Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption.
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.
Feng
Haotian
H
Molecular Orthopaedic Laboratory, Centre for Orthopaedic Research, School of Surgery and Pathology, University of Western Australia, Nedlands, Western Australia 6009, Australia.
Cheng
Taksum
T
Pavlos
Nathan J
NJ
Yip
Kirk H M
KH
Carrello
Amerigo
A
Seeber
Ruth
R
Eidne
Karin
K
Zheng
Ming H
MH
Xu
Jiake
J
D000818:Animals; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D051379:Mice; D010010:Osteoclasts / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
C484226:Atp6ap1 protein, mouse; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases