeptide synthesis [4,9]. Yeast ribosomes contain more than two acidic proteins: one laboratory has already characterized three members of the A-protein family in S a c c h a r o m y c e s ceret'isiae [9] and their gene sequences have been determined [10], while another group has reported the amino acid sequence of an acidic ribosomal protein from yeast [11] which differs from the three mentioned above. The cukaryotic A-proteins contain a 22 amino acid sequence near the carboxyl end of the protein which is highly conserved among eukaryotes [7,10]. The C-terminal sequence includes an epitope recognized by autoantibodies from systemic lupus erythematosus patients [12]. We report here the identification of four proteins from ribosomes of the lower cukaryote D i c t y o s t e l i u m d i s c o i d e u m which we consider as members of the Aprotein family, according to their characteristics of size, p i values, phosphorylated state and tendency to
form dimers in solution. The proteins identified in this work are specifically phosphorylatcd by a type !I casein kinase from Dictyostelium. Several observations, such as the higher p l values determined fi~r the Dictyostelium A-proteins, or the absence of immunological cross-reactivity between these proteins and acidic ribosoma.', proteins from yeast, reflect the existence of distinctive features in Dictyostelium A-proteins. The data presented corroborate those obtained by analysis of the primary, structure deduced from the nucleotide sequence tff eDNA clones encoding two of thcsc Dictyostelium A-proteins: P I and P2, which we have reported recently [13,14]. Materials
and
Methods
Materials, All reagents used were of chcmical analytical grade. Acrylamide, methylenebisacrylamide, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine (Tcmed), all of elcctrophoresis purity were obtained from Bio-Rad laboratories. Ampholincs 3.5-5 were from LKB. Urea and sucrose wcre ultrapure grades from Schwarz/Mann. [y-~2p]ATP was purchased from Amersham. Tosyllysylchloromethyiketone, casein, nucleotides, sodium o"thovanadate and alkaline phosphatase were supplied by Sigma Chemicals. Yeast ribosomal acidic protein samples, as well as polycional antibodies against these proteins, were the generous gift of J.P.G. Ballesta. Organism and growth conditions. Dict.vostelitvn discoideum strain AX-2 was grown in axenic HL-5 medium
[15]. Ribosomes. Standard procedures were used to lyse Dic~ostelium cells and to pellet ribosomal particles [16], Purification of ribosomes was carried out by resuspension of ribosomal pellets in ().5 M ammonium chloride, 311 mM Tris-HCI (pH 7.2), 0.1 M magnesium acetate and 5 mM 2-mercaptocthanol, This suspension of ribosomes was kept at 0°C for 1-3 h with gentle stirring and was then centrifuged through discontinuous sucrose gradients [17] in tl.5 M ammonium chloride, Ill mM Tris-HCI (pH 7.8), 50 mM KCI, 10 mM magnesium acetate and 5 mM 2-mercaptocthanol. The resulting pellets were resuspended in 80 mM KCI. 10 mM magnesium acetate, 20 mM Tris-HCl, and stored at - 7 0 ° C . The steps of ribosome purification as well as the extraction of ribosomal proteins described below were always performed in the presence of 500 p,M phenylmethanesulfonyl fluoride and 5(1 g M tosyllysylchloromethyl ketone, to prevent proteolysis. When indicated, 25(1 ~tM sodium orthovanadate was also added to inhibit phosphatases.
Ribosomal spill .tax,rein (SPa ,_ fr:wtion) and ribosomal core partich's (Po_, cores). Purified Dictyostelium ribo-
somes (4 m g / m l ) were treated with 0.2 M ammonium chloride and 5()% ethanol (v/v) at 0°C t\~r 15 min, as described previously [18]. The particlcs (Pc~._, corcs) were recovered by centrifugation at 12000 x g for 15 rain, and :,:suspended in I0 mM Tris-HC1 (pl-t 7.4), t0 mM magnc,~,um acetate and fiO mM KC1 at approx. 2(I mg/ml, l'hc split protein fractions (SPql,), contaiaing mainly the acidic ribosomal proteins, were prccipi,atcd by addition of 2.25 vol. of acetone at - 2i)°C. I-xtraction ~J.!rihosomat p.,~teins. Extraction of ribosomal proteins from 8()S ribosomal particles or P,,_, cores was performed as described by Ramagopai and Ennis [1{}]. Ribosomes or P~2 cores (5{1-2{1{} ,q_,f~} units/ml) wcrc adjusted to {1.i M magnesiur~ acctate, and 2.2 vol. of glacial acetic acid were added dropwise with constant stirring in the cold. Stirring was continued for 1 h and rRNA was rcLqovcd by centrifugation at 27{}{){}x g for ll} rain. The pellet was rccxtractcd for an additional 2{}-3{I min with 67c~ acetic acid {v/v}, 0.1 M magnesium acetate, and the acetic extracts from both centrilugations were combined. The ribosomal proteins wcrc concentrated from the acetic acid extracts by acetone precipitation.
Preparation of ribosomal proteins for eh'ctrophoresis. Solutions of ribosomal proteins suitable for elcctrophoretic separation of proteins wcrc prcparcd from acetone precipitates by resuspension in buffer for Laemmli gels [19] and heating at t}{}°C for 1(} rain. When isoclectr;c focusing or two-dimensional separation was required, acetone precipitates were resuspended in 6 M urea and 2¢~- ampholines {w/vl from 3.5 -5. Gel electrophoresis. Electrophoresis was carried out in sodium dodecyl sulphate, 5-2{}% polyacrylamide {w/v} linear gradient slab gels, according to Laemmli
[19]. lsoelectric fi}cusing. Isoelcctric focusing was performed in 5ok polyacrylamide {w/v} slab gels with 6 M urea and 2% LKB ampholines {w/v) between pH 3.5-5. Gels were run in a Multiphore System {LKB), using l f~- sulfuric acid (v/v) and 0.113 M sodium hydroxide in the electrode strips, according to the procedure described by Vidales et al. [2]. At the end of the run, the actual pH gradient ranged from pH 3 to 7, as determined by monitoring pH values along the slab gels, Two-dbnensional electrophoresis. Two-dimensional separation of proteins was performed according to O'Farreli [20]. The first dimension consisted of isoelcctric focusing in cylindrical gels, using the conditions described above, after equilibrating in 80 mM Tris-C1H (pH 6.8), 2% SDS (w/v), I0 mM 2-mercaptoethanol and 0.004c~. bromofenot blue (w/v) during 2-3 h at room temperature. Separation in the second dimension was by molecular weight on sodium dodecyI sulphate, 5-20% lincar polyacrylamide (w/v) gradient gels.
Protein kinase assay. Thc standard incubation mixture contained in 50 /,!: ILl M Tris-HCI (pH 7.5), 5 mM MgCI 2. 10-50 tzg of casein (or ribosomal proteins). (I.05 mM [y-32P]ATP (specific activity 8(I-140 cpm pmol ~), and kinase. Purification of caseht kinase. Purified fractions of type 11 casein kinase were obtained from vegetative l)ictyostelium nuclei or soluble protein fractions, as dcscribcd previously [21 ]. 7)'eatment of acidic rthosomal proteins with alkaline phosphatase. This treatment was carried out according to the procedure dcscribcd by van Agthovcn ct al. [22]. 15 ~.g of acidic proteins werc rcsuspendcd in 1 mM ammonium carbonate (pH 8.7), and were incubated with 5(I U of calf intestine alkaline phosphatasc (Sigma) at 37°C for ! h. After inct, bation, proteins wcrc recovered by lyophilization. Protein &'termim~tion. Protein concentration was detcrmincd by thc Bradford procedure [23]. using bovine serum albumin its a standard. Antisera and imnmntJh~.eic,tl r('u('tion.~~ to #nmoi#lized ptvteins. Antisera dgainsl lJi('/vo.st('littnt r i l ~ o s t ) m a l acidic proteins wcrc t~blaincd ttsing :l method reported by Springer ct al. [24]. ('oomltssic blt,c .,,rained protcins bands of 12(1()() from 5-2(Vi polyacrylamidc (w/v) gradient gcls of SP,,_, fractions wcrc cut out and injected in rabbits to obtain polyclonal antibodies. Before intramuscular injection, the ~,el slices were homogenized with 75C/- Frcund's adjuvant (v/v). The firsl immunization used Frcund's complete adjttvant, and subsequent in.jcctitms wcrc repeated weekly with Frcund's incomplete adjuvant. After five injections, rabbit scram was tested t'~)r anti-A-proteins reactivity. AntiA-proteins IgG was purified using the previously described method [25]. Electrophorctic transfer of protcins resolved in polyacrylamidc gels onto nitrocellulose sheets wits pcrl\)rmcd essentially as described [26]. After incubation of thc nitrocellulose, sheets with rabbit polyclonal antibody, the reaction was visualized by treatment with pcroxidasc-labclled goat anti-rabbit antibody (Nordic, Tilburg, Tlac Nctherlands). followed by incubation with substratc. Results
Identification oi" Dictyostefimn discoideum ribmomai acidic proteins Fractions of ribosomal proteins were extracted from l)ictvostelium ribosomes with 50~4 ethanol (v/v) and 0.2 M NHaCI washes (SP,~2 fractions). Under these conditions, the rnost acidic proteins from yeast ribosomes [25q and other cukaryotic organisms arc selectively rclcascd [27], and thc resulting particles (P,., cores) can bc used to reconstitute functional 8()S ribosomal particles upon addition of their split proteins [2].
I
2
3
-
-""
(*) I D (isoefectric focusingX-)
lad 92.5 K ~
bb,'eK~
.,:
~e"19tJt ElsevierScience Publishers B.V. All rights reserved [13(14-4165/t~t/$[13.511
BBAGEN 236(12
Dictyostelium discoideum acidic ribosomal phosphoproteins: Identification and in vitro phosphorylation Jesfis Prieto, E i e n a C a n d e l , M a r g a r i t a F e r n f i n d e z - R e n a r t
and Antonio Coloma
Departaownto de Bioqufmica. l"acultad de Medici~'a de la Unit'er.~idad Aut{mcmta de Madrid and Institttto ~h' hwestigaciotws Biott~tlicas del CSIC, Madrid tSpain)
{Received 19 April 1991)
Key words: R;bt~somal:,cidicpht~spht~protcin;Casein kinase type ii: In vitro phosphorylation:( D. di.~cokh.um
Four acidic phosphoproteins from the ribosomes of the slime mold Dictyostelium discoideum have been identified and partially characterized. These proteins are selectively released from ribosomal particles by salt/ethanol washes, have low molecular weight and acidic p l, and tend to aggregate in solution to form homodimers. These features correspond to proteins of different origins that have been included in the conserved family of eukaryotic A-ribosomal proteins, and, therefore, we have named them Dictyostelium ribosomal proteins AI, A2, A3 and A4. We also demonstrate that Dictyostelium ribosomal A-proteins are specifically phosphorylated in vitro by a type !i casein kinase previously identified in Dictyostelium. lsoelectric focusing separation has permitted us to identify four proteins (or P-proteins) that may consist of the phosphorylated forms of A-proteins. A-proteins from Dictyostelium and yeast do not present immunological cross-reactivity. Dictyostelium A-proteins contain, therefore, some specific features in their amino acid sequence that distinguish them from other members of the conserved eukaryoti¢ A-protein family; this conclusion is coherent with data deduced from the nucleotide sequence of eDNA clones encoding two Dictyostelium A-proteins (PI and P2) which we have recently reported.
Introduction Acidic proteins have been identified in the large ribosomal subunit from diverse species [1,2]. These proleins, called A-proteins (acidic) or P-proteins (the phosphorylated eukaryotic A proteins), appear to be involved in the interaction of supernatant factors with the ribosome [3,4] in the translation of the genetic message, In E s c h e r i c h i a coli ribosomes, two acidic proteins (L7 and LI2) were identified which correspond to a unique polypeptide in which the amino terminus can appcar either in free (protein L12) or acetylated (L7) form. They form a complex in which two L 7 / L I 2 dimers arc mounted on a single L10 ribosomal protein, constituting a characteristic structure on the large ribosomal subunit [5]. In higher organisms, two proteins analogous to L 7 / L I 2 are generally tk~und. The A eukaryotic pro-
('orrespt~ndencc: A. Coloma, Dcpartamento de Bioquimica, FaculIr.td de Mcdicinade la UnivcrsidadAut6noma dc Madrid. Arzobispo Morcillo. 4. 2802~)Madrid, Spain.
teins are coded by independent genes, have a molecular weight of about 12000 and show important analogies in their amino acid sequence [6-8]. These two proteins interact with eukaryotic elongation factors EFI and EF2, and are required for aminoacyl-tRNA binding, EF2-dependent GTPase activity and polypeptide synthesis [4,9]. Yeast ribosomes contain more than two acidic proteins: one laboratory has already characterized three members of the A-protein family in S a c c h a r o m y c e s ceret'isiae [9] and their gene sequences have been determined [10], while another group has reported the amino acid sequence of an acidic ribosomal protein from yeast [11] which differs from the three mentioned above. The cukaryotic A-proteins contain a 22 amino acid sequence near the carboxyl end of the protein which is highly conserved among eukaryotes [7,10]. The C-terminal sequence includes an epitope recognized by autoantibodies from systemic lupus erythematosus patients [12]. We report here the identification of four proteins from ribosomes of the lower cukaryote D i c t y o s t e l i u m d i s c o i d e u m which we consider as members of the Aprotein family, according to their characteristics of size, p i values, phosphorylated state and tendency to
form dimers in solution. The proteins identified in this work are specifically phosphorylatcd by a type !I casein kinase from Dictyostelium. Several observations, such as the higher p l values determined fi~r the Dictyostelium A-proteins, or the absence of immunological cross-reactivity between these proteins and acidic ribosoma.', proteins from yeast, reflect the existence of distinctive features in Dictyostelium A-proteins. The data presented corroborate those obtained by analysis of the primary, structure deduced from the nucleotide sequence tff eDNA clones encoding two of thcsc Dictyostelium A-proteins: P I and P2, which we have reported recently [13,14]. Materials
and
Methods
Materials, All reagents used were of chcmical analytical grade. Acrylamide, methylenebisacrylamide, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine (Tcmed), all of elcctrophoresis purity were obtained from Bio-Rad laboratories. Ampholincs 3.5-5 were from LKB. Urea and sucrose wcre ultrapure grades from Schwarz/Mann. [y-~2p]ATP was purchased from Amersham. Tosyllysylchloromethyiketone, casein, nucleotides, sodium o"thovanadate and alkaline phosphatase were supplied by Sigma Chemicals. Yeast ribosomal acidic protein samples, as well as polycional antibodies against these proteins, were the generous gift of J.P.G. Ballesta. Organism and growth conditions. Dict.vostelitvn discoideum strain AX-2 was grown in axenic HL-5 medium
[15]. Ribosomes. Standard procedures were used to lyse Dic~ostelium cells and to pellet ribosomal particles [16], Purification of ribosomes was carried out by resuspension of ribosomal pellets in ().5 M ammonium chloride, 311 mM Tris-HCI (pH 7.2), 0.1 M magnesium acetate and 5 mM 2-mercaptocthanol, This suspension of ribosomes was kept at 0°C for 1-3 h with gentle stirring and was then centrifuged through discontinuous sucrose gradients [17] in tl.5 M ammonium chloride, Ill mM Tris-HCI (pH 7.8), 50 mM KCI, 10 mM magnesium acetate and 5 mM 2-mercaptocthanol. The resulting pellets were resuspended in 80 mM KCI. 10 mM magnesium acetate, 20 mM Tris-HCl, and stored at - 7 0 ° C . The steps of ribosome purification as well as the extraction of ribosomal proteins described below were always performed in the presence of 500 p,M phenylmethanesulfonyl fluoride and 5(1 g M tosyllysylchloromethyl ketone, to prevent proteolysis. When indicated, 25(1 ~tM sodium orthovanadate was also added to inhibit phosphatases.
Ribosomal spill .tax,rein (SPa ,_ fr:wtion) and ribosomal core partich's (Po_, cores). Purified Dictyostelium ribo-
somes (4 m g / m l ) were treated with 0.2 M ammonium chloride and 5()% ethanol (v/v) at 0°C t\~r 15 min, as described previously [18]. The particlcs (Pc~._, corcs) were recovered by centrifugation at 12000 x g for 15 rain, and :,:suspended in I0 mM Tris-HC1 (pl-t 7.4), t0 mM magnc,~,um acetate and fiO mM KC1 at approx. 2(I mg/ml, l'hc split protein fractions (SPql,), contaiaing mainly the acidic ribosomal proteins, were prccipi,atcd by addition of 2.25 vol. of acetone at - 2i)°C. I-xtraction ~J.!rihosomat p.,~teins. Extraction of ribosomal proteins from 8()S ribosomal particles or P,,_, cores was performed as described by Ramagopai and Ennis [1{}]. Ribosomes or P~2 cores (5{1-2{1{} ,q_,f~} units/ml) wcrc adjusted to {1.i M magnesiur~ acctate, and 2.2 vol. of glacial acetic acid were added dropwise with constant stirring in the cold. Stirring was continued for 1 h and rRNA was rcLqovcd by centrifugation at 27{}{){}x g for ll} rain. The pellet was rccxtractcd for an additional 2{}-3{I min with 67c~ acetic acid {v/v}, 0.1 M magnesium acetate, and the acetic extracts from both centrilugations were combined. The ribosomal proteins wcrc concentrated from the acetic acid extracts by acetone precipitation.
Preparation of ribosomal proteins for eh'ctrophoresis. Solutions of ribosomal proteins suitable for elcctrophoretic separation of proteins wcrc prcparcd from acetone precipitates by resuspension in buffer for Laemmli gels [19] and heating at t}{}°C for 1(} rain. When isoclectr;c focusing or two-dimensional separation was required, acetone precipitates were resuspended in 6 M urea and 2¢~- ampholines {w/vl from 3.5 -5. Gel electrophoresis. Electrophoresis was carried out in sodium dodecyl sulphate, 5-2{}% polyacrylamide {w/v} linear gradient slab gels, according to Laemmli
[19]. lsoelectric fi}cusing. Isoelcctric focusing was performed in 5ok polyacrylamide {w/v} slab gels with 6 M urea and 2% LKB ampholines {w/v) between pH 3.5-5. Gels were run in a Multiphore System {LKB), using l f~- sulfuric acid (v/v) and 0.113 M sodium hydroxide in the electrode strips, according to the procedure described by Vidales et al. [2]. At the end of the run, the actual pH gradient ranged from pH 3 to 7, as determined by monitoring pH values along the slab gels, Two-dbnensional electrophoresis. Two-dimensional separation of proteins was performed according to O'Farreli [20]. The first dimension consisted of isoelcctric focusing in cylindrical gels, using the conditions described above, after equilibrating in 80 mM Tris-C1H (pH 6.8), 2% SDS (w/v), I0 mM 2-mercaptoethanol and 0.004c~. bromofenot blue (w/v) during 2-3 h at room temperature. Separation in the second dimension was by molecular weight on sodium dodecyI sulphate, 5-20% lincar polyacrylamide (w/v) gradient gels.
Protein kinase assay. Thc standard incubation mixture contained in 50 /,!: ILl M Tris-HCI (pH 7.5), 5 mM MgCI 2. 10-50 tzg of casein (or ribosomal proteins). (I.05 mM [y-32P]ATP (specific activity 8(I-140 cpm pmol ~), and kinase. Purification of caseht kinase. Purified fractions of type 11 casein kinase were obtained from vegetative l)ictyostelium nuclei or soluble protein fractions, as dcscribcd previously [21 ]. 7)'eatment of acidic rthosomal proteins with alkaline phosphatase. This treatment was carried out according to the procedure dcscribcd by van Agthovcn ct al. [22]. 15 ~.g of acidic proteins werc rcsuspendcd in 1 mM ammonium carbonate (pH 8.7), and were incubated with 5(I U of calf intestine alkaline phosphatasc (Sigma) at 37°C for ! h. After inct, bation, proteins wcrc recovered by lyophilization. Protein &'termim~tion. Protein concentration was detcrmincd by thc Bradford procedure [23]. using bovine serum albumin its a standard. Antisera and imnmntJh~.eic,tl r('u('tion.~~ to #nmoi#lized ptvteins. Antisera dgainsl lJi('/vo.st('littnt r i l ~ o s t ) m a l acidic proteins wcrc t~blaincd ttsing :l method reported by Springer ct al. [24]. ('oomltssic blt,c .,,rained protcins bands of 12(1()() from 5-2(Vi polyacrylamidc (w/v) gradient gcls of SP,,_, fractions wcrc cut out and injected in rabbits to obtain polyclonal antibodies. Before intramuscular injection, the ~,el slices were homogenized with 75C/- Frcund's adjuvant (v/v). The firsl immunization used Frcund's complete adjttvant, and subsequent in.jcctitms wcrc repeated weekly with Frcund's incomplete adjuvant. After five injections, rabbit scram was tested t'~)r anti-A-proteins reactivity. AntiA-proteins IgG was purified using the previously described method [25]. Electrophorctic transfer of protcins resolved in polyacrylamidc gels onto nitrocellulose sheets wits pcrl\)rmcd essentially as described [26]. After incubation of thc nitrocellulose, sheets with rabbit polyclonal antibody, the reaction was visualized by treatment with pcroxidasc-labclled goat anti-rabbit antibody (Nordic, Tilburg, Tlac Nctherlands). followed by incubation with substratc. Results
Identification oi" Dictyostefimn discoideum ribmomai acidic proteins Fractions of ribosomal proteins were extracted from l)ictvostelium ribosomes with 50~4 ethanol (v/v) and 0.2 M NHaCI washes (SP,~2 fractions). Under these conditions, the rnost acidic proteins from yeast ribosomes [25q and other cukaryotic organisms arc selectively rclcascd [27], and thc resulting particles (P,., cores) can bc used to reconstitute functional 8()S ribosomal particles upon addition of their split proteins [2].
I
2
3
-
-""
(*) I D (isoefectric focusingX-)
lad 92.5 K ~
bb,'eK~
.,:
