The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

ABSTRACT The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.

[1]  S. Rospert.  Ribosome Function: Governing the Fate of a Nascent Polypeptide , 2004, Current Biology.

[2]  Peter J McCormick,et al.  Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins , 2004, Cell.

[3]  M. Rodnina,et al.  Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. , 2004, Molecular cell.

[4]  R. Altman,et al.  Ribosomal dynamics inferred from variations in experimental measurements. , 2003, RNA.

[5]  M. Gautschi,et al.  The Yeast Nα-Acetyltransferase NatA Is Quantitatively Anchored to the Ribosome and Interacts with Nascent Polypeptides , 2003, Molecular and Cellular Biology.

[6]  Ulrike M K Böttcher,et al.  TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes , 2003, The EMBO journal.

[7]  V. Ramakrishnan,et al.  Insights into the decoding mechanism from recent ribosome structures. , 2003, Trends in biochemical sciences.

[8]  O. Namy,et al.  Identification of stop codon readthrough genes in Saccharomyces cerevisiae , 2003, Nucleic acids research.

[9]  E. Craig,et al.  Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding? , 2003, Current opinion in microbiology.

[10]  D. Masison,et al.  Deletion of the Hsp70 chaperone gene SSB causes hypersensitivity to guanidine toxicity and curing of the [ PSI +] prion by increasing guanidine uptake in yeast , 2003, Molecular Genetics and Genomics.

[11]  V. Ramakrishnan,et al.  Selection of tRNA by the Ribosome Requires a Transition from an Open to a Closed Form , 2002, Cell.

[12]  T. Kinzy,et al.  Identification of a role for actin in translational fidelity in yeast , 2002, Molecular Genetics and Genomics.

[13]  M. Rodnina,et al.  Structural dynamics of ribosomal RNA during decoding on the ribosome. , 2002, Biochimie.

[14]  M. Gautschi,et al.  A functional chaperone triad on the yeast ribosome , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Wiedmann,et al.  The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  F. Hartl,et al.  Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein , 2002, Science.

[17]  J. Weissman,et al.  The utility of prions. , 2002, Developmental cell.

[18]  Isabelle Hatin,et al.  Impact of the six nucleotides downstream of the stop codon on translation termination , 2001, EMBO reports.

[19]  Y. Chernoff,et al.  Mechanism of Prion Loss after Hsp104 Inactivation in Yeast , 2001, Molecular and Cellular Biology.

[20]  J Frank,et al.  The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. , 2001, Molecular cell.

[21]  T. Lithgow,et al.  RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Murtiashaw,et al.  The role of active site residue arginine 218 in firefly luciferase bioluminescence. , 2001, Biochemistry.

[23]  S. Joseph,et al.  Conformational changes in the ribosome induced by translational miscoding agents. , 2000, Journal of molecular biology.

[24]  A. Chacińska,et al.  Prion-Dependent Switching between Respiratory Competence and Deficiency in the Yeast nam9-1Mutant , 2000, Molecular and Cellular Biology.

[25]  H. True,et al.  A yeast prion provides a mechanism for genetic variation and phenotypic diversity , 2000, Nature.

[26]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[27]  O. Namy,et al.  Nonsense-mediated decay mutants do not affect programmed -1 frameshifting. , 2000, RNA.

[28]  B. Roberts,et al.  Prions of yeast as heritable amyloidoses. , 2000, Journal of structural biology.

[29]  S. Liebman,et al.  Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable. , 2000, Biochemistry.

[30]  Roger A. Garrett,et al.  The Ribosome, Structure, Function, Antibiotics, and Cellular Interactions , 2000 .

[31]  T. Zoladek,et al.  The growth of mdp1/rsp5 mutants of Saccharomyces cerevisiae is affected by mutations in the ATP-binding domain of the plasma membrane H+ -ATPase. , 2000, Gene.

[32]  Y. Chernoff,et al.  Evidence for a Protein Mutator in Yeast: Role of the Hsp70-Related Chaperone Ssb in Formation, Stability, and Toxicity of the [PSI] Prion , 1999, Molecular and Cellular Biology.

[33]  J. Frank,et al.  Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA , 1999, The EMBO journal.

