Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae

  • Vijayakanth Pagadala
  • Luke Vistain
  • Jindrich Symersky
  • David M. Mueller
Article

Abstract

The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His6 tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F1Fo ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.

Keywords

ATP synthase F1Fo ATPase Mitochondrion Oxidative phosphorylation 

References

  1. Abrahams JP, Leslie AGW (1996) Acta Crystallogr D 52:30–42CrossRefGoogle Scholar
  2. Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Nature 370:621–628CrossRefGoogle Scholar
  3. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schägger H (1998) EMBO J 17:7170–7178CrossRefGoogle Scholar
  4. Bateson M, Devenish RJ, Nagley P, Prescott M (1996) Anal Biochem 238(1):14–18CrossRefGoogle Scholar
  5. Bianchet MA, Hullihen J, Pedersen PL, Amzel LM (1998) Proc Natl Acad Sci USA 95:11065–11070CrossRefGoogle Scholar
  6. Cherezov V, Peddi A, Muthusubramaniam L, Zheng YF, Caffrey M (2004) Acta Crystallogr D Biol Crystallogr 60(Pt 10):1795–1807CrossRefGoogle Scholar
  7. Cipriano DJ, Bi Y, Dunn SD (2002) J Biol Chem 277(19):16782–16790CrossRefGoogle Scholar
  8. Cipriano DJ, Dunn SD (2006) J Biol Chem 281(1):501–507CrossRefGoogle Scholar
  9. Dautant A, Velours J, Giraud MF (2010) J Biol Chem 285(38):29502–29510CrossRefGoogle Scholar
  10. Duvezin-Caubet S, Caron M, Giraud MF, Velours J, di Rago JP (2003) Proc Natl Acad Sci U S A 100(23):13235–13240CrossRefGoogle Scholar
  11. Eble KS, Coleman WB, Hantgan RR, Cunningham CC (1990) J Biol Chem 265(32):19434–19440Google Scholar
  12. Ekman P, Jager O (1993) Anal Biochem 214(1):138–141CrossRefGoogle Scholar
  13. Gietz D, St. Jean A, Woods RA, Schiestl RH (1992) Nucleic Acids Res 20:1425CrossRefGoogle Scholar
  14. Ibrahim NG, Stuchell RN, Beattie DS (1973) Eur J Biochem 36(2):519–527CrossRefGoogle Scholar
  15. Joshi AS, Zhou J, Gohil VM, Chen S, Greenberg ML (2009) Biochim Biophys Acta 1793(1):212–218CrossRefGoogle Scholar
  16. Kabaleeswaran V, Puri N, Walker JE, Leslie AG, Mueller DM (2006) EMBO J 25:5433–5442CrossRefGoogle Scholar
  17. Kabaleeswaran V, Shen H, Symersky J, Walker JE, Leslie AG, Mueller DM (2009) J Biol Chem 284:10546–10551CrossRefGoogle Scholar
  18. Kane Dickson V, Silvester JA, Fearnley IM, Leslie AG, Walker JE (2006) EMBO J 25(12):2911–2918CrossRefGoogle Scholar
  19. Kim IC, Beattie DS (1973) Eur J Biochem 36(2):509–518CrossRefGoogle Scholar
  20. Knowles AF, Penefsky HS (1997) Biochim Biophys Acta Bio-Membr 1329:311–320CrossRefGoogle Scholar
  21. Laemmli UK (1970) Nature 227:680–685CrossRefGoogle Scholar
  22. Lai-Zhang J, Mueller DM (2000) Eur J Biochem 267(8):2409–2418CrossRefGoogle Scholar
  23. Lai-Zhang J, Xiao Y, Mueller DM (1999) EMBO J 18(1):58–64CrossRefGoogle Scholar
  24. Laird DM, Parce JW, Montgomery RI, Cunningham CC (1986) J Biol Chem 261(31):14851–14856Google Scholar
  25. Lange C, Nett JH, Trumpower BL, Hunte C (2001) EMBO J 20(23):6591–6600CrossRefGoogle Scholar
  26. Lau WC, Rubinstein JL (2010) Proc Natl Acad Sci U S A 107(4):1367–1372CrossRefGoogle Scholar
  27. Mileykovskaya E, Dowhan W (2009) Biochim Biophys Acta 1788(10):2084–2091CrossRefGoogle Scholar
  28. Mueller DM (1988) J Biol Chem 263:5634–5639Google Scholar
