Letter

Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase

  • Nature volume 521, pages 241245 (14 May 2015)
  • doi:10.1038/nature14365
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Abstract

Eukaryotic vacuolar H+-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions1,2. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases3,4,5, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H+ ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6284, EMD-6285, and EMD-6286. Atomic models have been deposited in the Protein Data Bank under accession numbers 3J9T, 3J9U, and 3J9V.

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Acknowledgements

We thank P. Rosenthal, R. Henderson, V. Kanelis, and L. Kay for comments on the manuscript. J.Z. was supported by a Doctoral Postgraduate Scholarship from the Natural Sciences and Engineering Research Council of Canada and a Mary Gertrude l’Anson Scholarship. J.L.R. is the Canada Research Chair in Electron Cryomicroscopy. This work was supported by operating grant MOP 81294 from the Canadian Institutes of Health Research.

Author information

Author notes

    • Jianhua Zhao
    •  & Samir Benlekbir

    These authors contributed equally to this work.

Affiliations

  1. Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada

    • Jianhua Zhao
    • , Samir Benlekbir
    •  & John L. Rubinstein
  2. Department of Medical Biophysics, The University of Toronto, Toronto Medical Discovery Tower, MaRS Centre, 101 College Street, Toronto, Ontario M5G 1L7, Canada

    • Jianhua Zhao
    •  & John L. Rubinstein
  3. Department of Biochemistry, The University of Toronto, 1 King’s College Circle, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada

    • John L. Rubinstein

Authors

  1. Search for Jianhua Zhao in:

  2. Search for Samir Benlekbir in:

  3. Search for John L. Rubinstein in:

Contributions

S.B. and J.L.R. initiated the project. J.Z. and S.B. collected images and performed pre-processing steps. J.Z. performed the image analysis. J.Z. and J.L.R. interpreted the data, prepared figures, and wrote the manuscript. J.L.R. and J.Z. contributed new computer algorithms used in image analysis.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John L. Rubinstein.

Extended data

Supplementary information

Videos

  1. 1.

    Cross sections through the three maps, each showing a different rotational state of the V-ATPase.

    Cross sections through the three maps, each showing a different rotational state of the V-ATPase.

  2. 2.

    Interpolation between the three observed rotational states of the V-ATPase

    Interpolation between the three observed rotational states of the V-ATPase.

  3. 3.

    Exploded view of subunits when interpolating between the three observed rotational states of the V-ATPase

    Exploded view of subunits when interpolating between the three observed rotational states of the V-ATPase.

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