[34]  S. Rospert,et al.  Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria. , 1999, Molecular biology of the cell.

[35]  J. Puglisi,et al.  Basis for prokaryotic specificity of action of aminoglycoside antibiotics , 1999, The EMBO journal.

[36]  E. Epping,et al.  Mutational Disruption of Plasma Membrane Trafficking of Saccharomyces cerevisiae Yor1p, a Homologue of Mammalian Multidrug Resistance Protein , 1999, Molecular and Cellular Biology.

[37]  W. V. Shaw,et al.  Missense translation errors in Saccharomyces cerevisiae. , 1998, Journal of molecular biology.

[38]  E. Craig,et al.  Zuotin, a ribosome‐associated DnaJ molecular chaperone , 1998, The EMBO journal.

[39]  M. Wiedmann,et al.  The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome–nascent chain complex , 1998, The EMBO journal.

[40]  M. Ter‐Avanesyan,et al.  Structure and Replication of Yeast Prions , 1998, Cell.

[41]  J. Puglisi,et al.  Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. , 1998, Journal of molecular biology.

[42]  T. Hallstrom,et al.  Regulation of Transcription Factor Pdr1p Function by an Hsp70 Protein in Saccharomyces cerevisiae , 1998, Molecular and Cellular Biology.

[43]  L. Alksne,et al.  Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin. , 1996, Biochimica et biophysica acta.

[44]  S. Lindquist,et al.  Maintenance and inheritance of yeast prions. , 1996, Trends in genetics : TIG.

[45]  D. Ruggero,et al.  Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea , 1996, Journal of bacteriology.

[46]  A. E. Dahlberg,et al.  The involvement of two distinct regions of 23 S ribosomal RNA in tRNA selection. , 1995, Journal of molecular biology.

[47]  J. Rousset,et al.  Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells. , 1995, Nucleic acids research.

[48]  N. Dean,et al.  Yeast glycosylation mutants are sensitive to aminoglycosides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[49]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[50]  Y. Chernoff,et al.  Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics. , 1994, The EMBO journal.

[51]  S. Liebman,et al.  An accuracy center in the ribosome conserved over 2 billion years. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Werner-Washburne,et al.  The translation machinery and 70 kd heat shock protein cooperate in protein synthesis , 1992, Cell.

[53]  M. Dante,et al.  Multifunctional yeast high-copy-number shuttle vectors. , 1992, Gene.

[54]  J. Heitman,et al.  FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[55]  F. Sherman,et al.  Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae. , 1990, Genetics.

[56]  S. Liebman,et al.  Isolation of omnipotent suppressors in an [eta+] yeast strain. , 1990, Genetics.

[57]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[58]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[59]  F. Sherman,et al.  Altered 40 S ribosomal subunits in omnipotent suppressors of yeast. , 1986, Journal of molecular biology.

[60]  G. Fink,et al.  A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance , 1984, Molecular and General Genetics MGG.

[61]  F. Sherman,et al.  Misreading of the ribosomal suppressor SUP46 due to an altered 40 S subunit in yeast. , 1981, Journal of molecular biology.

[62]  J. Davies,et al.  Phenotypic suppression and misreading in Saccharomyces cerevisiae , 1979, Nature.

[63]  F. Sherman,et al.  Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics , 1979, Nature.

[64]  M. Rodnina,et al.  Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. , 2001, Annual review of biochemistry.

[65]  J. Puglisi,et al.  Aminoglycoside Antibiotics and Decoding , 2000 .

[66]  T. Pape,et al.  Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G , 2000 .

[67]  C. Kurland,et al.  Translational accuracy and the fitness of bacteria. , 1992, Annual review of genetics.

[68]  P. Walter,et al.  In vitro protein translocation across microsomal membranes of Saccharomyces cerevisiae. , 1991, Methods in enzymology.

[69]  A. Haid,et al.  [13] Immunochemical identification of membrane proteins after sodium dodecyl sulfate—polyacrylamide gel electrophoresis , 1983 .

[70]  A. Haid,et al.  Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , 1983, Methods in enzymology.

[71]  G. Fink,et al.  Methods in yeast genetics , 1979 .