Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut.
The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.
Grüber
G
G
Universität Osnabrück, Fachbereich Biologie/Chemie, Osnabrück, Germany. ggrueber@biologie.uni-osnabrueck.de
Radermacher
M
M
Ruiz
T
T
Godovac-Zimmermann
J
J
Canas
B
B
Kleine-Kohlbrecher
D
D
Huss
M
M
Harvey
W R
WR
Wieczorek
H
H
D000595:Amino Acid Sequence; D000818:Animals; D003300:Copper; D003432:Cross-Linking Reagents; D007091:Image Processing, Computer-Assisted; D007454:Iodides; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D020836:Protein Structure, Quaternary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D007454:Iodides; D010446:Peptide Fragments; D003300:Copper; D006180:Proton-Translocating ATPases; C029892:cupric chloride