Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Nature volume 539, pages 118–122 (2016)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Zhao, J., Benlekbir, S. & Rubinstein, J. L. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Allegretti, M. et al. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237–240 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Zhou, A. et al. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. eLife 4, e10180 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  4. Morales-Rios, E., Montgomery, M. G., Leslie, A. G. W. & Walker, J. E. Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution. Proc. Natl Acad. Sci. USA 112, 13231–13236 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Schep, D. G., Zhao, J. & Rubinstein, J. L. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance. Proc. Natl Acad. Sci. USA 113, 3245–3250 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kane, P. M. Disassembly and reassembly of the yeast vacuolar H+-ATPase in vivo. J. Biol. Chem . 270, 17025–17032 (1995)

    Article  CAS  PubMed  Google Scholar 

  7. Sumner, J. P. et al. Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits. J. Biol. Chem. 270, 5649–5653 (1995)

    Article  CAS  PubMed  Google Scholar 

  8. Couoh-Cardel, S., Milgrom, E. & Wilkens, S. Affinity purification and structural features of the yeast vacuolar ATPase Vo membrane sector. J. Biol. Chem. 290, 27959–27971 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Benlekbir, S., Bueler, S. A. & Rubinstein, J. L. Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11-Å resolution. Nat. Struct. Mol. Biol. 19, 1356–1362 (2012)

    Article  CAS  PubMed  Google Scholar 

  10. Qi, J. & Forgac, M. Function and subunit interactions of the N-terminal domain of subunit a (Vph1p) of the yeast V-ATPase. J. Biol. Chem. 283, 19274–19282 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Powell, B., Graham, L. A. & Stevens, T. H. Molecular characterization of the yeast vacuolar H+-ATPase proton pore. J. Biol. Chem. 275, 23654–23660 (2000)

    Article  CAS  PubMed  Google Scholar 

  12. Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H. & Anraku, Y. VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase. J. Biol. Chem. 272, 4795–4803 (1997)

    Article  CAS  PubMed  Google Scholar 

  13. Nishi, T., Kawasaki-Nishi, S. & Forgac, M. The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function. J. Biol. Chem. 278, 5821–5827 (2003)

    Article  CAS  PubMed  Google Scholar 

  14. Couoh-Cardel, S., Hsueh, Y.-C., Wilkens, S. & Movileanu, L. Yeast V-ATPase proteolipid ring acts as a large-conductance transmembrane protein pore. Sci. Rep. 6, 24774 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Matthies, D. et al. High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na+-coupled ATP synthase. Nat. Commun. 5, 5286 (2014)

    Article  ADS  PubMed  Google Scholar 

  16. Vik, S. B. & Antonio, B. J. A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit. J. Biol. Chem. 269, 30364–30369 (1994)

    Article  CAS  PubMed  Google Scholar 

  17. Junge, W., Lill, H. & Engelbrecht, S. ATP synthase: an electrochemical transducer with rotatory mechanics. Trends Biochem. Sci. 22, 420–423 (1997)

    Article  CAS  PubMed  Google Scholar 

  18. Neale, C., Chakrabarti, N., Pomorski, P., Pai, E. F. & Pomès, R. Hydrophobic gating of ion permeation in magnesium channel CorA. PLOS Comput. Biol. 11, e1004303 (2015)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  19. Angevine, C. M., Herold, K. A. & Fillingame, R. H. Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane. Proc. Natl Acad. Sci. USA 100, 13179–13183 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Toei, M., Toei, S. & Forgac, M. Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase. J. Biol. Chem. 286, 35176–35186 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kühlbrandt, W. & Davies, K. M. Rotary ATPases: a new twist to an ancient machine. Trends Biochem. Sci. 41, 106–116 (2016)

    Article  PubMed  CAS  Google Scholar 

  22. Pellegrini-Calace, M., Maiwald, T. & Thornton, J. M. PoreWalker: a novel tool for the identification and characterization of channels in transmembrane proteins from their three-dimensional structure. PLOS Comput. Biol. 5, e1000440 (2009)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  23. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360, 376 (1996)

    Article  CAS  PubMed  Google Scholar 

  24. Kawasaki-Nishi, S., Nishi, T. & Forgac, M. Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation. Proc. Natl Acad. Sci. USA 98, 12397–12402 (2001)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cain, B. D. & Simoni, R. D. Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli. J. Biol. Chem. 261, 10043–10050 (1986)

