Analysis of V-ATPase complexes in osteoclasts by Native Blue Electrophoresis

Introduction: In yeast, tobacco hornworm and mammalian dendritic cells, one means of controlling vacuolar H+-ATPase (V-ATPase) activity is by regulated assembly of V-ATPase from disassembled peripheral (V1) and integral (V0) subdomains. To test whether this occurs during osteoclast activation, detergent extracts from unactivated osteoclasts were subjected to Native-Blue gel electrophoresis, which separates native protein complexes by size. The assembly hypothesis predicts that complexes the size of V1 (550,000 kD) and V0 (250,000) should be detected. Methods: Mouse marrow osteoclasts were extracted with 1% Triton X-100 to solubilize membranes and total extracts were subjected to Native Blue gel electrophoresis on a 3-18% acrylamide gradient in the first dimension and SDS-PAGE on 15% gels in the second dimension. Gels were stained with Coomassie Blue to detect total proteins or blotted and probed with antibodies to the V1 domain (anti- anti-E and anti-B2) or V0 domain (anti-a3). Results: Both B2 and a3 were detected only as components of complexes of around 800,000 kD, a size consistent with being the intact enzyme. In contrast, only about 1/3 of the E subunit was present in the intact complex. The additional 2/3 of E was detected as monomers. Conclusions: These data do not support the simple hypothesis that cytosolic V1 assembles onto membrane bound V0 during osteoclast activation. However, the unassembled pool of subunit E hints that partial assembly may be required during the activation process. Supported by a UFCD Student Summer Research Fellowship and a grant from the NIH (R01 AR47959) (LSH)