FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2004090241> ?p ?o ?g. }

• W2004090241 endingPage "31961" @default.
• W2004090241 startingPage "31956" @default.
• W2004090241 abstract "Protein L12, together with the P0/P1/P2 protein complex, forms the protein moiety of the GTPase domain in the eukaryotic ribosome. In Saccharomyces cerevisiae protein L12 is encoded by a duplicated gene, rpL12A and rpL12B. Inactivation of both copies has been performed and confirmed by Southern and Western analyses. The resulting strains are viable but grow very slowly. Growth rate is recovered upon transformation with an intact copy of the L12 gene. Ribosomes from the disrupted strain lack protein L12 but are able to carry out translationin vitro at about one fourth of the control rate. The L12-deficient ribosomes have also a defective stalk containing standard amounts of the 12-kDa acidic proteins P1β and P2α, but proteins P1α and P2β are drastically reduced. Moreover, the affinity of P0 is reduced in the defective ribosomes. Footprinting of the 26 S rRNA GTPase domain indicates that protein L12 protects in different extent residues G1235, G1242, A1262, A1270, and A1272 from chemical modification. The results in this report indicate that protein L12 is not essential for cell viability but has a relevant role in the structure and stability of the eukaryotic ribosomal stalk. Protein L12, together with the P0/P1/P2 protein complex, forms the protein moiety of the GTPase domain in the eukaryotic ribosome. In Saccharomyces cerevisiae protein L12 is encoded by a duplicated gene, rpL12A and rpL12B. Inactivation of both copies has been performed and confirmed by Southern and Western analyses. The resulting strains are viable but grow very slowly. Growth rate is recovered upon transformation with an intact copy of the L12 gene. Ribosomes from the disrupted strain lack protein L12 but are able to carry out translationin vitro at about one fourth of the control rate. The L12-deficient ribosomes have also a defective stalk containing standard amounts of the 12-kDa acidic proteins P1β and P2α, but proteins P1α and P2β are drastically reduced. Moreover, the affinity of P0 is reduced in the defective ribosomes. Footprinting of the 26 S rRNA GTPase domain indicates that protein L12 protects in different extent residues G1235, G1242, A1262, A1270, and A1272 from chemical modification. The results in this report indicate that protein L12 is not essential for cell viability but has a relevant role in the structure and stability of the eukaryotic ribosomal stalk. dimethyl sulfate 2-keto-3-ethoxybutyraldehyde 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate 5-fluoro-orotic acid polymerase chain reaction base pair(s) polyacrylamide gel electrophoresis. The ribosomal region involved in the hydrolysis of the elongation factor-bound GTP molecule upon its interaction with the ribosome during translation is generally called the ribosomal GTPase domain. In bacteria, a number of elements, RNA and proteins, have been shown to participate in this process to a different extent (see Ref. 1Wilson K.S. Noller H.F. Cell. 1998; 92: 337-349Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar for a review). At least two RNA components have been identified, which include the α-sarcin loop in domain VI of the 23 S rRNA and a highly conserved double hairpin in domain II, frequently referred to as the RNA GTPase center (2Schmidt F.J. Thompson J. Lee K. Dijk J. Cundliffe E. J. Biol. Chem. 1981; 256: 12301-12305Abstract Full Text PDF PubMed Google Scholar, 3Egebjerg J. Douthwaite S.D. Liljas A. Garrett R.A. J. Mol. Biol. 1990; 213: 275-288Crossref PubMed Scopus (122) Google Scholar). Two proteins, L10 and L11, which bind to partially overlapping sites in the conserved domain II region, are also structural components of this active domain. On the other hand, protein L10 forms a very stable association with two dimers of the acidic protein L7/L12, the 6 m urea-resistant pentameric complex L10-(L7/L12)4, which is the main component of a typical protuberance of the large ribosomal subunit called the ribosomal stalk. The stalk is directly involved in the interaction of the elongation factors, as has been clearly shown by electron microscopy (4Stark H. Rodnina M.V. Rinke A.J. Brimacombe R. Wintermeyer W. van Heel M. Nature. 1997; 389: 403-406Crossref PubMed Scopus (311) Google Scholar), participating in the translocation mechanism. Protein L11, which binds to the rRNA in a cooperative way with pentameric protein complex L10-(L7/L12)4 (5Rosendahl G. Douthwaite S. J. Mol. Biol. 1993; 234: 1013-1020Crossref PubMed Scopus (74) Google Scholar), is important for the GTPase activity, and in determining the right conformation of the GTPase center (6Xing Y. Draper D.E. J. Mol. Biol. 1995; 249: 319-331Crossref PubMed Scopus (67) Google Scholar). This protein also has a key role in the mechanism by which thiostrepton and similar antibiotics block the elongation factor functions (7Cundliffe E. Dixon P. Stark M. Stöffler G. Ehrlich R. Stöffler-Meilicke M. Cannon M. J. Mol. Biol. 1979; 132: 235-252Crossref PubMed Scopus (60) Google Scholar, 8Porse B.T. Leviev I. Mankin A.S. Garrett R.A. J. Mol. Biol. 1998; 276: 391-404Crossref PubMed Scopus (101) Google Scholar). Protein L11 is not essential for cell viability, and L11-defective bacterial strains have been obtained that are also resistant to thiostrepton (7Cundliffe E. Dixon P. Stark M. Stöffler G. Ehrlich R. Stöffler-Meilicke M. Cannon M. J. Mol. Biol. 1979; 132: 235-252Crossref PubMed Scopus (60) Google Scholar).Despite the large amount of data accumulating on its structure and function, we are still far from understanding the molecular mechanism supporting the bacterial GTPase center function. Much less is obviously known in the case of the eukaryotic organisms. Prokaryotic and eukaryotic ribosomes carry out the same basic functions, and, consequently, data obtained about the former have usually been extrapolated to the latter. Although the individual components show substantial differences (P0 is larger than L10, and L7/L12 has evolved to two families of closely related proteins, P1 and P2), they play similar roles. In fact, some of the data available on the structure of the eukaryotic GTPase domain fit nicely with those from bacterial ribosomes. Thus, there is a pentameric complex, P0-(P1)2-(P2)2 (9Uchiumi T. Wahba A.J. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5580-5584Crossref PubMed Scopus (112) Google Scholar), and a L11-like protein (10Juan-Vidales F. Sanchez-Madrid F. Saenz-Robles M. Ballesta J.P.G. Eur. J. Biochem. 1983; 136: 275-281Crossref PubMed Scopus (18) Google Scholar), presently called L12 (11Mager W.H. Planta R.J. Ballesta J.G. Lee J.C. Mizuta K. Suzuki K. Warner J.R. Woolford J. Nucleic Acids Res. 1997; 25: 4872-4875Crossref PubMed Scopus (123) Google Scholar), both binding to the 26 S/28 S GTPase RNA domain at sites that are equivalent to those found in bacteria (12El-Baradi T.T.A.L. de Regt V.C.H.F. Einerhand S.W.C. Teixido J. Planta R.J. Ballesta J.P.G. Raué H.A. J. Mol. Biol. 1987; 195: 909-917Crossref PubMed Scopus (60) Google Scholar). Moreover, both components are required for the correct conformation of the rRNA as determined by in vitro binding studies (13Uchiumi T. Kominami R. J. Biol. Chem. 1997; 272: 3302-3308Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).Nevertheless, the eukaryotic organisms have developed new regulatory elements missing from less evolved systems. In this sense, the GTPase domain, and more specifically the ribosomal stalk, might be a paradigmatic example. In eukaryotes, the stalk also seems to be involved in the interaction and activity of the elongation factors, but, in addition, a number of interesting features not present in the bacterial ribosome strongly suggest its implication in a regulatory mechanism of the ribosomal activity (see Ref. 14Ballesta J.P.G. Remacha M. Progr. Nucleic Acids Res. Mol. Biol. 1996; 55: 157-193Crossref PubMed Google Scholar for a recent review).In general, the data underline the lower stability of the eukaryotic stalk as compared with the equivalent bacterial structure, and this feature is in the base of the mechanism that eukaryotes seem to have developed to regulate the ribosome translational activity in certain conditions (15Remacha M. Jimenez-Diaz A. Santos C. Zambrano R. Briones E. Rodriguez Gabriel M.A. Guarinos E. Ballesta J.P.G. Biochem. Cell Biol. 1995; 73: 959-968Crossref PubMed Scopus (86) Google Scholar). The structural differences existent between bacterial L10 and eukaryotic P0 must be to a great extent responsible for the dissimilar stability of their respective ribosomal stalks. In fact, it has been found that the eukaryotic specific C-terminal extension plays an important role in the formation of the pentameric complex (16Santos C. Ballesta J.P.G. J. Biol. Chem. 1995; 270: 20608-20614Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Moreover, the evolution from a unique L7/L12 bacterial protein to a family of proteins, P1 and P2, apart from introducing potentially useful structural diversity into the system, seems to have affected the self-association properties of these eukaryotic proteins, which, in contrast to the bacterial equivalents, can be found as monomers in solution (17Zurdo J. Sanz J.M. Gonzalez C. Rico M. Ballesta J.P.G. Biochemistry. 1997; 36: 9625-9635Crossref PubMed Scopus (43) Google Scholar).The two other components of the active domain, the rRNA and protein L12, although not directly forming part of the stalk, can obviously affect its stability. In the first case, however, the full in vivo functional interchangeability of the bacterial and yeast domain II GTPase RNA (18Musters W. Gonzalves P.M. Boon K. Raué H.A. van Heerikhuizen H. Planta R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1469-1473Crossref PubMed Scopus (46) Google Scholar, 19Thompson J. Muster W. Cundliffe E. Dahlberg A.E. EMBO J. 1993; 12: 1499-1504Crossref PubMed Scopus (54) Google Scholar) excludes this component as being important in order to explain their functional differences. The second component, protein L12, has an important role in determining thein vitro RNA GTPase center structure (13Uchiumi T. Kominami R. J. Biol. Chem. 1997; 272: 3302-3308Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).The Saccharomyces cerevisiae protein L12, formerly called L15, is encoded by an intron-less duplicated gene (20Pucciarelli M.G. Remacha M. Vilella M.D. Ballesta J.P.G. Nucleic Acids Res. 1990; 18: 4409-4416Crossref PubMed Scopus (13) Google Scholar). In contrast to the other yeast GTPase domain components, protein L12 has been studied in less detail. As a way to carry out an in vivo analysis of its function, especially its role in the GTPase structure and function, the obtention of S. cerevisiae strains defective in protein L12 was undertaken. The results of this study are shown in this report.MATERIALS AND METHODSChemicalsDimethyl sulfate (DMS)1was from Merck, kethoxal (2-keto-3-ethoxybutyraldehyde) was from U. S. Biochemical Corp., and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) was from Sigma.Strains and Growth ConditionsEither S. cerevisiae W303 (a/α, leu2–3, 112/leu2–3, 112, trp1–1/trp1–1, ura3–1/ura3–1, his 3–11, 15/his 3–11, 15, ade2–1/ade2–1, can1–100/can1–100) or S. cerevisiae W303–1b (α, leu2–3, 112, trp1–1, ura3–1, his 3–11, 15, ade2–1, can1–100) was used as a wild-type strain in the experiments performed in this report. Yeasts were grown in either YEP medium or minimal SD medium (21Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar) supplemented with the appropriate nutritional requirements. The carbon source was either 2% glucose or 2% galactose. When required, 5-fluoro-orotic acid (5-FOA) and G418 were present at 1 mg/ml and 200 μg/ml, respectively.Escherichia coli DH5α was used for handling cloning vectors and was grown in LB medium. Bacteria were transformed according to standard procedures (22Hanahan D. Glover D.M. DNA Cloning: A Practical Approach. IRL Press, Oxford1985: 109-136Google Scholar).Genetic ManipulationsS. cerevisiae mating, sporulation, and tetrad analysis were performed following published methods (21Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar).Recombinant DNA TechniquesRestriction endonucleases, T4 DNA ligase, Klenow DNA polymerase I fragment, and other enzymes were purchased from Boehringer Mannheim, New England Biolabs, or Amersham Pharmacia Biotech.DNA preparation, restriction enzyme digestions, agarose gel electrophoresis, ligation of DNA fragments, Southern blots, etc., were carried out according to standard techniques (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA was sequenced by the dideoxy chain termination method using universal primers and complementary oligonucleotides. Probes were labeled by the random initiation method using Klenow DNA polymerase fragment and [α-32P]dCTP.PCR was performed in 100–50-μl reaction mixtures in the conditions optimal for the used polymerase. The PCR products were purified by electrophoresis in agarose gels.Gene Disruption StrategiesInactivation of the rpL12 genes was performed by transforming S. cerevisiae with appropriate disruption cassettes following the methods described by Philippsen and co-workers (24Wach A. Brachat A. Pölhmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2224) Google Scholar, 25Wach A. Yeast. 1996; 12: 259-265Crossref PubMed Scopus (703) Google Scholar). Transformation was carried out by the lithium acetate method as described previously (26Gietz R. Woods R. Johnston J.R. Molecular Genetics of Yeast: A Practical Approach. IRL Press, Oxford1994: 121-134Google Scholar).The disruption cassettes contained either the KanMX4 or theSchizosaccharomyces pombe HIS3 genes as selection markers, flanked by DNA fragments homologous to regions next to the fragment that has to be deleted. Cassettes with either 40–45-bp flanking fragments (SFH) or 400–560-bp flanking fragments (LFH) were obtained by PCR using chimeric oligonucleotides (24Wach A. Brachat A. Pölhmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2224) Google Scholar, 25Wach A. Yeast. 1996; 12: 259-265Crossref PubMed Scopus (703) Google Scholar).PlasmidspUG7-L12AA 1.4-kilobase pair fragment, including the coding region, 528 bp from the 5′ flanking region and 367 bp from the 3′ flanking region of the rpL12A gene, was obtained from genomic S. cerevisiae W303 DNA by PCR using PfuDNA polymerase. The PCR fragment was subcloned by blunt end ligation into the EcoRV site of the pUG7 polylinker. The absence of PCR induced mutations was confirmed by DNA sequencing.pFL36-L12A SacI-XhoI 1.4-kilobase pair insert containing the rpL12A gene was cut from pUG7-L12A and inserted in the corresponding sites of plasmid pFL36 (27Bonneaud N. Ozier-Kalogeropoulos O. Li G. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (500) Google Scholar).pYES2-L12A 498-bp XhoI-BamHI PCR fragment containing the rpL12A gene coding region was inserted in the corresponding sites of plasmid pYES2 (Invitrogen) under the control of the GAL1 promoter.DNA Blots (Southern)Yeast DNA was prepared as described (21Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). DNA, after digestion with restriction enzymes, was resolved by electrophoresis in 0.8% agarose gels, and blotted to nylon membranes (Amersham Pharmacia Biotech). Hybridization was performed according to standard procedures (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).RNA FootprintingRibosomes (20 μg) were preincubated at 30 °C for 20 min in 100 μl of 50 mm Hepes-KOH (pH 7.8), 15 mmKCl, 15 mm NH4Cl, 10 mmMgCl2, 1 mm dithiothreitol, and 0.1 mm EDTA (buffer 1). As a control, 12 μg of naked renatured RNA were preincubated in the same conditions in 70 mm Hepes-KOH (pH 7.8), 270 mm KCl, 10 mm MgCl2, and 1 mm dithiothreitol. The samples were treated as described previously (28Christiansen J. Egebjerg J. Larsen N. Garrett R.A. Speding G. Ribosomes and Protein Synthesis: A Practical Approach. Oxford University Press, Oxford1990: 229-252Google Scholar) with one of the following chemical probes: 1 μl of DMS (1:1 dilution in absolute ethanol), 5 μl of kethoxal (35 mg/ml in 20% ethanol), and 100 μl of CMCT (42 mg/ml in buffer 1), and incubated at 30 °C for 10 min (DMS and kethoxal) or 20 min (CMCT).Primer extension was performed according to Ref. 29Moazed D. Stern S. Noller H.F. J. Mol. Biol. 1986; 187: 399-416Crossref PubMed Scopus (445) Google Scholar. The end-labeled oligonucleotide primer 5′-TGCCTACTCGTCAGGGC-3′, complementary to residues 1356–1372 of S. cerevisiae 26 S rRNA, was used as primer.Cell FractionationCells were broken with glass beads in 20 mmTris-HCl, pH 7.4, 80 mm KCl, 10 mmMgCl2, including a mixture of protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 2 mg/ml leupeptin, 2 mg/ml pepstatin). The extracts were centrifuged for 15 min. at 15,000 rpm in a Sorvall SS34 rotor, producing a S-30 fraction.The S-30 was centrifuged to obtain the ribosomes and supernatant fractions as described previously (30Sanchez-Madrid F. Reyes R. Conde P. Ballesta J.P.G. Eur. J. Biochem. 1979; 98: 409-416Crossref PubMed Scopus (94) Google Scholar). As a source of supernatant factors for in vitro protein synthesis, the fraction precipitated between 20% and 50% saturation of ammonium sulfate, and called S-100, was used. The acidic P proteins (SP fraction) were extracted from the ribosomes by ammonium-ethanol treatment (30Sanchez-Madrid F. Reyes R. Conde P. Ballesta J.P.G. Eur. J. Biochem. 1979; 98: 409-416Crossref PubMed Scopus (94) Google Scholar); the extracted fraction was dialyzed against 10 mm Hepes, pH 7.4, and 0.5 mm phenylmethylsulfonyl fluoride and concentrated by filtration through Centricon SR3 membranes (Amicon).Electrophoretic MethodsProteins were analyzed by either SDS-PAGE or by isoelectrofocusing. SDS-PAGE was performed according to standard procedures. Isoelectrofocusing was carried out on vertical 5% polyacrylamide, 8 m urea isoelectrofocusing gels in the 2.5–5.0 pH range as described previously (31Zambrano R. Briones E. Remacha M. Ballesta J.P.G. Biochemistry. 1997; 36: 14439-14446Crossref PubMed Scopus (62) Google Scholar).Proteins were either detected by silver staining or blotted to either PVDF or nitrocellulose membranes by electrophoresis in a semidry system using Novablot LKB buffer. Proteins in membranes were immunodetected following standard procedures (32Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar) and using specific antibodies. Antibodies to L11, L12, and P proteins have been previously described (10Juan-Vidales F. Sanchez-Madrid F. Saenz-Robles M. Ballesta J.P.G. Eur. J. Biochem. 1983; 136: 275-281Crossref PubMed Scopus (18) Google Scholar, 33Saenz-Robles M.T. Vilella M.D. Pucciarelli G. Polo F. Remacha M. Ortiz B.L. Vidales F. Ballesta J.P.G. Eur. J. Biochem. 1988; 177: 531-537Crossref PubMed Scopus (14) Google Scholar, 34Vilella 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).Activity Tests: Polyphenylalanine SynthesisThe reaction was performed in 50-μl samples containing 10 pmol of 80 S ribosomes, 5 μl of S-100, 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.DISCUSSIONProtein L12 is encoded in S. cerevisiae by a duplicated gene, rpL12A and rpL12B, which express identical polypeptides (20Pucciarelli M.G. Remacha M. Vilella M.D. Ballesta J.P.G. Nucleic Acids Res. 1990; 18: 4409-4416Crossref PubMed Scopus (13) Google Scholar). Sporulation and tetrad analysis of a heterozygous diploid strain carrying a plasmidic L12 gene under the control of the GAL1 promoter yielded haploid strains carrying either one or both L12 gene copies simultaneously inactivated and a galactose-inducible copy.Expression of both L12 gene copies is required for optimal growth indicating that none of them provides enough ribosomal protein and suggesting the absence of a compensatory regulatory mechanism reported in other instances (37Woolford J.J. Adv. Genet. 1991; 29: 63-118Crossref PubMed Scopus (63) Google Scholar). It is interesting, however, that the absence of rpL12A can be almost completely compensated by increasing the growth temperature of the corresponding disrupted strain. This might indicate that either a real temperature-dependent regulatory process takes place or, simply, that the expression of rpL12B copy is temperature-sensitive. Further experimental work, now in progress, is required to clear up different pending questions related to the expression of the two yeast protein L12 genes.Transfer of the cells to a glucose medium to repress the plasmidic L12 gene expression drastically reduced but did not abolish the growth of the double L12 disruptant. In fact, it was possible to obtain viable double disrupted strains in which the L12 carrying plasmid had been cured by growing in 5′-FOA. It seems, therefore, clear that protein L12 is not an absolute requirement for ribosome activity and cell viability. However, the protein must have an important role in ribosome function as cells lacking L12 have a doubling time close to 6 h.In contrast to E. coli, in which a significant number of ribosomal proteins are dispensable for cell viability (38Dabbs E.R. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag, New York1986: 733-748Google Scholar), most proteins seem to be essential in the yeast ribosome. Excluding the exchangeable acidic proteins P1/P2, out of 32 proteins so far studied, only three, S31 (UBI3) (39Finley D. Bartel B. Varshavsky A. Nature. 1989; 338: 394-401Crossref PubMed Scopus (552) Google Scholar), L24 (formerly L30) (40Baronas-Lowell D.M. Warner J.R. Mol. Cell. Biol. 1990; 10: 5235-5243Crossref PubMed Scopus (55) Google Scholar), and L39 (UBI1/UBI2) (41Sachs A.B. Davis R.W. Science. 1990; 247: 1077-1079Crossref PubMed Scopus (151) Google Scholar), are shown to be dispensable for cell viability. These results indicate that eukaryotic ribosomes have tighter structural requirements than the bacterial one, and the changes resulting from the protein absence are lethal for the ribosome activity.Protein L11 is also dispensable for viability in bacteria (7Cundliffe E. Dixon P. Stark M. Stöffler G. Ehrlich R. Stöffler-Meilicke M. Cannon M. J. Mol. Biol. 1979; 132: 235-252Crossref PubMed Scopus (60) Google Scholar). There are, however, significant differences in the role played by bacterial L11 and eukaryotic L12, the most important being the different involvement in the stability of the stalk. Thus, although the bacterial mutants lacking protein L11 contain standard amounts of L7/12 (42Stöffler G. Cundliffe E. Stöffler-Meilicke M. Dabbs E.R. J. Biol. Chem. 1980; 255: 10517-10522Abstract Full Text PDF PubMed Google Scholar), in the L12-defective yeast strains two of the four acidic proteins, P1α and P2β, are missing from the ribosomes. These results indicate that protein L12 is either directly or indirectly involved in the interaction of the 12-kDa acidic proteins with the eukaryotic ribosome. Physical proximity of the C-terminal domain of bacterial L7/12 to protein L11 has been reported, confirming the high flexibility of the acidic proteins (43Traut R.R. Dey D. Bochkarlov D.E. Oleinikov A.V. Jokhadze G.G. Hamman B. Jameson D. Biochem. Cell Biol. 1995; 73: 949-958Crossref PubMed Scopus (56) Google Scholar). However, these interactions are probably irrelevant to the binding of the acidic proteins to the ribosome, which takes place only through the N-terminal domain of the proteins in bacteria (44Marquis D.M. Fahnestock S.R. Henderson E. Woo D. Schwinge D. Clark M. Lake J.A. J. Mol. Biol. 1981; 150: 121-132Crossref PubMed Scopus (16) Google Scholar) as well as in yeast (45Payo J.M. Santana-Roman H. Remacha M. Ballesta J.P.G. Zinker S. Biochemistry. 1995; 34: 7941-7948Crossref PubMed Scopus (34) Google Scholar). In any case, although the acidic proteins have been cross-linked to protein P0 (46Uchiumi T. Kikuchi M. Terao K. Ogata K. J. Biol. Chem. 1985; 260: 5675-5682Abstract Full Text PDF PubMed Google Scholar), a direct interaction between L12 and proteins P1/P2 has not been so far reported in the ribosome, although they have been shown to be able to associate in solution (33Saenz-Robles M.T. Vilella M.D. Pucciarelli G. Polo F. Remacha M. Ortiz B.L. Vidales F. Ballesta J.P.G. Eur. J. Biochem. 1988; 177: 531-537Crossref PubMed Scopus (14) Google Scholar).The preferential release of P1α and P2β from L12-defective ribosomes indicates the asymmetrical structure of the yeast stalks as compared with the bacterial one, and stresses the different structural role of each protein of the same type, P1α/P1β and P2α/P2β. Previously, the analysis of different disrupted mutants had shown that the effects caused by the absence of one of the proteins cannot be suppressed by an excess of the other one (47Remacha M. Santos C. Ballesta J.P.G. Mol. Cell. Biol. 1990; 10: 2182-2190Crossref PubMed Scopus (32) Google Scholar, 48Remacha M. Santos C. Bermejo B. Naranda T. Ballesta J.P.G. J. Biol. Chem. 1992; 267: 12061-12067Abstract Full Text PDF PubMed Google Scholar, 49Remacha 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). All the functional and structural data confirm, therefore, that the two members of the same protein family are performing specific functions.Independent of its physiological meaning, which is not obvious, the existence of four acidic proteins in yeast ribosomes together with the assumed dimeric character of these proteins raises some interesting structural questions. Thus, as only four copies of acidic proteins have been detected per eukaryotic ribosome (9Uchiumi T. Wahba A.J. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5580-5584Crossref PubMed Scopus (112) Google Scholar, 50Saenz-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), in the cell there must be either a homogeneous population of ribosomes carrying one monomer of each protein type or a heterogeneous population of particles carrying two dimers in different combinations, (P1α)2/(P2α)2, (P1α)2/(P2β)2, (P1β)2/(P2α)2,(P1β)2/(P2β)2. The specific release of P1α and P2β by the absence of L12 fits in better with an asymmetrical ribosomal stalk made by one protein of each type, P0, P1α, P2β, P1β, P2α, in which the four 12-kDa proteins do not have the same role.Contrary to what could be expected, there seems to be a closer relationship between two proteins of a different type, P1α/P2β and P1β/P2α, the binding of the first pair being more directly affected by the presence of protein L12. These two pairs might play the same function than the standard P1 and P2 dimers in mammals. The P1α/P2β and P1β/P2α associations are, however, not an absolute requirement for the formation of the stalk, as it is possible to obtain, by gene disruption, yeast mutant strains with ribosomes exclusively containing the P1α/P2α and P1β/P2β pairs (48Remacha M. Santos C. Bermejo B. Naranda T. Ballesta J.P.G. J. Biol. Chem. 1992; 267: 12061-12067Abstract Full Text PDF PubMed Google Scholar, 49Remacha 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). In any case, the P1α/P2β pair seem to be especially relevant for ribosome activity as its absence is more harmful for cell growth than the absence of P1β/P2α (49Remacha 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).Protein L12 also affects the interaction of protein P0, although not so obviously as in the case of the acidic proteins. P0 is present in similar amounts in disruptant and in wild-type ribosomes, but the protein can be released from the particles more easily in the first case. Thus, although P0, unlike bacterial L10, is hardly washed off the eukaryotic ribosomes by ammonium/ethanol buffers (30Sanchez-Madrid F. Reyes R. Conde P. Ballesta J.P.G. Eur. J. Biochem. 1979; 98: 409-416Crossref PubMed Scopus (94) Google Scholar, 35Towbin G. Ramjoue H.P. Kaster H. Liverani D. Gordon J. J. Biol. Chem. 1982; 257: 12709-12715Abstract Full Text PDF PubMed Google Scholar), the protein can be detected in the mutant ribosome washes at relatively low ammonium chloride concentrations.The effect of protein L12 in the protein P0 interaction is in agreement with the binding of both proteins to partially overlapping sites in the mammalian rRNA (51Uchiumi T. Kominami R. J. Biol. Chem. 1992; 267: 19179-19185Abstract Full Text PDF PubMed Google Scholar). Our footprinting results with ribosomes, in addition to confirm the absence of L12 from the double disrupted strain ribosome, clearly show that this" @default.
• W2004090241 created "2016-06-24" @default.
• W2004090241 creator A5014990435 @default.
• W2004090241 creator A5016076694 @default.
• W2004090241 creator A5030268170 @default.
• W2004090241 creator A5043124468 @default.
• W2004090241 date "1998-11-01" @default.
• W2004090241 modified "2023-09-27" @default.
• W2004090241 title "The GTPase Center Protein L12 Is Required for Correct Ribosomal Stalk Assembly but Not for Saccharomyces cerevisiaeViability" @default.
• W2004090241 cites W1487537952 @default.
• W2004090241 cites W1493809750 @default.
• W2004090241 cites W1504695628 @default.
• W2004090241 cites W1527440198 @default.
• W2004090241 cites W1540325573 @default.
• W2004090241 cites W1546031408 @default.
• W2004090241 cites W1549587154 @default.
• W2004090241 cites W1563632225 @default.
• W2004090241 cites W1566884392 @default.
• W2004090241 cites W1568474642 @default.
• W2004090241 cites W1594797965 @default.
• W2004090241 cites W1605208286 @default.
• W2004090241 cites W174730266 @default.
• W2004090241 cites W1969517561 @default.
• W2004090241 cites W1969937718 @default.
• W2004090241 cites W1977018957 @default.
• W2004090241 cites W1980272310 @default.
• W2004090241 cites W1998172433 @default.
• W2004090241 cites W2007067255 @default.
• W2004090241 cites W2007728632 @default.
• W2004090241 cites W2011906599 @default.
• W2004090241 cites W2013778327 @default.
• W2004090241 cites W2017269218 @default.
• W2004090241 cites W2018550609 @default.
• W2004090241 cites W2021383324 @default.
• W2004090241 cites W2023864622 @default.
• W2004090241 cites W2024270310 @default.
• W2004090241 cites W2028860842 @default.
• W2004090241 cites W2030412343 @default.
• W2004090241 cites W2031557479 @default.
• W2004090241 cites W2044253175 @default.
• W2004090241 cites W2046290726 @default.
• W2004090241 cites W2046363945 @default.
• W2004090241 cites W2070756319 @default.
• W2004090241 cites W2080138031 @default.
• W2004090241 cites W2096817012 @default.
• W2004090241 cites W2100057937 @default.
• W2004090241 cites W2101108802 @default.
• W2004090241 cites W2115898224 @default.
• W2004090241 cites W2118950202 @default.
• W2004090241 cites W2124166994 @default.
• W2004090241 cites W2161859426 @default.
• W2004090241 cites W2239952390 @default.
• W2004090241 cites W340017914 @default.
• W2004090241 doi "https://doi.org/10.1074/jbc.273.48.31956" @default.
• W2004090241 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9822666" @default.
• W2004090241 hasPublicationYear "1998" @default.
• W2004090241 type Work @default.
• W2004090241 sameAs 2004090241 @default.
• W2004090241 citedByCount "50" @default.
• W2004090241 countsByYear W20040902412012 @default.
• W2004090241 countsByYear W20040902412013 @default.
• W2004090241 countsByYear W20040902412014 @default.
• W2004090241 countsByYear W20040902412016 @default.
• W2004090241 countsByYear W20040902412018 @default.
• W2004090241 countsByYear W20040902412019 @default.
• W2004090241 countsByYear W20040902412020 @default.
• W2004090241 countsByYear W20040902412021 @default.
• W2004090241 countsByYear W20040902412022 @default.
• W2004090241 crossrefType "journal-article" @default.
• W2004090241 hasAuthorship W2004090241A5014990435 @default.
• W2004090241 hasAuthorship W2004090241A5016076694 @default.
• W2004090241 hasAuthorship W2004090241A5030268170 @default.
• W2004090241 hasAuthorship W2004090241A5043124468 @default.
• W2004090241 hasBestOaLocation W20040902411 @default.
• W2004090241 hasConcept C104317684 @default.
• W2004090241 hasConcept C144027150 @default.
• W2004090241 hasConcept C185592680 @default.
• W2004090241 hasConcept C207332259 @default.
• W2004090241 hasConcept C2777576037 @default.
• W2004090241 hasConcept C2778447961 @default.
• W2004090241 hasConcept C2779222958 @default.
• W2004090241 hasConcept C2779560860 @default.
• W2004090241 hasConcept C38062823 @default.
• W2004090241 hasConcept C55493867 @default.
• W2004090241 hasConcept C67705224 @default.
• W2004090241 hasConcept C67905577 @default.
• W2004090241 hasConcept C70721500 @default.
• W2004090241 hasConcept C86803240 @default.
• W2004090241 hasConcept C88478588 @default.
• W2004090241 hasConceptScore W2004090241C104317684 @default.
• W2004090241 hasConceptScore W2004090241C144027150 @default.
• W2004090241 hasConceptScore W2004090241C185592680 @default.
• W2004090241 hasConceptScore W2004090241C207332259 @default.
• W2004090241 hasConceptScore W2004090241C2777576037 @default.
• W2004090241 hasConceptScore W2004090241C2778447961 @default.
• W2004090241 hasConceptScore W2004090241C2779222958 @default.
• W2004090241 hasConceptScore W2004090241C2779560860 @default.
• W2004090241 hasConceptScore W2004090241C38062823 @default.

• next