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Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase

Abstract

Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V1 region drives proton translocation through the membrane-embedded VO region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase1,2,3,4,5. We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae6,7, allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac8c′c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.

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Figure 1: The intact VO complex.
Figure 2: Asymmetry of the c-ring.
Figure 3: Cytoplasmic half-channel and subunit a/c-ring interaction.
Figure 4: Irregularly spaced Glu residues require protonation before deprotonation.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers 8363, 8364, 8367, and 8409. The atomic model has been deposited in the Protein Data Bank under accession number 5TJ5.

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Acknowledgements

We thank N. Grigorieff for providing access to the Titan Krios electron microscope and H. Urlaub for providing C.S. with access to mass spectrometry instrumentation while in Göttingen. We thank Z. Ripstein, P. Tieleman, R. Pomès, and J.-P. Julien for discussions and J. Zhao, J.-P. Julien and V. Kanelis for a critical reading of the manuscript. M.T.M.-J. was supported by a Postdoctoral Fellowship from the Canadian Institutes of Health Research (CIHR), C.V.R. is a Royal Society Professor and J.L.R. holds a Canada Research Chair. This work was supported by operating grant MOP81294 from the Canadian Institutes of Health Research (J.L.R.), Wellcome Trust grants WT008150 and WT099141 (C.V.R.), and European Research Council IMPRESS grant ERC268851 (C.V.R.).

Author information

Authors and Affiliations

Authors

Contributions

M.T.M.-J. prepared yeast strains, purified protein, prepared cryo-EM specimens, performed 200 kV cryo-EM and Relion image analysis, and built the atomic models. A.R. collected and pre-processed data with the 300-kV microscope and performed image analysis with Frealign. C.S. performed the mass spectrometry analysis. S.A.B. prepared yeast strains and assisted with protein purification. S.B. assisted with cryo-EM specimen preparation and screening. C.V.R. supervised mass spectrometry experiments, and J.L.R. supervised the other aspects of the work and coordinated experiments. J.L.R. and M.T.M.-J. wrote the manuscript and prepared the figures with input from the other authors.

Corresponding author

Correspondence to John L. Rubinstein.

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The authors declare no competing financial interests.

Additional information

Reviewer Information

Nature thanks A. Hack, K. Parra, H. Saibil and J. Weber for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Subunit composition of the intact V-ATPase and dissociated V1 and VO regions.

The rotor is outlined in black and the two half-channels in the VO region are indicated with dashed lines. The intact V-ATPase (left) dissociates into the auto-inhibited V1 and VO complexes upon nutrient starvation. Figure adapted from ref. 1.

Extended Data Figure 2 Cryo-EM map generation.

a, An example micrograph with protein particles circled in red. Scale bar, 500 Å. b, Fourier shell correlation (FSC) curve. The highest-resolution information used in image alignment (6 Å) and the overall resolution of the map at FSC = 0.143 (3.9 Å) are indicated. c, Local resolution assessment. Scale bar, 25 Å. d, Image orientation distribution. e, Example 2D class average images.

Extended Data Figure 3 Model building.

af, Example regions of the atomic model built for subunits a (a), c″ and c′ (b), c(1) (c), d (d), e (e), and f (f). g, The different α-helices from the c-ring bearing conserved Glu residues show variable resolution. An α-helix from the N-terminal domain of subunit a has poor resolution. Residue numbers are shown in brackets.

Extended Data Figure 4 VO complex lacking subunit d.

a, The VO complex map from all of the particle images shows subunit d. b, VO complex map from a 3D class, containing 24,744 particle images, that lacks subunit d was determined at 7.8-Å resolution. Scale bar, 25 Å.

Extended Data Figure 5 VO complex is in rotational state 3

. a–c, rotational states 1, 2, and 3 of the intact V-ATPase show the two α helices of subunit c" within the c-ring1. d, The two α-helices of subunit c″ within the c-ring show the ring to be in the same orientation as in rotational state 3 of the intact V-ATPase. Scale bar, 25 Å.

Extended Data Figure 6 Identification of subunit f.

a, SDS–PAGE gel (left) and western blot (right) against a 3×FLAG-tag for the affinity purification of 3×FLAG-tagged YPR170W-B (subunit f) and Vma1p (subunit A) show that both proteins are components of the V-ATPase. b, Surface-rendered 3D maps (upper) and map cross-sections (lower) showing the wild-type VO complex (left) and the VO complex from a yeast strain with the YPR170W-B gene deleted (right). Density from YPR170W-B is indicated with a red arrow. Scale bar, 25 Å. c, Yeast strains with the STV1 and VPH1 genes deleted, the STV1 and YPR170W-B gene deleted, and only STV1 gene deleted were grown on both YPD medium (left) and YPD medium with zinc (right), demonstrating that deletion of YPR170W-B does not cause the VMA phenotype.

Supplementary information

Supplementary Tables

This file contains Supplementary Tables 1-2. Table 1 contains data acquisition, processing, and model statistics and Table 2 contains a summary of the mass spectrometry results for candidate proteins identified in the membrane region of the V-ATPase. (PDF 602 kb)

Supplementary Data

This file contains a spreadsheet showing the mass spectrometry database search results. (XLSX 32 kb)

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Mazhab-Jafari, M., Rohou, A., Schmidt, C. et al. Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase. Nature 539, 118–122 (2016). https://doi.org/10.1038/nature19828

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