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Abstract
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 Vo proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast Vo The structure indicated that, when V1 is released from Vo, the N-terminal cytoplasmic domain of subunit a (aNT) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the aNT-d interface. Here we reconstituted Vo into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between aNT and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the aNT-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 Vo revealed that Vo 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 Vo functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.
Highlights
Eukaryotic vacuolar H؉-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space
We reconstituted Vo into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ϳ15-Å resolution revealed two sites of contact between an N-terminal cytoplasmic domain (aNT) and d that are mediated by highly conserved charged residues
The ϳ320-kDa Vo contains subunits acc’c’’de, which are organized in a membrane-integral “proteolipid” ring (c8c’c’’ [22, 23]), a membrane-bound subunit a with an integral C-terminal domain that is bound at the periphery of the proteolipid ring, and an N-terminal cytoplasmic domain that is bound to subunit d (Fig. 1)
Summary
Eukaryotic vacuolar H؉-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast Vo. The structure indicated that, when V1 is released from Vo, the N-terminal cytoplasmic domain of subunit a (aNT) changes conformation to bind rotor subunit d. We recently obtained a cryo-EM reconstruction of yeast Vo [38], and a comparison with EM models of holo V-ATPase showed that aNT undergoes a large structural change to bind the rotor subunit d in free Vo, the resolution of the model was insufficient to precisely define the aNT-d interface. We showed previously that autoinhibited, membrane-detached V1-ATPase is halted in state 2 [45], and we propose that this conformational mismatch to state 3 Vo could function to prevent unintended reassembly of holo V-ATPase under conditions when the proton pumping activity of the enzyme is not needed
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