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Abstract: The P-type Mg2+-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (−318/+193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (−1026/+193). Serial 5′ deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells. [Copyright &y& Elsevier]

Abstract: Phospholipid asymmetry is a fundamental feature of the plasma membrane of most eukaryotic cells and its regulation is linked to diverse physiological processes such as apoptosis and blood clotting [P. Williamson, R.A. Schlegel, Biochim. Biophys. Acta 1585 (2002) 53–63; R.F. Zwaal, A.J. Schroit, Blood 89 (1997) 1121–1132]. In addition, the phospholipid translocases (flippases) that are thought to establish asymmetry are also implicated in vesicle-mediated protein transport throughout the secretory and endocytic pathways [T.R. Graham, Trends Cell Biol. 14 (2004) 670–677]. However, the biochemical properties of phospholipid translocases in membranes of the Golgi complex and endosomes have received much less attention than translocases in the plasma membrane. We describe here a method for purifying yeast Golgi membranes and assaying an ATP-dependent phospholipid translocase activity in these membranes using fluorescent lipid analogues. This assay detects ATP-dependent translocation of labeled phosphatidylserine across late Golgi membranes, which requires the activity of a P-type ATPase called Drs2p [P. Natarajan, J. Wang, Z. Hua, T.R. Graham, Proc. Natl. Acad. Sci. USA 101 (2004) 10614–10619]. [Copyright &y& Elsevier]

Abstract: Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl2, cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes. [Copyright &y& Elsevier]

Abstract: Multiple lines of evidence indicate that the P-type Mg2+-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon–intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position −143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5′-flanking sequence (−1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5′ end of this sequence up to nucleotide −291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to −217 caused a drastic decrease in activity in all three cell lines, but a −148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFκB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein. [Copyright &y& Elsevier]

The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters (scramblases) that shuttle phospholipids non-specifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells’ outer surface. Since PS promotes blood coagulation, defective scramblase activity upon platelet stimulation causes a bleeding disorder (Scott syndrome). PS exposure also plays a pivotal role in the recognition and removal of apoptotic cells via a PS-recognizing receptor on phagocytic cells. Furthermore, expression of PS at the cell surface can occur in a wide variety of disorders. This review aims at highlighting how PS expression in different cells may complicate a variety of pathological conditions, including those that promote thromboembolic complications or produce aberrations in apoptotic cell removal. [ABSTRACT FROM AUTHOR]

Selective oxidation of phosphatidylserine (PS) during apoptosis precedes its externalization in plasma membrane and is essential for the engulfment of apoptotic cells. To experimentally test whether PS oxidation stimulates its externalization via its effects on aminophospholipid translocase (APT) or by enhanced PS scrambling, action of oxidized PS (PSox) was studied using leukemia HL-60 cells and lymphoma Raji cells. Both PS and PSox were equally well recognized by APT. PSox did not inhibit APT. Rate of transmembrane PS diffusion was fourfold higher in cells with integrated PSox than with PS. Thus, PSox acts as a “non-enzymatic scramblase” likely contributing to PS externalization. [Copyright &y& Elsevier]

A balance of the activities of multiple enzymes maintains the typical asymmetry of plasma membrane lipids in healthy cells. Such enzyme activities are (a) the aminophopholipid translocase (APTL) (a lipid-selective P-type ATPase that catalyzes inward movement of aminophospholipids), (b) the scramblase (a calcium-dependent and ATP-independent enzyme that catalyzes both inward and outward movement of lipids), (c) the floppase (an ATP-dependent enzyme that catalyzes only outward movement of lipids). Activation or inhibition of any one of these enzymes would lead to a loss in this asymmetry. Apoptosis-associated externalization of phophatidylserine has been reported for many different cell-types, but the exact mechanism involved in this loss of membrane asymmetry has not been identified yet. In this report we demonstrate concurrence of APTL inhibition, caspase-3 activation and apoptosis in CNS-derived HN2-5 and HOG cells. Additionally, we provide data to demonstrate that the phagocytosis of apoptotic, CNS-derived HN2-5 cells by the microglial cells requires recognition through phosphatidylserine (PS). Thus the enzyme aminopholipid translocase is inhibited during apoptosis of CNS-derived cells and this alone could account for the loss of plasma membrane lipid-asymmetry observed in these cells. [Copyright &y& Elsevier]