Asymmetric Interactions between the Acidic P1 and P2 Proteins in the Saccharomyces cerevisiae Ribosomal Stalk
2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês
10.1074/jbc.m103229200
ISSN1083-351X
AutoresEsther Guarinos, Miguel Remacha, Juan P. G. Ballesta,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe Saccharomyces cerevisiaeribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1α, P1β, P2α, and P2β. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1α, P0-1β, P0-2α, and P0-2β. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1α·P2β, P0-1β·P2α, P0-2α·P1β, and P0-2β·P1α. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1α·P2β and P1β·P2α, which have different structural and functional roles. The Saccharomyces cerevisiaeribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1α, P1β, P2α, and P2β. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1α, P0-1β, P0-2α, and P0-2β. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1α·P2β, P0-1β·P2α, P0-2α·P1β, and P0-2β·P1α. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1α·P2β and P1β·P2α, which have different structural and functional roles. The ribosomal stalk is an important structural element of the large ribosomal subunit directly associated with the interaction of the elongation factors during the protein synthesis elongation step in bacteria (for a review, see Ref. 1Möller W. Maassen J.A. Hardesty B. Kramer G. Structure, Function and Genetics of Ribosomes. Springer-Verlag, New York1986: 309-325Google Scholar). A direct confirmation of this association has recently been shown by cryoelectron microscopy (2Stark H. Rodnina M.V. Rinke-Appel J. Brimacombe R. Wintermeyer W. van Heel M. Nature. 1997; 389: 403-406Crossref PubMed Scopus (312) Google Scholar, 3Agrawal R.K. Penzek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar). Involvement of the stalk components has also been reported in initiation (4Kay A. Sander G. Grunberg-Manago M. Biochem. Biophys. Res. Commun. 1973; 51: 979-986Crossref PubMed Scopus (30) Google Scholar, 5Fakunding J.L. Traut R.R. Hershey J.W.B. J. Biol. Chem. 1973; 248: 8555-8559Abstract Full Text PDF PubMed Google Scholar) and termination (6Moffat J.G. Timms K.M. Trotman C.N. Tate W.P. 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Biochem. 1999; 262: 606-611Crossref PubMed Scopus (76) Google Scholar), which in the case of Saccharomyces cerevisiae EF-2 has been confirmed by electron microscopy (12Gómez-Lorenzo M.G. Spahn C.M.T. Agrawal R.K. Grassucci R.A. Penczek P. Chakraburtty K. Ballesta J.P.G. Lavandera J.L. Garcia-Bustos J.F. Frank J. EMBO J. 2000; 19: 2710-2718Crossref PubMed Scopus (147) Google Scholar). In addition to this function, the eukaryotic stalk, at least the yeast stalk, might participate in a translation regulatory mechanism not reported in bacteria (13Ballesta J.P.G. Remacha M. Prog. Nucleic Acid Res. Mol. Biol. 1996; 55: 157-193Crossref PubMed Google Scholar). The bacterial stalk is made of protein L10 and two dimers of proteins L7/L12, the amino-terminal acetylated and nonacetylated forms of a unique polypeptide. The pentamer L10-((L7/L12)2)2 is extraordinarily stable (14Pettersson I. Liljas A. FEBS Lett. 1979; 98: 139-144Crossref PubMed Scopus (27) Google Scholar) and binds directly to the highly conserved GTPase-related site in the 23 S rRNA (15Beauclerk A.D. Cundliffe E. Dijk J. J. Biol. Chem. 1984; 259: 6559-6563Abstract Full Text PDF PubMed Google Scholar, 16Egebjerg J. Douthwaite S.D. Liljas A. Garrett R.A. J. Mol. Biol. 1990; 213: 275-288Crossref PubMed Scopus (123) Google Scholar) through the L10 amino-terminal domain (17Gudkov A.T. Tumanova L.G. Gongadze G.M. Bushuev U.N. FEBS Lett. 1980; 109: 34-38Crossref PubMed Scopus (39) Google Scholar). Although the three-dimensional structure of the bacterial acidic proteins has recently been resolved (18Wahl M.C. Bourenkov G.P. Bartunik