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Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase

2012; Elsevier BV; Volume: 287; Issue: 16 Linguagem: Inglês

10.1074/jbc.m112.343962

1083-351X

Rebecca A. Oot, Stephan Wilkens,

Advanced Electron Microscopy Techniques and Applications

Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V1-ATPase and Vo-proton channel domains. We found previously that the head domain of the single copy C subunit (Chead) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654–24664). Here we generated a water-soluble construct of the N-terminal domain of the Vo "a" subunit composed of amino acid residues 104–372 (aNT(104–372)). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that aNT(104–372) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the aNT(104–372) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that aNT(104–372) binds the Cfoot and EG heterodimer with dissociation constants of 22 and 33 μm, respectively. We speculate that the spatial closeness of the aNT, Cfoot, and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the aNT(104–372)-Cfoot or aNT(104–372)-EG interaction by an as-yet unknown signaling mechanism. Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V1-ATPase and Vo-proton channel domains. We found previously that the head domain of the single copy C subunit (Chead) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654–24664). Here we generated a water-soluble construct of the N-terminal domain of the Vo "a" subunit composed of amino acid residues 104–372 (aNT(104–372)). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that aNT(104–372) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the aNT(104–372) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that aNT(104–372) binds the Cfoot and EG heterodimer with dissociation constants of 22 and 33 μm, respectively. We speculate that the spatial closeness of the aNT, Cfoot, and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the aNT(104–372)-Cfoot or aNT(104–372)-EG interaction by an as-yet unknown signaling mechanism.