Molecular and General Genetics MGG

, Volume 242, Issue 4, pp 383–390 | Cite as

Reduced but accurate translation from a mutant AUA initiation codon in the mitochondrial COX2 mRNA of Saccharomyces cerevisiae

  • Julio J. Mulero
  • Thomas D. Fox
Article

Abstract

We have changed the translation initiation codon of the COX2 mRNA of Saccharomyces cerevisiae from AUG to AUA, generating a mutation termed cox2-10. This mutation reduced translation of the COX2 mRNA at least five-fold without affecting the steady-state level of the mRNA, and produced a leaky nonrespiratory growth phenotype. To address the question of whether residual translation of the cox2-10 mRNA was initiating at the altered initiation codon or at the next AUG codon downstream (at position 14), we took advantage of the fact that the mature coxll protein is generated from the electrophoretically distinguishable coxII precursor by removal of the amino-terminal 15 residues, and that this processing can be blocked by a mutation in the nuclear gene PET2858. We constructed a pet2858, cox2-10 double mutant strain using a pet2858 allele from our mutant collection. The double mutant accumulated low levels of a polypeptide which comigrated with the coxII precursor protein, not the mature species, providing strong evidence that residual initiation was occurring at the mutant AUA codon. Residual translation of the mutant mRNA required the COX2 mRNA-specific activator PET111. Furthermore, growth of cox2-10 mutant strains was sensitive to alterations in PET111 gene dosage: the respiratory-defective growth phenotype was partially suppressed in haploid strains containing PET111 on a high-copy-number vector, but became more severe in diploid strains containing only one functional copy of PET111.

Key words

Mitochondria Translation Yeast PET111 PET2858 

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References

  1. Behrens M, Michaelis G, Pratje E (1991) Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase. Mol Gen Genet 228:167–176Google Scholar
  2. Chen X, Kindle K, Stern D (1993) Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth. EMBO J 12:3627–3635Google Scholar
  3. Conde J, Fink GR (1976) A mutant of S. cerevisiae defective for nuclear fusion. Proc Natl Acad Sci USA 73:3651Google Scholar
  4. Coruzzi G, Tzagoloff A (1979) Assembly of the mitochondrial membrane system: DNA sequence of subunit II of yeast cytochrome c oxidase. J Biol Chem 254:9324–9330Google Scholar
  5. Costanzo MC, Fox TD (1990) Control of mitochondrial gene expression in Saccharomyces cerevisiae. Annu Rev Genet 24:91–113Google Scholar
  6. Costanzo MC, Fox TD (1993) Suppression of a defect in the 5′-untranslated leader of the mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein. Mol Cell Biol 13:4806–4813Google Scholar
  7. Costanzo MC, Seaver EC, Fox TD (1986) At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA. EMBO J 5:3637–3641Google Scholar
  8. Dekker PJT, Papadopoulou B, Grivell LA (1993) In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition. Curr Genet 23:22–27Google 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. Elble R (1992) A simple and efficient procedure for transformation of yeast. Biotechniques 13:18–20Google Scholar
  11. Folley LS, Fox TD (1991) Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon. Genetics 129:659–668Google Scholar
  12. Fox TD (1979a) Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II. Proc Natl Acad Sci USA 76:6534–6538Google Scholar
  13. Fox TD (1979b) Genetic and physical analysis of the mitochondrial gene for subunit II of yeast cytochrome c oxidase. J Mol Biol 130:63–82Google Scholar
  14. Fox TD (1987) Natural variation in the genetic code. Annu Rev Genet 21:67–91Google Scholar
  15. 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
  16. Fox TD, Sanford JC, McMullin TW (1988) Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA. Proc Natl Acad Sci USA 85:7288–7292Google 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. 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
  19. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167Google Scholar
  20. Hinnebusch AG, Liebman SW (1991) Protein synthesis and translational control in Saccharomyces cerevisiae. In: Broach JR, Pringle JR, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces: genome dynamics, protein synthesis, and energetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 627–735Google Scholar
  21. Hudspeth MES, Ainley WM, Shumard DS, Butow RA, Grossman LI (1982) Location and structure of the var1 gene on yeast mitochondrial DNA: nucleotide sequence of the 40.0 allele. Cell 30:617–626Google Scholar
  22. Johnston SA, Anziano PQ, Shark K, Samord JC, Butow RA (1988) Mitochondrial transformation in yeast by bombardment with microprojectiles. Science 240:1538–1541Google Scholar
  23. Marykwas DL, Fox TD (1989) Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen. Mol Cell Biol 9:484–491Google Scholar
  24. Mannhaupt G, Michaelis G, Pratje E, Schweizer E, Hawthorne DC (1983) A precursor to subunit II of cytochrome oxidase in Saccharomyces cerevisiae. In: Schweyen RJ, Wolf K, Kaudewitz F (eds) Mitochondria 1983: Nucleo-mitochondrial interactions. Walter der Gruyter, Berlin-New York, pp. 449–454Google Scholar
  25. 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
  26. Mulero JJ, Fox TD (1993) PET111 acts in the 5′-leader of the Saccharomyces cerevisiae mitochondrial COX2 mRNA to promote its translation. Genetics 133:509–516Google Scholar
  27. Pon L, Schatz G (1991) Biogenesis of yeast mitochondria. In: Broach JR, Pringle JR, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces: genome dynamics, protein synthesis and energetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 333–406Google Scholar
  28. Poutre CG, Fox TD (1987) PET111 a Saccharomyces cerevisiae nuclear gene required for translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit II. Genetics 115:637–647Google Scholar
  29. Pratje E, Guiard B (1986) One nuclear gene controls the removal of transient pre-sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. EMBO J 5:1313–1317Google Scholar
  30. Pratje E, Mannhaupt G, Michaelis G, Beyreuther K (1983) A nuclear mutation prevents processing of a mitochondrially encoded membrane protein in Saccharomyces cerevisiae. EMBO J 2:1049–1054Google Scholar
  31. Rogers D, Bussey H (1978) Fidelity of conjugation in Saccharomyces cerevisiae. Mol Gen Genet 162:173–182Google Scholar
  32. Rose MD, Winston F, Hieter P (1988) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, New YorkGoogle Scholar
  34. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  35. Schneider A, Behrens M, Scherer P, Pratje E, Michaelis G, Schatz G (1991) Inner membrane protease 1, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO J 10:247–254Google Scholar
  36. Sevarino KA, Poyton RO (1980) Mitochondrial biogenesis: identification of a precursor to yeast cytochrome c oxidase subunit II, an integral polypeptide. Proc Natl Acad Sci USA 77:142–146Google Scholar
  37. Shen Z, Fox TD (1989) Substitution of an invariant nucleotide at the base of the highly conserved “530-loop” of 15S rRNA causes suppression of mitochondrial ochre mutations. Nucleic Acids Res 17:4535–4539Google Scholar
  38. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  39. Strick CA, Fox TD (1987) Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5′ leader. Mol Cell Biol 7:2728–2734Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Julio J. Mulero
    • 1
  • Thomas D. Fox
    • 2
  1. 1.Section of BiochemistryMolecular and Cell Biology Cornell UniversityIthacaUSA
  2. 2.Section of Genetics and DevelopmentCornell UniversityIthacaUSA

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