Resolution of the V1ATPase fromManduca sextainto Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Vincenzo F. Rizzo, Ünal Coskun, Michael Radermacher, Teresa Ruiz, Andrea Armbrüster, Gerhard Grüber
2002 Journal of Biological Chemistry  
The effect of the ATPase activity of Manduca sexta V 1 ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V 1 were studied. The V 1 was highly activated in the presence of 0.04 -0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A 3 B 3 HEGF and A 3 B 3 EG with a remaining ATPase activity of 52 and
more » ... tivity of 52 and 65%, respectively. The hydrolytic-active A 3 B 3 EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V 1 provide an organization of the subunits in the enzyme in the framework of the known threedimensional reconstruction of the V 1 ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grü ber, G. (2001) J. Struct. Biol. 135, 26 -37). Vacuolar ATPases (V 1 V O ATPases) define an ubiquitous class of proton pumps, which utilize ATP hydrolysis to maintain an acidic pH inside the vacuole (1). The electrochemical ion gradient created across the vacuolar membrane is used for the accumulation of positively charged substrates such as calcium and basic amino acids (2). In addition to this storage function, the vacuolar compartment has secretory and proteolytic functions (3, 4). The V-ATPases, consisting of at least thirteen distinct subunits (A 3 :B 3 :C:D:E:F:G y :H z :a:d:c:cЈ:cЉ) are morphologically subdivided in two components: a membrane-bound domain, V O , that contains the ion channel, and an extrinsic domain, V 1 , in which ATP hydrolysis takes place (4, 5). The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8, 9) , revealed a molecule with a single, compact stalk. The V 1 ATPase from M. sexta, which reversibly dissociates EXPERIMENTAL PROCEDURES Materials-All chemicals were at least of analytical grade and were obtained from BIOMOL (Hamburg, Germany), Merck (Darmstadt, Germany), Promega (Madison, WI), ROTH (Karlsruhe, Germany), Sigma, or Serva (Heidelberg, Germany). Purification of V 1 ATPase-Tobacco hornworms were reared as described at the web site manduca.entomology.wisc.edu/manual/cover.html. The Manduca eggs were a generous gift of Dr. J. Dolzer and PD Dr. M. Stengl, Philipps-University of Marburg, and Prof. Trenczek, University of Giessen. The V 1 ATPase from M. sexta was isolated according to Gräf et al. (15) with the following differences. (i) V 1 ATPase purification occurred in the presence of the protease inhibitors Rotistapi
doi:10.1074/jbc.m208623200 pmid:12414800 fatcat:stou2c3inrhflivhawalnf32fe