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Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

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Abstract

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.

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Acknowledgments

We thank Ferhan Ayaydin (Cellular Imaging Laboratory, Biological Research Centre Szeged, Hungary) for his help in taking the transmitted light microscopy images of yeast vacuoles. We also thank E. Kónya for excellent technical assistance. Financial support was received from the Hungarian National Science Fund (OTKA K68804 and K101633). D.M. thanks Christian Griesinger and the Department for NMR-based structural biology for financial assistance.

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It is declared that the experiments presented in this manuscript comply with the current laws of the country in which they were performed.

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  1. Elfrieda Fodor-Ayaydin

    Present address: Institute of Biochemistry, Biological Research Centre, Temesvári krt. 62, 6726, Szeged, Hungary

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  1. Institute of Biophysics, Biological Research Centre, Temesvári krt. 62, 6726, Szeged, Hungary

    Csilla Ferencz, Pál Petrovszki, Zoltán Kóta, Elfrieda Fodor-Ayaydin, András Dér & Tibor Páli

  2. Institute of Genetics, Biological Research Centre, Temesvári krt. 62, 6726, Szeged, Hungary

    Lajos Haracska

  3. Department of Biological Nanochemistry, Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Pusztaszeri u. 59-67, 1025, Budapest, Hungary

    Attila Bóta & Zoltán Varga

  4. Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

    Derek Marsh

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Ferencz, C., Petrovszki, P., Kóta, Z. et al. Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes. Eur Biophys J 42, 147–158 (2013). https://doi.org/10.1007/s00249-012-0871-z

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  • DOI: https://doi.org/10.1007/s00249-012-0871-z

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