From Lysosomes to the Plasma Membrane

Abstract

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 κB 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 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 κB 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 (1Ash P. Loutit J.F. Townsend K.M. Nature. 1980; 283: 669-670Crossref PubMed Scopus (235) Google Scholar, 2Scheven B.A. Visser J.W. Nijweide P.J. Nature. 1986; 321: 79-81Crossref PubMed Scopus (218) Google Scholar) that form an extracellular compartment (resorption lacunae) between the plasma membrane (ruffled border) and the bone surface. The acidic pH of the lacunae (3Blair H.C. Teitelbaum S.L. Ghiselli R. Gluck S. Science. 1989; 245: 855-857Crossref PubMed Scopus (721) Google Scholar, 4Väänänen H.K. Karbukorpi E.K. Sundquist K. Wallmark B. Roininen I. Hentunen T. Tuukkanen J. Lakkakorpi P. J. Cell Biol. 1990; 111: 1131-1305Crossref Scopus (322) Google Scholar) is essential for mineral solubilization and hydrolysis of bone matrix by enzymes, including collagenase and cathepsin K (5James I.E. Lark M.W. Zembryki D. Lee-Rykaczewski E.V. Hwang S.M. Tomaszek T.A. Belfiore P. Gowen M. J. Bone Miner. Res. 1999; 14: 1562-1569Crossref PubMed Scopus (42) Google Scholar). The degraded matrix is transported in luminal acidic vesicles (organelles) to the secretory domain facing the extracellular space (6Salo J. Lehenkair P. Murali M. Metsikkö K. Vääbänen H.K. Science. 1997; 276: 270-273Crossref PubMed Scopus (340) Google Scholar). Vacuolar type H+-ATPase (V-ATPase) 1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; NF-κB, nuclear factor kappa B; RANK, receptor activator of NF-κB; RANKL, RANK ligand; sRANKL, extracellular domain of RANKL; MCSF, macrophage colony-stimulating factor; DMEM, Dulbecco's modified Eagle's medium; MEM, minimal essential medium; PBS, phosphate-buffered saline; lamp2, lysosome-associated membrane protein 2; TRAP, tartrate-resistant acid phosphatase.1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; NF-κB, nuclear factor kappa B; RANK, receptor activator of NF-κB; RANKL, RANK ligand; sRANKL, extracellular domain of RANKL; MCSF, macrophage colony-stimulating factor; DMEM, Dulbecco's modified Eagle's medium; MEM, minimal essential medium; PBS, phosphate-buffered saline; lamp2, lysosome-associated membrane protein 2; TRAP, tartrate-resistant acid phosphatase. 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 (7Futai M. Oka T. Moriyama Y. Wada Y. J. Biochem. 1998; 124: 259-267Crossref PubMed Scopus (39) Google Scholar). V-ATPase is a multisubunit complex formed from a catalytic V1 sector and a membrane-spanning VO sector (7Futai M. Oka T. Moriyama Y. Wada Y. J. Biochem. 1998; 124: 259-267Crossref PubMed Scopus (39) Google Scholar, 8Anraku Y. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics. 2. Elsevier Science, New York1996: 93-109Google Scholar, 9Futai M. Oka T. Sun-Wada G.-H. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar, 10Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (363) Google Scholar, 11Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (516) Google Scholar, 12Wieczorek H. Brown D. Grinstein S. Ehrenfeld J. Harvey W.R. Bioessays. 1999; 21: 637-648Crossref PubMed Scopus (223) Google Scholar, 13Nishi T. Forgac M. Nat. Rev. 2002; 3: 94-103Crossref Scopus (977) Google Scholar). The VO sector may have a pertinent role in localizing V-ATPases to various cellular membranes. The yeast VO sector is composed of five subunits (a, c, c′, c″, and d) and has two subunit a isoforms, Stv1p and Vph1p. The nematode Caenorhabditis elegans has two c (14Oka T. Yamamoto R. Futai M. J. Biol. Chem. 1997; 272: 24387-24392Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Oka T. Yamamoto R. Futai M. J. Biol. Chem. 1998; 273: 22570-22576Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 16Oka T. Futai M. J. Biol. Chem. 2000; 275: 29556-29561Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and four a (17Oka T. Toyomura T. Honjyo K. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 33079-33085Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) isoforms with different localizations. On the other hand, a single gene for the c subunit has been detected in mammals (18Hanada H. Hasebe M. Moriyama Y. Maeda M. Futai M. Biochem. Biophys. Res. Commun. 1991; 176: 1062-1067Crossref PubMed Scopus (49) Google Scholar, 19Hayami K. Noumi T. Inoue H. Sun-Wada G.-H. Yoshimizu T. Kanazawa H. Gene (Amst.). 2001; 273: 199-206Crossref PubMed Scopus (17) Google Scholar), and no subunit c′ gene has been found so far. Only one gene for the c″ subunit was found in three organisms (14Oka T. Yamamoto R. Futai M. J. Biol. Chem. 1997; 272: 24387-24392Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 21Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2001; 276: 34122-34130Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 22Sun-Wada G.-H. Murakami H. Nakai H. Wada Y. Futai M. Gene (Amst.). 2001; 274: 93-99Crossref PubMed Scopus (8) Google Scholar). Mouse embryos lacking the c subunit die shortly after the blastocyst stage during early development (23Sun-Wada G.-H. Murata Y. Yamamoto A. Kanazawa H. Wada Y. Futai M. Dev. Biol. 2000; 228: 315-325Crossref PubMed Scopus (97) Google Scholar). In contrast to the c subunit, three subunit a isoforms (a1, a2, and a3) have been identified in mouse (24Nishi T. Forgac M. J. Biol. Chem. 2000; 275: 6824-6830Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and a fourth isoform, a4, was recently detected in mouse (26Oka T. Murata Y. Namba M. Yoshimizu T. Toyomura T. Yamamoto A. Sun-Wada G.-H. Hamasaki N. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 40050-40054Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 27Smith A.N. Finberg K.E. Wagner C.A. Lifton R.P. Devonald M.A. Su Y. Karet F.E. J. Biol. Chem. 2001; 276: 42382-42388Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and humans (28Smith A.N. Skaug J. Choate K.A. Nayir A. Bakkaloglu A. Ozen S. Hulton S.A. Sanjad S.A. Al-Sabban E.A. Lifton R.P. Scherer S.W. Karet F.E. Nat. Genet. 2000; 26: 71-75Crossref PubMed Scopus (5) Google Scholar). V-ATPase with the a3 isoform was localized to the osteoclast plasma membrane (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). In addition, disruption of the Atp6i gene for mouse a3 causes severe osteopetrosis due to the loss of osteoclast-mediated acidification of resorption lacunae (29Li Y.-P. Chen W. Liang Y. Li E. Stachenko P. Nat. Genet. 1999; 23: 447-451Crossref PubMed Scopus (411) Google Scholar). A spontaneous mouse osteopetrotic mutation (30Nakamura N. Takahashi N. Udagawa N. Moriyama Y. Kurokawa T. Jimi E. Sasaki T. Suda T. FEBS Lett. 1997; 401: 207-212Crossref PubMed Scopus (49) Google Scholar) has been mapped to the same gene (31Scimeca J.-C. Franchi A. Trojani C. Parrinello H. Grosgeorge J. Robert C. Jaillon O. Poirier C. Gaudray P. Carle G.F. Bone. 2000; 26: 207-213Crossref PubMed Scopus Google Scholar). These that the a3 isoform is an essential subunit for the osteoclast plasma membrane the a3 isoform is in so and it is not to osteoclasts (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). the localization of a3 in other cell to In we found that the V-ATPase with the a3 isoforms was localized to late endosomes and in and RAW RAW can form multinuclear cells cultured with the extracellular domain of RANKL (receptor activator of nuclear factor κB J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The cells osteoclast as tartrate-resistant acid and cathepsin K. After RANKL stimulation, the a3 isoform with lysosome are localized on organelles with the of to the cell and on the plasma membrane of mature multinuclear osteoclast-like These findings that V-ATPase is to the ruffled border osteoclast differentiation. subunit a isoforms were and as (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, T. Murata Y. Namba M. Yoshimizu T. Toyomura T. Yamamoto A. Sun-Wada G.-H. Hamasaki N. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 40050-40054Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The for lamp2 membrane protein and were from the and are also from and and Dulbecco's modified Eagle's minimal essential and extracellular domain of RANKL mouse and and Cell and cells were in and and osteoclast RAW cells were with a and for in were cultured on a for in sRANKL, and and by the for tartrate-resistant acid was as T. K. Takahashi N. T. Udagawa N. Sasaki T. Suda T. Cell Physiol. 1990; Scopus Google Scholar). the osteoclasts to the bone were and with in and were out and in the same After with PBS, the were with an for were with in PBS, in and were on and with were immunochemically as H. Moriyama Y. Futai M. H. PubMed Scopus Google Scholar). were with phosphate-buffered and for in After with and for in and the cells were with the were with the same and with the for were and finally on a with of acidic organelles, osteoclast-like cells were with for were for with with in PBS, and with and in The cells were with for and with the for were finally with were with a was used as K. H. A. H. Wada M. H. M. A. T. T. Y. S. Y. J. Cell Biol. 1997; PubMed Scopus Google Scholar). cells were with and with for Y. N. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar). were with by with were with and were with and by an of V-ATPase with the a3 in the and the of V-ATPase with the a3 isoform was localized to the plasma membrane and the periphery of mouse osteoclasts derived from bone cells (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). To we of and shown in osteoclasts were identified after with tartrate-resistant acid The V-ATPase with the a3 isoform was found in the plasma membrane of the cells facing the bone of the a3 in and of RAW the of whether the V-ATPase localization is in the RAW can differentiate into osteoclast-like multinuclear cells J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast to those in the and no was on the plasma membrane or shown in the a3 was colocalized with lamp2, a marker for late endosomes and S.A. C.A. I. A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, S. Biol. Res. Google Scholar). the same was for mouse cells These that V-ATPase with the a3 isoform is localized to the late endosomes and in the osteoclast cells and of the a1 and a2 were not with that of lamp2 as shown in that V-ATPases with a1 or a2 are not in The for a marker N. M. C. 1997; Full Text Full Text PDF PubMed Scopus (340) Google Scholar), was not with those of a3 with a1 and a2, that isoforms are localized to the Golgi complex in RAW a1 was also localized to organelles other than endosomes or the Golgi of V-ATPase with the a3 in during of interest to the cellular localization or of during the a3 isoform was localized to stimulation RAW cells were cultured in the of RANKL and and osteoclast-like multinuclear cells were identified by for The of cells a after stimulation for not The localization of the organelles with after stimulation, the organelles were in the they were found the and on the to the plasma membrane After a3 localized to the plasma membrane and to the osteoclasts derived from bone cells (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). We found that lamp2 was also localized on the to the plasma membrane after stimulation and on the plasma membrane and after The lamp2 was with that of a3 in and and with the that the a3 isoform and lamp2 were localized to lamp2 was on membranes and or inside the in the a3 localization was to the membranes in an of luminal acidic organelles, the of the organelles a3 The signals of during the of we a of a3 for A of in the of a V-ATPase that the were by These that V-ATPase with the a3 isoform in endosomes membranes was transported to the and of it was with the plasma membrane to the cell proton from the of the of a3 about stimulation with V1 subunit that V-ATPase with the a3 isoform on the to the was as to the a into osteoclast-like On the other hand, no in a1 or a2 was with for osteoclasts derived from bone cells (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The isoform, is in (26Oka T. Murata Y. Namba M. Yoshimizu T. Toyomura T. Yamamoto A. Sun-Wada G.