  29. Mueller DM (2000) J Bioener Biomem 32(4):391–400CrossRefGoogle Scholar
  30. Mueller DM, Getz GS (1986) J Biol Chem 261(25):11816–11822Google Scholar
  31. Mueller DM, Puri N, Kabaleeswaran V, Terry C, Leslie AGW, Walker JE (2004) Protein Expr Purif 37(2):479–485CrossRefGoogle Scholar
  32. Noji H, Yasuda R, Yoshida M, Kinosita K Jr (1997) Nature 386:299–302CrossRefGoogle Scholar
  33. Ogur M, St. John R, Nagai S (1957) Science 125(3254):928–929CrossRefGoogle Scholar
  34. Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326CrossRefGoogle Scholar
  35. Penefsky HS (1979) Methods Enzymol 56:527–530CrossRefGoogle Scholar
  36. Prescott M, Higuti T, Nagley P, Devenish RJ (1995) BiochemBiophysResCommun 207:943–949Google Scholar
  37. Robinson NC (1993) J Bioenerg Biomembr 25(2):153–163CrossRefGoogle Scholar
  38. Robinson NC, Zborowski J, Talbert LH (1990) Biochemistry 29(38):8962–8969CrossRefGoogle Scholar
  39. Rondelez Y, Tresset G, Nakashima T, Kato-Yamada Y, Fujita H, Takeuchi S, Noji H (2005) Nature 433(7027):773–777CrossRefGoogle Scholar
  40. Rubinstein JL, Walker JE, Henderson R (2003) EMBO J 22(23):6182–6192CrossRefGoogle Scholar
  41. Schägger H, von Jagow G (1987) Anal Biochem 166:368–379CrossRefGoogle Scholar
  42. Sedlak E, Panda M, Dale MP, Weintraub ST, Robinson NC (2006) Biochemistry 45(3):746–754CrossRefGoogle Scholar
  43. Sikorski RS, Hieter P (1989) Genetics 122:19–27Google Scholar
  44. Stock D, Leslie AGW, Walker JE (1999) Science 286(5445):1700–1705CrossRefGoogle Scholar
  45. Suzuki T, Murakami T, Iino R, Suzuki J, Ono S, Shirakihara Y, Yoshida M (2003) J Biol Chem 278(47):46840–46846CrossRefGoogle Scholar
  46. Swanljung P, Frigeri L, Ohlson K, Ernster L (1973) Biochim Biophys Acta 305(3):519–533CrossRefGoogle Scholar
  47. Towbin H, Staehelin T, Gordin J (1979) Proc Natl Acad Sci U S A 76:4350–4354CrossRefGoogle Scholar
  48. Tsukihara T, Shimokata K, Katayama Y, Shimada H, Muramoto K, Aoyama H, Mochizuki M, Shinzawa-Itoh K, Yamashita E, Yao M, Ishimura Y, Yoshikawa S (2003) Proc Natl Acad Sci U S A 100(26):15304–15309CrossRefGoogle Scholar
  49. Tsunoda SP, Rodgers AJ, Aggeler R, Wilce MC, Yoshida M, Capaldi RA (2001) Proc Natl Acad Sci U S A 98(12):6560–6564CrossRefGoogle Scholar
  50. Vagin A, Teplyakov A (2010) Acta Crystallogr D Biol Crystallogr 66(Pt 1):22–25CrossRefGoogle Scholar
  51. Van Veldhoven PP, Mannaerts GP (1987) Anal Biochem 161(1):45–48CrossRefGoogle Scholar
  52. Velours J, Vailler J, Paumard P, Soubannier V, Lai-Zhang J, Mueller DM (2001) J Biol Chem 276:8602–8607CrossRefGoogle Scholar
  53. Watt IN, Montgomery MG, Runswick MJ, Leslie AG, Walker JE (2010) Proc Natl Acad Sci U S A 107(39):16823–16827CrossRefGoogle Scholar
  54. Xiao Y, Metzl M, Mueller DM (2000) J Biol Chem 275(10):6963–6968CrossRefGoogle Scholar
  55. Yaffe MP (1991) Methods Enzymol 194:627–643CrossRefGoogle Scholar
  56. Yasuda R, Noji H, Kinosita K Jr, Yoshida M (1998) Cell 93:1117–1124CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Vijayakanth Pagadala
    • 1
  • Luke Vistain
    • 1
  • Jindrich Symersky
    • 1
  • David M. Mueller
    • 1
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyRosalind Franklin University of Medicine and Science, The Chicago Medical SchoolNorth ChicagoUSA
  2. 2.North ChicagoUSA

Personalised recommendations