    Article  CAS  PubMed  Google Scholar 

  26. DeCoursey, T. E. The voltage-gated proton channel: a riddle, wrapped in a mystery, inside an enigma. Biochemistry 54, 3250–3268 (2015)

    Article  CAS  PubMed  Google Scholar 

  27. Bueler, S. A. & Rubinstein, J. L. Vma9p need not be associated with the yeast V-ATPase for fully-coupled proton pumping activity in vitro. Biochemistry 54, 853–858 (2015)

    Article  CAS  PubMed  Google Scholar 

  28. Nelson, H. & Nelson, N. Disruption of genes encoding subunits of yeast vacuolar H+-ATPase causes conditional lethality. Proc. Natl Acad. Sci. USA 87, 3503–3507 (1990)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Compton, M. A., Graham, L. A. & Stevens, T. H. Vma9p (subunit e) is an integral membrane VO subunit of the yeast V-ATPase. J. Biol. Chem. 281, 15312–15319 (2006)

    Article  CAS  PubMed  Google Scholar 

  30. MacCallum, J. L., Bennett, W. F. D. & Tieleman, D. P. Distribution of amino acids in a lipid bilayer from computer simulations. Biophys. J. 94, 3393–3404 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996)

    Article  CAS  PubMed  Google Scholar 

  32. Olsen, J. V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021 (2005)

    Article  CAS  PubMed  Google Scholar 

  33. Marr, C. R., Benlekbir, S. & Rubinstein, J. L. Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device. J. Struct. Biol. 185, 42–47 (2014)

    Article  CAS  PubMed  Google Scholar 

  34. Tivol, W. F., Briegel, A. & Jensen, G. J. An improved cryogen for plunge freezing. Microsc. Microanal. 14, 375–379 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007)

    Article  CAS  PubMed  Google Scholar 

  36. Rubinstein, J. L. & Brubaker, M. A. Alignment of cryo-EM movies of individual particles by optimization of image translations. J. Struct. Biol. 192, 188–195 (2015)

    Article  PubMed  Google Scholar 

  37. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  PubMed  Google Scholar 

  38. Zhao, J., Brubaker, M. A., Benlekbir, S. & Rubinstein, J. L. Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images. J. Struct. Biol. 192, 209–215 (2015)

    Article  PubMed  Google Scholar 

  39. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005)

    Article  PubMed  Google Scholar 

  41. Grant, T. & Grigorieff, N. Automatic estimation and correction of anisotropic magnification distortion in electron microscopes. J. Struct. Biol. 192, 204–208 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  42. Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  43. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  44. Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007)

    Article  CAS  PubMed  Google Scholar 

  45. Grigorieff, N. Frealign: an exploratory tool for single-particle Cryo-EM. Methods Enzymol . 579, 191–226 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Penczek, P. A. et al. CTER-rapid estimation of CTF parameters with error assessment. Ultramicroscopy 140, 9–19 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013)

    Article  PubMed  Google Scholar 

  48. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006)

    Article  CAS  PubMed  Google Scholar 

  49. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr . 60, 2126–2132 (2004)

    Article  PubMed  CAS  Google Scholar 

  50. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr . 66, 213–221 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Author notes

  1. Carla Schmidt

    Present address: †Present address: Interdisciplinary Research Center HALOmem, Martin Luther University, Halle-Wittenberg, 06120 Halle (Saale), Germany.,

Authors and Affiliations

  1. Molecular Structure and Function Program, The Hospital for Sick Children, Toronto, M5G 0A4, Ontario, Canada

    Mohammad T. Mazhab-Jafari, Stephanie A. Bueler, Samir Benlekbir & John L. Rubinstein

  2. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, 20147, Virginia, USA

    Alexis Rohou

  3. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, OX1 3QZ, UK

    Carla Schmidt & Carol V. Robinson

  4. Department of Medical Biophysics, University of Toronto, Toronto, M5G 1L7, Ontario, Canada

    John L. Rubinstein

  5. Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    John L. Rubinstein

Authors
  1. Mohammad T. Mazhab-Jafari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexis Rohou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carla Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephanie A. Bueler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Samir Benlekbir
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carol V. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John L. Rubinstein
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

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.

a–f, 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)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature19828

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing