The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H+-ATPase

Hayami, Keiko; Noumi, Takato; Inoue, Hiroki; Sun‐Wada, Ge‐Hong; Yoshimizu, Takao; Kanazawa, Hiroshi

doi:10.1016/s0378-1119(01)00590-x

Cited by 16 publications

(16 citation statements)

References 30 publications

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“…For the V O membrane-intrinsic sector, only single genes for the small, highly hydrophobic 16 kDa and 23 kDa subunits are functional in mammals (Hayami et al, 2001;Sun-Wada et al, 2001;Sun-Wada et al, 2000). The a subunit is a protein with a relative molecular mass of approximately 100 kDa, with an N-terminal cytosolic portion and 9 membrane-spanning segments Nelson and Harvey, 1999;Nishi and Forgac, 2002;Sun-Wada et al, 2003).…”

Section: Expression and Localization Of A Subunit Isoforms In Mouse Pmentioning

confidence: 99%

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

Sun‐Wada

Toyomura

Murata

et al. 2006

Journal of Cell Science

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Vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit enzyme that has important roles in the acidification of a variety of intracellular compartments and some extracellular milieus. Four isoforms for the membrane-intrinsic subunit (subunit a) of the V-ATPase have been identified in mammals, and they confer distinct cellular localizations and activities on the proton pump. We found that V-ATPase with the a3 isoform is highly expressed in pancreatic islets, and is localized to membranes of insulin-containing secretory granules in β-cells. oc/oc mice, which have a null mutation at the a3 locus, exhibited a reduced level of insulin in the blood, even with high glucose administration. However, islet lysates contained mature insulin, and the ratio of the amount of insulin to proinsulin in oc/oc islets was similar to that of wild-type islets, indicating that processing of insulin was normal even in the absence of the a3 function. The insulin contents of oc/oc islets were reduced slightly, but this was not significant enough to explain the reduced levels of the blood insulin. The secretion of insulin from isolated islets in response to glucose or depolarizing stimulation was impaired. These results suggest that the a3 isoform of V-ATPase has a regulatory function in the exocytosis of insulin secretion.

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Section: Expression and Localization Of A Subunit Isoforms In Mouse Pmentioning

confidence: 99%

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

Sun‐Wada

Toyomura

Murata

et al. 2006

Journal of Cell Science

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199

175

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“…The nematode Caenorhabditis elegans has two c (14 -16) and four a (17) isoforms with different localizations. On the other hand, a single gene for the c subunit has been detected in mammals (18,19), and no subunit cЈ gene has been found so far. Only one gene for the cЉ subunit was found in three organisms (14, 20 -22).…”

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confidence: 99%

From Lysosomes to the Plasma Membrane

Toyomura

Murata

Yamamoto

et al. 2003

Journal of Biological Chemistry

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253

121

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Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H ؉ -ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760 -8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor B ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.Osteoclasts are multinuclear bone-resorbing cells derived from hematopoietic stem cells (1, 2) that form an extracellular compartment (resorption lacunae) between the plasma membrane (ruffled border) and the bone surface. The acidic pH of the lacunae (3, 4) is essential for mineral solubilization and hydrolysis of bone matrix by enzymes, including collagenase and cathepsin K (5). The degraded matrix is transported in luminal acidic vesicles (organelles) to the secretory domain facing the extracellular space (6). Vacuolar type H ϩ -ATPase (V-ATPase) 1 functions in the ruffled border as a proton pump that acidifies the resorption lacunae. Although the important role of V-ATPase in bone resorption has been established, much less is known about the mechanism of targeting the enzyme during osteoclast differentiation. Meanwhile, V-ATPases function in the membranes of ubiquitous organelles, including secretory vesicles, endosomes, the Golgi apparatus, and lysosomes. Thus, it is of interest to know how V-ATPases are localized to the various membranes and whether or not they have different compositions depending on the cellular location (7).V-ATPase is a multisubunit complex formed from a catalytic V 1 sector and a membrane-spanning V O sector (7-13). The V O sector may have a pertinent role in localizing V-ATPases to various cellular membranes. The yeast V O sector is composed of five subunits (a, c, cЈ, cЉ, and d) and has two subunit a isoforms, Stv1p and Vph1p. The nematode Caenorhabditis elegans has two c (14 -16) ...

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“…In fungi all three proteolipids are essential components of each V-ATPase molecule. It is not yet clear if the V-ATPase in animal cells has a cЈ subunit (26,27).Previously, we proposed a simple model to explain bafilomycin inhibition of the V-ATPase; binding occurs at the interface of the rotating c subunits and the stationary a subunit, blocking rotation (20). In the current paper we identify additional bfr mutations in N. crassa and describe strains with increased resistance to both bafilomycin and concanamycin.…”

mentioning

confidence: 99%

“…In fungi all three proteolipids are essential components of each V-ATPase molecule. It is not yet clear if the V-ATPase in animal cells has a cЈ subunit (26,27).…”

mentioning

confidence: 99%

The Bafilomycin/Concanamycin Binding Site in Subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae

Bowman

Graham

Stevens

et al. 2004

Journal of Biological Chemistry

137

114

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The vacuolar H؉ -ATPase is inhibited with high specificity and potency by bafilomycin and concanamycin, macrolide antibiotics with similar structures. We previously reported that mutation at three residues in subunit c of the vacuolar ATPase from Neurospora crassa conferred strong resistance to bafilomycin but little or no resistance to concanamycin (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). We have identified additional mutated sites in subunit c that confer resistance to bafilomycin. Furthermore, by subjecting a resistant mutant to a second round of mutation we isolated strains with increased resistance to both bafilomycin and concanamycin. In all of these strains the second mutation is also in subunit c, suggesting it forms at least part of the concanamycin binding site. Sitedirected mutagenesis of the gene encoding subunit c in Saccharomyces cerevisiae showed that single mutations in each of the residues identified in one of the double mutants of N. crassa conferred resistance to both bafilomycin and concanamycin. Mutations at the corresponding sites in the VMA11 and VMA16 genes of S. cerevisiae, which encode the c and c؆ subunits, did not confer resistance to the drugs. In all, nine residues of subunit c have been implicated in drug binding. The positions of these residues support a model in which the drug binding site is a pocket formed by helices 1, 2, and 4. We hypothesize that the drugs inhibit by preventing the rotation of the c subunits. Vacuolar proton-translocating ATPases (V-ATPases)1 are large, complex enzymes responsible for acidification of many internal compartments in eukaryotic cells. They also occur on plasma membranes of specialized cells, where they acidify the surrounding milieu. Numerous physiological processes depend on the activity of V-ATPases, including protein processing, transport of metabolites, receptor-mediated endocytosis, neurotransmitter uptake, and apoptosis (1, 2). V-ATPases are implicated as a contributing factor in multiple diseases such as osteoporosis (3), deafness (4), and cancer (5). Because of its vital role in so many processes and diseases, the V-ATPase is an attractive target for the development of therapeutic agents (6 -10).The bafilomycins and concanamycins have been particularly important in elucidating the physiological role of V-ATPases and are under investigation as possible lead compounds for drug therapy directed toward this enzyme (9, 11). These macrolide antibiotics, which are very similar in structure, are specific, highly potent inhibitors of all eukaryotic V-ATPases in vitro and in vivo (12)(13)(14). Despite the widespread use of these compounds, however, our understanding of how they inhibit is rudimentary. Early reports indicated that bafilomycin and concanamycin acted on the Vo segment of the enzyme (15-18). Using a radiolabeled derivative of concanamycin, Huss et al. (19) identified subunit c as the site bound by the inhibitor in the Manduca sexta enzyme.We have used a genetic approach to identify specific amino aci...

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The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H+-ATPase

Cited by 16 publications

References 30 publications

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

From Lysosomes to the Plasma Membrane

The Bafilomycin/Concanamycin Binding Site in Subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae

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