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Biochemistry (Moscow)

, Volume 75, Issue 8, pp 1055–1063 | Cite as

Point mutations in Pma1 H+-ATPase of Saccharomyces cerevisiae: Influence on its expression and activity

  • V. V. PetrovEmail author
Accelerated Publication

Abstract

Yeast Pma1 H+-ATPase is a key enzyme of cell metabolism generating electrochemical proton gradient across the plasma membrane, thus playing an important role in the maintenance of ion homeostasis in the cell. Using site-directed mutagenesis, we have previously replaced all 21 amino acid residues in the transmembrane segment M8 with Ala (Guerra et al. (2007) Biochim. Biophys. Acta, 1768, 2383–2392). In this work, we present new data on the role of these amino acid residues in the structure-function relationship in the enzyme and cell tolerance to heat shock. Mutations Q798A and I799A are lethal for cells regardless of expression of the enzyme in secretory vesicles or plasma membrane. The F796A mutation causes enzyme and cell sensitivity to heat shock when expressed in secretory vesicles. The I794A mutation increases temperature sensitivity of cells when the enzyme is expressed either in secretory vesicles or, to a lesser extent, in plasma membrane. The E803A mutation has no significant influence on the ATPase and cell sensitivity to heat shock; however, it causes a shift in the equilibrium between E1 and E2 conformations of the enzyme towards E1.

Key words

yeast plasma membrane secretory vesicles ATPase transmembrane segment heat shock site-directed mutagenesis 

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References

  1. 1.
    Lutsenko, S., and Kaplan, J. H. (1995) Biochemistry, 34, 15607–15613.CrossRefPubMedGoogle Scholar
  2. 2.
    Goffeau, A., and Slayman, C. W. (1981) Biochim. Biophys. Acta, 639, 197–223.PubMedGoogle Scholar
  3. 3.
    Serrano, R., Kielland-Brandt, M. C., and Fink, G. R. (1986) Nature, 319, 689–693.CrossRefPubMedGoogle Scholar
  4. 4.
    Zhang, P., Toyoshima, C., Yonekura, K., Green, N. M., and Stokes, D. L. (1998) Nature, 392, 835–839.CrossRefPubMedGoogle Scholar
  5. 5.
    Toyoshima, C., Nakasano, M., Nomura, H., and Ogawa, H. (2000) Nature, 405, 647–655.CrossRefPubMedGoogle Scholar
  6. 6.
    Toyoshima, C., and Nomura, H. (2002) Nature, 418, 605–611.CrossRefPubMedGoogle Scholar
  7. 7.
    Toyoshima, C., and Muzutani, T. (2004) Nature, 430, 529–535.CrossRefPubMedGoogle Scholar
  8. 8.
    Morth, J. P., Pedersen, B. P., Toustrup-Jensen, M. S., Sorensen, T. L.-M., Petersen, J., Andersen, J. P., Vilsen, B., and Nissen, P. (2007) Nature, 450, 1043–1050.CrossRefPubMedGoogle Scholar
  9. 9.
    Shinoda, T., Ogawa, H., Cornelius, F., and Toyoshima, C. (2009) Nature, 459, 446–450.CrossRefPubMedGoogle Scholar
  10. 10.
    Auer, M., Scarborough, G. A., and Kuhlbrandt, W. (1998) Nature, 392, 840–843.CrossRefPubMedGoogle Scholar
  11. 11.
    Pedersen, B. P., Buch-Pedersen, M. J., Morth, J. P., Palmgren, M. G., and Nissen, P. (2007) Nature, 450, 1111–1115.CrossRefPubMedGoogle Scholar
  12. 12.
    Guerra, G., Petrov, V. V., Allen, K. E., Miranda, M., Pardo, J. P., and Slayman, C. W. (2007) Biochim. Biophys. Acta, 1768, 2383–2392.CrossRefPubMedGoogle Scholar
  13. 13.
    Petrov, V. V., Padmanabha, K. P., Nakamoto, R. K., Allen, K. E., and Slayman, C. W. (2000) J. Biol. Chem., 275, 15709–15716.CrossRefPubMedGoogle Scholar
  14. 14.
    Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem., 266, 7940–7949.PubMedGoogle Scholar
  15. 15.
    Harris, S. L., Perlin, D. S., Seto-Yong, D., and Haber, J. E. (1991) J. Biol. Chem., 266, 24439–24445.PubMedGoogle Scholar
  16. 16.
    Petrov, V. V. (2009) Biochemistry (Moscow), 74, 1155–1163.CrossRefGoogle Scholar
  17. 17.
    Novick, P., Field, C., and Schekman, R. (1980) Cell, 21, 205–215.CrossRefPubMedGoogle Scholar
  18. 18.
    Sarkar, G., and Sommer, S. S. (1990) BioTechniques, 8, 404–407.PubMedGoogle Scholar
  19. 19.
    Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem., 264, 21857–21864.PubMedGoogle Scholar
  20. 20.
    Fabiato, A., and Fabiato, F. (1979) J. Physiol., 75, 463–505.Google Scholar
  21. 21.
    Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem., 66, 375–400.Google Scholar
  22. 22.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265–275.PubMedGoogle Scholar
  23. 23.
    Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem., 70, 241–250.CrossRefPubMedGoogle Scholar
  24. 24.
    Ferreira, T., Mason, A. B., Pypaert, M., Allen, K. E., and Slayman, C. W. (2002) J. Biol. Chem., 277, 21027–21040.CrossRefPubMedGoogle Scholar
  25. 25.
    Ambesi, A., Miranda, M., Petrov, V. V., and Slayman, C. W. (2000) J. Exp. Biol., 203, 155–160.PubMedGoogle Scholar
  26. 26.
    Lecchi, S., Allen, K. E., Pardo, J. P., Mason, A. B., and Slayman, C. W. (2005) Biochemistry, 44, 16624–16632.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  1. 1.Skryabin Institute of Biochemistry and Physiology of MicroorganismsRussian Academy of SciencesPushchino, Moscow RegionRussia

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