The asymmetrical lipid composition of biomembranes is due to the activity of a class of membrane bound ATPases (or 'flippases') that translocates selectively phospholipids from one side of a membrane to the other. In cancer cells, the P-glycoprotein, responsible for multidrug resistance, acts also as a flippase, carrying hydrophobic drugs from the inner to the outer cell surface. The objectives of this project were to identify such phospholipid (or drug) translocators in the membranes of eukaryotic cells, to study their biochemical properties, to purify them to homogeneity, to produce antibodies, reconstitute their activity in proteoliposomes. We hoped to apprehend the physical consequences of lipid asymmetry and lipid transport on membranes either in eukaryotic cells or in liposomes. Our objectives included the understanding of the biological functions of these ATPases. The aminophospholipid translocase for example may be important for cell recognition, cell fusion, membrane curvature and possibly endocytosis. Illustrative examples of cases where phospholipid redistribution plays a physiological role are blood coagulation and elimination of aged or apoptotic cells in the circulation. Finally a long run objective was to suggest new ideas for the control and regulation of these proteins responsible for the asymmetrical lipid distribution in biomembranes.
First evidence of an aminophospholipid translocase activity in proteoliposomes using a protein purified fraction from the human erythrocyte membrane with a molecular weight of 110-120 kDa (Lab A). Partial proteolysis of this fraction could not be overcome and cloning of the protein has not be achieved. The role of the red cell Rh-protein in the aminophospholipid translocation could be excluded using RhNull cells (Labs A& B). However attempts to express the Rh polypetide in cellular lineages transfected by vectors expressing various Rh-cDNA failed. Lab B has achieved the molecular cloning of Mg-ATPase, from a partial sequence made available to us by Williamson & Schelegel (USA). Progress were made at a quantitative level in the detection of translocase activity in the plasma membrane of various cell lines in labs A & C (fibroblasts, hepatocyte, epithelial cells, ram sperm cells). Difficulties arose on the other hand with yeast (Lab C). Progress were made also on the characterization of the translocation of lipids stimulated by an ATP-independent 'flippase', in Endoplasmic Reticulum and in Golgi fractions (Labs A & D). In particular, the catalysis of glycosphingolipid transmembrane movement was demonstrated in purified Golgi and ER fractions. Experiments on liposomes considered as model systems for biological membranes were carried (Lab A) by various physical techniques including fluorescence and magnetic resonance. The influence of lipid redistribution on membrane curvature and surface tension was demonstrated. Important results were obtained by Lab C in Utrecht on the P-gp (see below). Cells overexpressing glycoprotein P were grown and used to purify the Pgp and reconstitution experiments started. Evidence were given that the Pgp and the Cl(-) channel protein are separate entities (labs E & F).
First reconstitution experiments showing a selective translocation of aminophospholipids with a MgATPase purified from human erythrocytes. A human Mg-ATPase has been cloned (but not yet expressed). The translocation of aminophospholipids in the plasma membrane of nucleated cells is at least one order of magnitude more efficient than in red cells suggesting a role of the translocase (or 'flippase') in lipid traffic, most probably in the bending of membranes required during endocytosis.
A major breakthrough is the discovery by the group of van Meer in Utrecht that MDR1 P-glycoprotein is a phospholipid translocase of broad specificity, while MDR3 specifically translocates phosphatidylcholine. This confirms the 'flippase hypothesis' put forward by Higgins (Oxford) a few years ago and which represented one of the challenge of this project. The role of the P-gp in the regulation of cell volme-activated chloride channnels has been ascertained. This identifies another important physiological role for this protein.
Funding SchemeCSC - Cost-sharing contracts
OX3 9DU Oxford
3508 TD Utrecht