Purification of functionally sealed cytoplasmic side-out plasma membrane vesicles from Saccharomyces cerevisiae.

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 microM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H(+)-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.