As shown in Figure 1, parts (b) and (c), ABCB1 can move, or “flip,” a hydrophobic or amphipathic substrate molecule from the inner leaflet of the membrane to the outer leaflet. This otherwise energetically unfavorable reaction is powered by the ATPase activity of the protein. Support for this so-called flippase model of transport by ABCB1 comes from experiments on ABCB4 (originally called MDR2), a protein homologous to ABCB1 that is present in the region of the liver-cell plasma membrane that faces the bile canaliculi. ABCB4 moves phosphatidylcholine from the cytosolic to the exoplasmic leaflet of the plasma membrane for subsequent release into the bile in combination with cholesterol and bile acids, which themselves are transported by other ABC super family members. Still other ABC superfamily members participate in the cellular export of various lipids, presumably by mechanisms similar to that of ABCB1 (see Table 1).
Fig1. The multidrug transporter ABCB1 (MDR1): Structure and model of ligand export. (a) Cross-sectional view through the center of an ABCB1 protein bound to two molecules of a drug ana log, qz59-sss (black), reveals the central location of the ligand-binding site in relation to the phospholipid bilayer: the central ligand-binding cavity is close to the leaflet-leaflet interface of the membrane. During transport, this binding cavity is alternately exposed to the exoplasmic and the cytosolic surface of the membrane. Serines 289 and 290 affect the ligand specificity of the transporter; they are shown as red spheres to highlight their juxtaposition to the bound ligand. Surface residues are colored yellow to denote hydrophobic and blue to denote hydrophilic amino acids. (b) Three-dimensional structure of ABCB1 with its ligand-binding site facing inward toward the cytosol. In this conformation, a hydrophilic ligand can bind directly from the cytosol. A more hydrophobic ligand can partition into the inner leaflet of the plasma membrane bilayer and then enter the ligand-binding site through a gap in the protein that is accessible directly from the hydrophobic core of the inner leaflet. (c) Model for the structure of ABCB1 with its ligand binding site facing outward, based on the structures of homologous bacterial ABC proteins. When the protein assumes this conformation, the ligand can either diffuse into the exoplasmic leaflet or directly into the aqueous extracellular medium. See D. Gutman et al., 2009, Trends Biochem. Sci. 35:36–42. [Data from S. G. Aller et al., 2009, Science 323:1718–1722, PDB ID 3g61.]
Table1. Selected Human ABC Proteins
ABCB4 was first suspected of having phospholipid flippase activity because mice with homozygous loss-of-function mutations in the ABCB4 gene exhibited defects in the secretion of phosphatidylcholine into bile. To determine directly if ABCB4 was in fact a flippase, researchers performed experiments on a homogeneous population of purified vesicles isolated from special mutant yeast cells with ABCB4 in the membrane and with the cytosolic face directed outward ( Figure2). After purifying these vesicles, investigators labeled them in vitro with a fluorescent phosphatidylcholine derivative. The fluorescence-quenching assay outlined in Figure2 was used to demonstrate that the vesicles containing ABCB4 exhibited an ATP-dependent flippase activity.
Fig2. An in vitro fluorescence quenching assay revealed the phospholipid flippase activity of ABCB4. A homogeneous population of secretory vesicles containing ABCB4 protein was obtained by introducing the cDNA encoding mammalian ABCB4 into a temperature-sensitive yeast sec mutant such that ABCB4 was localized to intracellular endoplasmic reticulum vesicles in its normal orientation and with the cytosolic face of the vesicles facing outward. Step 1: When synthetic phospholipids containing a fluorescently modified head group (blue) were added to the medium surrounding the purified vesicles, they were incorporated primarily into the outer, cytosolic leaflets of the vesicles. Step 2: If ABCB4 acted as a flippase, then on addition of ATP to the medium, a small fraction of the outward-facing labeled phospholipids would be flipped to the inside leaflet. Step 3: Flipping was detected by adding a non-membrane-permeating quenching compound called dithionite to the medium. Dithionite reacts with the fluorescent head groups, destroying their ability to fluoresce (gray). In the presence of the quencher, only labeled phospholipids in the protected environment of the inner leaflet will fluoresce. Subsequent to the addition of the quenching agent, the total fluorescence decreases with time until it plateaus at the point at which all external fluorescence is quenched and only the internal phospholipid fluorescence can be detected. The observation of greater fluorescence (less quenching) in the presence of ATP than in its absence indicates that ABCB4 has flipped some of the labeled phospholipid to the inside leaflet. Not shown here are “control” vesicles isolated from cells that did not express ABCB4 and that exhibited no flippase activity. Step 4: Addition of detergent to the vesicles generates micelles and makes all fluorescent lipids accessible to the quenching agent, lowering the fluorescence to baseline values. See S. Ruetz and P. Gros, 1994, Cell 77:1071.
