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

Abstract

The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low 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 it is not fixed on a solid support, instead it is functioning in its native membrane environment.Special Issue: Structure, function, folding and assembly of membrane proteins -Insight from Biophysics.

Oscillating Electric Field Measures the Rotation Rate in a Native Rotary Enzyme

“…2, Top). It should be noted that the high ConcA sensitive fraction of the ATPase activity proves that most of the V-ATPases are properly oriented in these vesicles15. We discovered a narrow peak in V-ATPase activity as a function of frequency of the AC field (Fig.…”

“…The specific and highly potent inhibitor concanamycin A (ConcA) was used to determine the V-ATPase contribution to total ATPase activity15. ConcA binds to the intramembranous domain of the V-ATPase, blocking rotation and hence proton transport and ATP hydrolysis15232428. Although V-ATPase sits in its native membrane environment, a static membrane potential is not present in our vesicles, as opposed to vacuoles in living yeast cells.…”

“…2, Top). In most but not all studies it is measured or assumed that m  = 6 in V-ATPase13615222332. Using the Lorentzian fits and taking m  = 6, the mean frequency of the full-cycle 360°-rotation is 13.2 ± 0.5 Hz (s.d., n  = 5).…”

“…Even the (so far) least invasive and least perturbing single molecule fluorescence resonance energy transfer approach to detect the rotation required the chemical modification of the rotary enzyme, namely binding of fluorescent donor and acceptor groups and reconstitution of the enzyme into artificial liposomes as “quasi-native environment”14. In the present study we use spherical single-layer membrane vesicles, with a mean diameter of 300 nm, formed from yeast vacuoles as described earlier15. These vesicles contain high concentration of properly oriented vacuolar proton-pumping adenosine-triphosphate (ATP) hydrolase (vacuolar proton-ATPase, V-ATPase), on which we measure ATPase activity15.…”

“…In the present study we use spherical single-layer membrane vesicles, with a mean diameter of 300 nm, formed from yeast vacuoles as described earlier15. These vesicles contain high concentration of properly oriented vacuolar proton-pumping adenosine-triphosphate (ATP) hydrolase (vacuolar proton-ATPase, V-ATPase), on which we measure ATPase activity15. In addition, since ATP is added to pre-formed stable vacuolar vesicles, reversely (inside-out) oriented V-ATPases, if any, remain inactive and do not contribute to the measured ATPase activity.…”