Abstract
Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex and the V1 complex lacking subunit C. On separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.
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Data availability
The cryo-EM maps and model coordinates have been deposited to the Electron Microscopy Data Bank (EMDB) (codes EMD-25996 to EMD-26002) and PDB (codes 7TMM, 7TMO, 7TMP, 7TMQ, 7TMR, 7TMS and 7TMT), respectively. Yeast strains and plasmids are available from the corresponding author. Starting models for the construction of atomic models came from the PDB (codes 3J9T, 1HO8, 1U7L and 6M0R). Source data are provided with this paper.
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Acknowledgements
We thank S. Benlekbir, Y. Z. Tan and H. Guo (The Hospital for Sick Children) for assistance with cryo-EM data collection. We thank P. Kane (SUNY Upstate Medical University) for the gift of the PMK549 yeast strain and for discussions about yeast growth, and S. Wilkens and R. Oot for discussions about the V1∆C crystal structure. T.V. was supported by an Ontario Graduate Scholarship, K.A.K. was supported by an Ontario Graduate Scholarship and a Canada Graduate Scholarship and J.L.R. was supported by the Canada Research Chairs program. This work was supported by Canadian Institutes of Health Research grant no. PJT166152 (J.L.R.). Cryo-EM data were collected at the Toronto High-Resolution High-Throughput cryo-EM facility and enzyme assays performed using infrastructure from the Hospital for Sick Children’s Structural and Biophysical Core Facility, both of which are supported by the Canada Foundation for Innovation and Ontario Research Fund. Mass spectrometry data were collected at the Network Biology Collaborative Center at the Lunenfeld-Tanenbaum Research Institute.
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T.V. purified V1, V1∆C, V1∆H and recombinant subunit H constructs, and performed enzyme assays, cryo-EM and the analysis associated with those specimens. K.A.K. purified intact V-ATPase and performed the cryo-EM and analysis for that specimen. S.A.B. prepared the VMA1-3×FLAG, vma5∆ yeast strain. M.C.J. prepared protein for early experiments and advised on protein purification. J.L.R. supervised and coordinated experiments. T.V. and J.L.R. wrote the manuscript and prepared figures with input from the other authors.
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Extended data
Extended Data Fig. 1 CryoEM of intact V-ATPase.
a, Example micrograph (from a dataset of 4015 micrographs) and 2D class average images. b, Fourier shell correlation curves after gold-standard refinement and correction for masking for the three rotational states of intact V-ATPase. c, Local resolution maps and orientation distribution plots for rotational State 1 (top), State 2 (middle), and State 3 (bottom). d, Masks (light blue density) used for local refinement of the V1 region (top), subunit a and the collar region (middle), and the VO region (bottom) for V-ATPase (shown for State 1).
Extended Data Fig. 2 CryoEM of the complete V1 complex.
a, Example micrograph (from a dataset of 3212 micrographs) and 2D class average images. b, Fourier shell correlation curves after gold-standard refinement and correction for masking for the complete V1 complex and V1∆C from the complete V1 protein preparation. c, Local resolution maps and orientation distribution plot for complete V1. d, Local resolution maps and orientation distribution plot for V1∆C in State 3.
Extended Data Fig. 3 Interface between subunit C and the central rotor in complete V1.
Subunit C (colored by sequence conservation) rotates toward the central rotor subunits F and D (gray) upon dissociation of V1 from VO. There is minimal sequence conservation on the surface of subunit C that interacts with the central rotor in the complete V1 complex. However, in the complete V1 complex subunit C is positioned to block rotation of the rotor subcomplex and prevent rotation from State 2 to State 3.
Extended Data Fig. 4 CryoEM of the V1ΔC complex.
a, Example micrograph (from a dataset of 11,175 micrographs), 2D class average images, and Fourier shell correlation curves after gold-standard refinement and correction for masking for V1∆C in States 2 and 3. b, Example micrograph (from a dataset of 4277 micrographs), 2D class average images, and Fourier shell correlation curves after gold standard refinement and correction for masking for V1∆C with 1 mM ATP in States 1, 2, and 3. c, Local resolution maps and orientation distribution plot for V1∆C in State 2 (top) and State 3 (bottom). d, Local resolution maps and orientation distribution plot for V1∆C with 1 mM ATP in State 1 (top), State 2 (middle), and State 3 (bottom).
Extended Data Fig. 5 Additional structure of V1ΔC complex that appears when the protein preparation is left at 4 °C.
a, CryoEM of the V1∆C sample showed an additional structure that increased in abundance the longer the sample was left at 4 °C. b, The additional structure fits the model of the V1∆C complex from X-ray crystallography30 (orange) along with an additional peripheral stalk and subunit H from an adjacent V1∆C complex in the crystal lattice (blue). c, The entire map of the additional structure is explained by V1∆C from the crystal (orange), an adjacent subunit H and peripheral stalk from the crystal (blue), and a third subunit H (red) not found in the crystal, but matching the conformation of subunit H observed in the cryo-EM maps. d, Replication of the assay results shown in Fig. 3b with separately purified batches of protein. V1ΔH (pink circles) has strong ATPase activity with a 5-fold excess of recombinant wildtype subunit H (green squares) inhibiting activity but a 5-fold excess of recombinant subunit H bearing the mutation I473D (blue triangles) unable to inhibit (mean ± s.d., n = 3 assays). Data for the graph in D are available as source data.
Supplementary information
Supplementary Video 1
Three-dimensional variability analysis shows flexibility in the collar region of the complete V1 complex.
Supplementary Video 2
Interpolation between the structure of V1 within intact V-ATPase and in the complete V1 complex shows the conformational changes that occur in V1 on separation from VO.
Supplementary Video 3
Three-dimensional variability analysis shows flexibility in subunit H and the peripheral stalks of the V1ΔC complex in all three rotational states.
Supplementary Video 4
Model for the conformational changes that occur during the cycle of dissociation and reassembly of the V1 and VO complexes.
Source data
Source Data Fig. 1
Unprocessed gel.
Source Data Fig. 2
Unprocessed gel.
Source Data Fig. 3
Unprocessed gel.
Source Data Fig. 3
Summary of mass spectrometry data for the bands identified in Fig. 3a.
Source Data Fig. 3
Source data for ATPase assays shown in Fig. 3b.
Source Data Extended Data Fig. 5
Source data for ATPase assays.
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Vasanthakumar, T., Keon, K.A., Bueler, S.A. et al. Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation. Nat Struct Mol Biol 29, 430–439 (2022). https://doi.org/10.1038/s41594-022-00757-z
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DOI: https://doi.org/10.1038/s41594-022-00757-z