Abstract
The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.
Highlights
The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks
The peripheral stalk of Na؉-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF subunits together with the N-terminal hydrophilic domain of NtpI
Purified NtpE-F-INterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry
Summary
V-ATPase, vacuolar ATPase; NtpINterm, N-terminal domain of NtpI subunit; SAXS, small-angle X-ray scattering; AUC, analytical ultracentrifugation; AUC/SE, analytical ultracentrifugation sedimentation equilibrium; AUC/SV, analytical ultracentrifuge sedimentation velocity; Req, equilibrium resonance units; NSD, normalized spatial discrepancy; Nterm, N-terminal. Electron microscopic studies have indicated that subunits E and G formed a rod-shaped heterodimer [8, 9] and are both present in two or three copies per complex (10 –12). These findings suggest that the peripheral stalk of V-ATPase is composed of two or three EG heterodimers together with subunits H and C, and the N-terminal domain of subunit a. NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpINterm) were individually purified, and the interactions between the three subunits were investigated. A low resolution structure model of the complex obtained by SAXS reveals an elongated “L”-shape
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