A Role for the Ppz Ser/Thr Protein Phosphatases in the Regulation of Translation Elongation Factor 1Bα
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m010824200
ISSN1083-351X
AutoresEulàlia de Nadal, Robert P. Fadden, Amparo Ruiz, Timothy Haystead, Joaquı́n Ariño,
Tópico(s)RNA modifications and cancer
ResumoIn vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Bα (formerly eEF1β). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased inppz cells. Pull-down experiments using a glutathioneS-transferase (GST)-EF1Bα fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Bα fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Bα in yeast, and this may result in modification of the translational accuracy. In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Bα (formerly eEF1β). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased inppz cells. Pull-down experiments using a glutathioneS-transferase (GST)-EF1Bα fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Bα fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Bα in yeast, and this may result in modification of the translational accuracy. elongation factor 1 protein phosphatase z synthetic minimal medium complete minimal medium 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate glutathione S-transferase open reading frame phenylmethylsulfonyl fluoride trifluoroacetic acid high pressure liquid chromatography polyacrylamide gel electrophoresis base pair(s) polymerase chain reaction kilobase pair(s) The elongation step of protein synthesis involves the binding of aminoacyl-tRNA to the ribosomal "A" site, formation of a peptide bond, and translocation of the newly formed peptidyl-tRNA to the "P" site. The elongation factor 1 (EF1)1 is responsible for the GTP-dependent binding of aminoacylated tRNA to the ribosomal A site in polypeptide chain elongation and participates in proofreading of the codon-anticodon match (1Hinnenbusch A. Liebmann S.W. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 627-735Google Scholar)In the budding yeast Saccharomyces cerevisiae, EF1 consist of different subunits: EF1A (formerly EF1α) is encoded by two different genes (TEF1 and TEF2), and it binds aminoacyl-tRNA in a GTP-dependent manner. The exchange of GDP for GTP on EF1A is stimulated by a member of the guanine nucleotide exchange factor family, EF1Bα, which is encoded by a single gene (TEF5). An additional subunit of uncertain function is encoded by genes TEF3 and TEF4. Although lack ofTEF3 and TEF4 results in no observable defects in translation (2Kinzy T.G. Ripmaster T.L. Woolford Jr., J.L. Nucleic Acids Res. 1994; 22: 2703-2707Crossref PubMed Scopus (33) Google Scholar), lack of TEF5 or simultaneous deletion ofTEF1 and TEF2 is lethal (3Hiraga K. Suzuki K. Tsuchiya E. Miyakawa T. FEBS Lett. 1993; 316: 165-169Crossref PubMed Scopus (46) Google Scholar, 4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). Components of EF1 have been shown to be phosphorylated in vitro by diverse protein kinases in species different from yeast (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar, 6Matsumoto S. Mizoguchi T. Oizumi N. Tsuruga M. Shinozaki K. Taira H. Ejiri S. Biosci. Biotechnol. Biochem. 1993; 57: 1740-1742Crossref PubMed Scopus (9) Google Scholar, 7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar).The function of EF1Bα on EF1A has been shown to be critical for an efficient and accurate translation. For example, cells with increased expression of the EF1A subunit can bypass the lethality of cells lacking EF1Bα. However, these cells present a number of defects, including higher sensitivity to inhibitors of translation elongation and changes in translational fidelity (4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). Alterations in translational fidelity have been also produced by specific mutations in EF1Bα, as it has been documented by evaluation of sensitivity to drugs such as paromomycin and analysis of translational fidelity at nonsense codons (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). The fidelity of translation may be related, at least in part, to the requirement for nucleotide exchange, as it has been tested by mutations in the GTP-binding motif of yeast EF1A (9Carr-Schmid A. Durko N. Cavallius J. Merrick W.C. Kinzy T.G. J. Biol. Chem. 1999; 274: 30297-30302Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).The yeast Ppz phosphatases are encoded by genes PPZ1 andPPZ2 (10Posas F. Casamayor A. Morral N. Ariño J. J. Biol. Chem. 1992; 267: 11734-11740Abstract Full Text PDF PubMed Google Scholar, 11Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar) and represent a novel type of Ser/Thr phosphatases characterized by a catalytic carboxyl-terminal half related to type 1 phosphatase. These phosphatases are involved in a variety of cell processes, including maintenance of cell integrity, in connection with the Pkc1/Mpk1 mitogen-activated protein kinase pathway (11Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar, 12Posas F. Casamayor A. Ariño J. FEBS Lett. 1993; 318: 282-286Crossref PubMed Scopus (72) Google Scholar), regulation of salt tolerance (13Posas F. Camps M. Ariño J. J. Biol. Chem. 1995; 270: 13036-13041Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and regulation of cell cycle at the G1/S transition (14Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (68) Google Scholar). In all cases, the function of Ppz1 appears to be more important than that of Ppz2. Recently, we have identified the halotolerant determinant Hal3 as a negative regulatory subunit of Ppz1 that modulates the diverse physiological functions of the phosphatase (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar).As an attempt to better understand the physiological role of the Ppz phosphatases, we have performed a two-dimensional electrophoretic analysis of proteins from in vivo 32P-labeled wild type and ppz strains, in search for polypeptides that might display an altered phosphorylation state in the absence of the phosphatases. This approach has led us to establish a previously unsuspected link between the Ppz phosphatases and the translation elongation factor 1Bα.DISCUSSIONIn this report we demonstrate that, in the yeast S. cerevisiae, translation elongation factor 1Bα is a phosphoprotein. Phosphorylation site mapping and sequence analysis indicates that the Ser-86 is the only phosphorylatable residue in this protein, at least under standard growth conditions. It is remarkable that data base search reveals that the equivalent Ser residue (as well as its acidic environment) is also found in a large variety of organisms, including Drosophila melanogaster,Caenorhabditis elegans, mouse, and human. Phosphorylation of EF1Bα has been reported in Artemia salina (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar), wheat (6Matsumoto S. Mizoguchi T. Oizumi N. Tsuruga M. Shinozaki K. Taira H. Ejiri S. Biosci. Biotechnol. Biochem. 1993; 57: 1740-1742Crossref PubMed Scopus (9) Google Scholar), and reticulocyte (7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar). In the former case, phosphorylation was ascribed to Ser-89, which is equivalent to Ser-86 in yeast EF1Bα. Interestingly, phosphorylation has been correlated to changes in its catalytic nucleotide exchange activity, although reports are somewhat contradictory (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar, 7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar).Our data indicate that deletion of the ppz genes and overexpression of Hal3, a negative regulatory subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar), result in increased phosphorylation of the EF1Bα protein, specifically at Ser-86. These results would be compatible with a role of Ppz1 in regulating the phosphorylation state of the translation factor and, possibly, its function. We also show here evidence that affinity-purified yeast EF1Bα contains significant amounts of bound Ppz1, by using an approach that was pivotal in the past to identify the Hal3 protein as a subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar). This could be taken as an indication that Ppz1 could be able to directly dephosphorylate EF1Bα. However, we have been unable to detect direct dephosphorylation of either in vivo labeled or CK-2 in vitrophosphorylated EF1Bα in the presence of bacterially expressed Ppz1. Although at this point we cannot provide direct evidence for the translation factor being a substrate for the phosphatase, this possibility formally remains. For instance, the phosphatase might require accessory proteins (absent in our in vitro assay) to effectively use EF1Bα as substrate. In this regard, there is a large body of evidence for the requirement of specific regulatory subunits (targeting subunits) for Ser/Thr phosphatases to localize at specific subcellular sites or to use a given phosphoprotein as an effective substrate (28Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2143) Google Scholar, 29Stark M.J. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) Google Scholar). It must be noted that dephosphorylation events have been previously related to the control of the accuracy of protein synthesis, as it is the case of the Ppq1/Sal6 Ser/Thr protein phosphatase (30Chen M.X. Chen Y.H. Cohen P.T. Eur. J. Biochem. 1993; 218: 689-699Crossref PubMed Scopus (29) Google Scholar, 31Vincent A. Newnam G. Liebman S.W. Genetics. 1994; 138: 597-608Crossref PubMed Google Scholar), the closest structural homologue of the Ppz phosphatases. However, the possible role of this phosphatase has not been worked out.We considered that if EF1Bα was a target (either direct or indirect) for Ppz1, it could be possible to establish some sort of functional connection between both proteins. Deletion of TEF5 is lethal for the cell, and high copy expression of TEF2 suppresses the lethal phenotype of tef5 mutants (4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). However, these cells are markedly sensitive to translational inhibitors, such as paromomycin and hygromycin B. It is remarkable that lack of Ppz phosphatases also results in a change in sensitivity to these compounds, although in this case yielding more tolerant cells. Because these drugs are aminoglycosides known to enter the yeast cell driven by the membrane potential, which is mostly maintained by the function of the membrane H+-ATPase (32Vallejo C.G. Serrano R. Yeast. 1989; 5: 307-319Crossref PubMed Scopus (95) Google Scholar), we considered the possibility that the increased tolerance could be an indirect effect due to altered proton efflux. However, this was ruled out by determining this parameter in wild type and ppz mutants and finding essentially identical values (data not shown).Changes in sensitivity to paromomycin have been related to altered translational fidelity (33Singh A. Ursic D. Davies J. Nature. 1979; 277: 146-148Crossref PubMed Scopus (172) Google Scholar, 34Palmer E. Wilhelm J.M. Sherman F. Nature. 1979; 277: 148-150Crossref PubMed Scopus (172) Google Scholar), a phenotype also produced by changes in the dosage of EF1A (35Song J.M. Picologlou S. Grant C.M. Firoozan M. Tuite M.F. Liebman S. Mol. Cell. Biol. 1989; 9: 4571-4575Crossref PubMed Scopus (66) Google Scholar). Recent evidence (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar) has been presented pointing out that mutations in the carboxyl-terminal region of EF1Bα results in increased sensitivity to translation inhibitors and that this effect was accompanied by enhanced translational fidelity (i.e. reduced readthrough at nonsense codons). These observations are in keeping with our finding that cells lacking Ppz phosphatases, which are more tolerant to certain translation inhibitors, show an increased readthrough at nonsense codons, most likely due to a decrease in translational fidelity.Further evidence for a functional interaction between EF1Bα and Ppz1 comes from the observation that overexpression of the translation factor strongly attenuates the growth defect, due to a delayed G1/S transition, of cells containing an excess of Ppz1 activity. Although we showed in the past that this defect correlates with a delay in G1/S cyclin mRNA expression (14Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (68) Google Scholar), immunoblot analysis of the protein level of different cyclins reveals that, at least in the case of Clb5, further post-transcriptional alterations (i.e. at the translation level) could exist. 2E. Nadal, R. P. Fadden, A. Ruiz, T. Haystead, and J. Ariño, unpublished results. Remarkably, a non-phosphorylatable version of EF1Bα was unable to counteract the effect of an excess of Ppz1, suggesting that in vivomodulation of the phosphorylation state of the factor is somehow involved in the regulation of its function. It has been reported that, when expressed from the powerful GAL promoter, a carboxyl-terminal fragment of EF1Bα, lacking Ser-86, was sufficient for normal growth and did not display dramatically altered drug or temperature sensitivity (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). Furthermore, a strain containing a S86A version of EF1Bα as the only source for the factor is viable. 3L. Valente and T. G. Kinzy, personal communication. Therefore, it must be concluded that regulation of EF1Bα by phospho-dephosphorylation at Ser-86 (which, at least in part, would involve Ppz1) must affect the function of the translation factor in a subtle way. From our data, it can be hypothesized that changes in the phosphorylation state of EF1Bα would result in altered nucleotide exchange on EF1A. However, alternative mechanisms cannot be excluded, because it has been postulated that EF1Bα may have additional regulatory effects on EF1A (9Carr-Schmid A. Durko N. Cavallius J. Merrick W.C. Kinzy T.G. J. Biol. Chem. 1999; 274: 30297-30302Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In any case, our data provides further support to the notion that phospho-dephosphorylation mechanisms are relevant for a proper regulation of protein synthesis. The elongation step of protein synthesis involves the binding of aminoacyl-tRNA to the ribosomal "A" site, formation of a peptide bond, and translocation of the newly formed peptidyl-tRNA to the "P" site. The elongation factor 1 (EF1)1 is responsible for the GTP-dependent binding of aminoacylated tRNA to the ribosomal A site in polypeptide chain elongation and participates in proofreading of the codon-anticodon match (1Hinnenbusch A. Liebmann S.W. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 627-735Google Scholar) In the budding yeast Saccharomyces cerevisiae, EF1 consist of different subunits: EF1A (formerly EF1α) is encoded by two different genes (TEF1 and TEF2), and it binds aminoacyl-tRNA in a GTP-dependent manner. The exchange of GDP for GTP on EF1A is stimulated by a member of the guanine nucleotide exchange factor family, EF1Bα, which is encoded by a single gene (TEF5). An additional subunit of uncertain function is encoded by genes TEF3 and TEF4. Although lack ofTEF3 and TEF4 results in no observable defects in translation (2Kinzy T.G. Ripmaster T.L. Woolford Jr., J.L. Nucleic Acids Res. 1994; 22: 2703-2707Crossref PubMed Scopus (33) Google Scholar), lack of TEF5 or simultaneous deletion ofTEF1 and TEF2 is lethal (3Hiraga K. Suzuki K. Tsuchiya E. Miyakawa T. FEBS Lett. 1993; 316: 165-169Crossref PubMed Scopus (46) Google Scholar, 4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). Components of EF1 have been shown to be phosphorylated in vitro by diverse protein kinases in species different from yeast (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar, 6Matsumoto S. Mizoguchi T. Oizumi N. Tsuruga M. Shinozaki K. Taira H. Ejiri S. Biosci. Biotechnol. Biochem. 1993; 57: 1740-1742Crossref PubMed Scopus (9) Google Scholar, 7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar). The function of EF1Bα on EF1A has been shown to be critical for an efficient and accurate translation. For example, cells with increased expression of the EF1A subunit can bypass the lethality of cells lacking EF1Bα. However, these cells present a number of defects, including higher sensitivity to inhibitors of translation elongation and changes in translational fidelity (4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). Alterations in translational fidelity have been also produced by specific mutations in EF1Bα, as it has been documented by evaluation of sensitivity to drugs such as paromomycin and analysis of translational fidelity at nonsense codons (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). The fidelity of translation may be related, at least in part, to the requirement for nucleotide exchange, as it has been tested by mutations in the GTP-binding motif of yeast EF1A (9Carr-Schmid A. Durko N. Cavallius J. Merrick W.C. Kinzy T.G. J. Biol. Chem. 1999; 274: 30297-30302Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The yeast Ppz phosphatases are encoded by genes PPZ1 andPPZ2 (10Posas F. Casamayor A. Morral N. Ariño J. J. Biol. Chem. 1992; 267: 11734-11740Abstract Full Text PDF PubMed Google Scholar, 11Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar) and represent a novel type of Ser/Thr phosphatases characterized by a catalytic carboxyl-terminal half related to type 1 phosphatase. These phosphatases are involved in a variety of cell processes, including maintenance of cell integrity, in connection with the Pkc1/Mpk1 mitogen-activated protein kinase pathway (11Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar, 12Posas F. Casamayor A. Ariño J. FEBS Lett. 1993; 318: 282-286Crossref PubMed Scopus (72) Google Scholar), regulation of salt tolerance (13Posas F. Camps M. Ariño J. J. Biol. Chem. 1995; 270: 13036-13041Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and regulation of cell cycle at the G1/S transition (14Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (68) Google Scholar). In all cases, the function of Ppz1 appears to be more important than that of Ppz2. Recently, we have identified the halotolerant determinant Hal3 as a negative regulatory subunit of Ppz1 that modulates the diverse physiological functions of the phosphatase (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar). As an attempt to better understand the physiological role of the Ppz phosphatases, we have performed a two-dimensional electrophoretic analysis of proteins from in vivo 32P-labeled wild type and ppz strains, in search for polypeptides that might display an altered phosphorylation state in the absence of the phosphatases. This approach has led us to establish a previously unsuspected link between the Ppz phosphatases and the translation elongation factor 1Bα. DISCUSSIONIn this report we demonstrate that, in the yeast S. cerevisiae, translation elongation factor 1Bα is a phosphoprotein. Phosphorylation site mapping and sequence analysis indicates that the Ser-86 is the only phosphorylatable residue in this protein, at least under standard growth conditions. It is remarkable that data base search reveals that the equivalent Ser residue (as well as its acidic environment) is also found in a large variety of organisms, including Drosophila melanogaster,Caenorhabditis elegans, mouse, and human. Phosphorylation of EF1Bα has been reported in Artemia salina (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar), wheat (6Matsumoto S. Mizoguchi T. Oizumi N. Tsuruga M. Shinozaki K. Taira H. Ejiri S. Biosci. Biotechnol. Biochem. 1993; 57: 1740-1742Crossref PubMed Scopus (9) Google Scholar), and reticulocyte (7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar). In the former case, phosphorylation was ascribed to Ser-89, which is equivalent to Ser-86 in yeast EF1Bα. Interestingly, phosphorylation has been correlated to changes in its catalytic nucleotide exchange activity, although reports are somewhat contradictory (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar, 7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar).Our data indicate that deletion of the ppz genes and overexpression of Hal3, a negative regulatory subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar), result in increased phosphorylation of the EF1Bα protein, specifically at Ser-86. These results would be compatible with a role of Ppz1 in regulating the phosphorylation state of the translation factor and, possibly, its function. We also show here evidence that affinity-purified yeast EF1Bα contains significant amounts of bound Ppz1, by using an approach that was pivotal in the past to identify the Hal3 protein as a subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar). This could be taken as an indication that Ppz1 could be able to directly dephosphorylate EF1Bα. However, we have been unable to detect direct dephosphorylation of either in vivo labeled or CK-2 in vitrophosphorylated EF1Bα in the presence of bacterially expressed Ppz1. Although at this point we cannot provide direct evidence for the translation factor being a substrate for the phosphatase, this possibility formally remains. For instance, the phosphatase might require accessory proteins (absent in our in vitro assay) to effectively use EF1Bα as substrate. In this regard, there is a large body of evidence for the requirement of specific regulatory subunits (targeting subunits) for Ser/Thr phosphatases to localize at specific subcellular sites or to use a given phosphoprotein as an effective substrate (28Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2143) Google Scholar, 29Stark M.J. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) Google Scholar). It must be noted that dephosphorylation events have been previously related to the control of the accuracy of protein synthesis, as it is the case of the Ppq1/Sal6 Ser/Thr protein phosphatase (30Chen M.X. Chen Y.H. Cohen P.T. Eur. J. Biochem. 1993; 218: 689-699Crossref PubMed Scopus (29) Google Scholar, 31Vincent A. Newnam G. Liebman S.W. Genetics. 1994; 138: 597-608Crossref PubMed Google Scholar), the closest structural homologue of the Ppz phosphatases. However, the possible role of this phosphatase has not been worked out.We considered that if EF1Bα was a target (either direct or indirect) for Ppz1, it could be possible to establish some sort of functional connection between both proteins. Deletion of TEF5 is lethal for the cell, and high copy expression of TEF2 suppresses the lethal phenotype of tef5 mutants (4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). However, these cells are markedly sensitive to translational inhibitors, such as paromomycin and hygromycin B. It is remarkable that lack of Ppz phosphatases also results in a change in sensitivity to these compounds, although in this case yielding more tolerant cells. Because these drugs are aminoglycosides known to enter the yeast cell driven by the membrane potential, which is mostly maintained by the function of the membrane H+-ATPase (32Vallejo C.G. Serrano R. Yeast. 1989; 5: 307-319Crossref PubMed Scopus (95) Google Scholar), we considered the possibility that the increased tolerance could be an indirect effect due to altered proton efflux. However, this was ruled out by determining this parameter in wild type and ppz mutants and finding essentially identical values (data not shown).Changes in sensitivity to paromomycin have been related to altered translational fidelity (33Singh A. Ursic D. Davies J. Nature. 1979; 277: 146-148Crossref PubMed Scopus (172) Google Scholar, 34Palmer E. Wilhelm J.M. Sherman F. Nature. 1979; 277: 148-150Crossref PubMed Scopus (172) Google Scholar), a phenotype also produced by changes in the dosage of EF1A (35Song J.M. Picologlou S. Grant C.M. Firoozan M. Tuite M.F. Liebman S. Mol. Cell. Biol. 1989; 9: 4571-4575Crossref PubMed Scopus (66) Google Scholar). Recent evidence (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar) has been presented pointing out that mutations in the carboxyl-terminal region of EF1Bα results in increased sensitivity to translation inhibitors and that this effect was accompanied by enhanced translational fidelity (i.e. reduced readthrough at nonsense codons). These observations are in keeping with our finding that cells lacking Ppz phosphatases, which are more tolerant to certain translation inhibitors, show an increased readthrough at nonsense codons, most likely due to a decrease in translational fidelity.Further evidence for a functional interaction between EF1Bα and Ppz1 comes from the observation that overexpression of the translation factor strongly attenuates the growth defect, due to a delayed G1/S transition, of cells containing an excess of Ppz1 activity. Although we showed in the past that this defect correlates with a delay in G1/S cyclin mRNA expression (14Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (68) Google Scholar), immunoblot analysis of the protein level of different cyclins reveals that, at least in the case of Clb5, further post-transcriptional alterations (i.e. at the translation level) could exist. 2E. Nadal, R. P. Fadden, A. Ruiz, T. Haystead, and J. Ariño, unpublished results. Remarkably, a non-phosphorylatable version of EF1Bα was unable to counteract the effect of an excess of Ppz1, suggesting that in vivomodulation of the phosphorylation state of the factor is somehow involved in the regulation of its function. It has been reported that, when expressed from the powerful GAL promoter, a carboxyl-terminal fragment of EF1Bα, lacking Ser-86, was sufficient for normal growth and did not display dramatically altered drug or temperature sensitivity (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). Furthermore, a strain containing a S86A version of EF1Bα as the only source for the factor is viable. 3L. Valente and T. G. Kinzy, personal communication. Therefore, it must be concluded that regulation of EF1Bα by phospho-dephosphorylation at Ser-86 (which, at least in part, would involve Ppz1) must affect the function of the translation factor in a subtle way. From our data, it can be hypothesized that changes in the phosphorylation state of EF1Bα would result in altered nucleotide exchange on EF1A. However, alternative mechanisms cannot be excluded, because it has been postulated that EF1Bα may have additional regulatory effects on EF1A (9Carr-Schmid A. Durko N. Cavallius J. Merrick W.C. Kinzy T.G. J. Biol. Chem. 1999; 274: 30297-30302Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In any case, our data provides further support to the notion that phospho-dephosphorylation mechanisms are relevant for a proper regulation of protein synthesis. In this report we demonstrate that, in the yeast S. cerevisiae, translation elongation factor 1Bα is a phosphoprotein. Phosphorylation site mapping and sequence analysis indicates that the Ser-86 is the only phosphorylatable residue in this protein, at least under standard growth conditions. It is remarkable that data base search reveals that the equivalent Ser residue (as well as its acidic environment) is also found in a large variety of organisms, including Drosophila melanogaster,Caenorhabditis elegans, mouse, and human. Phosphorylation of EF1Bα has been reported in Artemia salina (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar), wheat (6Matsumoto S. Mizoguchi T. Oizumi N. Tsuruga M. Shinozaki K. Taira H. Ejiri S. Biosci. Biotechnol. Biochem. 1993; 57: 1740-1742Crossref PubMed Scopus (9) Google Scholar), and reticulocyte (7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar). In the former case, phosphorylation was ascribed to Ser-89, which is equivalent to Ser-86 in yeast EF1Bα. Interestingly, phosphorylation has been correlated to changes in its catalytic nucleotide exchange activity, although reports are somewhat contradictory (5Janssen G.M. Maessen G.D. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar, 7Peters H.I. Chang Y.W. Traugh J.A. Eur. J. Biochem. 1995; 234: 550-556Crossref PubMed Scopus (46) Google Scholar). Our data indicate that deletion of the ppz genes and overexpression of Hal3, a negative regulatory subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar), result in increased phosphorylation of the EF1Bα protein, specifically at Ser-86. These results would be compatible with a role of Ppz1 in regulating the phosphorylation state of the translation factor and, possibly, its function. We also show here evidence that affinity-purified yeast EF1Bα contains significant amounts of bound Ppz1, by using an approach that was pivotal in the past to identify the Hal3 protein as a subunit of Ppz1 (15De Nadal E. Clotet J. Posas F. Serrano R. Gómez N. Ariño J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar). This could be taken as an indication that Ppz1 could be able to directly dephosphorylate EF1Bα. However, we have been unable to detect direct dephosphorylation of either in vivo labeled or CK-2 in vitrophosphorylated EF1Bα in the presence of bacterially expressed Ppz1. Although at this point we cannot provide direct evidence for the translation factor being a substrate for the phosphatase, this possibility formally remains. For instance, the phosphatase might require accessory proteins (absent in our in vitro assay) to effectively use EF1Bα as substrate. In this regard, there is a large body of evidence for the requirement of specific regulatory subunits (targeting subunits) for Ser/Thr phosphatases to localize at specific subcellular sites or to use a given phosphoprotein as an effective substrate (28Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2143) Google Scholar, 29Stark M.J. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) Google Scholar). It must be noted that dephosphorylation events have been previously related to the control of the accuracy of protein synthesis, as it is the case of the Ppq1/Sal6 Ser/Thr protein phosphatase (30Chen M.X. Chen Y.H. Cohen P.T. Eur. J. Biochem. 1993; 218: 689-699Crossref PubMed Scopus (29) Google Scholar, 31Vincent A. Newnam G. Liebman S.W. Genetics. 1994; 138: 597-608Crossref PubMed Google Scholar), the closest structural homologue of the Ppz phosphatases. However, the possible role of this phosphatase has not been worked out. We considered that if EF1Bα was a target (either direct or indirect) for Ppz1, it could be possible to establish some sort of functional connection between both proteins. Deletion of TEF5 is lethal for the cell, and high copy expression of TEF2 suppresses the lethal phenotype of tef5 mutants (4Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). However, these cells are markedly sensitive to translational inhibitors, such as paromomycin and hygromycin B. It is remarkable that lack of Ppz phosphatases also results in a change in sensitivity to these compounds, although in this case yielding more tolerant cells. Because these drugs are aminoglycosides known to enter the yeast cell driven by the membrane potential, which is mostly maintained by the function of the membrane H+-ATPase (32Vallejo C.G. Serrano R. Yeast. 1989; 5: 307-319Crossref PubMed Scopus (95) Google Scholar), we considered the possibility that the increased tolerance could be an indirect effect due to altered proton efflux. However, this was ruled out by determining this parameter in wild type and ppz mutants and finding essentially identical values (data not shown). Changes in sensitivity to paromomycin have been related to altered translational fidelity (33Singh A. Ursic D. Davies J. Nature. 1979; 277: 146-148Crossref PubMed Scopus (172) Google Scholar, 34Palmer E. Wilhelm J.M. Sherman F. Nature. 1979; 277: 148-150Crossref PubMed Scopus (172) Google Scholar), a phenotype also produced by changes in the dosage of EF1A (35Song J.M. Picologlou S. Grant C.M. Firoozan M. Tuite M.F. Liebman S. Mol. Cell. Biol. 1989; 9: 4571-4575Crossref PubMed Scopus (66) Google Scholar). Recent evidence (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar) has been presented pointing out that mutations in the carboxyl-terminal region of EF1Bα results in increased sensitivity to translation inhibitors and that this effect was accompanied by enhanced translational fidelity (i.e. reduced readthrough at nonsense codons). These observations are in keeping with our finding that cells lacking Ppz phosphatases, which are more tolerant to certain translation inhibitors, show an increased readthrough at nonsense codons, most likely due to a decrease in translational fidelity. Further evidence for a functional interaction between EF1Bα and Ppz1 comes from the observation that overexpression of the translation factor strongly attenuates the growth defect, due to a delayed G1/S transition, of cells containing an excess of Ppz1 activity. Although we showed in the past that this defect correlates with a delay in G1/S cyclin mRNA expression (14Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (68) Google Scholar), immunoblot analysis of the protein level of different cyclins reveals that, at least in the case of Clb5, further post-transcriptional alterations (i.e. at the translation level) could exist. 2E. Nadal, R. P. Fadden, A. Ruiz, T. Haystead, and J. Ariño, unpublished results. Remarkably, a non-phosphorylatable version of EF1Bα was unable to counteract the effect of an excess of Ppz1, suggesting that in vivomodulation of the phosphorylation state of the factor is somehow involved in the regulation of its function. It has been reported that, when expressed from the powerful GAL promoter, a carboxyl-terminal fragment of EF1Bα, lacking Ser-86, was sufficient for normal growth and did not display dramatically altered drug or temperature sensitivity (8Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). Furthermore, a strain containing a S86A version of EF1Bα as the only source for the factor is viable. 3L. Valente and T. G. Kinzy, personal communication. Therefore, it must be concluded that regulation of EF1Bα by phospho-dephosphorylation at Ser-86 (which, at least in part, would involve Ppz1) must affect the function of the translation factor in a subtle way. From our data, it can be hypothesized that changes in the phosphorylation state of EF1Bα would result in altered nucleotide exchange on EF1A. However, alternative mechanisms cannot be excluded, because it has been postulated that EF1Bα may have additional regulatory effects on EF1A (9Carr-Schmid A. Durko N. Cavallius J. Merrick W.C. Kinzy T.G. J. Biol. Chem. 1999; 274: 30297-30302Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In any case, our data provides further support to the notion that phospho-dephosphorylation mechanisms are relevant for a proper regulation of protein synthesis. The skillful technical help of Anna Vilalta and Yolanda Prado are acknowledged. We thank T. G. Kinzy, M. Remacha, and J. P. Garcı́a-Ballesta for fruitful discussion.
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