Abstract
The vacuolar H+ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase’s membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits’ cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ~8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.
Highlights
The vacuolar H+ -ATPase (V-ATPase; V1Vo-ATPase) is a large multisubunit enzyme complex responsible for ATP hydrolysis-driven acidification of subcellular compartments in all eukaryotic organisms and the extracellular space in some tissues[1,2,3]
Vo20, and aNT21–24, have been shown to participate in trans-SNARE pairing in a calcium- and calmodulin-dependent manner[26], and it has been speculated that these Vo-SNARE interactions may assist in vacuole fusion in yeast[21] or spontaneous neurotransmitter release[26]
Using single-molecule electrophysiology measurements, we further show that the c-ring functions as a transmembrane protein pore with a large unitary conductance of ~8.3 nS, a finding that is in accord with the c-ring’s pore diameter of ~3.5 nm, as estimated from cryo-electron microscopy images of c-ring two-dimensional (2-D) crystals and homology modeling
Summary
The vacuolar H+ -ATPase (V-ATPase; V1Vo-ATPase) is a large multisubunit enzyme complex responsible for ATP hydrolysis-driven acidification of subcellular compartments in all eukaryotic organisms and the extracellular space in some tissues[1,2,3]. The V-ATPase from S. cerevisiae, a well-characterized model system for the enzyme from higher organisms, is composed of 14 different polypeptides with varying stoichiometries and can be divided into a V1 catalytic sector (A3B3(C)DE3FG3H), and a membrane-integral Vo proton translocating sector (ac8c’c”de) (Fig. 1)[4,5,6]. Besides its canonical proton-pumping function in the holo V-ATPase, the free Vo or parts thereof, have been implicated in membrane fusion and neurotransmitter release[20,21,22,23,24,25,26] (reviewed in references[27,28]). This work provides the first direct evidence that eukaryotic V-ATPase c-ring exhibits some of the properties required for membrane fusion and neurotransmitter release
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