Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae |
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Authors: | Menendez A Larsson C Ugalde U |
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Abstract: | 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 μM) 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. |
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