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Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês

10.1074/jbc.274.8.4869

1083-351X

Michael Justice, Theresa Ku, Ming‐Jo Hsu, Karen Carniol, Dennis M. Schmatz, Jennifer Nielsen,

Mycotoxins in Agriculture and Food

The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form. We have previously described a class of Saccharomyces cerevisiaemutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance. We now report on a second class of sordarin-resistant mutants. Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S. cerevisiae. Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified. Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e. Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex. To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation. These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2. The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form. We have previously described a class of Saccharomyces cerevisiaemutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance. We now report on a second class of sordarin-resistant mutants. Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S. cerevisiae. Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified. Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e. Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex. To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation. These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2. Eukaryotic elongation factor 2 (eEF2) 1The abbreviations eEF2eukaryotic elongation factor 2EF-Gelongation factor GPCRpolymerase chain reactionSCsynthetic complete medium 1The abbreviations eEF2eukaryotic elongation factor 2EF-Gelongation factor GPCRpolymerase chain reactionSCsynthetic complete mediumand its prokaryotic counterpart, elongation factor G (EF-G), promote the translocation of the ribosome along messenger RNA during the elongation phase of protein synthesis. Hydrolysis of GTP to GDP drives translocation and is associated with a presumed conformational change in eEF2. Sordarin (1Hauser D. Sigg H.P. Helv. Chim. Acta. 1971; 54: 1178-1190Crossref PubMed Scopus (102) Google Scholar) and its analogs are fungal-specific translation inhibitors (2Dominguez J.M. Kelly V.A. Kinsman O.S. Marriott M.S. Gomez de las Heras F. Martin J.J. Antimicrob. Agents Chemother. 1998; 42: 2274-2278Crossref PubMed Google Scholar, 3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 4Dominguez J.M. Martin J.J. Antimicrob. Agents Chemother. 1998; 42: 2279-2283Crossref PubMed Google Scholar, 5Capa L. Mendoza A. Lavandera J.L. Gomez de las Heras F. Garcia-Bustos J.F. Antimicrob. Agents Chemother. 1998; 42: 2694-2699Crossref PubMed Google Scholar). The observation that eEF2 is the major determinant of sordarin specificity was confirmed by the identification of 15 unique sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae. In our original characterization of 21 sordarin-resistant mutants, five mutations were not linked to the EFT1 or EFT2 genes. In this work, we show that these five mutations map to the essential ribosomal protein L10e.The ribosome, although not contributing significantly to the fungal specificity of sordarin, is a critical partner in forming the stabilized post-translocational complex (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Detection of a complex between fungal eEF2 and a labeled sordarin analog is strongly dependent upon the presence of ribosomes. L10, the prokaryotic counterpart ofS. cerevisiae L10e, has been localized to the base of the stalk structure conserved in all large ribosomal subunits (6Liljas A. Prog. Biophys. Mol. Biol. 1982; 40: 161-228Crossref PubMed Scopus (109) Google Scholar, 7Gudkov A.T. FEBS Lett. 1997; 407: 253-256Crossref PubMed Scopus (48) Google Scholar, 8Rich B.E. Steitz J.A. Mol. Cell. Biol. 1987; 7: 4065-4074Crossref PubMed Scopus (232) Google Scholar). The eukaryotic ribosomal stalk proteins L10e, L12eIA, L12eIIA, L12eIB, and L12eIIB in S. cerevisiae (9Mitsui K. Nakagawa T. Tsurugi K. J. Biochem. (Tokyo). 1989; 106: 223-227Crossref PubMed Scopus (15) Google Scholar, 10Hunter Newton C. Shimmin L.C. Yee J. Dennis D.P. J. Bacteriol. 1990; 172: 579-588Crossref PubMed Google Scholar) comprise a pentameric structure that is similar to the L10 and L7/L12 complex inEscherichia coli ribosomes. The conservation of the stalk structure has been visualized in recent cryoimages of both 70S (11Agrawal R.K. Penczek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar), in which the binding position of the EF-G-GDP-fusidic acid complex is observed in detail, and 80S (12Verschoor A. Warner J.R. Srivastava S. Grassucci R.A. Frank J. Nucleic Acids Res. 1998; 26: 655-661Crossref PubMed Scopus (77) Google Scholar) ribosomes. The prokaryotic L7/L12 ribosomal proteins have been studied extensively by many physical and biological techniques, but much less is known about the structure and function of prokaryotic or eukaryotic L10. L10 is among the proteins reported to be cross-linked to eEF2 in 80S ribosomes by bifunctional reagents (13Uchiumi T. Kikuchi M. Ogata K. J. Biol. Chem. 1986; 261: 9663-9667Abstract Full Text PDF PubMed Google Scholar). In yeast, mutational analysis of the L10e ribosomal protein gene has shown that only L10e is essential, and that the carboxyl-terminal 132 amino acids of the L10e protein are required for viability. However, the L12e proteins that comprise the L10e/L12e pentameric complex are not essential (14Remacha M. Jimenez-Diaz A. Santos C. Briones E. Zambrano R. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Biochem. Cell Biol. 1995; 73: 959-968Crossref PubMed Scopus (86) Google Scholar). Several findings suggest that there are associations between the stalk proteins and elongation factors. Mutations in L7/L12 perturb both EF-Tu and EF-G functions inE. coli (15Bilgin N. Kirsebom L.A. Ehrenberg M. Kurland C.G. Biochimie (Paris). 1988; 70: 611-618Crossref PubMed Scopus (41) Google Scholar). Chemical cross-links have been observed between EF-Tu and EF-G and the L7/L12 complex (reviewed in Ref. 16Czworkowski J. Moore P.B. Prog. Nucleic Acid Res. Mol. Biol. 1996; 54: 293-332Crossref PubMed Google Scholar). Of particular relevance to the present case, cross-links are observed between the EF-G and L7/L12 proteins in the presence of the nonhydrolyzable GTP analog GMPPCP, but not in the presence of GDP and fusidic acid (17Acharya A.S. Moore P.B. Richards F.M. Biochemistry. 1973; 12: 3108-3114Crossref PubMed Scopus (39) Google Scholar, 18Maasen J.A. Moller W. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1277-1280Crossref PubMed Scopus (60) Google Scholar). L7/L12 proteins in the EF-G-fusidic acid-ribosome complex are resistant to trypsin proteolysis (19Gudkov A.T. Gongadze S. FEBS Lett. 1984; 176: 32-36Crossref PubMed Scopus (35) Google Scholar). Our current results add to the body of information implicating L10e and eEF2 interactions to be important in translocation. These studies define a role for a small region of L10e in mediating inhibition by a new class of natural product with unprecedented selectivity for fungal protein synthesis and provide evidence for a functional interaction between L10e and eEF2. Eukaryotic elongation factor 2 (eEF2) 1The abbreviations eEF2eukaryotic elongation factor 2EF-Gelongation factor GPCRpolymerase chain reactionSCsynthetic complete medium 1The abbreviations eEF2eukaryotic elongation factor 2EF-Gelongation factor GPCRpolymerase chain reactionSCsynthetic complete mediumand its prokaryotic counterpart, elongation factor G (EF-G), promote the translocation of the ribosome along messenger RNA during the elongation phase of protein synthesis. Hydrolysis of GTP to GDP drives translocation and is associated with a presumed conformational change in eEF2. Sordarin (1Hauser D. Sigg H.P. Helv. Chim. Acta. 1971; 54: 1178-1190Crossref PubMed Scopus (102) Google Scholar) and its analogs are fungal-specific translation inhibitors (2Dominguez J.M. Kelly V.A. Kinsman O.S. Marriott M.S. Gomez de las Heras F. Martin J.J. Antimicrob. Agents Chemother. 1998; 42: 2274-2278Crossref PubMed Google Scholar, 3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 4Dominguez J.M. Martin J.J. Antimicrob. Agents Chemother. 1998; 42: 2279-2283Crossref PubMed Google Scholar, 5Capa L. Mendoza A. Lavandera J.L. Gomez de las Heras F. Garcia-Bustos J.F. Antimicrob. Agents Chemother. 1998; 42: 2694-2699Crossref PubMed Google Scholar). The observation that eEF2 is the major determinant of sordarin specificity was confirmed by the identification of 15 unique sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae. In our original characterization of 21 sordarin-resistant mutants, five mutations were not linked to the EFT1 or EFT2 genes. In this work, we show that these five mutations map to the essential ribosomal protein L10e. eukaryotic elongation factor 2 elongation factor G polymerase chain reaction synthetic complete medium eukaryotic elongation factor 2 elongation factor G polymerase chain reaction synthetic complete medium The ribosome, although not contributing significantly to the fungal specificity of sordarin, is a critical partner in forming the stabilized post-translocational complex (3Justice M.C. Hsu M.-J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Detection of a complex between fungal eEF2 and a labeled sordarin analog is strongly dependent upon the presence of ribosomes. L10, the prokaryotic counterpart ofS. cerevisiae L10e, has been localized to the base of the stalk structure conserved in all large ribosomal subunits (6Liljas A. Prog. Biophys. Mol. Biol. 1982; 40: 161-228Crossref PubMed Scopus (109) Google Scholar, 7Gudkov A.T. FEBS Lett. 1997; 407: 253-256Crossref PubMed Scopus (48) Google Scholar, 8Rich B.E. Steitz J.A. Mol. Cell. Biol. 1987; 7: 4065-4074Crossref PubMed Scopus (232) Google Scholar). The eukaryotic ribosomal stalk proteins L10e, L12eIA, L12eIIA, L12eIB, and L12eIIB in S. cerevisiae (9Mitsui K. Nakagawa T. Tsurugi K. J. Biochem. (Tokyo). 1989; 106: 223-227Crossref PubMed Scopus (15) Google Scholar, 10Hunter Newton C. Shimmin L.C. Yee J. Dennis D.P. J. Bacteriol. 1990; 172: 579-588Crossref PubMed Google Scholar) comprise a pentameric structure that is similar to the L10 and L7/L12 complex inEscherichia coli ribosomes. The conservation of the stalk structure has been visualized in recent cryoimages of both 70S (11Agrawal R.K. Penczek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar), in which the binding position of the EF-G-GDP-fusidic acid complex is observed in detail, and 80S (12Verschoor A. Warner J.R. Srivastava S. Grassucci R.A. Frank J. Nucleic Acids Res. 1998; 26: 655-661Crossref PubMed Scopus (77) Google Scholar) ribosomes. The prokaryotic L7/L12 ribosomal proteins have been studied extensively by many physical and biological techniques, but much less is known about the structure and function of prokaryotic or eukaryotic L10. L10 is among the proteins reported to be cross-linked to eEF2 in 80S ribosomes by bifunctional reagents (13Uchiumi T. Kikuchi M. Ogata K. J. Biol. Chem. 1986; 261: 9663-9667Abstract Full Text PDF PubMed Google Scholar). In yeast, mutational analysis of the L10e ribosomal protein gene has shown that only L10e is essential, and that the carboxyl-terminal 132 amino acids of the L10e protein are required for viability. However, the L12e proteins that comprise the L10e/L12e pentameric complex are not essential (14Remacha M. Jimenez-Diaz A. Santos C. Briones E. Zambrano R. Rodriguez-Gabriel M.A. Guarinos E. Ballesta J.P.G. Biochem. Cell Biol. 1995; 73: 959-968Crossref PubMed Scopus (86) Google Scholar). Several findings suggest that there are associations between the stalk proteins and elongation factors. Mutations in L7/L12 perturb both EF-Tu and EF-G functions inE. coli (15Bilgin N. Kirsebom L.A. Ehrenberg M. Kurland C.G. Biochimie (Paris). 1988; 70: 611-618Crossref PubMed Scopus (41) Google Scholar). Chemical cross-links have been observed between EF-Tu and EF-G and the L7/L12 complex (reviewed in Ref. 16Czworkowski J. Moore P.B. Prog. Nucleic Acid Res. Mol. Biol. 1996; 54: 293-332Crossref PubMed Google Scholar). Of particular relevance to the present case, cross-links are observed between the EF-G and L7/L12 proteins in the presence of the nonhydrolyzable GTP analog GMPPCP, but not in the presence of GDP and fusidic acid (17Acharya A.S. Moore P.B. Richards F.M. Biochemistry. 1973; 12: 3108-3114Crossref PubMed Scopus (39) Google Scholar, 18Maasen J.A. Moller W. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1277-1280Crossref PubMed Scopus (60) Google Scholar). L7/L12 proteins in the EF-G-fusidic acid-ribosome complex are resistant to trypsin proteolysis (19Gudkov A.T. Gongadze S. FEBS Lett. 1984; 176: 32-36Crossref PubMed Scopus (35) Google Scholar). Our current results add to the body of information implicating L10e and eEF2 interactions to be important in translocation. These studies define a role for a small region of L10e in mediating inhibition by a new class of natural product with unprecedented selectivity for fungal protein synthesis and provide evidence for a functional interaction between L10e and eEF2. We thank Dr. Jonathan Dinman of the University of Medicine and Dentistry of New Jersey and Dr. James Bodley of the University of Minnesota for helpful discussions and comments. We thank Drs. Paul Liberator and Mythili Shastry of Merck Research Laboratories for assistance and comments on the manuscript.