RECONSTITUTION OF ATP-DEPENDENT AMINOPHOSPHOLIPID TRANSLOCATION IN PROTEOLIPOSOMES

In addition to ion-pumping ATPases, most plasma membranes of animal ce lls contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from hum an erythrocyte membranes by a series of column chromatographic procedu res after solubilization in Triton X-100. When reincorporated into art ificial bilayers formed from phosphatidylcholine, it was able to trans port a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosp hatidylinositol) in the outer membrane leaflet. In contrast the transm embrane distribution of spin-labeled phosphatidylcholine was unaffecte d in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoli posomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid tr anslocase activity, which is responsible for the inward transport of p hosphatidylserine acid phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by the se membranes.