The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site

Vacuolar H+-ATPase (V-ATPase) binds actin filaments with high affinity (K-d = 55 nM; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Che m. 274, 29164-29171). We have proposed that this interaction is an importan t mechanism controlling transport of V-ATPase from the cytoplasm to the pla sma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament bindi ng site in their amino-terminal domain. In pelleting assays containing acti n filaments and partially disrupted V-ATPase, B subunits were found in grea ter abundance in actin pellets than were other V-ATPase subunits, suggestin g that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tigh tly, but fusion proteins containing the carboxyl-terminal half of B1 subuni t, or the full-length E subunit, did not bind F-actin. Fusion proteins cont aining the amino-terminal 106 amino acids of the B1 isoform or the amino-te rminal 112 amino acids of the 112 isoform bound filamentous actin with K-d values of 130 and 190 nM, respectively, and approached saturation at 1 mel of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fu sion proteins competed with V-ATPase for binding to filamentous actin. In s ummary, binding sites for F-actin are present in the aminoterminal domains of both isoforms of the B subunit, and likely are responsible for the inter action between V-ATPase and actin filaments in vivo.