Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump.
In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.
Adachi
I
I
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Puopolo
K
K
Marquez-Sterling
N
N
Arai
H
H
Forgac
M
M
D001693:Biological Transport, Active; D002846:Chromatography, Affinity; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D006031:Glycosylation; D037102:Lectins; D008961:Models, Structural; D009439:Neuraminidase / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons
D003432:Cross-Linking Reagents; D037102:Lectins; D011522:Protons; D009439:Neuraminidase; D006180:Proton-Translocating ATPases