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1
Russell, V. E., U. Klein, M. Reuveni, D. D. Spaeth, M. G. Wolfersberger, and W. R. Harvey. "Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes." Journal of Experimental Biology 166, no. 1 (May 1, 1992): 131–43. http://dx.doi.org/10.1242/jeb.166.1.131.
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In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.
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Parra, Karlett J., Chun-Yuan Chan, and Jun Chen. "Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals." Eukaryotic Cell 13, no. 6 (April 4, 2014): 706–14. http://dx.doi.org/10.1128/ec.00050-14.
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ABSTRACTVacuolar H+-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeastSaccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.
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Collaco, Anne M., Peter Geibel, Beth S. Lee, John P. Geibel, and Nadia A. Ameen. "Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR." American Journal of Physiology-Cell Physiology 305, no. 9 (November 1, 2013): C981—C996. http://dx.doi.org/10.1152/ajpcell.00067.2013.
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Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR−/−mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR−/−intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHiin cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.
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Sautin, Yuri Y., Ming Lu, Andrew Gaugler, Li Zhang, and Stephen L. Gluck. "Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells." Molecular and Cellular Biology 25, no. 2 (January 15, 2005): 575–89. http://dx.doi.org/10.1128/mcb.25.2.575-589.2005.
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ABSTRACT Vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps. They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm. Mechanisms of extrinsic control of V-ATPase are poorly understood. Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae. Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function. Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K). Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK1. Glucose induces rapid ATP-independent assembly of the V1 and Vo domains of V-ATPase and extensive translocation of the V-ATPase V1 and Vo domains between different membrane pools and between membranes and the cytoplasm. In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane. The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110α of PI3K. Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling.
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Gogarten, J. P., T. Starke, H. Kibak, J. Fishman, and L. Taiz. "Evolution and isoforms of V-ATPase subunits." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 137–47. http://dx.doi.org/10.1242/jeb.172.1.137.
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The structure of V- and F-ATPases/ATP synthases is remarkably conserved throughout evolution. Sequence analyses show that the V- and F-ATPases evolved from the same enzyme that was already present in the last common ancestor of all known extant life forms. The catalytic and non-catalytic subunits found in the dissociable head groups of both V-ATPases and F-ATPases are paralogous subunits, i.e. these two types of subunits evolved from a common ancestral gene. The gene duplication giving rise to these two genes (i.e. those encoding the catalytic and non-catalytic subunits) pre-dates the time of the last common ancestor. Similarities between the V- and F-ATPase subunits and an ATPase-like protein that is implicated in flagellar assembly are evaluated with regard to the early evolution of ATPases. Mapping of gene duplication events that occurred in the evolution of the proteolipid, the non-catalytic and the catalytic subunits onto the tree of life leads to a prediction of the likely quaternary structure of the encoded ATPases. The phylogenetic implications of V-ATPases found in eubacteria are discussed. Different V-ATPase isoforms have been detected in some higher eukaryotes, whereas others were shown to have only a single gene encoding the catalytic V-ATPase subunit. These data are analyzed with respect to the possible function of the different isoforms (tissue-specific, organelle-specific). The point in evolution at which the different isoforms arose is mapped by phylogenetic analysis.
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Kane, Patricia M. "The Where, When, and How of Organelle Acidification by the Yeast Vacuolar H+-ATPase." Microbiology and Molecular Biology Reviews 70, no. 1 (March 2006): 177–91. http://dx.doi.org/10.1128/mmbr.70.1.177-191.2006.
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SUMMARY All eukaryotic cells contain multiple acidic organelles, and V-ATPases are central players in organelle acidification. Not only is the structure of V-ATPases highly conserved among eukaryotes, but there are also many regulatory mechanisms that are similar between fungi and higher eukaryotes. These mechanisms allow cells both to regulate the pHs of different compartments and to respond to changing extracellular conditions. The Saccharomyces cerevisiae V-ATPase has emerged as an important model for V-ATPase structure and function in all eukaryotic cells. This review discusses current knowledge of the structure, function, and regulation of the V-ATPase in S. cerevisiae and also examines the relationship between biosynthesis and transport of V-ATPase and compartment-specific regulation of acidification.
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Nelson, Nathan, and William R. Harvey. "Vacuolar and Plasma Membrane Proton-Adenosinetriphosphatases." Physiological Reviews 79, no. 2 (April 1, 1999): 361–85. http://dx.doi.org/10.1152/physrev.1999.79.2.361.
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The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPases have similar structure and mechanism of action with F-ATPase and several of their subunits evolved from common ancestors. In eukaryotic cells, F-ATPases are confined to the semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The mechanistic and structural relations between the two enzymes prompted us to suggest similar functional units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine. It was the yeast genetics that allowed the identification of special properties of individual subunits and the discovery of factors that are involved in the enzyme biogenesis and assembly. The V-ATPases play a major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial cells. This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney. The list of animals with plasma membranes that are energized by V-ATPases now includes members of most, if not all, animal phyla. This includes the classical Na+absorption by frog skin, male fertility through acidification of the sperm acrosome and the male reproductive tract, bone resorption by mammalian osteoclasts, and regulation of eye pressure. V-ATPase may function in Na+uptake by trout gills and energizes water secretion by contractile vacuoles in Dictyostelium. V-ATPase was first detected in organelles connected with the vacuolar system. It is the main if not the only primary energy source for numerous transport systems in these organelles. The driving force for the accumulation of neurotransmitters into synaptic vesicles is pmf generated by V-ATPase. The acidification of lysosomes, which are required for the proper function of most of their enzymes, is provided by V-ATPase. The enzyme is also vital for the proper function of endosomes and the Golgi apparatus. In contrast to yeast vacuoles that maintain an internal pH of ∼5.5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red alga maintain internal pH as low as 0.1 in their vacuoles. One of the outstanding questions in the field is how such a conserved enzyme as the V-ATPase can fulfill such diverse functions.
