ATPaseII,avanadate-sensitiveandphosphatidylserinedependent Mg 2؉ -ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

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ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either halftransporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "insideout" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5-and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP Ͼ Ͼ CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer. Members of the superfamily of ATP-binding cassette (ABC)2 transporters actively translocate a wide variety of substances, including anions, lipids, peptides, and other compounds across membranes (1). Two ABC half-transporters, ABCG5 (G5) and ABCG8 (G8), expressed in the absorptive cells of the intestine and in hepatocytes play critical roles in the trafficking of cholesterol and other neutral sterols (2). G5 and G8 form heterodimers in the endoplasmic reticulum and are transported to the apical membranes (3, 4). In enterocytes, the G5G8 complex limits the amount of dietary sterols that are incorporated into lipoproteins and delivered to the liver (3, 4). In hepatocytes, G5 and G8 are required for efficient cholesterol secretion into bile, the major pathway for cholesterol elimination in mammals (5). Mutations inactivating either G5 or G8 cause sitosterolemia, a recessive disorder characterized by hypercholesterolemia and phytosterolemia because of increased fractional absorption and reduced biliary secretion of sterols (2, 6). Detailed metabolic studies in genetically modified mice in which G5 and G8 are overexpressed or inactivated confirm the central role of G5 and G8 in sterol trafficking (5, 7). The fractional absorption of sterols is increased and biliary sterol secretion decreased in the G5G8 Ϫ/Ϫ mice, whereas overexpression of G5 and G8 reduces the fractional absorption of dietary cholesterol and promotes biliary cholesterol secretion (5, 7).All ABC transporters share a common molecular architecture, which includes a pair of nucleotide binding domains ...

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