Reconstitution of a Plasma-Membrane H+-ATPase Into Bilayer Lipid Membrane

The plasma membrane H + -ATPase of Neurospora has been reconsti­ tuted into planar lipid bilayer membranes by means of the vesicle-fusion technique described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was first transferred from iso­ lated plasma membrane fragments into asolectin vesicles by a detergent-dialysis procedure (Perlin et al., 1984). After H + -pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into preformed bilayers. Critical features of the fusion process include (i) attachment of the vesicles to the bilayer in the presence of divalent cations (Mg ++ ), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or freeze-thawin g of the vesicles, and/or by inclusions of physiologic channels. Enough proton pumps could be thus incorporated into bilayers to achieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress con­ ductance fluctuations due to the channels, and when that precaution was taken, we could demonstrate the proton pump the work against membrane potentials of at least 50 mV.