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Forgac, M. "Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 71–80. http://dx.doi.org/10.1242/jeb.203.1.71.
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The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c”) that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(−) conductance that is necessary for vacuolar acidification.
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Abe, Michiko, Mayu Saito, Ayana Tsukahara, Shuka Shiokawa, Kazuma Ueno, Hiroki Shimamura, Makoto Nagano, Junko Y. Toshima, and Jiro Toshima. "Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs." Journal of Biological Chemistry 294, no. 20 (April 5, 2019): 8273–85. http://dx.doi.org/10.1074/jbc.ra118.006192.
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Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.
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Imada, Katsumi, Tohru Minamino, Yumiko Uchida, Miki Kinoshita, and Keiichi Namba. "Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator." Proceedings of the National Academy of Sciences 113, no. 13 (March 16, 2016): 3633–38. http://dx.doi.org/10.1073/pnas.1524025113.
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FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3β3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2–FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.
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Schep, Daniel G., Jianhua Zhao, and John L. Rubinstein. "Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance." Proceedings of the National Academy of Sciences 113, no. 12 (March 7, 2016): 3245–50. http://dx.doi.org/10.1073/pnas.1521990113.
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Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.
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Rawson, Shaun, Michael A. Harrison, and Stephen P. Muench. "Rotating with the brakes on and other unresolved features of the vacuolar ATPase." Biochemical Society Transactions 44, no. 3 (June 9, 2016): 851–55. http://dx.doi.org/10.1042/bst20160043.
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The rotary ATPase family comprises the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and archaeal ATPase (A-ATPase). These either predominantly utilize a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in EM has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F- and V-ATPases, the secondary structure organization of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases, there are still a number of unresolved questions about the mechanism, regulation and overall architecture, which this mini-review aims to highlight and discuss.
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Smardon, Anne M., Heba I. Diab, Maureen Tarsio, Theodore T. Diakov, Negin Dehdar Nasab, Robert W. West, and Patricia M. Kane. "The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast." Molecular Biology of the Cell 25, no. 3 (February 2014): 356–67. http://dx.doi.org/10.1091/mbc.e13-05-0231.
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The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H+-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma− growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma− growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma− growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma− phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.
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Kane, P. M., and K. J. Parra. "Assembly and regulation of the yeast vacuolar H(+)-ATPase." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 81–87. http://dx.doi.org/10.1242/jeb.203.1.81.
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The yeast vacuolar H(+)-ATPase (V-ATPase) consists of a complex of peripheral subunits containing the ATP binding sites, termed the V(1) sector, attached to a complex of membrane subunits containing the proton pore, termed the V(o) sector. Interaction between the V(1) and V(o) sectors is essential for ATP-driven proton transport, and this interaction is manipulated in vivo as a means of regulating V-ATPase activity. When yeast (Saccharomyces cerevisiae) cells are deprived of glucose for as little as 5 min, up to 75% of the assembled V-ATPase complexes are disassembled into cytoplasmic V(1) sectors and membrane-bound V(o) sectors. Remarkably, this disassembly is completely reversible. Restoration of glucose to the growth medium results in quantitative reassembly of the disassembled complexes in as little as 5 min, even in the absence of any new protein synthesis. Cells also appear to regulate the extent of V(1)V(o) assembly on a long-term basis. Yeast cells grown for extended periods in a poor carbon source contain a high proportion of free V(1) and V(o) sectors, and these sectors remain poised for reassembly when growth conditions improve. Parallel experiments on the Manduca sexta V-ATPase suggest that reversible disassembly may be a general regulatory mechanism for V-ATPases. These results imply that V-ATPases are surprisingly dynamic structures, and their unique ‘regulated instability’ raises a number of interesting physiological and structural questions. How are extracellular conditions such as carbon source communicated to V-ATPase complexes present on intracellular membranes? How are such major structural changes in the V-ATPase generated and how are V(1) sectors ‘silenced’ in vivo to prevent unproductive hydrolysis of cytoplasmic ATP by the dissociated enzyme? We are addressing these questions using a combination of genetic and biochemical approaches.
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Sennoune, Souad R., Karina Bakunts, Gloria M. Martínez, Jenny L. Chua-Tuan, Yamina Kebir, Mohamed N. Attaya, and Raul Martínez-Zaguilán. "Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity." American Journal of Physiology-Cell Physiology 286, no. 6 (June 2004): C1443—C1452. http://dx.doi.org/10.1152/ajpcell.00407.2003.
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Tumor cells thrive in a hypoxic microenvironment with an acidic extracellular pH. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. We hypothesize that vacuolar H+-ATPases (V-ATPases) that normally reside in acidic organelles are also located at the cell surface, thus regulating cytosolic pH and exacerbating the migratory ability of metastatic cells. Immunocytochemical data revealed for the first time that V-ATPase is located at the plasma membrane of human breast cancer cells: prominent in the highly metastatic and inconspicuous in the lowly metastatic cells. The V-ATPase activities in isolated plasma membranes were greater in highly than in lowly metastatic cells. The proton fluxes via V-ATPase evaluated by fluorescence spectroscopy in living cells were greater in highly than in lowly metastatic cells. Interestingly, lowly metastatic cells preferentially used the ubiquitous Na+/H+exchanger and HCO3−-based H+-transporting mechanisms, whereas highly metastatic cells used plasma membrane V-ATPases. The highly metastatic cells were more invasive and migratory than the lowly metastatic cells. V-ATPase inhibitors decreased the invasion and migration in the highly metastatic cells. Altogether, these data indicate that V-ATPases located at the plasma membrane are involved in the acquisition of a more metastatic phenotype.
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Al-bataineh, Mohammad M., Fan Gong, Allison L. Marciszyn, Michael M. Myerburg, and Núria M. Pastor-Soler. "Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase." American Journal of Physiology-Renal Physiology 306, no. 9 (May 1, 2014): F981—F995. http://dx.doi.org/10.1152/ajprenal.00362.2013.
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The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.
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Banerjee, Subhrajit, and Patricia M. Kane. "Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast." Molecular Biology of the Cell 28, no. 19 (September 15, 2017): 2518–30. http://dx.doi.org/10.1091/mbc.e17-05-0316.
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Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H+-ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae. Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of Vo a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases.
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Taiz, L. "THE PLANT VACUOLE." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 113–22. http://dx.doi.org/10.1242/jeb.172.1.113.
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Plant cells are unique in containing large acidic vacuoles which occupy most of the cell volume. The vacuolar H+-ATPase (V-ATPase) is the enzyme responsible for acidifying the central vacuole, although it is also present on Golgi and coated vesicles. Many secondary transport processes are driven by the proton-motive force generated by the V-ATPase, including reactions required for osmoregulation, homeostasis, storage, plant defense and many other functions. However, a second proton pump, the V-PPase, serves as a potential back-up system and may, in addition, pump potassium. The plant V-ATPase is structurally similar to other eukaryotic V-ATPases and its subunits appear to be encoded by small multigene families. These multigene families may play important roles in the regulation of gene expression and in the sorting of V-ATPase isoforms to different organelles.
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Obrdlik, Petr, Kerstin Diekert, Natalie Watzke, Christine Keipert, Ulrich Pehl, Catrin Brosch, Nicole Boehm, et al. "Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes." Biochemical Journal 427, no. 1 (March 15, 2010): 151–59. http://dx.doi.org/10.1042/bj20091380.
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Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51±7 μM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2±0.6 μM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.
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Granger, D., M. Marsolais, J. Burry, and R. Laprade. "V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration." American Journal of Physiology-Cell Physiology 282, no. 6 (June 1, 2002): C1454—C1460. http://dx.doi.org/10.1152/ajpcell.00319.2001.
Full textAbstract:
We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO[Formula: see text]-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpHi/d t) following an acid load. Oligomycin (25 μM), an inhibitor of F-type ATPases, decreased dpHi/d t by 88 ± 6%, suggesting a role for an ATP-dependent process involved in pHi recovery. Moreover, dpHi/d t was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 μM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpHi/d t by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.
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Grüber, G. "Structural features and nucleotide-binding capability of the C subunit are integral to the regulation of the eukaryotic V1Vo ATPases." Biochemical Society Transactions 33, no. 4 (August 1, 2005): 883–85. http://dx.doi.org/10.1042/bst0330883.
Full textAbstract:
V-ATPases (vacuolar ATPases) are responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are composed of a catalytic V1 sector, in which ATP hydrolysis takes place, and the Vo sector, which functions in proton conduction. The best established mechanism for regulating the V-ATPase activity in vivo involves reversible dissociation of the V1 and Vo domains, in which subunit C is intimately involved. In the last year, impressive progress has been made in elucidating the structure of the C subunit and its arrangement inside the V-ATPase. Nucleotide occupancy by subunit C, followed by conformational changes of this subunit has shed light on the mechanism of V-ATPase regulation.
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Allan, Adrian K., Juan Du, Shireen A. Davies, and Julian A. T. Dow. "Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles." Physiological Genomics 22, no. 2 (July 14, 2005): 128–38. http://dx.doi.org/10.1152/physiolgenomics.00233.2004.
Full textAbstract:
V-ATPases are ubiquitous, vital proton pumps that play a multiplicity of roles in higher organisms. In many epithelia, they are the major energizer of cotransport processes and have been implicated in functions as diverse as fluid secretion and longevity. The first animal knockout of a V-ATPase was identified in Drosophila, and its recessive lethality demonstrated the essential nature of V-ATPases. This article surveys the entire V-ATPase gene family in Drosophila, both experimentally and in silico. Adult expression patterns of most of the genes are shown experimentally for the first time, using in situ hybridization or reporter gene expression, and these results are reconciled with published expression and microarray data. For each subunit, the single gene identified previously by microarray, as upregulated and abundant in tubules, is shown to be similarly abundant in other epithelia in which V-ATPases are known to be important; there thus appears to be a single dominant “plasma membrane” V-ATPase holoenzyme in Drosophila. This provides the most comprehensive view of V-ATPase expression yet in a multicellular organism. The transparent Malpighian tubule phenotype first identified in lethal alleles of vha55, the gene encoding the B-subunit, is shown to be general to those plasma membrane V-ATPase subunits for which lethal alleles are available, and to be caused by failure to accumulate uric acid crystals. These results coincide with the expression view of the gene family, in which 13 of the genes are specialized for epithelial roles, whereas others have spatially or temporally restricted patterns of expression.
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Holliday, L. Shannon. "Vacuolar H+-ATPase: An Essential Multitasking Enzyme in Physiology and Pathophysiology." New Journal of Science 2014 (January 23, 2014): 1–21. http://dx.doi.org/10.1155/2014/675430.
Full textAbstract:
Vacuolar H+-ATPases (V-ATPases) are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (pro)renin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.
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Li, Sheena Claire, Theodore T. Diakov, Tao Xu, Maureen Tarsio, Wandi Zhu, Sergio Couoh-Cardel, Lois S. Weisman, and Patricia M. Kane. "The signaling lipid PI(3,5)P2 stabilizes V1–Vo sector interactions and activates the V-ATPase." Molecular Biology of the Cell 25, no. 8 (April 15, 2014): 1251–62. http://dx.doi.org/10.1091/mbc.e13-10-0563.
Full textAbstract:
Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane Vo domain. Multiple stresses induce changes in V1-Vo assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-Vo assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified Vo complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of Vo-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-Vo interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-Vo assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1∆ and vac14∆ mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.
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Wieczorek, H. "The insect V-ATPase, a plasma membrane proton pump energizing secondary active transport: molecular analysis of electrogenic potassium transport in the tobacco hornworm midgut." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 335–43. http://dx.doi.org/10.1242/jeb.172.1.335.
Full textAbstract:
Goblet cell apical membranes in the larval midgut of Manduca sexta are the site of active and electrogenic K+ secretion. They possess a vacuolar-type ATPase which, in its immunopurified form, consists of at least nine polypeptides. cDNAs for the A and B subunits screened by monoclonal antibodies to the A subunit of the Manduca V-ATPase or by hybridisation with a cDNA probe for a plant V-ATPase B subunit have been cloned and sequenced. There is a high degree of identity to the sequences of the respective subunits of other V-ATPases. The M. sexta plasma membrane V-ATPase is an electrogenic proton pump which energizes, by the electrical component of the proton-motive force, electrogenic K+/nH+ antiport, resulting in net electrogenic K+ secretion. Since the midgut lacks a Na+/K(+)-ATPase, all solute fluxes in this epithelium seem to be energized by the V-ATPase. Thus, the midgut provides an alternative to the classical concept of animal plasma membrane energization by the Na(+)-motive force generated by the Na+/K(+)-ATPase.
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Pérez-Castiñeira, José R., Agustín Hernández, Rocío Drake, and Aurelio Serrano. "A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast." Biochemical Journal 437, no. 2 (June 28, 2011): 269–78. http://dx.doi.org/10.1042/bj20110447.
Full textAbstract:
V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.
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Sze, H., JM Ward, S. Lai, and I. Perera. "VACUOLAR-TYPE H+-TRANSLOCATING ATPases IN PLANT ENDOMEMBRANES: SUBUNIT ORGANIZATION AND MULTIGENE FAMILIES." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 123–35. http://dx.doi.org/10.1242/jeb.172.1.123.
Full textAbstract:
Acidification of endomembrane compartments by the vacuolar-type H+-translocating ATPase (V-ATPase) is vital to the growth and development of plants. The V-ATPase purified from oat roots is a large complex of 650x10(3 )Mr that contains 10 different subunits of 70, 60, 44, 42, 36, 32, 29, 16, 13 and 12x10(3 )Mr. This set of ten polypeptides is sufficient to couple ATP hydrolysis to proton pumping after reconstitution of the ATPase into liposomes. Unlike some animal V-ATPases, the purified and reconstituted V-ATPase from oat is directly stimulated by Cl-. The peripheral complex of the ATPase includes the nucleotide-binding subunits of 70 and 60x10(3 )Mr and polypeptides of 44, 42, 36 and 29x10(3 )Mr. Six copies of the 16x10(3 )Mr proteolipid together with three other polypeptides are thought to make up the integral sector that forms the H+-conducting pathway. Release of the peripheral complex from the native membrane completely inactivates the pump; however, the peripheral subunits can be reassembled with the membrane sector to form a functional H+ pump. Comparison of V-ATPases from several plants indicates considerable variations in subunit composition. Hence, several forms of the V-ATPase may exist among, and probably within, plant species. At least four distinct cDNAs encode the 16x10(3 )Mr proteolipid subunit in oat. Multiple genes could encode different subtypes of the H+ pump that are regulated by the developmental stage and physiological function specific to the cell or tissue type.
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Sakai, Hiromu, Yoshie Moriura, Takuya Notomi, Junko Kawawaki, Keiko Ohnishi, and Miyuki Kuno. "Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts." American Journal of Physiology-Cell Physiology 299, no. 3 (September 2010): C570—C578. http://dx.doi.org/10.1152/ajpcell.00486.2009.
Full textAbstract:
In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance ( Cm) simultaneously. Cm is a measure of cell surface. Extracellular Ca2+ (2.5–40 mM) decreased Cm and the V-ATPase current simultaneously. The decreased Cm, together with the enhanced uptake of a lipophilic dye (FM1–43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A1, a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C γ2 subunit. Holding the cytosolic Ca2+ at either high (0.5–5 μM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.
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Bowman, E. J., and B. J. Bowman. "Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 97–106. http://dx.doi.org/10.1242/jeb.203.1.97.
Full textAbstract:
Vacuolar ATPases (V-ATPases) are large complex enzymes that are structural and mechanistic relatives of F(1)F(o)-ATPases. They hydrolyze ATP and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. The proton gradients generated are used to drive the movement of various compounds across membranes. V-ATPases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the plasma membranes of many specialized eukaryotic cells. They have been implicated in a wide variety of cellular processes and are associated with several diseases. Bafilomycin and concanamycin, specific inhibitors of V-ATPases, have been instrumental in implicating the V-ATPase in many of these roles. To understand further the mechanism of inhibition by these antibiotics and the physiological role of the enzyme in the cell, we have isolated mutants of the filamentous fungus Neurospora crassa that are resistant to concanamycin. Concanamycin has a dramatic effect on hyphal morphology at acid pH and is lethal at basic pH. In the resistant mutants, the cells can germinate and grow, although abnormally, in basic medium. Thus far, none of the mutants we have characterized is mutated in a gene encoding a subunit of the V-ATPase. Instead, the largest class of mutants is mutated in the gene encoding the plasma-membrane H(+)-ATPase. Mutations in at least four uncharacterized genes can also confer resistance. Inactivation of the V-ATPase by disruption of vma-1, which encodes the catalytic subunit (A) of the enzyme, causes a much more severe phenotype than inhibition by concanamycin. A strain lacking vma-1 is seriously impaired in rate of growth, differentiation and capacity to produce viable spores. It is also completely resistant to concanamycin, indicating that the inhibitory effects of concanamycin in vivo are due to inhibition of the V-ATPase. How the multiplicity of ATPases within a cell is regulated and how their activity is integrated with other metabolic reactions is poorly understood. Mutant analysis should help unravel this puzzle.
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Drory, Omri, and Nathan Nelson. "The Emerging Structure of Vacuolar ATPases." Physiology 21, no. 5 (October 2006): 317–25. http://dx.doi.org/10.1152/physiol.00017.2006.
Full textAbstract:
Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.
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Wieczorek, H., G. Grber, W. R. Harvey, M. Huss, H. Merzendorfer, and W. Zeiske. "Structure and regulation of insect plasma membrane H(+)V-ATPase." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 127–35. http://dx.doi.org/10.1242/jeb.203.1.127.
Full textAbstract:
H(+) V-ATPases (V-ATPases) are found in two principal locations, in endomembranes and in plasma membranes. The plasma membrane V-ATPase from the midgut of larval Manduca sexta is the sole energizer of all transepithelial secondary transport processes. At least two properties make the lepidopteran midgut a model tissue for studies of general aspects of V-ATPases. First, it is a rich source for purification of the enzyme and therefore for structural studies: 20 larvae provide up to 0.5 mg of holoenzyme, and soluble, cytosolic V(1) complexes can be obtained in even greater amounts of up to 2 mg. Second, midgut ion-tranport processes are strictly controlled by the regulation of the V-ATPase, which is the sole energizer of all ion transport in this epithelium. Recent advances in our understanding the structure of the V(1) and V(o) complexes and of the regulation of the enzyme's biosynthesis and ion-transport activity will be discussed.
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Forgac, M. "Structure, function and regulation of the coated vesicle V-ATPase." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 155–69. http://dx.doi.org/10.1242/jeb.172.1.155.
Full textAbstract:
The coated vesicle V-ATPase plays an important role in both receptor-mediated endocytosis and intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling. The coated vesicle V-ATPase is a macromolecular complex of relative molecular mass 750,000 composed of nine subunits arranged in two structural domains. The peripheral V1 domain, which has a relative molecular mass of 500,000, has the subunit structure 73(3)58(3)40(1)34(1)33(1) and possesses all the nucleotide binding sites of the V-ATPase. The integral Vo domain of relative molecular mass 250,000 has a subunit composition of 100(1)38(1)19(1)17(6) and possesses the pathway for proton conduction across the membrane. Reassembly studies have allowed us to probe the role of specific subunits in the V-ATPase complex while chemical labeling studies have allowed us to identify specific residues which play a critical role in catalysis. From both structural analysis and sequence homology, the vacuolar-type H(+)-ATPases resemble the F-type H(+)-ATPases. Unlike the F1 and Fo domains of the F-type ATPases, however, the V1 and Vo domains do not appear to function independently. The possible relevance of these observations to the regulation of vacuolar acidification is discussed.
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Nelson, N., N. Perzov, A. Cohen, K. Hagai, V. Padler, and H. Nelson. "The cellular biology of proton-motive force generation by V-ATPases." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 89–95. http://dx.doi.org/10.1242/jeb.203.1.89.
Full textAbstract:
The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps. The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus. In contrast to yeast vacuoles, which maintain an internal pH of approximately 5. 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles. It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly. Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function). The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered. We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S. cerevisiae that belong to the family. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5. Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes. We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital. The interaction between V-ATPase and the secretory pathway is investigated.
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Hinton, Ayana, Souad R. Sennoune, Sarah Bond, Min Fang, Moshe Reuveni, G. Gary Sahagian, Daniel Jay, Raul Martinez-Zaguilan, and Michael Forgac. "Function of a Subunit Isoforms of the V-ATPase in pH Homeostasis and in Vitro Invasion of MDA-MB231 Human Breast Cancer Cells." Journal of Biological Chemistry 284, no. 24 (April 14, 2009): 16400–16408. http://dx.doi.org/10.1074/jbc.m901201200.
Full textAbstract:
It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in ∼18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.
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Gluck, S. L., R. D. Nelson, B. S. Lee, Z. Q. Wang, X. L. Guo, J. Y. Fu, and K. Zhang. "Biochemistry of the renal V-ATPase." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 219–29. http://dx.doi.org/10.1242/jeb.172.1.219.
Full textAbstract:
In most eukaryotic cells, vacuolar H(+)-ATPases (V-ATPases) are present primarily or exclusively in intracellular membrane compartments, functioning in the acidification of the endocytic and secretory vacuolar apparatus necessary for constitutive cell function. V-ATPases also participate in renal hydrogen ion secretion in both the proximal and distal nephron, residing at high concentrations on the plasma membrane, where they are regulated physiologically to maintain the acid-base balance of the organism. Recent experiments have begun to reveal how the kidney controls transcellular proton transport while still maintaining acidification of intracellular compartments. Control may occur by recruitment of proton pumps to or away from the plasma membrane. The proton-transporting plasma membrane of intercalated cells is a specialized apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiological stimuli. Regulation may also occur by changes in the kinetics of the V-ATPase. V-ATPases are a family of structurally similar enzymes which differ in the composition of specific subunits. Cytosolic regulatory enzymes present in renal cells may preferentially affect V-ATPases in selective membrane compartments.
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Klein, U. "THE INSECT V-ATPase, A PLASMA MEMBRANE PROTON PUMP ENERGIZING SECONDARY ACTIVE TRANSPORT: IMMUNOLOGICAL EVIDENCE FOR THE OCCURRENCE OF A V-ATPase IN INSECT ION-TRANSPORTING EPITHELIA." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 345–54. http://dx.doi.org/10.1242/jeb.172.1.345.
Full textAbstract:
Active electrogenic K+ transport in insects serves as the energy source for secretion or absorption in gastrointestinal epithelia or for the receptor current in sensory epithelia. In the larval midgut of the tobacco hornworm Manduca sexta, a vacuolar-type proton pump (V-ATPase) and a K+/nH+ antiport represent the functional elements of the potassium pump. Several immunological findings support the hypothesis that active K+ transport in other insect epithelia may also be energized by a V-ATPase. In immunoblots, crude homogenates of sensilla-rich antennae and Malpighian tubules of M. sexta cross reacted with an immune serum directed to the purified plasma membrane V-ATPase from the midgut; the M. sexta midgut V-ATPase cross reacted with polyclonal antibodies to endomembrane V-ATPases from xenic origin. In immunocytochemical investigations of larvae of M. sexta and adults of Antheraea pernyi, monoclonal antibodies to defined subunits of the purified midgut V-ATPase or polyclonal antibodies to xenic endomembrane V-ATPase labelled the sites of active K+ transport: the goblet cell apical membrane in the midgut, the brush border of Malpighian tubules and the apical projections of the auxiliary cells in antennal sensilla. The functional mechanism of a primary H+-pumping V-ATPase and a secondary H+-dependent K+ transport postulated for K+-transporting insect epithelia may be further applicable to active Na+ or Cl- transport and would provide a unifying concept for all ouabain-insensitive electrogenic ion transport in insects. The findings from the midgut investigations, however, are the first instance in which a V-ATPase provides an alternative to the Na+/K+-ATPase in energizing secondary active transport in animal plasma membranes.
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Dane, Michaela, Kerstin Steinert, Kordula Esser, Susanne Bickel-Sandkötter, and Francisco Rodriguez-Valera. "Properties Of The Plasma Membrane Atpases Of The Halophilic Archaebacteria Haloferax Mediterranei And Haloferax Volcanii." Zeitschrift für Naturforschung C 47, no. 11-12 (December 1, 1992): 835–44. http://dx.doi.org/10.1515/znc-1992-11-1209.
Full textAbstract:
Both, Haloferax mediterranei and Haloferax volcanii membranes contain ATPases which are capable of hydrolyzing ATP in presence of Mg2+ or Mn2+. The ATPases require high concentrations of NaCl, a pH value of 9, and high temperatures up to 60 °C. Free manganese ions inhibited the enzyme activity of either ATPase. The ATPases of Hf. mediterranei and Hf. volcanii, respectively, show different sensitivities to inhibitors of ATP hydrolysis. ATP hydrolysis of isolated Hf. mediterranei ATPase was inhibited by NaN3, which was reported to be specific for F-ATPases, by nitrate and N-ethylmaleimide (NEM), which are specific inhibitors of V-ATPases. ATP hydrolysis of Haloferax mediterranei membranes was not inhibited by DCCD , but [14C]DCCD was bound to a 14 kDa peptide of the isolated, partially purified enzyme. Furthermore, the ATPase was inactivated by preincubation with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The ATPase activity of Hf. volcanii membranes was inhibited by NEM but not by nitrate and NaN3. SDS gel electrophoresis of the partially purified enzyme of Haloferax mediterranei showed putative ATPase subunits of 53.5, 49, 42, 22, 21, 14, 12, and 7.5 kDa. Immunoblots showed cross reactivity between a 53 kDa peptide and anti-β (chloroplast F1), as well as between 53, 50 and 47 kDa peptides and an ATPase antibody of Methanosarcina barkeri. The results will be discussed in context with the placement of the archaebacterial ATPases (A-ATPases) between F- and V-ATPases
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Casare, Fernando, Daiane Milan, and Ricardo Fernandez. "Stimulation of calcium-sensing receptor increases biochemical H+-ATPase activity in mouse cortex and outer medullary regions." Canadian Journal of Physiology and Pharmacology 92, no. 3 (March 2014): 181–88. http://dx.doi.org/10.1139/cjpp-2013-0256.
Full textAbstract:
The aim of this project was to investigate the interaction between the calcium-sensing receptor (CaSR) and proton extrusion by the V-ATPase and gastric-like isoform of the H+/K+-ATPase in the mouse nephron. Biochemical activity of H+- ATPases was analysed using a partially purified membrane fraction of mouse cortex and outer medullary region. The V-ATPase activity (sensitive to 10−7 mol·L−1 bafilomycin) from the cortical and outer medullary region was significantly stimulated by increasing the [Formula: see text] (outside Ca2+), in a dose-dependent pattern. Gastric H+/K+-ATPase activity (sensitive to 10−5 mol·L−1 Schering 28080) was also sensitive to changes in [Formula: see text] levels. A significant increase in V-ATPase activity was also observed when CaSR was stimulated with agonists such as 300 μmol·L−1 Gd3+ and 200 μmol·L−1 neomycin, both in the cortex and outer medulla. The cortical and outer medullary gastric H+/K+-ATPase activity was also stimulated by Gd3+ and neomycin. Finally, cortical V-ATPase activity was significantly stimulated by 10−9 mol·L−1 angiotensin II, and the stimulation of CaSR in the presence of angiotensin significantly enhanced this effect, suggesting that an interaction in the intracellular signaling pathways is involved. In summary, CaSR stimulation enhances the biochemical activity of V-ATPase and gastric H+/K+-ATPase in both the cortical and outer medullary region of mouse kidney.
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Nolta, K. V., H. Padh, and T. L. Steck. "An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum." Journal of Cell Science 105, no. 3 (July 1, 1993): 849–59. http://dx.doi.org/10.1242/jcs.105.3.849.
Full textAbstract:
Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.
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Liberman, Rachel, Sarah Bond, Mara G. Shainheit, Miguel J. Stadecker, and Michael Forgac. "Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway." Journal of Biological Chemistry 289, no. 3 (November 22, 2013): 1355–63. http://dx.doi.org/10.1074/jbc.m113.524561.
Full textAbstract:
The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.
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Păunescu, Teodor G., Leileata M. Russo, Nicolas Da Silva, Jana Kovacikova, Nilufar Mohebbi, Alfred N. Van Hoek, Mary McKee, Carsten A. Wagner, Sylvie Breton, and Dennis Brown. "Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice." American Journal of Physiology-Renal Physiology 293, no. 6 (December 2007): F1915—F1926. http://dx.doi.org/10.1152/ajprenal.00160.2007.
Full textAbstract:
Mice deficient in the ATP6V1B1 (“B1”) subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 (“B2”) isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1−/− mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1−/− mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of “rod-shaped” intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28–40% of normal) V-ATPase function is preserved in medullary ICs from B1−/− mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.
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FINBOW, Malcolm E., and Michael A. HARRISON. "The vacuolar H+-ATPase: a universal proton pump of eukaryotes." Biochemical Journal 324, no. 3 (June 15, 1997): 697–712. http://dx.doi.org/10.1042/bj3240697.
Full textAbstract:
The vacuolar H+-ATPase (V-ATPase) is a universal component of eukaryotic organisms. It is present in the membranes of many organelles, where its proton-pumping action creates the low intra-vacuolar pH found, for example, in lysosomes. In addition, there are a number of differentiated cell types that have V-ATPases on their surface that contribute to the physiological functions of these cells. The V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the familiar FoF1 ATP synthase (F-ATPase), having the same basic architectural construction, and many of the subunits from the two display identity with one another. All the core subunits of the V-ATPase have now been identified and much is known about the assembly, regulation and pharmacology of the enzyme. Recent genetic analysis has shown the V-ATPase to be a vital component of higher eukaryotes. At least one of the subunits, i.e. subunit c (ductin), may have multifunctional roles in membrane transport, providing a possible pathway of communication between cells. The structure of the membrane sector is known in some detail, and it is possible to begin to suggest how proton pumping is coupled to ATP hydrolysis.
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Sarafian, V., M. Potier, and R. J. Poole. "Radiation-inactivation analysis of vacuolar H+-ATPase and H+-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport." Biochemical Journal 283, no. 2 (April 15, 1992): 493–97. http://dx.doi.org/10.1042/bj2830493.
Full textAbstract:
The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.
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Dow, J. A., S. A. Davies, Y. Guo, S. Graham, M. E. Finbow, and K. Kaiser. "Molecular genetic analysis of V-ATPase function in Drosophila melanogaster." Journal of Experimental Biology 200, no. 2 (January 1, 1997): 237–45. http://dx.doi.org/10.1242/jeb.200.2.237.
Full textAbstract:
V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps. Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia. While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context. For such purposes, Drosophila melanogaster is a uniquely suitable model. Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D. melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles. Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction. These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D. melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.
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Zhou, Long, and Leonid A. Sazanov. "Structure and conformational plasticity of the intact Thermus thermophilus V/A-type ATPase." Science 365, no. 6455 (August 22, 2019): eaaw9144. http://dx.doi.org/10.1126/science.aaw9144.
Full textAbstract:
V (vacuolar)/A (archaeal)-type adenosine triphosphatases (ATPases), found in archaea and eubacteria, couple ATP hydrolysis or synthesis to proton translocation across the plasma membrane using the rotary-catalysis mechanism. They belong to the V-type ATPase family, which differs from the mitochondrial/chloroplast F-type ATP synthases in overall architecture. We solved cryo–electron microscopy structures of the intact Thermus thermophilus V/A-ATPase, reconstituted into lipid nanodiscs, in three rotational states and two substates. These structures indicate substantial flexibility between V1 and Vo in a working enzyme, which results from mechanical competition between central shaft rotation and resistance from the peripheral stalks. We also describe details of adenosine diphosphate inhibition release, V1-Vo torque transmission, and proton translocation, which are relevant for the entire V-type ATPase family.
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Li, Ru, Shan Bai, Yuanyuan He, Qi Chen, Yanping Yao, Jinzi Wang, and Baoshan Chen. "Cpvma1, a Vacuolar H+-ATPase Catalytic Subunit of Cryphonectria parasitica, is Essential for Virulence and Hypovirus RNA Accumulation." Phytopathology® 109, no. 8 (August 2019): 1417–24. http://dx.doi.org/10.1094/phyto-08-18-0289-r.
Full textAbstract:
The vacuolar H+-ATPases (V-ATPases) are conserved ATP-dependent proton pumps that acidify intracellular compartments in eukaryotic cells. The role of Cpvma1, a V-ATPase catalytic subunit A of Cryphonectria parasitica, was investigated by generating cpvma1-overexpressing and cpvma1-silenced strains. The mutant strains were evaluated for phenotypic characteristics, V-ATPase activity, response to elevated pH and Ca2+ in the medium, virulence on chestnut, and accumulation of hypovirus RNA in the cells. Compared with the wild-type strain, cpvma1-overexpressing strains showed no significant difference in phenotype; however, cpvma1-silenced strains exhibited a phenotype of reduced growth rate, lower level of sporulation, and a marked decrease in V-ATPase activity and virulence. In addition, silencing of cpvma1 increased sensitivity to elevated pH and Ca2+, implicating an important role for Cpvma1 in pH adaptation and Ca2+ homeostasis. Furthermore, silencing of cpvma1 resulted in significantly decreased accumulation of hypoviral RNA. Taken together, our results indicate that Cpvma1 plays an important role in the regulation of phenotypic traits and virulence and the accumulation of hypovirus RNA in C. parasitica.
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Pérez-Sayáns, M., JM Suárez-Peñaranda, F. Barros-Angueira, PG Diz, JM Gándara-Rey, and A. García-García. "An update in the structure, function, and regulation of V-ATPases: the role of the C subunit." Brazilian Journal of Biology 72, no. 1 (February 2012): 189–98. http://dx.doi.org/10.1590/s1519-69842012000100023.
Full textAbstract:
Vacuolar ATPases (V-ATPases) are present in specialized proton secretory cells in which they pump protons across the membranes of various intracellular organelles and across the plasma membrane. The proton transport mechanism is electrogenic and establishes an acidic pH and a positive transmembrane potential in these intracellular and extracellular compartments. V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic sector (V1) and a hydrophobic membrane sector (V0) responsible for driving protons. V-ATPase activity is regulated by three different mechanisms, which control pump density, association/dissociation of the V1 and V0 domains, and secretory activity. The C subunit is a 40-kDa protein located in the V1 domain of V-ATPase. The protein is encoded by the ATP6V1C gene and is located at position 22 of the long arm of chromosome 8 (8q22.3). The C subunit has very important functions in terms of controlling the regulation of the reversible dissociation of V-ATPases.
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Vedovelli, Luca, John T. Rothermel, Karin E. Finberg, Carsten A. Wagner, Anie Azroyan, Eric Hill, Sylvie Breton, Dennis Brown, and Teodor G. Păunescu. "Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting." American Journal of Physiology-Renal Physiology 304, no. 5 (March 1, 2013): F522—F532. http://dx.doi.org/10.1152/ajprenal.00394.2012.
Full textAbstract:
Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.
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Mccarty, RE. "A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP SYNTHASES." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 431–41. http://dx.doi.org/10.1242/jeb.172.1.431.
Full textAbstract:
My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).
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Xie, Xiao-Song, David Padron, Xibin Liao, Jin Wang, Michael G. Roth, and Jef K. De Brabander. "Salicylihalamide A Inhibits the V0Sector of the V-ATPase through a Mechanism Distinct from Bafilomycin A1." Journal of Biological Chemistry 279, no. 19 (March 3, 2004): 19755–63. http://dx.doi.org/10.1074/jbc.m313796200.
Full textAbstract:
The newly identified specific V-ATPase inhibitor, salicylihalamide A, is distinct from any previously identified V-ATPase inhibitors in that it inhibits only mammalian V-ATPases, but not those from yeast or other fungi (Boyd, M. R., Farina, C., Belfiore, P., Gagliardi, S., Kim, J. W., Hayakawa, Y., Beutler, J. A., McKee, T. C., Bowman, B. J., and Bowman, E. J. (2001)J. Pharmacol. Exp. Ther.297, 114–120). In addition, salicylihalamide A does not compete with concanamycin or bafilomycin for binding to V-ATPase, indicating that it has a different binding site from those classic V-ATPase inhibitors (Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H. (2002)J. Biol. Chem.277, 40544–40548). By using purified bovine brain V-pump and its dissociated V1and V0sectors, we identified the recognition and binding site for salicylihalamide to be within the V0domain. Salicylihalamide does not inhibit the ATP hydrolysis activity of the dissociated V1-ATPase but inhibits the ATPase activity of the holoenzyme by inhibiting the V0domain. Salicylihalamide causes a dramatic redistribution of cytosolic V1from soluble to membrane-associated form, a change not observed in cells treated with either bafilomycin or NH4Cl. By synthesizing and characterizing a series of salicylihalamide derivatives, we investigated the structural determinants of salicylihalamide inhibition in terms of potency and reversibility, and used this information to suggest a possible binding mechanism.