Reconstitution of the Neurospora crassa plasma membrane H(+)-adenosine triphosphatase.
The purified H(+)-ATPase of the Neurospora crassa plasma membrane has been reconstituted by a gel filtration method into lipidic vesicles using sodium deoxycholate as the detergent. Reconstitution was performed for lipid/ATPase ratios ranging from 1000:1 to 5:1 (w/w). Whatever the lipid/ATPase ratio, the ATPase molecules completely associate with the lipid vesicles. The ATPase specific activity is identical for all proteoliposomes regardless of the lipid/ATPase ratio, but the H+ transport decreases at high protein/lipid ratios, suggesting that the proteoliposomes are more leaky to H+ as the amount of protein inserted into the lipidic membrane increases. Analysis of the fragments generated by trypsin proteolysis in the presence and in the absence of MgATP+ vanadate indicate that most of the reconstituted ATPase molecules are able to assume the transition state of the enzyme dephosphorylation reaction, and are therefore functional. The orientation (inside-out or rightside-out) of the ATPase molecules in the vesicles is independent of the lipid/ATPase ratio chosen for the reconstitution. For all the lipid/ATPase ratios tested, most of the ATPase molecules (> 99%) expose their cytoplasmic side to the outside of the reconstituted proteoliposomes. The size of the vesicles increases parallel to the ATPase amount. Although the H+ leakiness of our preparation at low lipid/protein ratios prevents proton pumping measurements, the reconstitution procedure described here has the main advantage on other procedures to allow the obtention of vesicles at high protein-to-lipid ratios, facilitating further structural characterization of the ATPase by biochemical and biophysical techniques. Therefore, the procedure described here could be of general interest in the field of membrane protein study.
Vigneron
L
L
Laboratoire des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Scarborough
G A
GA
Ruysschaert
J M
JM
Goormaghtigh
E
E
D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel / Q000379:methods; D003902:Detergents; D004789:Enzyme Activation; D008081:Liposomes / Q000138:chemical synthesis; D009492:Neurospora crassa / Q000201:enzymology* / Q000648:ultrastructure; D011510:Proteolipids / Q000138:chemical synthesis; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin
D003902:Detergents; D008081:Liposomes; D011510:Proteolipids; D014357:Trypsin; D006180:Proton-Translocating ATPases