Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells

Abstract

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A 2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.The distribution of lipids across the eukaryotic plasma membrane bilayer is asymmetric with the choline phospholipids sphingomyelin and phosphatidylcholine (PC) 1 at the cell surface and the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) at the inside (1, 2). PS and PE are continuously translocated from the exoplasmic to the cytoplasmic leaflet of cellular membranes by proteins belonging to a subfamily of P-type ATPases (3, 4). Inward translocation of PS is essential to prevent PS cell surface signaling, which induces blood coagulation and serves as a signal for cell-cell recognition, e.g. the removal of apoptotic cells by macrophages. An additional function of lipid translocation has emerged recently. Evidence from mammalian and yeast cells suggests that ATP-dependent inward translocation of phospholipids by the aminophospholipid translocase affects membrane curvature and is a, or the, driving force for the formation of endocytic vesicles at the plasma membrane (4 -6). The fact that yeast expresses five P-type ATPase family members in different compartments of the exocytic and endocytic transport pathways suggests the possibility that all membrane budding from sphingolipid-and cholesterol-rich membranes depends on the mass translocation of membrane phospholipids.The bulk membrane phospholipid in mammalian cells is PC, which constitutes 25-50 mol % of the membrane lipids. However, it is unclear if cells possess mechanisms for ...

Yeast ABC transporters – A tale of sex, stress, drugs and aging

“…However, facts are modest at best, as most studies addressing membrane lipid asymmetry employed short-chain fluorescent lipids as transport or ''flippase'' substrates, which may be recognized as ''drugs'' rather than natural membrane lipids. Hence, reconstituted in vitro systems [92], as well as natural lipid substrates [89], will be necessary to unequivocally demonstrate functions for ABC transporters in phospholipid homeostasis. During the past years, intensive research efforts led to a better understanding of the molecular mechanisms of members of the ABC protein family.…”

Headgroup-specific Exposure of Phospholipids in ABCA1-expressing Cells

“…We cannot preclude that endogenous PC is transported by ABCA1. Recently, it has been suggested that ABCB1 and ABCB4 are involved in cell surface translocation of natural PC (31). However, these transporters have been shown to recognize NBD analogues of PC as well.…”

“…ABCB4 (MDR3) and its mouse homologue (mdr2 Pgp) transport PC (31,(33)(34)(35)(36)(37). In contrast, ABCB1 (MDR1 Pgp), giving rise to a frequent form of pleiotropic resistance in tumor cells, transports various lipid analogues (18,23,36,38) and probably also the endogenous lipids platelet-activating factor (a short-chain PC), phosphatidylcholine, phosphatidylserine, and glucosylceramide (18,31,39,40) from the cytoplasmic to the exoplasmic plasma membrane leaflet. ABCC1 (MRP1), belonging to the ABCC family, was reported to mediate outward transport of C6-NBD-PC and -PS in murine erythrocytes (41,42) and NBD-sphingolipids in epithelial cells (43).…”

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

“…In turn, lipid transfer could be altered through the presence or conformational change of peptides (24) or translocase proteins (25,26), as has been observed for alamethicin and PS lipids (27). In Fig.…”