A novel role for subunit C in mediating binding of the H+-V-ATPase to the actin cytoskeleton.
Primary proton transport by V-ATPases is regulated via the reversible dissociation of the V(1)V(0) holoenzyme into its V(1) and V(0) subcomplexes. Laser scanning microscopy of different tissues from the tobacco hornworm revealed co-localization of the holoenzyme and F-actin close to the apical membranes of the epithelial cells. In midgut goblet cells, no co-localization was observed under conditions where the V(1) complex detaches from the apical membrane. Binding studies, however, demonstrated that both the V(1) complex and the holoenzyme interact with F-actin, the latter with an apparently higher affinity. To identify F-actin binding subunits, we performed overlay blots that revealed two V(1) subunits as binding partners, namely subunit B, resembling the situation in the osteoclast V-ATPase (Holliday, L. S., Lu, M., Lee, B. S., Nelson, R. D., Solivan, S., Zhang, L., and Gluck, S. L. (2000) J. Biol. Chem. 275, 32331-32337), but, in addition, subunit C, which gets released during reversible dissociation of the holoenzyme. Overlay blots and co-pelleting assays showed that the recombinant subunit C also binds to F-actin. When the V(1) complex was reconstituted with recombinant subunit C, enhanced binding to F-actin was observed. Thus, subunit C may function as an anchor protein regulating the linkage between V-ATPase and the actin-based cytoskeleton.
Vitavska
Olga
O
Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, D-49069 Osnabrück, Germany.
Wieczorek
Helmut
H
Merzendorfer
Hans
H
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D018402:Manduca; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012469:Salivary Glands / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology*
D000199:Actins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases