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. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma 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 binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting 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 tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.
Holliday
L S
LS
Departments of Medicine and Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA. hollils@medicine.ufl.edu
Lu
M
M
Lee
B S
BS
Nelson
R D
RD
Solivan
S
S
Zhang
L
L
Gluck
S L
SL
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D001921:Brain / Q000201:enzymology; D002352:Carrier Proteins; D002417:Cattle; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D058619:Maltose-Binding Proteins; D051379:Mice; D010010:Osteoclasts / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases
D000199:Actins; D002352:Carrier Proteins; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases