Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Rizzo, Vincenzo F.; Coskun, Ünal; Radermacher, Michael; Ruíz, Teresa; Armbrüster, Andrea; Grüber, Gerhard

doi:10.1074/jbc.m208623200

Cited by 31 publications

(25 citation statements)

References 51 publications

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“…Therefore, this density can indicate the presence of subunit G. Such close proximity of subunit G to E has been described from binding (24) and zero-length cross-link experiments (50). In line with these findings are the most recent data of the V 1 -ATPase from M. sexta demonstrating that the "core" complex consists at least of the four subunits A, B, E, and G and that it still retains 58% of hydrolytic ATPase activity (26). Averaged projections of this core complex show a hexagonal arrangement of the major subunits A and B surrounding a seventh mass composed of both subunits E and G. Furthermore, subunit G of the yeast vacuolar ATPase exists in solution as an elongated dimer (8.0 Ϯ 0.3 nm) (33).…”

Section: Structure Of a F 1 -/V 1 -Atpase Hybrid Complexsupporting

confidence: 75%

Structural Characterization of an ATPase Active F1-/V1 -ATPase (α3β3EG) Hybrid Complex

Chaban

Coskun

Keegstra

et al. 2004

Journal of Biological Chemistry

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Vacuolar-type ATPases (V-ATPases)1 are membrane-bound proteins functioning in active ion pumping at the expense of ATP hydrolysis. They are present in the membranes of all eukaryotic cells and the plasma membrane of some bacteria (1-3). V-ATPases are responsible for acidification of intracellular compartments and generation of an electrochemical gradient (ion motive force) (4, 5). These enzymes consist of two structural and functional parts, a hydrophilic, V 1 , and hydrophobic, V O , portion. The V 1 part contains eight (A-H) and seven subunits (A-G) in the cases of the eukaryotic and prokaryotic enzymes, respectively (3, 4). V O in eukaryotes consists of subunits a, c, and d, corresponding to subunits I, K, and C in prokaryotic V-ATPases (Enterococcus hirae nomenclature). Phylogenetic (6, 7), biochemical (8), and structural studies of V-ATPases (reviewed in Refs. 3, 9, and 10) show its relation to the well characterized F-ATPases (11, 12). Because of the structural similarity, it is believed that the energy-coupling mechanism of ATP hydrolysis and ion translocation is similar to that in F-ATPases (13). Therefore, the ATP hydrolysis in the A 3 B 3 hexamer of V 1 might trigger the rotational movement(s) of the central stalk subunit(s). Rotation of the central stalk transfers a generated torque to the ring of the c (K) subunits in the V O part (13), resulting in the pumping of ions over the membrane. The stability of the A 3 B 3 hexamer against rotation is provided by peripheral stalk(s) connecting static subunits of V 1 and V O .Despite assigned similarity in the catalytic mechanism, a growing amount of evidence suggests that some structural features and regulatory mechanisms of both enzymes are different, reflecting diversities in their functions and evolution (for review, see Ref. 14). The most significant structural difference is shown in the stalk(s) region. Unlike F-ATPases, where the presence of only one peripheral stalk was reported (15, 16), two or three peripheral stalks were described for the V-ATPase (17-19). The structure of the central stalk in the V-ATPase also seems to be more complicated. Electron microscopy (20) and small angle x-ray scattering studies (21) indicated that the central stalk of V-ATPase is rather elongated when compared with that in the F-ATPase. The exact assignment of the subunits involved in these stalk region formations is a matter of debate, and it is difficult to identify the V-ATPase stalk subunits that have significant sequence similarity to the stalk subunits of the F-ATPase (3). According to secondary structure prediction, the V-ATPase subunits D and E can be candidates for the homologue role of the rotating ␥ subunit in F-ATPase (22,23). Experiments on assembly of the V-ATPase from yeast indicate that either the D or E subunits, or both, might be a functional homologue of subunit ␥ (24). Recently, hydrolytically active A 3 B 3 EC and A 3 B 3 EG subcomplexes from Caloramator fervidus (25) and Manduca sexta (26) V-ATPase, respectively, were visualized by electron microsco...

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Section: Structure Of a F 1 -/V 1 -Atpase Hybrid Complexsupporting

confidence: 75%

Structural Characterization of an ATPase Active F1-/V1 -ATPase (α3β3EG) Hybrid Complex

Chaban

Coskun

Keegstra

et al. 2004

Journal of Biological Chemistry

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“…Previous data from photoactivated cross-linking of unique cysteine residues on subunit B suggested that subunits E and G are part of the peripheral stalk connecting the V 1 and V 0 domains and that subunit D is located in the interior of the A 3 B 3 hexameric head, thus making it the likely homolog to the ␥ subunit in F 1 (14,15). The peripheral location of subunit E has been challenged by electron microscopy data obtained on the V 1 domain of the Manduca sexta V-ATPase (50). One question in attempting to reconcile these two sets of data is the accuracy of the structural model of the V 1 domain on which the interpretation of the cross-linking data is based.…”

Section: Position Of Subunit H In the Yeastmentioning

confidence: 98%

Three-Dimensional Structure of the Vacuolar ATPase

Wilkens

Inoué

Forgac

2004

Journal of Biological Chemistry

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To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V 1 in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V 1 and V 0 domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V 0 . For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F 1 domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido)benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V 1 and V 0 domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.The vacuolar ATPase (V-ATPase, V 1 V 0 -ATPase) 1 is an essential component of all eukaryotic cells (1-4). The complex is found in both the endomembrane system of subcellular organelles and the plasma membrane of certain specialized cells. The function of the V-ATPase is to pump protons across the membrane bilayer, a process powered by hydrolysis of ATP. The V-ATPase is a large, multisubunit complex that can be divided into three domains: a water-soluble V 1 , which is responsible for binding and hydrolysis of ATP; a membrane embedded V 0 , which contains the proton translocation pore; and a stalk domain, which functions as a structural and functional connection between the V 1 and V 0 . The subunit composition of the bovine V-ATPase has been determined by quantitative amino acid analysis to be A 3 B 3 CDEFG 2 H 2 for the V 1 and a(ccЈ) 4 -5 cЉd for the V 0 (5, 6). Overall, the structure and mechanism of the V-ATPase is similar to the structure and mechanism of the related F 1 F 0 -ATPsynthase (7,8). According to the current mechanistic model of V-ATPase function, ATP hydrolysis taking place on the V 1 A subunits drives rotation of a central domain composed of subunits D, F, d, and the ring of proteolipids against the remainder of the complex (9, 10). Proton translocation occurs at the interface of the membrane bound domain of the V 0 a subunit and the lipid exposed glutamate sidechains from the c subunits. As in the F-ATPase motor, a peripheral stalk prevents rotation of the static domain during enzyme functioning. However, electron microsc...

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“…The gross structure and tentative subunit positions in the complex have emerged through cross-linking studies (Graham et al 2000;Gru¨ber et al 2001) complemented by lowresolution structural information from electron microscopy (Boekema et al 1997(Boekema et al , 1999Wilkens et al 1999;Ubbink-Kok et al 2000;Domgall et al 2002). Electron microscopy has confirmed that the isolated V 1 domain is a hetero hexametric complex of alternating A and B subunits (Radermacher et al 1999(Radermacher et al , 2001Gru¨ber et al 2000;Rizzo et al 2003). The asymmetric protein ring, as well as the 7-fold symmetry of the c ring, has been visualized by electron microscopy, demonstrating that the V o domain is the rotor responsible for proton translocation (Wilkens and Forgac 2001;Murata et al 2003).…”

Section: Introductionmentioning

confidence: 93%

Electron-microscopic structure of the V-ATPase from mung bean

Zhang

2004

Planta

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The vacuolar H(+)-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V1 and Vo with overall dimensions of 25 nm x 13.7 nm and a stalk region connecting the V1 and Vo domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.

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Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Cited by 31 publications

References 51 publications

Structural Characterization of an ATPase Active F1-/V1 -ATPase (α3β3EG) Hybrid Complex

Structural Characterization of an ATPase Active F1-/V1 -ATPase (α3β3EG) Hybrid Complex

Three-Dimensional Structure of the Vacuolar ATPase

Electron-microscopic structure of the V-ATPase from mung bean

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