ATP-dependent and drug-inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins

A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface.