Structure, subunit function and regulation of the coated vesicle and yeast vacuolar (H+)-ATPases

Arata, Yoichiro; Nishi, Tsuyoshi; Kawasaki-Nishi, Shoko; Shao, Elim; Wilkens, Stephan; Forgac, Michael

doi:10.1016/s0005-2728(02)00257-8

Cited by 41 publications

(26 citation statements)

References 38 publications

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“…This binding does take place (Fig.·2), which means that subunit G is exposed in the VATPase complex, which supports previous results obtained from studies on accessibility to trypsin cleavage (Gruber et al, 2000). The bound antibody did not inhibit the activities of the Vma10p-HA-containing V-ATPase (Figs·3, 4) which is in agreement with Vma10p being a part of the stator, and not participating in the rotor apparatus, as previously suggested (Nelson and Harvey, 1999;Charsky et al, 2000;Arata et al, 2002). It appears that the G subunit is present at 2-3 copies per complex (Supekova et al, 1995;Hunt and Bowman, 1997).…”

supporting

confidence: 90%

“…Further analysis showed that the N terminus of the G subunit might fold into an α helix in which one face has highly conserved residues in both the V1 G and F0 b subunits (Hunt and Bowman, 1997). Because of this structural resemblance, it was suggested that subunit G, together with subunit E, act like a 'hook' or a 'stator' similar to b in F-ATPase (Nelson and Harvey, 1999;Arata et al, 2002). Mutational analysis of the conserved face of the G subunit α helix revealed that several mutations were tolerated and even stabilized the complex, while others rendered the complex unstable (Charsky et al, 2000).…”

mentioning

confidence: 99%

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Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity

Aviezer-Hagai

Padler‐Karavani

Nelson

2003

Journal of Experimental Biology

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SUMMARYV-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits. This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available. In this system, the effect of antibody on the activity of the enzyme can be analyzed. Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants. The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme. Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme. These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus. The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane. This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA. Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four. Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices.

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supporting

confidence: 90%

mentioning

confidence: 99%

Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity

Aviezer-Hagai

Padler‐Karavani

Nelson

2003

Journal of Experimental Biology

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“…3B). We and others 3 have noted that all the available antibodies to a1 show a weak signal 3 U. Kornak, personal communication. (lanes 2, 4, 6, and 8) using an affinitypurified antibody raised against two different G3 peptide sequences.…”

Section: Confirmation Of G3-a4 Interaction and Analysis Of Possible Imentioning

confidence: 96%

“…In the current structural model of the H ϩ -ATPase, the peripheral V 1 (hydrolytic) domain contains 3A, 3B, one each of C, D, E, F, and H, and two G subunits. This is loosely linked to the membrane-anchored V 0 domain by two stalks; the central stalk consists of the D and F subunits, and the peripheral stalk contains the C, E, G, and H subunits together with the N terminus of the a subunit (1)(2)(3)(4). The a subunit is present as a single copy in the V 0 domain along with single copies of the d and e subunits and a ring structure comprising multiple numbers of c and cЈ (in yeast cells) and a single cЉ subunit (1,3,5,6).…”

mentioning

confidence: 99%

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

Norgett

Borthwick

Al‐Lamki

et al. 2007

Journal of Biological Chemistry

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The specialized H ؉ -ATPases found in the inner ear and acidhandling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown widespread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H ؉ -ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H ؉ -ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V 1 and V 0 domains in man, which is known to be required for H ؉ -ATPase assembly and regulation.

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“…The role of the other V 1 subunits is not so clear. It is thought that the central stalk may be composed of D (32 kDa) and F (14 kDa) subunits (8,9). The stator region is thought to be composed of E (27 kDa), G (13 kDa), C (42 kDa), and H (54 kDa) subunits (9 -13).…”

mentioning

confidence: 99%

Topological Characterization of the c, c′, and c″ Subunits of the Vacuolar ATPase from the Yeast Saccharomyces cerevisiae

Flannery

Graham

Stevens

2004

Journal of Biological Chemistry

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The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V 1 and V 0 . Hydrolysis of ATP in the V 1 subcomplex is tightly coupled to proton translocation accomplished by the V 0 subcomplex, which is composed of five unique subunits (a, d, c, c , and c؆). Three of the subunits, subunit c (Vma3p), c (Vma11p), and c؆ (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c؆ (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c have four transmembrane segments with their N and C termini in the lumen and that c؆ has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.

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Structure, subunit function and regulation of the coated vesicle and yeast vacuolar (H+)-ATPases

Cited by 41 publications

References 38 publications

Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity

Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

Topological Characterization of the c, c′, and c″ Subunits of the Vacuolar ATPase from the Yeast Saccharomyces cerevisiae

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