The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

Lu, Ming; Vergara, Sandra; Zhang, Li; Holliday, L. Shannon; Aris, John P.; Gluck, Stephen L.

doi:10.1074/jbc.m203521200

Cited by 71 publications

(69 citation statements)

References 28 publications

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“…Crucially, to unequivocally confirm the requirement for the H + -V-ATPase, we augmented drug experiments with specific constructs targeting the H + -V-ATPase. Misexpression of the dominant-negative E subunit (Lu et al, 2002) in Xenopus and in zebrafish embryos causes randomization, confirming the requirement for H + -V-ATPase for correct asymmetry. Though changing V mem and pH by overexpression can randomize, only the V-ATPase is implicated endogenously in setting LR-relevant values of these physiological parameters, as specific inhibitors of the Na + /K + -ATPase (Levin et al, 2002) and other H + pumps (Fig.…”

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confidence: 61%

“…To test unequivocally the requirement for H + -V-ATPase activity using specific molecular reagents, we analyzed asymmetry in embryos injected with YCHE78 mRNA, which encodes a wellcharacterized dominant negative H + -V-ATPase subunit E (Lu et al, 2002). Misexpression of this dominant-negative construct specifically induced 20% heterotaxia [(n=191), controls=4% (n=422), 2 =40.6, PӶ0.001].…”

Section: Inhibition Of H + -V-atpase Specifically Causes Heterotaxiamentioning

confidence: 99%

“…1K with 1L) in the absence of changes in dorsoanterior character or general toxicity (Danos and Yost, 1995;Danos and Yost, 1996). For example, the potent and highly-specific V-ATPase blockers concanamycin (Huss et al, 2002) To test unequivocally the requirement for H + -V-ATPase activity using specific molecular reagents, we analyzed asymmetry in embryos injected with YCHE78 mRNA, which encodes a wellcharacterized dominant negative H + -V-ATPase subunit E (Lu et al, 2002). Misexpression of this dominant-negative construct specifically induced 20% heterotaxia [(n=191), controls=4% (n=422), 2 =40.6, PӶ0.001].…”

Section: Inhibition Of H + -V-atpase Specifically Causes Heterotaxiamentioning

confidence: 99%

“…Specific and dose-dependent ( Fig. 1M) randomization of LR asymmetry, induced by loss-of-function reagents targeting the H + -V-ATPase, but not other H + pumps or a wide range of other ion transporters, implicate endogenous H + -VATPase in the LR pathway in Xenopus.To test unequivocally the requirement for H + -V-ATPase activity using specific molecular reagents, we analyzed asymmetry in embryos injected with YCHE78 mRNA, which encodes a wellcharacterized dominant negative H + -V-ATPase subunit E (Lu et al, 2002). Misexpression of this dominant-negative construct specifically induced 20% heterotaxia [(n=191), controls=4% (n=422), 2 =40.6, PӶ0.001].…”

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Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Adams

Robinson

Fukumoto³

et al. 2006

Development

251

306

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Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H + flux. A pharmacological screen implicated the H + -pump H + -V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H + -V-ATPase subunits during the first three cleavages. H + -flux across plasma membranes is also asymmetric at the four-and eight-cell stages, and this asymmetry requires H + -V-ATPase activity. Abolishing the asymmetry in H + flux, using a dominant-negative subunit of the H + -V-ATPase or an ectopic H + pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H + -V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (V mem ) regulation. Varying either pH or V mem , independently of direct manipulation of H + -V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H + -V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H + -V-ATPase induces heterotaxia in both species; in chick, H + -V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H + -V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH-and V mem -dependent model of the early physiology of LR patterning. Development 133, 1657Development 133, -1671Development 133, (2006 DEVELOPMENT 1658 necessary to characterize the endogenous behavior of the relevant pumps in embryos and to place their function in the context of known LR patterning mechanisms. Here, we explore the properties of H + -V-ATPase function in several vertebrate embryos. Through endogenous localization of the H + -V-ATPase and gain-and loss-offunction experiments in chick, frog and zebrafish, we identify the H + -V-ATPase as a novel, conserved, obligate component of LR patterning upstream of asymmetric gene expression. KEY WORDS: Left-right asymmetry, H + -V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage MATERIALS AND METHODS Animal husbandryXenopus embryos were collected according to standard protocols (Sive et al., 2000) in 0.1ϫ Modified Marc's Ringers (MMR) pH 7.8 + 0.1% Gentamicin. Xenopus embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Chick embryos from Charles River Laboratories, maintained at 38°C, were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1992). Zebrafish embryos (Westerfield, 1995) were maintained at 28.5°C in water containing 1 drop per gallon Methyl Blue. Assaying organ situsXenopus e...

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confidence: 61%

Section: Inhibition Of H + -V-atpase Specifically Causes Heterotaxiamentioning

confidence: 99%

Section: Inhibition Of H + -V-atpase Specifically Causes Heterotaxiamentioning

confidence: 99%

mentioning

confidence: 99%

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Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Adams

Robinson

Fukumoto³

et al. 2006

Development

251

306

View full text Add to dashboard Cite

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“…A more detailed structural model, mainly derived from studies of both yeast and bovine orthologs, is that H ϩ -ATPases can better be defined by several regions. They are, as before, a catalytic core composed of A and B subunits; a central rotor or stalk, suggested to be composed of D and F subunits; a peripheral "stator" likely to be composed of the N terminus of the a-subunit together with C, E, G, and H, and a proton-translocating domain composed of the C terminus of a, with c, cЈ, and cЉ subunits (3)(4)(5). However, to date, mammalian orthologs of yeast cЈ have not been identified.…”

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confidence: 99%

The a-Subunit of the V-type H+-ATPase Interacts with Phosphofructokinase-1 in Humans

Zhou

Al‐Lamki

et al. 2003

Journal of Biological Chemistry

108

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V-type or H؉ -ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in ␣-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H ؉ -ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzymelinked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pulldown assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in ␣-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H ؉ -ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H ؉ -ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.The H ϩ -, vacuolar-type or V-ATPases (hereafter abbreviated H ϩ -ATPases) are a family of ATP-dependent proton pumps that acidify intracellular compartments in all cells. They also pump protons across the plasma membranes of osteoclasts and of certain epithelial cells in the kidney and male genital tract. H ϩ -ATPases from fungi, plants, and animals are structurally very similar to each other (1), consisting of two major functional domains known as V 1 and V 0 . The V 1 domain is composed of at least eight different subunits (A-H) and contains ATP binding sites involving the A and B subunits. This domain is mainly responsible for ATP hydrolysis. The V 0 domain contains up to five additional subunits (a, c, cЈ, cЉ, and d) and is mainly responsible for proton translocation across the membrane in which it is anchored (2).

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Structure and Regulation of Plant Vacuolar H+-ATPase

Seidel

Progress in Botany

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The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

Cited by 71 publications

References 28 publications

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

The a-Subunit of the V-type H+-ATPase Interacts with Phosphofructokinase-1 in Humans

Structure and Regulation of Plant Vacuolar H+-ATPase

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