Proteolysis and orientation on reconstitution of the coated vesicle proton pump.
We recently proposed a structural model for the ATP-dependent proton pump from clathrin-coated vesicles (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802). To test this model further, we have carried out additional structural analysis of the (H+)-ATPase in both the detergent-solubilized and reconstituted states in this and the following paper (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). The orientation of the reconstituted proton pump was determined by analyzing the effect of detergent on ATP hydrolysis and by quantitating the extent of labeling of luminally oriented subunits using a membrane-impermeant reagent. Greater than 90% of the reconstituted (H+)-ATPase is oriented with the cytoplasmic surface facing outward. Treatment of the reconstituted (H+)-ATPase with trypsin results in rapid cleavage of the 100-, 73-, 58-, 38-, and 34-kDa subunits and slower cleavage of the 40- and 33-kDa subunits, consistent with our previous results indicating that all of these polypeptides have some portion of their mass exposed to the cytoplasmic surface. The 19- and 17-kDa subunits, by contrast, appear resistant to cleavage by trypsin in both the detergent-solubilized and reconstituted states, consistent with their being buried extensively in the hydrophobic phase of the bilayer. Treatment of the enzyme with trypsin under conditions in which the 100-, 73-, 58-, 38-, and 34-kDa subunits have been cleaved results in a species which is virtually inactive with respect to proton transport but retains 50% of the original ATPase activity, suggesting that proteolysis has resulted in uncoupling of these two activities. Cleavage of both the 73- and 58-kDa subunits by trypsin at a site 1-2 kDa from the amino terminus is inhibited in the presence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP, consistent with the suggestion that both the 73- and 58-kDa subunits may be nucleotide binding proteins.
Adachi
I
I
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Arai
H
H
Pimental
R
R
Forgac
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D001693:Biological Transport, Active; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D006868:Hydrolysis; D008961:Models, Structural; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons; D013501:Surface-Active Agents; D014357:Trypsin
D002966:Clathrin; D011522:Protons; D013501:Surface-Active Agents; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases