Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

Abstract

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport 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 phosphatidylethanlamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidyicholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and 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 these membranes.The rapid passage of aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the outer to the inner leaflet of the human erythrocyte membrane is an ATP-dependent process that involves the activity of a

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2

“…M embers of the P4 subfamily of P-type ATPases are known as flippases, because they transport (flip) specific phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane bilayer (1)(2)(3). Thus, they establish and maintain a physiologically essential asymmetry between the two leaflets, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) being concentrated in the cytoplasmic leaflet and phosphatidylcholine (PC) and sphingomyelin in the exoplasmic leaflet.…”

“…This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. E-mail: alve@fi.au.dk or jpa@fi.au.dk.…”

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

“…Determination of Mg 2+ ATPase activity in sample sets of the PMI study was completed as a representative of the membrane-bound ATP-requiring enzyme flippase, as highly specific antibodies to flippase have not yet been obtained (Auland et al, 1994). This indirect measure of activity was performed by measurement of the net amount of inorganic phosphate (P i ) formed (Sadrzadeh et al, 1993).…”

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

“…This asymmetry seems to be generated by an aminophospholipid translocase (APLT), or flippase activity in the plasma membrane (1,2). Seigneuret and Devaux (3) defined this APLT activity by using spin-labeled derivatives of PE and PS and reconstituted an APLT activity with a Mg 2ϩ -ATPase purified from the erythrocyte membrane (4). A similar ATP-dependent APLT activity was found in the membrane of secretory granules from chromaffin cells and was suggested to be equivalent to ATPase II purified from the same source (5).…”