Current Genetics

, Volume 28, Issue 1, pp 26–31 | Cite as

Cloning and analysis of the nuclear gene MRP-S9 encoding mitochondrial ribosomal protein S9 of Saccharomyces cerevisiae

  • Peter Kötter
  • Karl-Diether Entian
Original Paper


The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153–278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho- or rhoo cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.

Key words

Ribosomal protein Mitochondria Yeast Pet phenotype 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1989) Current protocols in molecular biology. John Wiley and Sons, New YorkGoogle Scholar
  3. Breining P, Piepersberg W (1986) Yeast omnipotent suppressor SUP1 (SUP45): nucleotide sequence of the wild-type and a mutant gene. Nucleic Acids Res 14:5187–5197Google Scholar
  4. Bennetzen JL, Hall BD (1982) The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase I. J Biol Chem 257:3018–3025Google Scholar
  5. Bucher P, Trifonov EN (1986) Compilation and analysis of eukaryotic POL II promotor sequences. Nucleic Acids Res 14:10009–10025Google Scholar
  6. Ciriacy M (1975) Genetics of alcohol dehydrogenase in Saccharomyces cereviasie. I. Isolation and genetic analysis of mutants. Mutat Res 29:315–326Google Scholar
  7. Dang H, Ellis SR (1990) Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli. Nucleic Acids Res 18:6895–6901Google Scholar
  8. Davis SC, Tzagoloff A, Ellis SR (1992) Characterization of a yeast mitochondrial ribosomal protein structurally related to the mammalian 68-kDa high-affinity laminin receptor. J Biol Chem 267:5508–5514Google Scholar
  9. Dohmen RJ, Strasser AWM, Höhner CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692Google Scholar
  10. Dujon B (1981) Mitochondrial genetics and functions. In: Strathern JN, Jones EW, Broach JR (eds) Molecular biology of the yeast Saccharomyces: life cycle and inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 505–635Google Scholar
  11. Fearon K, Mason TL (1988) Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7 a protein of the large subunit of the mitochondrial ribosome. Mol Cell Biol 8:3636–3646Google Scholar
  12. Fearon K, Mason TL (1992) Structure and function of MRP20 and MRP49 the nuclear genes for two proteins of the 54 subunit of the yeast mitochondrial ribosome. J Biol Chem 267:5162–5170Google Scholar
  13. Gancedo JM, Gancedo C (1971) Fructose-1–6-bisphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non-fermenting yeast. Arch Microbiol 76:132–138Google Scholar
  14. Grohmann L, Graack H-R, Kruft V, Choli T, Goldschmidt-Reisin S, Kitakawa M (1991) Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria. FEBS Lett 284:51–56Google Scholar
  15. Grosveld GC, Rosenthal A, Flavell RA (1982) Sequence requirements for the transcription of the rabbit β-globin gene in vivo: the-80 region. Nucleic Acids Res 10:4951–4971Google Scholar
  16. Gunteski-Hamblin A-M, Clarke DM, Shull GE (1992) Molecular cloning and tissue distribution of alternatively spliced mRNAs encoding possible mammalian homologues of the yeast secretory pathway calcium pump. Biochemistry 31:7600–7608Google Scholar
  17. Hanahan D (1985) Techniques for transformation of Escherichia coli. In: Glover DM (ed) DNA-Cloning I. IRL Press, Oxford, pp 109–135Google Scholar
  18. Hansen RJ, Hinze H, Holzer H (1976) Assay of phosphoenol-pyruvate carboxikinase in crude yeast extracts. Anal Biochem 74:576–584Google Scholar
  19. Harrer R, Schwank S, Schüler HJ, Schweizer E (1993) Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae. Cur Genet 24:136–140Google Scholar
  20. Haußmann P, Zimmermann FK (1971) The role of mitochondria in carbon catabolite repression in yeast. Mol Gen Genet 148:205–211Google Scholar
  21. Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359Google Scholar
  22. Isono S, Thamm S, Kitakawa M, Isono K (1985) Cloning and nucleotide sequencing of the genes for ribosomal proteins S9 (rspI) and L13 (rplM) of Escherichia coli. Mol Gen Genet 198:279–282Google Scholar
  23. Kang W, Matsushita Y, Grohmann L, Graack H-R, Kitakawa M, Isono K (1991) Cloning and analysis of the nuclear gene for YmL33 a protein of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. J Bacteriol 173:4013–4020Google Scholar
  24. Kimura M, Chow CK (1984) The complete amino-acid sequences of ribosomal proteins L17, L27, and S9 from Bacillus stearothermophilus. Eur J Biochem 139:225–234Google Scholar
  25. Kitakawa M, Grohmann L, Graack H-R, Isono K (1990) Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae. Nucleic Acids Res 18:1521–1529Google Scholar
  26. Kozak M (1983) Comparison of initiation of protein synthesis in prokaryotes, eukaryotes, and organelles, Microbiol Rev 47:1–45Google Scholar
  27. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Biol Chem 27:105–132Google Scholar
  28. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  29. Matsushita Y, Kitakawa M, Isono K (1989) Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast. Mol Gen Genet 219:119–124Google Scholar
  30. McEwen JE, Cameron VL, Poyton RO (1985) Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. J Bacteriol 161:831–835Google Scholar
  31. Myers AM, Pape LK, Tzagoloff A (1985) Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J 4:2087–2092Google Scholar
  32. Myers AM, Crivellone MD, Tzagoloff A (1987) Assembly of the mitochondrial membrane system: MRP1 and MRP2 two yeast nuclear genes coding for mitochondrial ribosomal proteins. J Biol Chem 262:3388–3397Google Scholar
  33. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448Google Scholar
  34. Rothstein RI (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211Google Scholar
  35. Rudolph HK, Antebi A, Fink GR, Buckley CM, Dorman TE, LeVitre J, Davidow LS, Mao JI, Moir DT (1989) The yeast secretory pathway is perturbed by mutations in PMR1 a member of a Ca2+ ATPase family. Cell 58:133–145Google Scholar
  36. Russel DW, Smith M, Williamson VM, Young ET (1983) Nuclcotide sequence of the yeast alcohol dehydrogenase II gene. J Biol Chem 258:2674–2682Google Scholar
  37. Schwartz RM, Dayhoff MO (1978) Origins of prokaryotes, cukaryotes, mitochondria, and chloroplasts. Science 199:395–403Google Scholar
  38. Sharp PM, Cowe E (1991) Synonymous codon usage in Saccharomyces cerevisiae. Yeast 7:657–678Google Scholar
  39. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039Google Scholar
  40. Theisen M, Potter AA (1992) Cloning, sequencing, expression, and functional studies of a 15000-molecular-weight Haemophilus somnus antigen similar to Escherichia coli ribosomal protein S9. J Bacteriol 174:17–23Google Scholar
  41. Tzagoloff A, Myers AM (1986) Genetics of mitochondrial biogenesis. Annu Rev Biochem 55:249–285Google Scholar
  42. Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194Google Scholar
  43. Wittmann HG (1983) Architecture of prokaryotic ribosomes. Annu Rev Biochem 52:35–65Google Scholar
  44. Moychik NA, Liao S-M, Kolodziej PA, Young RA (1990) Subunits shared by eukaryotic nuclear RNA polymerases. Genes Dev 4:313–323Google Scholar
  45. Young ET, Pilgrim DB (1985) Isolation and DNA sequence of ADH3 a nuclear gene encoding the mitochondrial isoenzyme of alcohol dehydrogenase in Saccharomyces cerevisiae. Mol Cell Biol 5:3024–3034Google Scholar
  46. Zamenhoff S (1957) Preparation and assay of deoxyribonucleic acids from animal tissue. Methods Enzymol 3:696–704Google Scholar
  47. Zaret KS, Sherman F (1982) DNA sequence required for efficient transcription termination in yeast. Cell 28:563–573Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Peter Kötter
    • 1
  • Karl-Diether Entian
    • 1
  1. 1.Institut für MikrobiologieJohann Wolfgang Goethe-Universität FrankfurtFrankfurtGermany

Personalised recommendations