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Boekema, Egbert J.; Ubbink-Kok, Trees; Lolkema, Juke S.; Brisson, Alain; Konings, Wn

doi:10.1023/a:1006044931980

Cited by 27 publications

(25 citation statements)

References 30 publications

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“…5, image 1, is very similar to averages obtained for the membrane-bound V-ATPase from Clostridium fervidus (12,13). In these membrane-bound samples, the orientation of the complex is apparently less influenced by the interaction with the carbon film, leading to a higher abundance of this projection.…”

Section: Figsupporting

confidence: 69%

“…In all the images showing a second stalk, there seems to be no clear connection of the stalk forming proteins into the membrane portion of the V 0 . A very similar looking picture, in which a weak density is running down on the side of the V 1 to end in a subunit bound to the central stalk, not in the membrane, has been obtained for the Na ϩ -transporting V-type ATPase from C. fervidus (12,13). Whether this clear difference in the appearance of the second stalk in the images of the V-versus F-type ATPase is due to staining artifacts, or a genuine structural and possibly functional difference between the two related enzymes, requires further study.…”

Section: Figmentioning

confidence: 53%

“…In this model, the two b subunits bound to the a subunit in the F 0 , and the ␦ subunit in the F 1 form a second stalk or stator keeping the two active domains of the complex in the correct spatial arrangement for energy coupling to occur. This second stalk or stator has only recently been observed by electron microscopy of negatively stained samples for both the V-and F-ATPase (12,13,22,37). Weak densities can be seen connecting the elongated proteins at the top of the V 1 to the stalk region in some images (see small black arrows in images 2 and 5 of Fig.…”

Section: Figmentioning

confidence: 84%

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Structure of the Vacuolar ATPase by Electron Microscopy

Wilkens¹,

Vasilyeva²,

Forgac³

1999

Journal of Biological Chemistry

134

142

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The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergentsolubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V 1 and V 0 , with overall dimensions of 28 ؋ 14 ؋ 14 nm. The V 1 is a more or less spherical molecule with a central cavity. The V 0 has the shape of a flattened sphere or doughnut with a radius of about 100 Å. The V 1 and V 0 are joined by a 60-Å long and 40-Å wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V 1 , and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30 -50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V 1 -V 0 interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F 1 F 0 -ATP synthase, confirming their common origin.A vacuolar ATPase (or V-ATPase) 1 is found in the membrane of subcellular compartments of eucaryotic cells, where it functions to acidify the interior and at the same time energize the membranes of organelles such as clathrin-coated vesicles, endosomes, lysosomes, chromaffin granules, and Golgi-derived vesicles (1, 2). ATP hydrolysis-driven acidification of these organelles plays an important role in processes like receptormediated endocytosis, neurotransmitter release, protein trafficking, pH maintenance, and storage of metabolites. Early electron microscopic images (3-5) show the V-ATPase complex organized in two parts, a membrane extrinsic V 1 and a membrane-embedded V 0 , named after the related F 1 F 0 -type ATP synthase. As in F 1 F 0 , ATP hydrolysis on the membrane extrinsic domain is coupled to proton translocation across the membrane bilayer, but unlike F 1 F 0 , the vacuolar enzyme cannot utilize the potential energy of a proton gradient to synthesize ATP. The V 1 V 0 -ATPase is composed of at least 12 different subunits. The V 1 contains subunits A-H with molecular weights of 73,000, 58,000, 40,000, 34,000, 33,000, 14,000, 10,000, and 50,000 -57,000, and the V 0 contains subunits a, cЈ, cЉ, and d having molecular weights of 100,000, 17,000, 19,000 and 38,000, respectively. The subunit stoichiometry of the complex is A 3 B 3 CDEF x G y H z ac (1) 6 cЉd, giving a calculated molecular weight of approximately 840,000 (assuming one copy of subunits F, G, and H each), in good agreement...

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Section: Figsupporting

confidence: 69%

Section: Figmentioning

confidence: 53%

Section: Figmentioning

confidence: 84%

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Structure of the Vacuolar ATPase by Electron Microscopy

Wilkens¹,

Vasilyeva²,

Forgac³

1999

Journal of Biological Chemistry

134

142

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“…Furthermore, DAMMIN allows to incorporate additional information about particle symmetry and anisometry. The V 1 ATPase is known to be an elongated particle possessing a quasi-3-fold symmetry (32,33), so these restrictions were implemented as described in the program manual.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Major Structural Changes in the Manduca sexta Midgut V1 ATPase Due to Redox Modulation

Grüber

Svergun

Godovac‐Zimmermann

et al. 2000

Journal of Biological Chemistry

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The shape and overall dimensions of the oxidized and reduced form of the V 1 ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6.20 ؎ 0.06 and 5.84 ؎ 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 ؎ 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V 1 molecule provide the first strong evidence for major structural changes in the V 1 ATPase because of redox modulation.Ion-translocating, vacuolar-type, ATPases (V-ATPases), which use ATP to acidify intracellular compartments and energize plasma membranes, consist of a hydrophilic, catalytic complex, V 1 , and a hydrophobic, ion-translocating channel complex, V O (1, 2). In the larval midgut of the tobacco hornworm, Manduca sexta, the V O complex is made up of four subunits with apparent molecular masses of 100 (a), 40 (d), 20 (e) and 17 (c) kDa. The hydrophilic portion, V 1 ATPase, consists of eight distinct subunits of molecular mass 67 (A), 56 (B), 54 (H), 40 (C), 32 (D), 28 (E), 16 (G), and 14 kDa (F). Three interdigitating copies each of the nucleotide-binding subunits A and B form a head piece at the top of the molecule, whereas the remaining V 1 subunits form a stalk connecting the head piece to the V O complex (3).Several mechanisms have been proposed for the regulation of V-ATPases, including disassembly and reassembly of peripheral V 1 and integral V O complexes (4 -7), regulation of parallel ion channels that compensate for the electrogenicity of the V-ATPase (8, 9), changes in the degree of coupling between ATP hydrolysis and proton pumping (9, 10), and reversible disulfide bond interchange between conserved cysteine residues near the catalytic site (reviewed in Ref. 11). Studies on the bovine clathrin-coated V-ATPase have shown that disulfide bond formation between the conserved residues Cys 254 and Cys 532 of the catalytic A subunit leads to reversible inactivation of the enzyme (12, 13). Such a reversibly inactivated, disulfidebonded state has been found in significant fractions of V-ATPase in native clathrin-coated vesicles (12). Disulfide bonds at the catalytic site of the V-ATPase are also induced by the nitric oxide-generating reagent S-nitrosoglutathione (14). This is consistent with the finding that in Neurospora crassa oxidizing and reducing agents have inhibitory and stabilizing effects, respectively, on the enzyme (15). Moreover, a mutation in the cysteine biosynthetic pathway in yeast leads to defective vacuolar acidification, which can be corrected by a C261V mutation, suggesting that disulfide bond formation can cause inhibition of V-ATPase activity ...

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“…The number of known subunits of the V-type ATPases exceeds that of the F-type ATPases. Also, recent evidence from electron microscopy suggests that the overall structure and shape of the two enzymes may be considerably different (Boekema et al, 1997(Boekema et al, , 1999.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H⁺-ATPase ofSaccharomyces cerevisiae

Sagermann

Matthews

2000

Acta Crystallogr D Biol Cryst

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The expression and crystallization of the VMA13p subunit of the vacuolar proton-translocating ATPase (V-ATPase) of Saccharomyces cerevisiae is described. This 478 amino-acid subunit is essential for activity but not for the assembly of this multisubunit complex. The protein has been recombinantly overexpressed in Escherichia coli and puri®ed. Diffraction-quality crystals have been obtained using the hanging-drop vapor-diffusion method with ammonium sulfate as precipitant. Several different crystal forms were obtained. The most suitable crystal form for crystallographic characterization belongs to space group P3 1 21 or its enantiomorph, with unit-cell parameters a = b = 118.8, c = 119.3 A Ê . Using an in-house X-ray source, the crystals diffract to about 3.5 A Ê resolution under rapidly frozen conditions.

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Cited by 27 publications

References 30 publications

Structure of the Vacuolar ATPase by Electron Microscopy

Structure of the Vacuolar ATPase by Electron Microscopy

Evidence for Major Structural Changes in the Manduca sexta Midgut V1 ATPase Due to Redox Modulation

Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H⁺-ATPase ofSaccharomyces cerevisiae

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Untitled

Cited by 27 publications

References 30 publications

Structure of the Vacuolar ATPase by Electron Microscopy

Structure of the Vacuolar ATPase by Electron Microscopy

Evidence for Major Structural Changes in the Manduca sexta Midgut V1 ATPase Due to Redox Modulation

Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase ofSaccharomyces cerevisiae

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Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H⁺-ATPase ofSaccharomyces cerevisiae