Abstract
Vacuolar-type ATPases (V-type ATPases) in eukaryotic cells are large membrane protein complexes that acidify various intracellular compartments. The enzymes are regulated by dissociation of the V1 and VO regions of the complex. Here we present the structure of the Saccharomyces cerevisiae V-type ATPase at 11-Å resolution by cryo-EM of protein particles in ice. The structure explains many cross-linking and protein interaction studies. Docking of crystal structures suggests that inhibition of ATPase activity by the dissociated V1 region involves rearrangement of the N- and C-terminal domains of subunit H and also suggests how this inhibition is triggered upon dissociation. We provide support for this model by demonstrating that mutation of subunit H to increase the rigidity of the linker between its two domains decreases its ability to inhibit ATPase activity.
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Acknowledgements
We thank V. Kanelis for advice on rigidifying the subunit H linker region and V. Kanelis, P. Rosenthal, E. Kunji and R. Henderson for discussions and a critical reading of this manuscript. Computations were done on the general-purpose cluster supercomputer at the SciNet High Performance Computing Consortium. J.L.R. was supported by a New Investigator Award from the Canadian Institutes of Health Research (CIHR) and an Early Researcher Award from the Ontario Ministry of Research and Innovation. This research was funded by operating grant MOP 81294 to J.L.R. from the CIHR.
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J.L.R. conceived the project and experimental approach, and S.B., S.A.B. and J.L.R. designed the experiments. S.A.B. constructed the S. cerevisiae strains and protein expression vectors, purified V1-ATPase complexes from yeast and recombinant proteins from E. coli, and carried out activity assays. S.B. purified the V-type ATPase complex from yeast, obtained the cryo-EM data, did the computational image analysis and constructed the 3D map. J.L.R. advised on all aspects of the work and wrote new computer programs used for the image analysis. J.L.R. and S.B. interpreted the data and J.L.R. wrote the manuscript with input from S.B. and S.A.B.
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Supplementary Video 1
Supplementary Movie 1. Cross sections through 3-D maps. (a) The final 3-D map at 11 Å resolution is shown as sharpened with B = –300 Å2 and filtered with a Cref filter 2. (b) The maps without sharpening or application of the Cref filter. (c) The final map filtered to 17 Å resolution. (d) A map of the M. sexta V-type ATPase 4 (EMD-1590) at 17 Å resolution. (AVI 513 kb)
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Benlekbir, S., Bueler, S. & Rubinstein, J. Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11-Å resolution. Nat Struct Mol Biol 19, 1356–1362 (2012). https://doi.org/10.1038/nsmb.2422
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DOI: https://doi.org/10.1038/nsmb.2422
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