Dimer formation of subunit G of the yeast V‐ATPase

Armbrüster, Andrea; Bailer, Susanne M.; Koch, Michel H. J.; Godovac‐Zimmermann, Jasminka; Grüber, Gerhard

doi:10.1016/s0014-5793(03)00643-4

Cited by 18 publications

(29 citation statements)

References 40 publications

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“…From earlier studies, it had been suggested that homodimers of subunits E, 12 H 13 and the related G subunit of the V-ATPase 14 are key structural elements of the A-and V-ATPase peripheral stalks, but these conclusions were all based on experimental data obtained for isolated, individual subunits. For example, isolated subunit H from M. jannaschii A-ATPase was shown to exist as a dimer in solution, a structural model of which was determined by small-angle X-ray scattering.…”

Section: Discussionmentioning

confidence: 99%

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

Kish-Trier

Briere

Dunn

et al. 2008

Journal of Molecular Biology

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Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.

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Section: Discussionmentioning

confidence: 99%

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

Kish-Trier

Briere

Dunn

et al. 2008

Journal of Molecular Biology

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“…It is notable that, unlike the other stalk components, two copies of subunit G are proposed to be present in the proton pump (18), with recombinant G protein spontaneously forming a dimer in solution (22). The presence of two copies of G within each pump may explain how the smallest pump subunit can interact with four other subunits.…”

Section: Discussionmentioning

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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“…Although the site within the a subunit that is in proximity to subunit C has not been determined, the cytoplasmic localization of the N-terminal hydrophilic domain of subunit a (63) makes this region a likely candidate. By contrast, the head domain of subunit C is in close proximity to subunit G. Subunit G is a 13-kDa protein present in two copies per complex (64) that appears to form a dimer (65), analogous to the coiled-coil structure observed for the b subunit in the peripheral stalk of F 1 F 0 (66). In fact subunit G and subunit b show partial sequence homology along one helical face (67) and both are tolerant to short deletions (68,69).…”

Section: Discussionmentioning

confidence: 99%

Cysteine-mediated Cross-linking Indicates That Subunit C of the V-ATPase Is in Close Proximity to Subunits E and G of the V1 Domain and Subunit a of the V0 Domain

Inoué

Forgac

2005

Journal of Biological Chemistry

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The vacuolar (H ϩ )-ATPases (V-ATPases) 1 are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments (such as endosomes, lysosomes, and secretory vesicles) as well as proton transport across the plasma membrane (1-11). V-ATPases present in intracellular compartments function in such processes as receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, coupled transport of small molecules and ions, and the entry of certain viruses and toxins from endocytic compartments into cells (1, 2). Plasma membrane V-ATPases are important for urinary acidification by renal intercalated cells, bone resorption by osteoclasts, K ϩ secretion by insect goblet cells, sperm maturation in the male reproductive tract, and the invasiveness of certain tumor cells (1, 7-11).The V-ATPases are composed of a peripheral V 1 domain responsible for ATP hydrolysis and an integral V 0 domain that carries out proton translocation (1, 2). V 1 is a 600 -650-kDa complex with the subunit composition A 3 B 3 C 1 D 1 E 1 F 1 G 2 H 1-2 (1, 2). The nucleotide binding sites are located at the interface of the A and B subunits (which form an alternating hexamer), with the catalytic sites located mainly on the A subunit and "non-catalytic" sites located mainly on the B subunit (12-14). The V 0 domain is a 250-kDa complex with the subunit composition a 1 d 1 e x c 4 cЈ 1 cЉ 1 (1, 15). The three proteolipid subunits (c, cЈ, and cЉ) each contain a buried glutamic acid residue that is essential for proton transport (16, 17) and together they form a hexameric ring. The V-ATPases thus resemble the F-ATPases (or ATP synthases), which are involved in ATP synthesis (18 -20). High resolution crystal structures have been obtained for both F 1 (21, 22) and F 1 bound to a proteolipid ring (23).The F-ATPases operate by a rotary mechanism (20). ATP hydrolysis in F 1 causes rotation of a central stalk composed of the ␥ and ⑀ subunits (23-26), which in turn causes rotation of the ring of proteolipid c subunits (27)(28)(29). A peripheral stalk (composed of the ␦ subunit and the soluble regions of subunit b, Ref. 30) functions as a stator, holding subunit a fixed relative to the ␣ 3 ␤ 3 head during ATP-driven rotation of the proteolipid ring. It is rotation of the ring of proteolipid c subunits relative to subunit a that is believed to drive proton transport (19,31,32). Comparison of amino acid sequences of the V-and FATPases reveals clear sequence homology both between the nucleotide binding subunits and between the proteolipid subunits of the two classes (33,34,16). Because there is virtually no sequence similarity for the remaining subunits, it has not been possible to deduce the function or localization of the remaining V-ATPase subunits from their primary sequence.Electron microscopy studies indicate that like the F-ATPases (35), the V-ATPases also contain multiple stalks connecting the peripheral and integral domains (36 -38). To determine the composition of the central and peripheral stalks...

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Dimer formation of subunit G of the yeast V‐ATPase

Cited by 18 publications

References 40 publications

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

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

Cysteine-mediated Cross-linking Indicates That Subunit C of the V-ATPase Is in Close Proximity to Subunits E and G of the V1 Domain and Subunit a of the V0 Domain

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