-H. Hamasaki N. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 40050-40054Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 27Smith A.N. Finberg K.E. Wagner C.A. Lifton R.P. Devonald M.A. Su Y. Karet F.E. J. Biol. Chem. 2001; 276: 42382-42388Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), was not of V-ATPase with the a3 organelles with and lamp2 were colocalized with not with in osteoclast-like cells as shown for osteoclasts derived from bone cells (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). and are known to M. J. M. Cell Biol. 1997; Scopus Google Scholar, J. R. T. J. Cell 1990; PubMed Google Scholar), and J. PubMed Scopus Google Scholar), with the a3 isoform and lamp2 were after the signals for the two The a3 localization was not by that were not in the of These that the are localized on the to the plasma membrane. of V-ATPase with the a3 Although V-ATPases with the a3 isoform were found in the plasma membrane the for indicate that it is localized to the facing the and the is also that the multinuclear cells not form a of found in osteoclasts T. H. Inoue S. K. J. Bone Miner. Res. 2001; PubMed Scopus (89) Google Scholar, H.K. M. J. Cell PubMed Google Scholar). The may a of the cells cultured on a surface. Thus, we RAW cells for to a bone and cultured for an The osteoclast-like cells on the bone a of the a3 isoform by a to A also that are formed on the bone and a3 is inside the The a3 was localized to the plasma and it was the bone whereas a plasma membrane with a3 in facing the bone not in membranes facing the that the a3 was in the cell periphery and plasma membrane facing the bone matrix A and indicate that V-ATPase with the a3 isoform is from to the ruffled border membrane resorption localization of the a3 isoform in osteoclast-like cells cultured on a bone localization of the a3 isoform in osteoclast-like cells is shown immunochemically by for the plasma membranes facing the bone are with an of the localization of a3 in organelles in osteoclast-like resorption lacunae. have luminal acidic organelles, including and endosomes, and extracellular acidic including resorption lacunae between the bone and osteoclasts. and extracellular acidic are by V-ATPase localized in and plasma (7Futai M. Oka T. Moriyama Y. Wada Y. J. Biochem. 1998; 124: 259-267Crossref PubMed Scopus (39) Google Scholar, 9Futai M. Oka T. Sun-Wada G.-H. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar, 11Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (516) Google Scholar). Thus, are whether V-ATPases in various have the same subunit and how the V-ATPases are transported to These are to the mechanism of membrane or (7Futai M. Oka T. Moriyama Y. Wada Y. J. Biochem. 1998; 124: 259-267Crossref PubMed Scopus (39) Google Scholar). We were in VO unique isoforms may in the localization of V-ATPases to membranes. The four mouse VO subunit a isoforms, a2, and a3 are (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). a3 was found in the plasma membrane and the osteoclasts derived from bone cells (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, A.N. Skaug J. Choate K.A. Nayir A. Bakkaloglu A. Ozen S. Hulton S.A. Sanjad S.A. Al-Sabban E.A. Lifton R.P. Scherer S.W. Karet F.E. Nat. Genet. 2000; 26: 71-75Crossref PubMed Scopus (5) Google Scholar). with a3 osteopetrosis in humans and mouse (29Li Y.-P. Chen W. Liang Y. Li E. Stachenko P. Nat. Genet. 1999; 23: 447-451Crossref PubMed Scopus (411) Google Scholar, J.-C. Franchi A. Trojani C. Parrinello H. Grosgeorge J. Robert C. Jaillon O. Poirier C. Gaudray P. Carle G.F. Bone. 2000; 26: 207-213Crossref PubMed Scopus Google Scholar, A. P.J. C. S. M. P. P. A. Nat. Genet. 2000; PubMed Scopus Google Scholar, A. W. S. B. T. C. C. Genet. 2000; PubMed Scopus Google Scholar). we also identified isoforms of mouse V1 and VO subunits and Y. Sun-Wada G.-H. Wada Y. Futai M. Gene (Amst.). 2002; PubMed Scopus (39) Google Scholar, Y. Sun-Wada G.-H. Yoshimizu T. Yamamoto A. Wada Y. Futai M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (59) Google Scholar, G.-H. Y. Murata Y. T. Wada Y. Futai M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (97) Google Scholar, G.-H. Yoshimizu T. Y. Wada Y. Futai M. Gene (Amst.). PubMed Scopus Google Scholar). In one isoform is and one or are in unique cells or The isoforms of the V1 sector may also in the localization of V-ATPase the cell with a a In isoforms and for the subunit are localized in the the catalytic domain with the VO sector (7Futai M. Oka T. Moriyama Y. Wada Y. J. Biochem. 1998; 124: 259-267Crossref PubMed Scopus (39) Google Scholar, 9Futai M. Oka T. Sun-Wada G.-H. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar, 11Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (516) Google Scholar, 13Nishi T. Forgac M. Nat. Rev. 2002; 3: 94-103Crossref Scopus (977) Google Scholar). Osteoclasts differentiate from hematopoietic cells of plasma membrane receptor with RANKL of (1Ash P. Loutit J.F. Townsend K.M. Nature. 1980; 283: 669-670Crossref PubMed Scopus (235) Google Scholar, 2Scheven B.A. Visser J.W. Nijweide P.J. Nature. 1986; 321: 79-81Crossref PubMed Scopus (218) Google Scholar, N. Takahashi N. T. H. Sasaki T. T. T. Suda T. S. A. 1990; PubMed Scopus Google Scholar). A murine macrophage line, RAW 264.7, can differentiate into osteoclast-like multinuclear cultured with the extracellular domain of RANKL J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. T. H. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The a3 is localized to the endosomes of RAW whereas of the a2 and a1 isoforms were in the Golgi complex and not colocalized with a late marker The subunit was not found in RAW These indicate that no a subunit no V-ATPase were localized to the plasma membrane differentiation into multinuclear a3 was found on the of organelles with lamp2, were on the to the plasma membrane. These indicate that V-ATPase with the a3 isoform is localized to the endosomes of cells and that organelles are transported the plasma membrane of osteoclast-like that the V-ATPase with the a3 isoform was finally to the plasma membrane. Thus, an is that or late endosomes, transported to the of the cell with the plasma membrane. indicate that a is essential for the of a proton pump on the cell surface. The the lysosome with plasma membrane in osteoclasts are by of and are the of of lysosome in cell including cells and Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Nat. Rev. Biol. 2002; 3: PubMed Scopus Google Scholar). is no in bone in and The of a3 into V-ATPase was by subunit A of the V1 sector and the c subunit of VO from the or RAW membranes. Y. A. T. Oka, G.-H. Y. Wada, and M. Futai, the of the a3 isoform and the A subunit of V1 to the same after that of V-ATPase with the a3 isoform was during osteoclast differentiation. the of the lysosome of V-ATPase to the plasma it is to out the that enzyme with the a3 isoform was to plasma membrane. We have to the protein protein to the plasma membrane in the protein as as other including and a isoforms to degraded or not the localization as the In is no on acid to a3 isoform that may In to a is by including and Y. P. Teitelbaum S.L. J. Cell Biol. 1997; PubMed Scopus Google Scholar). has been shown that a role in osteoclast is with a with N. Science. 1998; PubMed Scopus Google Scholar). The V-ATPase is colocalized with in osteoclasts (25Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, N. Science. 1998; PubMed Scopus Google Scholar), that the organelles in bone resorption are transported to the cell the of the a3 localization with the has also been shown that the subunit of V-ATPase a M. S. Gluck S.L. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The luminal acidic organelles to the of the plasma membrane may to form an resorption domain an with finally that endosomes V-ATPases localization during the of in the of V-ATPase, the subunit a3 isoform a pertinent other isoforms and are not assembled into the plasma membrane. Thus, ubiquitous V-ATPase a plasma membrane pump in osteoclasts. We I. for with the RAW cell A. for on and S. and M. for in the