H.D. Huber R. EMBO J. 2000; 19: 174-186Crossref PubMed Scopus (79) Google Scholar), the detailed structure of the stalk protein complex is unknown. In fact, the ribosomal stalk is not present in the recently reported 2.3-Å resolution atomic structure of the large ribosomal subunit probably because of its high flexibility (19Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2796) Google Scholar). Nevertheless a symmetric structure for the pentameric complex has been proposed (20Bocharov E.V. Gudkov A.T. Budovskaya E.V. Arseniev A.S. FEBS Lett. 1998; 423: 347-350Crossref PubMed Scopus (32) Google Scholar), although the function of the two L7/L12 dimers in the complex might not be the same (21Traut R.R. Dey D. Bochkarlov D.E. Oleinikov A.V. Jokhadze G.G. Hamman B.D. Jameson D.M. Biochem. Cell Biol. 1995; 73: 949-958Crossref PubMed Scopus (56) Google Scholar), and a physical separation of both dimers has been proposed (22Matadeen R. Patwardhan A. Gowen B. Orlova E.V. Pape T. Cuff M. Mueller F. Brimacombe R. van Heel M. Struct. Fold. Des. 1999; 7: 1575-1583Abstract Full Text Full Text PDF Scopus (116) Google Scholar). The new role proposed for the ribosomal stalk in eukaryotic organisms correlates with a higher structural complexity and dynamism. Proteins P0 and P1/P2 are the eukaryotic counterparts of L10 and L7/L12, respectively. As in bacteria, one copy of P0 and four copies of P1/P2 seem to form a complex (23Uchiumi T. Wahba A.J. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5580-5584Crossref PubMed Scopus (115) Google Scholar). However, the eukaryotic pentamer is less stable and, in contrast to the bacterial pentamer, is readily disassembled by treating the ribosome with ammonium/ethanol buffers (8Sanchez-Madrid F. Reyes R. Conde P. Ballesta J.P.G. Eur. J. Biochem. 1979; 98: 409-416Crossref PubMed Scopus (95) Google Scholar). The amino acid sequence similarity of the prokaryotic and eukaryotic stalk components is rather low (24Shimmin L.C. Ramirez G. Matheson A.T. Dennis P.P. J. Mol. Evol. 1989; 29: 448-462Crossref PubMed Scopus (79) Google Scholar). Their structural differences are especially remarkable in the case of P0, which is larger than L10 and contains a carboxyl-end extension of around 100 amino acids not present in the bacterial polypeptide (24Shimmin L.C. Ramirez G. Matheson A.T. Dennis P.P. J. Mol. Evol. 1989; 29: 448-462Crossref PubMed Scopus (79) Google Scholar). This extension resembles the structure of the acidic P1/P2 proteins, containing a flexible hinge and the same highly conserved 13-amino acid terminal peptide (see Fig. 1). In fact, the P0 carboxyl extension plays the same role as the 12-kDa proteins when these acidic proteins are not present in the ribosome (25Santos C. Ballesta J.P.G. J. Biol. Chem. 1995; 270: 20608-20614Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 26Remacha M. Santos C. Bermejo B. Naranda T. Ballesta J.P.G. J. Biol. Chem. 1992; 267: 12061-12067Abstract Full Text PDF PubMed Google Scholar). As a consequence of this functional similarity, the proteins P1/P2, in contrast to bacterial L7/L12, are not essential for ribosome activity and cell viability (27Remacha M. Jimenez-Diaz A. Bermejo B. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Mol. Cell. Biol. 1995; 15: 4754-4762Crossref PubMed Google Scholar). The acidic proteins have also evolved notably. In eukaryotes they are found as a set of genetically independent polypeptides, which can be grouped in two families, P1 and P2, and are made up of a different number of components depending on the species. Only one protein of each type has been found in mammals, insects, and fungi (28Möller W. Slobin L.I. Amons R. Richter D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4744-4748Crossref PubMed Scopus (54) Google Scholar, 29Lin A. Wittman-Leibold B. McNelly T. Wool I.G. J. Biol. Chem. 1982; 257: 9189-9197Abstract Full Text PDF PubMed Google Scholar, 30Prieto J. Candel E. Fernandez-Renart M. Coloma A. Biochim. Biophys. Acta. 1991; 115: 6-14Crossref Scopus (4) Google Scholar), whereas in protozoa (31Schijman A.G. Vazquez M.P. Ben Dov C. Ghio S. Lorenzi H. Levin M.J. Biochim. Biophys. Acta. 1995; 1264: 15-18Crossref PubMed Scopus (7) Google Scholar) and yeast (32Newton C.H. Shimmin L.C. Yee J. Dennis P.P. J. Bacteriol. 1990; 172: 579-588Crossref PubMed Google Scholar, 33Beltrame M. Bianchi M.E. Mol. Cell. Biol. 1990; 10: 2341-2348Crossref PubMed Scopus (36) Google Scholar) there are several. In plants, a third type of acidic protein, P3, has been reported (34Szick K. Springer M. Bailey-Serres J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2378-2383Crossref PubMed Scopus (52) Google Scholar). The presence of several acidic proteins of the same type in the stalk raises a number of questions regarding their respective structural and functional roles. In S. cerevisiae there are four acidic proteins, two of the P1 type, P1α and P1β, and two of the P2 type, P2α and P2β. The estimation of a total of four copies of acidic proteins per yeast ribosome (35Saenz-Robles M.T. Remacha M. Vilella M.D. Zinker S. Ballesta J.P.G. Biochim. Biophys. Acta. 1990; 1050: 51-55Crossref PubMed Scopus (67) Google Scholar) seems to exclude the presence of one dimer of each polypeptide as reported in eukaryotes having only one acidic protein of each type, P1 and P2 (23Uchiumi T. Wahba A.J. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5580-5584Crossref PubMed Scopus (115) Google Scholar). There is some evidence suggesting that the acidic proteins are present as monomers in theS. cerevisiae ribosomal stalk (36Ballesta J.P.G. Guarinos E. Zurdo J. Parada P. Nusspaumer G. Lalioti V.S. Perez-Fernandez J. Remacha M. Garrett R. Douthwaite S. Matheson A. Liljas A. Moore P.B. Noller H.F. The Ribosome. Structure, Function, Antibiotics and Cellular Interactions. American Society for Microbiology, Washington, D. C.2000: 115-125Google Scholar), although this issue is still not totally settled because at least the P2 proteins are able to form dimers in solution (37Zurdo J. Sanz J.M. Gonzalez C. Rico M. Ballesta J.P.G. Biochemistry. 1997; 36: 9625-9635Crossref PubMed Scopus (43) Google Scholar). In addition, a number of questions regarding the mutual relationship between different stalk components are still open. In an attempt to explore further the structural and functional role of the P0 carboxyl-end domain as well as its interaction with the 12-kDa acidic proteins, a series of chimeras were prepared that carry a whole acidic protein replacing the last 112 amino acids forming the original P0 carboxyl terminus. These artificial constructs have been shown to be functional in yeast, and the results obtained from strains expressing them have provided relevant information on the structure of theS. cerevisiae ribosomal stalk. S. cerevisiae W303dGP0 (MAT α,leu2-3, 112, ura3-1,trp1-1, his3-11, 15,ade2-1, can1-100,RPP0::URA3-GAL1-RPP0) and S. cerevisiaeD67dGP0 (MAT α, leu2-3, 112, ura3-1,trp1-1, his3-11, 15,ade2-1, can1-100,RPP1A::LEU2, RPP1β::TRP1,RPP0::URA3-GAL1-RPP0) were derived from S. cerevisiae W303 and D67, respectively, by integration through homologous recombination in the RPP0 locus of a construct carrying the P0 coding region fused to theGAL1 promoter (38Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Abstract Full Text PDF PubMed Google Scholar). D45dGP0 (MAT α, leu2-3,112, ura3-1, trp1-1,his3-11, 15, ade2-1,can1-100, RPP2A::URA3,RPP2B::HIS3,RPP0::kanMx4-GAL1-RPP0) and D4567dGP0 (MAT α,leu2-3, 112, ura3-1,trp1-1, his3-11, 15,ade2-1, can1-100,RPP1A::LEU2, RPP1B::TRP1,RPP2A::URA3, RPP2B::HIS3,RPP0::kanMx4-GAL1-RPP0) were constructed in a similar way from S. cerevisiae D45 and D4567 (27Remacha M. Jimenez-Diaz A. Bermejo B. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Mol. Cell. Biol. 1995; 15: 4754-4762Crossref PubMed Google Scholar), respectively, but using a gentamycin resistance gene as a genetic marker for the P0 gene replacement. Yeasts were grown in either YEP medium (1% yeast extract, 2% peptone) or minimal YNB medium (0.67% yeast nitrogen base, 2% carbon source) supplemented with the necessary nutritional requirements. In both cases, the carbon source was either 2% glucose or 2% galactose as required. Escherichia coli DH5α was used for the maintenance and preparation of plasmids and was grown in LB medium. Restriction endonucleases were purchased from Roche Molecular Biochemicals, MBI Fermentas, New England Biolabs, and Amersham Pharmacia Biotech and were used as recommended by the suppliers. T4 DNA ligase, calf intestinal alkaline phosphatase, and the DNA polymerase I Klenow fragment were from Roche Molecular Biochemicals. DNA manipulations were performed basically as described previously (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Polymerase chain reaction was carried out using Pfu DNA polymerase from Stratagene and custom-made oligonucleotides from Isogen following the recommendations of Dieffenbach and Dveksler (40Dieffenbach C.W. Dveksler G.S. PCR Primer. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995Google Scholar). Bacterial transformations were performed according to the procedure of Hanahan (41Hanahan D. Glover D.M. DNA Cloning: A Practical Approach. IRL Press, Oxford1985: 109-136Google Scholar). Yeasts were transformed using the lithium acetate method as described previously (42Hill H. Donald I.G. Griffiths D.E. Nucleic Acids Res. 1991; 19: 5791Crossref PubMed Scopus (459) Google Scholar). Using appropriate oligonucleotides as primers, the genes RPP1A,RPP2A, and RPP2B encoding the acidic protein P1α, P2α and P2β, respectively, were obtained by polymerase chain reaction using Pfu polymerase from plasmids in which they were previously cloned (32Newton C.H. Shimmin L.C. Yee J. Dennis P.P. J. Bacteriol. 1990; 172: 579-588Crossref PubMed Google Scholar, 43Remacha M. Saenz-Robles M.T. Vilella M.D. Ballesta J.P.G. J. Biol. Chem. 1988; 263: 9094-9101Abstract Full Text PDF PubMed Google Scholar). At the same time, a newNheI restriction site was introduced downstream from the termination codon (see Fig. 1). On the other hand, taking advantage of the presence in the RPP0 gene of an EcoRV site in the position corresponding to amino acids 203 and 204 and aNheI site in the 3′ region, the coding sequence encoding the last 112 residues was removed from RRP0 in plasmid BSP0 (38Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Abstract Full Text PDF PubMed Google Scholar). Afterward the corresponding polymerase chain reaction fragments encoding the acidic proteins were subcloned in the same sites of BSP0 using the NheI site in one end and blunt end ligation in the other. The chimeric gene was then subcloned as a 2.9-kilobase pair EcoRI-XhoI fragment in the centromeric pFL37 plasmid, which was derived from pFL38 (44Bonneaud N. Ozier-Kalogeropoulos O. Li G. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (500) Google Scholar) as reported previously (45Rodriguez-Gabriel M.A. Remacha M. Ballesta J.P.G. J. Biol. Chem. 2000; 275: 2130-2136Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). A similar cloning strategy was used in this case, but the intron present in the P1β protein-encoding RPP1B gene (43Remacha M. Saenz-Robles M.T. Vilella M.D. Ballesta J.P.G. J. Biol. Chem. 1988; 263: 9094-9101Abstract Full Text PDF PubMed Google Scholar) was removed beforehand by overlap extension polymerase chain reaction (46Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6830) Google Scholar). Yeasts were grown exponentially in rich YEP medium up toA 600 = 0.6, and cells were collected by centrifugation and washed with buffer 1 (100 mm Tris-HCl, pH 7.4, 20 mm KCl, 12.5 mm MgCl2, 5 mm β-mercaptoethanol). Cells in buffer 1 were supplemented with protease inhibitors (0.5 µmol of phenylmethylsulfonyl fluoride, 1.25 µg of leupeptin, aprotinin, and pepstatin/g of cells) and broken with glass beads. The extract was centrifuged in a Beckman SS-34 rotor (12,000 rpm, 15 min, 4 °C) yielding the supernatant S30 fraction, which was afterward submitted to high speed centrifugation at 90,000 rpm for 30 min at 4 °C in a Beckman TL100.3 rotor. The supernatant S100 fraction was stored at −80 °C, and the crude ribosome pellet was resuspended in buffer 2 (20 mm Tris-HCl, pH 7.4, 500 mmNH4Ac, 100 mm MgCl2, 5 mm β-mercaptoethanol). When required, ribosomes were centrifuged through a discontinuous sucrose gradient (20%, 40%) in buffer 2 at 90,000 rpm for 120 min at 4 °C in a TL100.3 rotor. The pellet of washed ribosomes was dissolved in buffer 1 and stored at −20 °C. Ribosomal proteins were analyzed either by 15% SDS-polyacrylamide gel electrophoresis or by isoelectrofocusing. Isoelectrofocusing was carried out as described previously (47Zambrano R. Briones E. Remacha M. Ballesta J.P.G. Biochemistry. 1997; 36: 14439-14446Crossref PubMed Scopus (62) Google Scholar). Particles were pretreated with RNase A (10 µg/mg of ribosomes) on ice for 30–45 min. After lyophilization, the samples were resuspended in a loading buffer (6% ampholytes, 8 m urea) and directly loaded into a standard vertical gel (5% acrylamide, 0.2% bisacrylamide, 6 m urea, 6% pH 2.5–5.0 ampholytes). 30 mm NaOH and 180 mmH2SO4 were used as cathode and anode solutions at the upper and lower part of the gel, respectively. Isoelectrofocusing was run in the cold room at 6-mA constant current until the voltage reached 600 V and then was lowered to 250 V for 16 h. Proteins were usually detected by standard silver staining. Alternatively gels were stained in a solution containing 0.25% Coomassie R-250 (Sigma) dissolved in 45% ethanol, 10% acetic acid. After 30 min, the gel was destained using the same solution without stain. Proteins in gels were transferred to membranes, which were treated with 5% skimmed milk dissolved in TBS (10 mm Tris-HCl, pH 7.4, 200 mm NaCl) for 30 min, and afterward they were incubated for 1 h with the antibody diluted in the same buffer. Subsequently the membranes were washed for 15 min in TBS containing 5% skimmed milk and 0.1% Tween 20, and then the second antibody (RαM/PO or DαR/PO), diluted in the former buffer, was added and incubated for 30 min. Finally the membrane was washed for 15 min with 0.1% Tween 20 in TBS. Bound antibodies were located by detecting peroxidase activity using the ECL system (Amersham Pharmacia Biotech) and then exposed to film. Specific antibodies to the different P proteins were described previously (38Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Abstract Full Text PDF PubMed Google Scholar, 48Vilella M.D. Remacha M. Ortiz B.L. Mendez E. Ballesta J.P.G. Eur. J. Biochem. 1991; 196: 407-414Crossref PubMed Scopus (62) Google Scholar). The reaction was performed in 50-µl samples containing 10 pmol of 80 S ribosomes, 5 µl of S100, 0.5 mg/ml tRNA, 0.3 mg/ml polyuridylic acid, 40 mm [3H]phenylalanine (120 cpm/pmol), 0.5 mm GTP, 1 mm ATP, 2 mm phosphocreatine, and 40 mg/ml creatine phosphokinase in 50 mm Tris-HCl, pH 7.6, 15 mmMgCl2, 90 mm KCl, 5 mmβ-mercaptoethanol. After incubation at 30 °C for 30 min, samples were precipitated with 10% trichloroacetic acid, boiled for 10 min, and filtered through glass fiber filters. Four protein P0 chimeras were constructed, P0-1α, P0-1β, P0-2α, and P0-2β in which the last 112 residues in the P0 amino acid sequence were replaced by each one of the acidic proteins, P1α, P1β, P2α, and P2β (Fig.1). The overall amino acid sequence identity of the acidic proteins and the replaced P0 segment ranges from 31.5 to 35.4%, which is reduced to around 25% if the identical last 13 residues are excluded. In addition, an alanine- and glycine-rich region, equivalent to the hinge of the eukaryotic acidic proteins (24Shimmin L.C. Ramirez G. Matheson A.T. Dennis P.P. J. Mol. Evol. 1989; 29: 448-462Crossref PubMed Scopus (79) Google Scholar,37Zurdo J. Sanz J.M. Gonzalez C. Rico M. Ballesta J.P.G. Biochemistry. 1997; 36: 9625-9635Crossref PubMed Scopus (43) Google Scholar), is found at an equivalent position in the P0 fragment (Fig. 1). The chimeric genes, subcloned in the plasmids pFL37P0/1α, pFL37P0/1β, pFL37P0/2α, and pFL37P0/2β were used to transform theS. cerevisiae P0 conditional null strains W303dGP0, D4567dGP0, D45dGP0, and D67dGP0. A summary of the strains and plasmids used is shown in Table I. In these strains, the genomic P0 gene was replaced by a gene copy under the control of the GAL1 promoter (38Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Abstract Full Text PDF PubMed Google Scholar). Consequently the strains depend on the P0 gene in the transforming plasmid to be able to grow in glucose media. As a control, the strains were also transformed with pFL37P0, which contains a wild-type copy of the P0 gene. S. cerevisiae W303dGP0 contains the nuclear genes for the four acidic stalk proteins, whereas strain D4567dGP0 lacks the four genes. D67P0 and D45dGP0 lack the nuclear genes for both P1 (P1α/P1β) and both P2 (P2α/P2β) proteins, respectively (26Remacha M. Santos C. Bermejo B. Naranda T. Ballesta J.P.G. J. Biol. Chem. 1992; 267: 12061-12067Abstract Full Text PDF PubMed Google Scholar, 27Remacha M. Jimenez-Diaz A. Bermejo B. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Mol. Cell. Biol. 1995; 15: 4754-4762Crossref PubMed Google Scholar).Table ISummary of S. cerevisiae strains and plasmids used in this studyStrainGenomic proteins1-aProtein encoded in the genome of the yeast strain and expressed in the cell. The wild-typeRPP0 gene is under the control of the GAL1promoter in all cases (38).Transforming plasmidPlasmid proteins1-bProtein chimeras encoded in the transforming plasmid. The fused genes are under the control of the native 5′ and 3′ untranslated region from the native RPP0gene (see "Materials and Methods").W303dGP0P1α, P1β, P2α, P2β, P0pFL37P0P0W303dGP0/P1αP1α, P1β, P2α, P2β, P0pFL37P0/1αP0-1αW303dGP0/P1βP1α, P1β, P2α, P2β, P0pFL37P0/1βP0-1βW303dGP0/P2αP1α, P1β, P2α, P2β, P0pFL37P0/2αP0-2αW303dGP0/P2βP1α, P1β, P2α, P0pFL37P0/2βP0-2βD67dGP0P2α, P2β, P0pFL37P0P0D67dGP0/P1αP2α, P2β, P0pFL37P0/1αP0-1αD67dGP0/P1βP2α, P2β, P0pFL37P0/1βP0-1βD67dGP0/P2αP2α, P2β, P0pFL37P0/2αP0-2αD67dGP0/P2βP2α, P2β, P0pFL37P0/2βP0-2βD45dGP0P2α, P2β, P0pFL37P0P0D45dGP0/P1αP1α, P1β, P0pFL37P0/1αP0-1αD45dGP0/P1βP1α, P1β, P0pFL37P0/1βP0-1βD45dGP0/P2αP1α, P1β, P0pFL37P0/2αP0-2αD45dGP0/P2βP1α, P1β, P0pFL37P0/2βP0-2βD4567dGP0P0pFL37P0P0D4567dGP0/P1αP0pFL37P0/1αP0-1αD4567dGP0/P1βP0pFL37P0/1βP0-1βD4567dGP0/P2αP0pFL37P0/2αP0-2αD4567dGP0/P2βP0pFL37P0/2βP0-2β1-a Protein encoded in the genome of the yeast strain and expressed in the cell. The wild-typeRPP0 gene is under the control of the GAL1promoter in all cases (38Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Abstract Full Text PDF PubMed Google Scholar).1-b Protein chimeras encoded in the transforming plasmid. The fused genes are under the control of the native 5′ and 3′ untranslated region from the native RPP0gene (see "Materials and Methods"). Open table in a new tab All the transformed strains were able to grow on glucose agar plates as well as in liquid media (Table II), indicating that the gene chimeras can complement the absence of the wild-type P0 expression in a glucose medium. In D4567dGP0, complementation depends exclusively on the plasmid-encoded P0 protein because there are no acidic proteins expressed in this strain (27Remacha M. Jimenez-Diaz A. Bermejo B. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Mol. Cell. Biol. 1995; 15: 4754-4762Crossref PubMed Google Scholar). As expected from previous reports (27Remacha M. Jimenez-Diaz A. Bermejo B. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Mol. Cell. Biol. 1995; 15: 4754-4762Crossref PubMed Google Scholar), wild-type P0, expressed from the control pFL37P0, allowed cell growth in glucose. In this case, replacement of the P0 carboxyl-terminal domain by either protein P2α or P2β did not significantly affect the functionality of the resulting P0-2α and P0-2β proteins, whereas the expression of both P1 chimeras, either P0-1α or P0-1β, caused a small but clear reduction of the protein-complementing activity. In contrast, the two P0/P1 chimeras were less damaging than the P0/P2 derivatives inS. cerevisiae W303dGP0. In the double disruptants D45dGP0 and D67dGP0, a stimulation of cell growth occurred when the chimera provided the acidic protein type missing in the corresponding strain. Thus, P0-1α and P0-1β stimulated growth in the D67 P1-defective cells, whereas P0-2α and P0-2β stimulated growth in the D45 strain lacking both P2 proteins.Table IIGrowth of S. cerevisiae strains transformed with protein P0 chimerasPlasmid-encoded proteinDoubling time of the transformed strainsW303dGP0 (P1α, P1β, P2α, P2β)2-aRibosomal stalk proteins expressed from the genomic genes.D67dGP0 (P2α, P2β)2-aRibosomal stalk proteins expressed from the genomic genes.D45dGP0 (P1α, P1β)2-aRibosomal stalk proteins expressed from the genomic genes.D4567dGP0 (none)2-aRibosomal stalk proteins expressed from the genomic genes.minP0100170231172P0-1α125150233196P0-1β123136231192P0-2α160212200177P0-2β160215208173Cells were grown in YEP liquid medium with 2% glucose as a carbon source, and the A 600 of samples taken every 60 min was measured. The average error of these values is around 4%.2-a Ribosomal stalk proteins expressed from the genomic genes. Open table in a new tab Cells were grown in YEP liquid medium with 2% glucose as a carbon source, and the A 600 of samples taken every 60 min was measured. The average error of these values is around 4%. Isoelectrofocusing showed that ribosomes from W303dGP0 transformed with the plasmids expressing the P0 chimeras were deprived of most of the 12-kDa proteins (Fig.2A). Only one of them was detected in each case. Thus, proteins P2β, P2α, P1β, and P1α were found in cells expressing P0-1α, P0-1β, P0-2α, and P0-2β, respectively (Fig. 2). P1β was mainly present in a processed form called P1β′ (49Santos C. Ortiz-Reyes B.L. Naranda T. Remacha M. Ballesta J.P.G. Biochemistry. 1993; 32: 4231-4236Crossref PubMed Scopus (24) Google Scholar). No traces of the remaining proteins were found in the washed ribosomes. In contrast, acidic proteins accumulated free in the S100 supernatant fraction of the cells expressing the chimeras (Fig. 2 B). A similar analysis of the P2-defective D45dGP0 transformants showed that ribosomes carrying P0-2α only bound protein P1β, whereas those containing P0-2β exclusively bound P1α. The ribosomes from the same strain transformed with the P0-1α and P0-1β constructs did not contain any acidic protein in the ribosome (Fig.3A). In the case of the P1-defective D67dGP0-derived strains the isoelectrofocusing gels showed the presence of P2β and P2α in the ribosomes from cells carrying the P0-1α and P0-1β chimeras and the absence of acidic proteins in the particles having P0-2α and P0-2β (Fig. 3 B). Therefore, regardless of the yeast strain transformed with each P0 chimera, the same associations were always detected in the ribosome, namely P0-1α·P2β, P0-1β·P2α, P0-2α·P1β, and P0-2β·P1α as summarized in Fig.4. Alterations in the ribosomal stalk have previously been shown to induce some specific phenotypes when grown at 37 °C (47Zambrano R. Briones E. Remacha M. Ballesta J.P.G. Biochemis
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