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J. Biol. Chem., Vol. 283, Issue 19, 13194-13204, May 9, 2008

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

Haotian Feng¹², Taksum Cheng¹³, Nathan J. Pavlos, Kirk H. M. Yip, Amerigo Carrello, Ruth Seeber, Karin Eidne, Ming H. Zheng, and Jiake Xu⁴

From the Molecular Orthopaedic Laboratory, Centre for Orthopaedic Research, School of Surgery and Pathology, University of Western Australia, Nedlands, Western Australia 6009, Australia and the Western Australia Institute for Medical Research, University of Western Australia, Nedlands, Western Australia 6009, Australia

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, V₁ and V₀. The peripheral cytoplasmically oriented V₁ domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V₀ domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V₀ 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 V₀ domain and efficient osteoclastic bone resorption.

Received for publication, November 28, 2007 , and in revised form, January 23, 2008.

^* This work was supported by the National Health and Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Recipient the Australian Postgraduate Award and a Jean Rogerson scholarship.

³ Recipient of the Australian Postgraduate Award.

⁴ To whom correspondence should be addressed: Molecular Orthopaedic Laboratory, Centre for Orthopaedic Research, School of Surgery and Pathology, University of Western Australia, QEII Medical Centre, 2^nd Floor M Block, Nedlands, 6009 WA Australia. Tel.: 618-9346-4051; Fax: 618-9346-3210; E-mail: jiake.xu{at}uwa.edu.au.

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