Cryo-electron Microscopy of the Vacuolar ATPase Motor Reveals its Mechanical and Regulatory Complexity

Muench, Stephen P.; Huss, Markus; Song, Chun; Phillips, Clair; Wieczorek, Helmut; Trinick, John; Harrison, Michael A.

doi:10.1016/j.jmb.2009.01.014

Cited by 94 publications

(131 citation statements)

References 64 publications

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“…Inconsistent with previous EM data for the eukaryotic V-ATPase, no portion of either stator contacts the C-subunit of the central rotor (21), and therefore would not impede rotation of the rotor. The bar that links the two peripheral stalks lies parallel to where the surface of the membrane would be.…”

Section: Resultssupporting

confidence: 80%

“…All of the features discussed below were apparent at any of these resolutions but most distinctly so at 16 Å, and consequently the figures rendered here are for a map built to 16 Å. The map has a clear handedness and the absolute hand of the map, which was inconsistent between previous published models ( [16][17][18][19][20][21], was found to be correct as represented here by using the Freehand test (41) (Fig. S4B).…”

Section: Resultsmentioning

confidence: 71%

“…This arrangement is different from the peripheral stalk of the F-type ATP synthase (11,12), which wraps around the long-axis of the complex and the eukaryotic V-ATPase (17,21), where the peripheral stalks have a noticeable curvature. While consistently observed, the role of this bending of the peripheral stalks in some F-and V-type ATPases is not known but could relate to the storage of elastic energy in this part of the assembly.…”

Section: Resultsmentioning

confidence: 85%

“…Because these earlier models were derived from negative stain data, they do not give reliable information about the internal structures of the complexes. A cryo-EM map of the V-ATPase from Manduca sexta also clearly showed three peripheral stalks for the eukaryotic enzyme (21). The model, although obtained from unstained protein in ice, did not show the central rotor penetrating into the A 3 B 3 hexamer and did not resolve significant internal features in the membrane region of the complex.…”

mentioning

confidence: 96%

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Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Lau

Rubinstein

2010

Proc. Natl. Acad. Sci. U.S.A.

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The eubacterium Thermus thermophilus uses a macromolecular assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 Å resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V O region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V O gives insight into the mechanism of torque generation from proton translocation. membrane protein | single particle analysis V acuolar-type ATPases (V-ATPases) in eukaryotes function as ATP-driven proton pumps that acidify intracellular compartments including lysosomes, endosomes, and secretory vesicles. This acidification, in turn, affects diverse processes including protein sorting and degradation, overall ion homeostasis, and protection of cells from oxidative stress (1). Extracellular acidification by V-ATPases is linked to tumor invasion and metastasis and osteoporosis (2). F-type ATP synthases and V-type ATPases are evolutionarily related but differ in the details of subunit composition and arrangement. Both F-and V-type ATPases use a rotary catalytic mechanism where proton translocation through the membranebound F O or V O region, respectively, generates a torque on a rotor subcomplex that drives ATP synthesis in the F 1 or V 1 region. The enzymes can also run in the opposite direction with ATP hydrolysis in the F 1 or V 1 region resulting in proton pumping through F O or V O . This mechanism has been the subject of a large body of research for F-type ATP synthases (e.g., 3-5), but there has also been direct demonstration of rotary catalysis for V-ATPases (6). Some archaea and eubacteria use a complex more closely related to VATPase than F-type ATP synthase, sometimes called an A-ATPase, to generate their supply of ATP (7).The V-ATPase from the eubacterium Thermus thermophilus is composed of nine different subunits with a stoichiometry of A 3 B 3 CDE 2 FG 2 IL 12 (Fig. S1). Subunit nomenclature for this family of enzymes differs between F-and V-type complexes and from organism to organism. Where the eukaryotic ATP synthase F 1 catalytic region consists of α 3 β 3 γδε, the V-ATPase V 1 catalytic region consists of B 3 A 3 DF with no equivalent of the ε-subunit. The catalytic A-subunit of V-ATPase is homologous to the F-type ATP synthas...

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 96%

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Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Lau

Rubinstein

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Moreover, according to several recent electron microscopy studies of V-ATPases from different organisms 16,[38][39][40] and small-angle X-ray scattering data of NtpI 1-341 , 26 it was determined that the N-terminal domain of the a-subunit forms an elongated 1-shaped structure. In these studies, it was also suggested that this part of the a-subunit lies parallel to the membrane, adjacent to its cytoplasmic side.…”

Section: Discussionmentioning

confidence: 99%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

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EPR Studies of V‐ATPase with Spin‐Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit c

et al. 2015

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Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.

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Cryo-electron Microscopy of the Vacuolar ATPase Motor Reveals its Mechanical and Regulatory Complexity

Cited by 94 publications

References 64 publications

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

EPR Studies of V‐ATPase with Spin‐Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit c

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Cryo-electron Microscopy of the Vacuolar ATPase Motor Reveals its Mechanical and Regulatory Complexity

Cited by 94 publications

References 64 publications

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

EPR Studies of V‐ATPase with Spin‐Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit c

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Product

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Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor