The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel.

The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.

Breaking up and making up: The secret life of the vacuolar H+ -ATPase.

The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION.

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-Šresolution 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.