Abstract 32

Ac45, a V-ATPase Accessory Subunit Interacts with Vo Domain Subunits and is necessary for Osteoclastic bone resorption

Taksum Cheng¹, Haotian Feng¹, Kirk H. M. Yip¹, Amerigo Carrello¹, Ruth Seeber², Karen M Kroeger², Karin Eidne², Ming H. Zheng¹, and Jiake Xu¹

¹Molecular Orthopaedic Laboratory, Orthopaedics Unit, School of Surgery and Pathology, ²Western Australia Institute for Medical Research, University of Western Australia, Nedlands WA 6009, Australia

Solubilization of bone mineral by osteoclasts is dependent on the acidification of the extracellular resorption lacuna by means of a multimeric vacuolar type proton pump (V-ATPases). Besides such specialized function the V-ATPases is also essential for acidification of diverse intracellular compartments that includes the Golgi apparatus, endosomes, lysosomes and secretoy granules. The core structure of V-ATPases comprises of two functionally and structurally distinct domains, V1 and V0. The peripheral cytoplasmically oriented V1 domain is responsible for ATP hydrolysis which provides the energy for the translocation of protons across the integral membrane bound V0 domain. Here, we have identified an accessory subunit, Ac45 that interacts with the V0 domain and is involved in V-ATPase-mediated function in osteoclasts. Ac45 was localized to the ruffled border region of polarized resorbing osteoclasts and partially colocalized with pH-dependent transferrin receptor and lysotracker, marker of lysosomal structures. Furthermore using bioluminescence resonance energy transfer (BRET) assays, we showed that Ac45 interacts with subunits a3, c, c" and the newly identified e, but not d of the V0 domain. Deletion of the 26 residue-cytoplasmic tail (aa437-463) of Ac45 distorted the interaction of Ac45 with subunits of V0 domain. Osteoclasts over-expressing the deletion mutant of Ac45 were smaller in size, exhibited an increase of intracellular pH and had impaired bone resorption capability. The effect of Ac45 gene knockdown in osteoclasts using retroviral-based RNA silencing (siRNA/RNAi) is currently being investigated. To conclude, our data suggest that Ac45 is involved in V0 domain interactions, acidification and bone resorption.

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