Functional mosaicism of membrane proteins in vesicles of Escherichia coli

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Abstract
Membrane vesicles of E. coli prepared by osmotic lysis of lysozyme EDTA spheroplasts have .apprx. 60% of the total membrane-bound NADH dehydrogenase (EC 1.6.99.3) and Mg2+-ATPase (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of .apprx. 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and .apprx. 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported Ca2+, using NADH or ATP as energy source. Neither D-lactate nor reduced phenazine methosulfate energized Ca uptake, suggesting that the observed Ca uptake was not due to a small population of everted vesicles. Transport of Ca driven by NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by NADH oxidation or ATP hydrolysis. The portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are probably functionally a homogeneous population but cannot be categorized as right-side-out or everted, since activities normally associated with only 1 side of the inner membrane can be found on both sides of the membrane of these vesicles. The oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase apparently establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.