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Journal of Bioenergetics and Biomembranes

, Volume 48, Issue 5, pp 509–520 | Cite as

Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae

  • Aaron Robertson
  • Kyle Schaltz
  • Karina Neimanis
  • James F. Staples
  • Allison E. McDonaldEmail author
Article

Abstract

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

Keywords

Mitochondria Respiration Mitochondrial disease Electron transport system Bioenergetics High-resolution respirometry 

Notes

Acknowledgments

This work was funded by Natural Sciences and Engineering Research Council Discovery Grants to JFS and AEM. AR and KS were supported by Wilfrid Laurier Undergraduate Student Assistantships and student awards from the Faculty of Science Students’ Association. KN was supported by an Ontario Graduate Scholarship.

References

  1. Affourtit C, Albury MS, Krab K, Moore AL (1999) Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe. J Biol Chem 274:6212–6218CrossRefGoogle Scholar
  2. Ajayi WU, Chaudhuri M, Hill GC (2002) Site-directed mutagenesis reveals the essentiality of the conserved residues in the putative diiron active site of the trypanosome alternative oxidase. J Biol Chem 277:8187–8193CrossRefGoogle Scholar
  3. Albury MS, Affourtit C, Moore A (1998) A highly conserved glutamate residue (glu-270) is essential for plant alternative oxidase activity. J Biol Chem 273:30301–30305CrossRefGoogle Scholar
  4. Antonicka H, Mattman A, Carlson CG, Glerum DM, Hoffbuhr KC, Leary SC, Kennaway NG, Shoubridge EA (2003) Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy. Am J Hum Genet 72:101–114CrossRefGoogle Scholar
  5. Baile MG, Claypool SM (2013) The power of yeast to model diseases of the powerhouse of the cell. Front Biosci 18:241–278CrossRefGoogle Scholar
  6. Bendall DS, Bonner WD (1971) Cyanide-insensitive respiration in plant mitochondria. Plant Physiol 47:236–245CrossRefGoogle Scholar
  7. Berthold DA, Voevodskaya N, Stenmark P, Graslund A, Nordlund P (2002) EPR studies of the mitochondrial alternative oxidase-evidence for a diiron carboxylate center. J Biol Chem 277:43608–43614CrossRefGoogle Scholar
  8. Chaudhuri M, Hill GC (1996) Cloning, sequencing, and functional activity of the Trypanosoma brucei brucei alternative oxidase. 83:125–129Google Scholar
  9. Chaudhuri M, Ajayi W, Hill GC (1998) Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase. Mol Biochem Parasitol 95:53–68CrossRefGoogle Scholar
  10. Crichton PG, Albury MS, Affourtit C, Moore AL (2010) Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis. Biochim Biophys Acta Bioenerg 1797:732–737CrossRefGoogle Scholar
  11. Dassa EP, Dufour E, Goncalves S, Jacobs HT, Rustin P (2009) The alternative oxidase, a tool for compensating cytochrome c oxidase deficiency in human cells. Physiol Plant 137:427–434CrossRefGoogle Scholar
  12. El-Khoury R, Dufour E, Rak M, Ramanantsoa N, Grandchamp N, Csaba Z, Duvillie B, Benit P, Gallego J, Gressens P, Sarkis C, Jacobs HT, Rustin P (2013) Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits mitochondrial ROS overproduction. PLoS Genet 9:e1003182CrossRefGoogle Scholar
  13. El-Khoury R, Kemppainen KK, Dufour E, Szibor M, Jacobs HT, Rustin P (2014) Engineering the alternative oxidase gene to better understand and counteract mitochondrial defects: state of the art and perspectives. Br J Pharmacol 171:2243–2249CrossRefGoogle Scholar
  14. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971CrossRefGoogle Scholar
  15. Fernandez-Ayala DJM, Sanz A, Vartiainen S, Kemppainen KK, Babusiak M, Mustalahti E, Costa R, Tuomela T, Zeviani M, Chung J, O’Dell KMC, Rustin P, Jacobs HT (2009) Expression of the Ciona intestinalis alternative oxidase (AOX) in drosophila complements defects in mitochondrial oxidative phosphorylation. Cell Metab 9:449–460CrossRefGoogle Scholar
  16. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274:546–567CrossRefGoogle Scholar
  17. Hakkaart GAJ, Dassa EP, Jacobs HT, Rustin P (2006) Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration. EMBO Rep 7:341–345CrossRefGoogle Scholar
  18. Holtzapffel RC, Castelli J, Finnegan PM, Millar AH, Whelan J, Day DA (2003) A tomato alternative oxidase protein with altered regulatory properties. Biochim Biophys Acta 1606:153–162CrossRefGoogle Scholar
  19. Huh WK, Kang SO (1999) Molecular cloning and functional expression of alternative oxidase from Candida albicans. J Bacteriol 181:4098–4102Google Scholar
  20. Johnston M, Carlson M (1992) Regulation of carbon and phoshate utilization. In: Jones EW, Pringle JR, Broach JR (eds) The molecular and cellular biology of the yeast saccharomyces: gene expression. Cold Spring Harbour Laboratory Press, New YorkGoogle Scholar
  21. Kido Y, Sakamoto K, Nakamura K, Harada M, Suzuki T, Yabu Y, Saimoto H, Yamakura F, Ohmori D, Moore A, Harada S, Kita K (2010) Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei. Biochim Biophys Acta Bioenerg 1797:443–450CrossRefGoogle Scholar
  22. Kirimura K, Yoda M, Usami S (1999) Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus Niger WU-2223 L. Genetics and Heredity 34:472–477Google Scholar
  23. Kumar AM, Soll D (1992) Arabidopsis alternative oxidase sustains Escherichia coli respiration. Proc Natl Acad Sci U S A 89:10842–10846CrossRefGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  25. Mathy G, Navet R, Gerkens P, Leprince P, De Pauw E, Sluse-Goffart CM, Sluse FE, Douette P (2006) Saccharomyces cerevisiae mitoproteome plasticity in response to recombinant alternative ubiquinol oxidase. J Proteome Res 5:339–348CrossRefGoogle Scholar
  26. McDonald AE (2008) Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed “cyanide-resistant” terminal oxidase. Funct Plant Biol 35:535–552CrossRefGoogle Scholar
  27. McDonald AE, Vanlerberghe GC, Staples JF (2009) Alternative oxidase in animals: unique characteristics and taxonomic distribution. J Exp Biol 212:2627–2634CrossRefGoogle Scholar
  28. Meisinger C, Pfanner N, Truscott KN (2006) Isolation of yeast mitochondria. Methods Mol Biol 313:33–40Google Scholar
  29. Minagawa N, Yoshimoto A (1986) The induction of cyanide-resistant respiration in Hansenula anomala. J Biochem 101:1141–1146Google Scholar
  30. Morán M, Moreno-Lastres D, Marin-Buera L, Arenas J, Martin MA, Ugalde C (2012) Mitochondrial respiratory chain dysfunction: implications in neurodegeneration. Free Radic Biol Med 53:595–609CrossRefGoogle Scholar
  31. Munnich A, Rustin P (2001) Clinical spectrum and diagnosis of mitochondrial disorders. Am J Med Genet 106:4–17CrossRefGoogle Scholar
  32. Nakamura K, Sakamoto K, Kido Y, Fujimoto Y, Suzuki T, Suzuki M, Yabu Y, Ohta N, Tsuda A, Onuma M, Kita K (2005) Mutational analysis of the Trypanosoma vivax alternative oxidase: the E(X)(6)Y motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity. Biochem Biophys Res Commun 334:593–600CrossRefGoogle Scholar
  33. Shiba T, Kido Y, Sakamoto K, Inaoka DK, Tsuge C, Tatsumi R, Takahashi G, Balogun EO, Nara T, Aoki T, Honma T, Tanaka A, Inoue M, Matsuoke S, Saimoto H, Moore A, Harada S, Kita K (2013) Structure of the trypanosome cyanide-insensitive alternative oxidase. Proc Natl Acad Sci U S A 110:4580–4585CrossRefGoogle Scholar
  34. Suzuki T, Hashimoto T, Yabu Y, Kido Y, Sakamoto K, Nihei C, Hato M, Suzuki S, Amano Y, Nagai K, Hosokawa T, Minagawa N, Ohta N, Kita K (2004a) Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications. Biochem Biophys Res Commun 313:1044–1052CrossRefGoogle Scholar
  35. Suzuki T, Nihei C, Yabu Y, Hashimoto T, Suzuki M, Yoshida A, Nagai K, Hosokawa T, Minagawa N, Suzuki S, Kita K, Ohta N (2004b) Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone. Parasitol Int 53:235–245CrossRefGoogle Scholar
  36. Timson DJ (2007) Galactose metabolism in Saccharomyces cerevisiae. Dyn Biochem 1:63–73Google Scholar
  37. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRefGoogle Scholar
  38. Vanlerberghe GC (2013) Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants. Int J Mol Sci 14:6805–6847CrossRefGoogle Scholar
  39. Vanlerberghe GC, McIntosh L (1994) Mitochondrial electron transport regulation of nuclear gene expression. Studies with the alternative oxidase gene of tobacco. Plant Physiol 105:867–874CrossRefGoogle Scholar
  40. Vanlerberghe GC, Day DA, Wiskich JT, Vanlerberghe AE, McIntosh L (1995) Alternative oxidase activity in tobacco leaf mitochondria (dependence on tricarboxylic acid cycle-mediated redox regulation and pyruvate activiation). Plant Physiol 109:353–361CrossRefGoogle Scholar
  41. Vishwakarma A, Dalal A, Tetali SD, Kirti PB, Padmasree K (2015) Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis. FEBS Open Bio 6:135–146CrossRefGoogle Scholar
  42. Williams BAP, Elliot C, Burri L, Kido Y, Kita K, Moore AL, Keeling PJ (2010) A broad distribution of alternative oxidase in microsporidian parasites. PLoS Pathog 6:e1000761CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Aaron Robertson
    • 1
  • Kyle Schaltz
    • 1
  • Karina Neimanis
    • 1
  • James F. Staples
    • 2
  • Allison E. McDonald
    • 1
    Email author
  1. 1.Department of BiologyWilfrid Laurier UniversityWaterlooCanada
  2. 2.Department of BiologyWestern UniversityLondonCanada

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