Experimental Relocation of the Mitochondrial Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution

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Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

Vyšlo v časopise: Experimental Relocation of the Mitochondrial Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution. PLoS Genet 8(8): e32767. doi:10.1371/journal.pgen.1002876
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002876

Souhrn

Zdroje

1. GrayMW, BurgerG, LangBF (1999) Mitochondrial evolution. Science 283 : 1476–1481.

2. Déquard-ChablatM, SellemCH, GolikP, BidardF, MartosA, et al. (2011) Two Nuclear Life Cycle-Regulated Genes Encode Interchangeable Subunits c of Mitochondrial ATP Synthase in Podospora anserina. Mol Biol Evol 28 : 2063–2075.

3. PalmerJD (1997) Organelle genomes: going, going, gone!. Science 275 : 790–791.

4. ClarosMG, PereaJ, ShuY, SamateyFA, PopotJL, et al. (1995) Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b. Eur J Biochem 228 : 762–771.

5. AllenJF (1993) Control of gene expression by redox potential and the requirement for chloroplast and mitochondrial genomes. J Theor Biol 165 : 609–631.

6. AllenJF (2003) Why chloroplasts and mitochondria contain genomes. Comp Funct Genomics 4 : 31–36.

7. RaceHL, HerrmannRG, MartinW (1999) Why have organelles retained genomes? Trends Genet 15 : 364–370.

8. AmiottEA, JaehningJA (2006) Mitochondrial transcription is regulated via an ATP “sensing” mechanism that couples RNA abundance to respiration. Mol Cell 22 : 329–338.

9. BanroquesJ, DelahoddeA, JacqC (1986) A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing. Cell 46 : 837–844.

10. SanchiricoM, TzellasA, FoxTD, Conrad-WebbH, PerimanPS, et al. (1995) Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus. Biochem Cell Biol 73 : 987–995.

11. NagleyP, FarrellLB, GearingDP, NeroD, MeltzerS, et al. (1988) Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle. Proc Natl Acad Sci U S A 85 : 2091–2095.

12. SupekovaL, SupekF, GreerJE, SchultzPG (2010) A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression. Proc Natl Acad Sci U S A 107 : 5047–5052.

13. MichonT, GalanteM, VeloursJ (1988) NH2-terminal sequence of the isolated yeast ATP synthase subunit 6 reveals post-translational cleavage. Eur J Biochem 172 : 621–625.

14. StockD, GibbonsC, ArechagaI, LeslieAG, WalkerJE (2000) The rotary mechanism of ATP synthase. Curr Opin Struct Biol 10 : 672–679.

15. BoyerPD (1997) The ATP synthase–a splendid molecular machine. Annu Rev Biochem 66 : 717–749.

16. SarasteM (1999) Oxidative phosphorylation at the fin de siecle. Science 283 : 1488–1493.

17. DyerMR, GayNJ, WalkerJE (1989) DNA sequences of a bovine gene and of two related pseudogenes for the proteolipid subunit of mitochondrial ATP synthase. Biochem J 260 : 249–258.

18. FarrellLB, GearingDP, NagleyP (1988) Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Expression in vitro from a chemically synthesized gene and import into isolated mitochondria. Eur J Biochem 173 : 131–137.

19. SteeleDF, ButlerCA, FoxTD (1996) Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation. Proc Natl Acad Sci U S A 93 : 5253–5257.

20. RakM, TetaudE, GodardF, SagotI, SalinB, et al. (2007) Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology. J Biol Chem 282 : 10853–10864.

21. SotoIC, FontanesiF, ValledorM, HornD, SinghR, et al. (2009) Synthesis of cytochrome c oxidase subunit 1 is translationally downregulated in the absence of functional F1F0-ATP synthase. Biochim Biophys Acta 1793 : 1776–1786.

22. ViebrockA, PerzA, SebaldW (1982) The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA. EMBO J 1 : 565–571.

23. Van DyckL, LangerT (1999) ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae. Cell Mol Life Sci 56 : 825–842.

24. Duvezin-CaubetS, CaronM, GiraudMF, VeloursJ, di RagoJP (2003) The two rotor components of yeast mitochondrial ATP synthase are mechanically coupled by subunit delta. Proc Natl Acad Sci U S A 100 : 13235–13240.

25. AckermanSH, TzagoloffA (2005) Function, structure, and biogenesis of mitochondrial ATP synthase. Prog Nucleic Acid Res Mol Biol 80 : 95–133.

26. RakM, ZengX, BriereJJ, TzagoloffA (2009) Assembly of F0 in Saccharomyces cerevisiae. Biochim Biophys Acta 1793 : 108–116.

27. MukhopadhyayA, UhM, MuellerDM (1994) Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media. FEBS Lett 343 : 160–164.

28. KucharczykR, EzkurdiaN, CouplanE, ProcaccioV, AckermanSH, et al. (2010) Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase. Biochim Biophys Acta 1797 : 1105–1112.

29. PelissierP, CamougrandN, VeloursG, GuerinM (1995) NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae. Curr Genet 27 : 409–416.

30. CouplanE, AiyarRS, KucharczykR, KabalaA, EzkurdiaN, et al. (2011) A yeast-based assay identifies drugs active against human mitochondrial disorders. Proc Natl Acad Sci U S A 108 : 11989–11994.

31. FlowerTR, ChesnokovaLS, FroelichCA, DixonC, WittSN (2005) Heat shock prevents alpha-synuclein-induced apoptosis in a yeast model of Parkinson's disease. J Mol Biol 351 : 1081–1100.

32. FunesS, DavidsonE, ClarosMG, van LisR, Perez-MartinezX, et al. (2002) The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii. J Biol Chem 277 : 6051–6058.

33. Bokori-BrownM, HoltIJ (2006) Expression of algal nuclear ATP synthase subunit 6 in human cells results in protein targeting to mitochondria but no assembly into ATP synthase. Rejuvenation Res 9 : 455–469.

34. Figueroa-MartinezF, Vazquez-AcevedoM, Cortes-HernandezP, Garcia-TrejoJJ, DavidsonE, et al. (2011) What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes. Mitochondrion 11 : 147–154.

35. Bittner-EddyP, MonroyAF, BramblR (1994) Expression of mitochondrial genes in the germinating conidia of Neurospora crassa. J Mol Biol 235 : 881–897.

36. HoustekJ, AnderssonU, TvrdikP, NedergaardJ, CannonB (1995) The expression of subunit c correlates with and thus may limit the biosynthesis of the mitochondrial F0F1-ATPase in brown adipose tissue. J Biol Chem 270 : 7689–7694.

37. KramarovaTV, ShabalinaIG, AnderssonU, WesterbergR, CarlbergI, et al. (2008) Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform. FASEB J 22 : 55–63.

38. FoxTD (1996) Translational control of endogenous and recoded nuclear genes in yeast mitochondria: regulation and membrane targeting. Experientia 52 : 1130–1135.

39. LangBF, GrayMW, BurgerG (1999) Mitochondrial genome evolution and the origin of eukaryotes. Annu Rev Genet 33 : 351–397.

40. Perez-MartinezX, BroadleySA, FoxTD (2003) Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J 22 : 5951–5961.

41. BarrientosA, ZambranoA, TzagoloffA (2004) Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J 23 : 3472–3482.

42. LaemmliUK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680–685.

43. GuerinB, LabbeP, SomloM (1979) Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol 55 : 149–159.

44. SchmittME, BrownTA, TrumpowerBL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18 : 3091–3092.

45. di RagoJP, NetterP, SlonimskiPP (1990) Pseudo-wild type revertants from inactive apocytochrome b mutants as a tool for the analysis of the structure/function relationships of the mitochondrial ubiquinol-cytochrome c reductase of Saccharomyces cerevisiae. J Biol Chem 265 : 3332–3339.

46. SchaggerH, PfeifferK (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19 : 1777–1783.

47. LowryOH, RosebroughNJ, FarrAL, RandallRJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193 : 265–275.

48. ArselinG, VaillierJ, GravesPV, VeloursJ (1996) ATP synthase of yeast mitochondria. Isolation of the subunit h and disruption of the ATP14 gene. J Biol Chem 271 : 20284–20290.

49. Lefebvre-LegendreL, SalinB, SchaefferJ, BrethesD, DautantA, et al. (2005) Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae. J Biol Chem 280 : 18386–18392.

50. TalbotJC, DautantA, PolidoriA, PucciB, Cohen-BouhacinaT, et al. (2009) Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability. J Bioenerg Biomembr 41 : 349–360.

51. GodardF, TetaudE, Duvezin-CaubetS, di RagoJP (2011) A genetic screen targeted on the FO component of mitochondrial ATP synthase in Saccharomyces cerevisiae. J Biol Chem 286 : 18181–18189.

52. Duvezin-CaubetS, RakM, Lefebvre-LegendreL, TetaudE, BonnefoyN, et al. (2006) A “petite obligate” mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase. J Biol Chem 281 : 16305–16313.

53. FouryF, RogantiT, LecrenierN, PurnelleB (1998) The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae. FEBS Lett 440 : 325–331.

54. KyteJ, DoolittleRF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157 : 105–132.