Certain ABC Proteins “Flip” Phospholipids and Other Lipid-Soluble Substrates from One Membrane Leaflet to the Other
As shown in Figure 11-15, 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 superfamily members. Still other ABC superfamily members participate in the cellular export of various lipids, presumably by mechanisms similar to that of ABCB1 (see Table 11-3).
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 (Figure 11-16). After purifying these vesicles, investigators labeled them in vitro with a fluorescent phosphatidylcholine derivative. The fluorescence-quenching assay outlined in Figure 11-16 was used to demonstrate that the vesicles containing ABCB4 exhibited an ATP-dependent flippase activity.
EXPERIMENTAL FIGURE 11-16 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 (see Figure 14-4). 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.