Molecular and General Genetics MGG

, Volume 235, Issue 1, pp 64–73 | Cite as

Suppression of carboxy-terminal truncations of the yeast mitochondrial mRNA-specific translational activator PET122 by mutations in two new genes, MRP17 and PET127

  • Pascal Haffter
  • Thomas D. Fox

Summary

The PET122 protein is one of three Saccharomyces cerevisiae nuclear gene products required specifically to activate translation of the mitochondrially coded COX3 mRNA. We have previously observed that mutations which remove the carboxy-terminal region of PET122 block translation of the COX3 mRNA but can be suppressed by unlinked nuclear mutations in several genes, two of which have been shown to code for proteins of the small subunit of mitochondrial ribosomes. Here we describe and map two more new genes identified as allele-specific suppressors that compensate for carboxy-terminal truncation of PET122. One of these genes, MRP17, is essential for the expression of all mitochondrial genes and encodes a protein of Mr 17343. The MRP17 protein is a component of the small ribosomal subunit in mitochondria, as demonstrated by the fact that a missense mutation, mrp17-1, predicted to cause a charge change indeed alters the charge of a mitochondrial ribosomal protein of the expected size. In addition, mrp17-1, in combination with some mutations affecting another mitochondrial ribosomal protein, caused a synthetic defective phenotype. These findings are consistent with a model in which PET122 functionally interacts with the ribosomal small subunit. The second new suppressor gene described here, PET127, encodes a protein too large (Mr 95900) to be a ribosomal protein and appears to operate by a different mechanism. PET127 is not absolutely required for mitochondrial gene expression and allele-specific suppression of pet122 mutations results from the loss of PET127 function: a pet127 deletion exhibited the same recessive suppressor activity as the original suppressor mutation. These findings suggest the possibility that PET127 could be a novel component of the mitochondrial translation system with a role in promoting accuracy of translational initiation.

Key words

Mitochondria Translation Ribosomal protein Suppression Genetic map 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baker A, Schatz G (1987) Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria. Proc Natl Acad Sci USA 84:3117–3121Google Scholar
  2. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346Google Scholar
  3. Cervantes E, Sharma SB, Maillet F, Vasse J, Truchet G, Rosenberg C (1989) The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors. Mol Microbiol 3:745–755Google Scholar
  4. Costanzo MC, Fox TD (1990) Control of mitochondrial gene expression in Saccharomyces cerevisiae. Annu Rev Genet 24:91–113Google Scholar
  5. Costanzo MC, Fox TD (1988a) Specific translational activation by nuclear gene products occurs in the 5′ untranslated leader of a yeast mitochondrial mRNA. Proc Natl Acad Sci USA 85:2677–2681Google Scholar
  6. Costanzo MC, Fox TD (1988b) Transformation of yeast by agitation with glass beads. Genetics 120:667–670Google Scholar
  7. Dang H, Franklin G, Darlak K, Spatola A, Ellis SR (1990) Discoordinate expression of the yeast mitochondrial ribosomal protein MRP1. J Biol Chem 265:7449–7454Google Scholar
  8. Daum G, Böhni P, Schatz G (1982) Import of proteins into mitochondria: cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257:13028–13033Google Scholar
  9. Douglas M, Butow RA (1976) Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA. Proc Natl Acad Sci USA 73:1083–1096Google Scholar
  10. Dunn TM, Shortle D (1990) Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisae. Mol Cell Biol 10:2308–2314Google Scholar
  11. Eckert KA, Kunkel TA (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res 18:3739–3744Google Scholar
  12. Faye G, Sor F (1977) Analysis of mitochondrial ribosomal proteins of Saccharomyces cerevisiae by two-dimensional polyacrylamide gel electrophoresis. Mol Gen Genet 155:27–34Google Scholar
  13. 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
  14. Fox TD, Folley LS, Mulero JJ, McMullin TW, Thorsness PE, Hedin LO, Costanzo MC (1991) Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol 194:149–165Google Scholar
  15. Gaber RF, Mathison L, Edelman I, Culbertson MR (1983) Frameshift suppression in Saccharomyces cerevisiae. VI. Complete genetic map of twenty-five suppressor genes. Genetics 103:389–407Google Scholar
  16. Haffter P, McMullin TW, Fox TD (1991) Functional interactions among two yeast mitochondrial ribosomal proteins and an mRNA-specific translational activator. Genetics 127:319–326Google Scholar
  17. Haffter P, McMullin TW, Fox TD (1990) A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria. Genetics 125:495–503Google Scholar
  18. Hartman PE, Roth JR (1973) Mechanisms of suppression. Adv Genet 17:1–105Google Scholar
  19. Huffaker TC, Hoyt MA, Botstein D (1987) Genetic analysis of the yeast cytoskeleton. Annu Rev Genet 21:259–284Google Scholar
  20. Jarvik J, Botstein D (1975) Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci USA 72:2738–2742Google Scholar
  21. 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
  22. Kitakawa M, Isono K (1991) The mitochondrial ribosomes. Biochimie 73:813–825Google Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  24. Lambowitz AM (1979) Preparation and analysis of mitochondrial ribosomes. Methods Enzymol 59:421–433Google Scholar
  25. Lemire BD, Fankhauser C, Baker A, Schatz G (1989) The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity. J Biol Chem 264:20206–20215Google Scholar
  26. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  27. 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
  28. McMullin TW, Haffter P, Fox TD (1990) A novel small subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator. Mol Cell Biol 10:4590–4595Google Scholar
  29. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78Google Scholar
  30. Mets LJ, Bogorad L (1974) Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins. Anal Biochem 57:200–210Google Scholar
  31. Mortimer RK, Schild D, Contopoulou CR, Kans JA (1989) Genetic map of Saccharomyces cerevisiae, edition 10. Yeast 5:321–403Google 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. 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
  34. Neff NF, Thomas JH, Grisafi P, Botstein D (1983) Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33:211–219Google Scholar
  35. Ohmen JD, Kloeckener-Gruissem B, McEwen JE (1988) Molecular cloning and nucleotide sequence of the nuclear PET122 gene required for expression of the mitochondrial COX3 gene in S. cerevisiae. Nucleic Acids Res 16:10783–10802Google Scholar
  36. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448Google Scholar
  37. Rose MD, Winston F, Hieter P (1988) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  38. Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243Google Scholar
  39. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  40. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27Google Scholar
  41. Singh A, Mason TL, Zimmermann RA (1978) A cold-sensitive cytoplasmic mutation of Saccharomyces cerevisiae affecting assembly of the mitochondrial 50S ribosomal subunit. Mol Gen Genet 161:143–151Google Scholar
  42. Stearns T, Hoyt MA, Botstein D (1990) Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics 124:251–262Google Scholar
  43. 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
  44. Thorsness PE, Fox TD (1990) Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae. Nature 346:376–379Google Scholar
  45. von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5:1335–1342Google Scholar
  46. Williams JF (1989) Optimization strategies for the polymerase chain reaction. BioTechniques 7:762–769Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Pascal Haffter
    • 1
  • Thomas D. Fox
    • 1
  1. 1.Section of Genetics and DevelopmentCornell UniversityIthacaUSA
  2. 2.Max-Planck-Institut für EntwicklungsbiologieTübingenGermany

Personalised recommendations