Evidence for rotation of V1-ATPase

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

[1]  J. Baleja,et al.  Localization of subunits D, E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B. , 2002, Biochemistry.

[2]  Kazuhiko Kinosita,et al.  Pause and rotation of F1-ATPase during catalysis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Stevens,et al.  V1-situated Stalk Subunits of the Yeast Vacuolar Proton-translocating ATPase* , 1997, The Journal of Biological Chemistry.

[4]  Kazuhiko Kinosita,et al.  Direct observation of the rotation of F1-ATPase , 1997, Nature.

[5]  T. Nishi,et al.  The vacuolar (H+)-ATPases — nature's most versatile proton pumps , 2002, Nature Reviews Molecular Cell Biology.

[6]  K. Yokoyama,et al.  Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase. , 1990, The Journal of biological chemistry.

[7]  K. Yokoyama,et al.  Isolation of prokaryotic V0V1-ATPase from a thermophilic eubacterium Thermus thermophilus. , 1994, The Journal of biological chemistry.

[8]  Ting Xu,et al.  Subunit Interactions in the Clathrin-coated Vesicle Vacuolar (H+)-ATPase Complex* , 1999, The Journal of Biological Chemistry.

[9]  Hiroyasu Itoh,et al.  Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase , 2001, Nature.

[10]  Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP. , 1994, The Journal of biological chemistry.

[11]  Masasuke Yoshida,et al.  V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP* , 1998, The Journal of Biological Chemistry.

[12]  F. Mayer,et al.  Subunit Structure and Organization of the Genes of the A1A0 ATPase from the Archaeon Methanosarcina mazei Gö1* , 1996, The Journal of Biological Chemistry.

[13]  P. Boyer,et al.  The binding change mechanism for ATP synthase--some probabilities and possibilities. , 1993, Biochimica et biophysica acta.

[14]  P. Boyer,et al.  Catalytic site forms and controls in ATP synthase catalysis. , 2000, Biochimica et biophysica acta.

[15]  T. Oshima,et al.  Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius. , 1987, Journal of biochemistry.

[16]  T. Stevens,et al.  Structure, function and regulation of the vacuolar (H+)-ATPase. , 1997, Annual review of cell and developmental biology.

[17]  Masasuke Yoshida,et al.  ATP synthase — a marvellous rotary engine of the cell , 2001, Nature Reviews Molecular Cell Biology.

[18]  H. Taguchi,et al.  V-Type H+-ATPase/Synthase from a Thermophilic Eubacterium, Thermus Thermophilus , 2000, The Journal of Biological Chemistry.

[19]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.