a4, a Unique Kidney-specific Isoform of Mouse Vacuolar H+-ATPase Subunit a

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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) ...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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From Lysosomes to the Plasma Membrane

Toyomura

Murata

Yamamoto

et al. 2003

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121

“…Vph1p is localized to the vacuole, whereas Stv1p is found primarily in Golgi (11,12). The mammalian a subunit has four isoforms, a1-4; a1, a2, and a3 are ubiquitously expressed, but to different degrees in different tissues and organelles, whereas a4 expression appears to be specific to plasma membranes of renal intercalated cells (9,(13)(14)(15). Although ubiquitously expressed, a3 appears to be most highly enriched in osteoclasts (9).…”

Section: V-atpasesmentioning

confidence: 99%

Osteopetrosis Mutation R444L Causes Endoplasmic Reticulum Retention and Misprocessing of Vacuolar H+-ATPase a3 Subunit

Bhargava

Voronov

Wang

et al. 2012

Background:The human V-ATPase a3 subunit mutation, R444L, causes infantile malignant osteopetrosis. Results: In mouse, the R444L equivalent, R445L, causes endoplasmic reticulum retention, misprocessing, and defective trafficking of a3 to the plasma membrane. Conclusion: Arginine 444/445 plays a critical role in mammalian a3 folding, or stability. Significance: R444L a3 infantile malignant osteopetrosis is a protein folding disease that may be amenable to protein rescue therapy.

“…The largest subunit (subunit a) of 100 kDa of the V 0 is a transmembrane glycoprotein displaying characteristics of an N-terminal hydrophilic domain and a C-terminal hydrophobic domain with multiple potential transmembrane helices (22). Four isoforms (a1, a2, a3, and a4) of the 100-kDa subunit of the vacuolar proton-translocating ATPase have been identified (23)(24)(25). The second largest single subunit (subunit d) of 38 kDa is a hydrophilic protein containing no membrane helices (26) and has been found to be tightly associated with V 0 (27).…”

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confidence: 99%

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Feng

Cheng

Pavlos

et al. 2008

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H؉)-ATPases (V-ATPases)polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V 1 and V 0 . The peripheral cytoplasmically oriented V 1 domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V 0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V 0 domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c؆, and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c؆, and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V 0 domain and efficient osteoclastic bone resorption.Osteoclasts are terminally differentiated multinucleated cells derived from the hematopoietic lineage that specialize in the solubilization of mineralized bone material (1). Upon attachment to the bone surface, the osteoclast undergoes a series of cytoskeletal reorganization and polarization events that culminate in the formation of a unique apical membrane known as the ruffled border. The ruffled border is the "resorptive organelle" of the osteoclast and is formed by the polarized targeting and fusion of acidified intracellular vesicles with the plasma membrane (2-4). These transport vesicles courier acidifying machineries as well as osteolytic enzymes such as, TRACP, cathepsin K, and matrix metalloproteinases (2, 5-7) to the ruffled border membrane domain where each cargo plays a discrete role in the resorptive function. Vacuolar H ϩ -adenosine triphosphatases (V-ATPases) 5 that line the ruffled border have long been established to play a vital role in the resorptive process. V-ATPases function to pump H ϩ into the underlying resorptive lacunae. This elevation in protons results in a highly acidified microenvironment that solubilizes the mineralized component of bone while providing optimal conditions for the degradation of the exposed organic phase by collagenolytic enzymes such cathepsin K (2, 7).The importance of V-ATPase in osteoclastic bone resorption is exemplified by studies employing specific inhibitors and gene knock-out strategies to impair V-ATPase functions (8 -11). Furthermore, mutations in the human TCIRG1 g...