Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.

Vacuolar H؉ -ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-B ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC 50 of ϳ1.2 M on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-B ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis. V-ATPases2 are proton pumps ubiquitous in eukaryotic cells, where they acidify numerous intracellular membrane compartments, including Golgi, endosomes, lysosomes, clathrin-coated vesicles, chromaffin granules, and insulin secretory granules (reviewed in Refs. 1-11). V-ATPases also pump protons across the plasma membrane into the extracellular space in a variety of specialized cells, including renal duct intercalated cells, clear cells of the epididymis, and osteoclasts. Here they are involved in functions, including pH homeostasis, sperm maturation, and bone resorption and remodeling. Mutations in V-ATPase subunits lead to diseases, such as renal tubular acidosis and osteopetrosis. Furthermore, their inappropriate activity can contribute to osteoporosis and tumor metastasis (12-15).V-ATPases are multisubunit molecular motors, structurally analogous to the F 1 F 0 -ATP synthases (F-ATPases), but working "in reverse" (16 -20). Thus, V-ATPases create proton gradients across membranes by utilizing the energy of ATP hydrolysis, rather than utilizing the potential energy of proton gradients to ...

The a subunit is the largest of 14 different subunits that make up the V-ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1-a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N-glycosylation sites, and the a3 isoform has been directly shown to be N-glycosylated. Here we ask if human a4 (ATP6V0A4) is N-glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression-vector system, to express cDNA constructs of epitope-tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase-treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4 , demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse-chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N-glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co-immunoprecipitation studies suggested that a4 does not assemble with the V-ATPase V domain. Taken together, these data show that N-glycosylation plays a crucial role in a4 stability, and in V-ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757-2768, 2016. © 2016 Wiley Periodicals, Inc.

V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl 2 , 200 mM CaCl 2 , or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30 -35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both V o and V 1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.Eukaryotic cells contain an evolutionarily conserved enzyme, the vacuolar proton pump, V-ATPase 2 that couples the energy of ATP hydrolysis to proton transport across membranes. Intracellular V-ATPases are found in compartments such as clathrin-coated vesicles, Golgi, endosomes, lysosomes, secretory vesicles, and the central vacuoles of yeast as reviewed previously (1). V-ATPases are also present in the plasma membranes of specialized cells such as osteoclasts, renal intercalated cells, spermatids, neutrophils, and macrophages, where they function in such processes as bone resorption, renal acidification, pH homeostasis, and coupled transport (2-7). V-ATPases are complexes composed of at least 13 different subunits. These subunits are organized into two domains, a cytoplasmic V 1 domain that hydrolyzes ATP and an integral membrane V o domain that translocates protons across membranes. The V 1 is composed of eight subunits, A-H, with three copies of the nucleotide binding subunits A and B, and possibly two copies of subunit E, and two copies of subunit G (8, 9). The yeast V o is composed of six different subunits, a, c, cЈ, cЉ, d, and e, with four copies of subunit c (10 -13). Subunits a, c, cЈ, and cЉ are thought to be responsible for proton translocation, but the functions of subunits d (14) and e (10) are unknown. Proton translocation through V o is driven by rotational catalysis of V 1 (15).Yeast V-ATPases fa...

The a subunit is the largest of 15 different subunits that make up the vacuolar H + -ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled VATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation.cDNA . Coimmunoprecipitation studies revealed an increase in association of a4 R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4 G820R , where stability, degradation and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa. Vacuolar H+ -ATPases (V-ATPases) are conserved, multisubunit rotary proton pumps that play crucial roles in regulating the pH of cells and their intracellular compartments (1-5). They can be categorized as endomembrane or plasma membrane V-ATPases, based on their subcellular localization (6,7). Endomembrane V-ATPases are expressed in all eukaryotic cells in the membranes of acidic organelles like lysosomes, endosomes and the Golgi apparatus, where they translocate protons to acidify the luminal compartments of the organelles (8). Plasma membrane V-ATPases traffic to the surfaces of some specialized cells, such as osteoclasts, kidney intercalated cells and metastatic cancer cells, where they secrete protons into the extracellular fluid (6,9-11).The V-ATPase complex consists of 15 different subunits arranged into two major sectors, the http://www.jbc.org/cgi