Eukaryotic vacuolar H⁺-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V₁-ATPase and membrane-integral V_o proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V_o The structure indicated that, when V₁ is released from V_o, the N-terminal cytoplasmic domain of subunit a (a_NT) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a_NT-d interface. Here we reconstituted V_o into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a_NT and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a_NT-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V_o revealed that V_o is halted in rotational state 3. Combined with our prior work that showed autoinhibited V₁-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V₁ and V_o functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.

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Journal Article

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Stam NJ, Wilkens S

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