Abcd1

Ubiquitous in peroxisomal membrane

Influences activity of peroxisomal enzyme that oxidizes very long chain fatty acids

Adrenoleukodystrophy (ADL)

ABCG5/8

Liver, intestine

Exports cholesterol and other sterols

P-Sitosterolemia

yeast sec mutant. At the permissive temperature, the ABCB4 protein is expressed by the transfected cells and moves through the secretory pathway to the cell surface (Chapter 17). At the nonpermissive temperature, however, secretory vesicles cannot fuse with the plasma membrane, as they do in wild-type cells; so vesicles containing ABCB4 and other yeast proteins accumulate in the cells. After purifying these secretory vesicles, investigators labeled them in vitro with a fluorescent phosphatidylcholine derivative. The fluorescence-quenching assay outlined in Figure 18-5 was used to demon

▲ EXPERIMENTAL FIGURE 18-5 In vitro fluorescence quenching assay can detect phospholipid flippase activity of ABCB4. A homogeneous population of secretory vesicles containing ABCB4 protein was purified from yeast sec mutants transfected with the ABCB4 gene. Step 1: Synthetic phospholipids containing a fluorescently modified head group (blue) were incorporated primarily into the outer, cytosolic leaflets of the purified vesicles. Step 2: If ABCB4 acted as a flippase, then on addition of ATP to the outside of the vesicles a small fraction of the outward-facing labeled phospholipids would be flipped to the inside leaflet. Step 3: Flipping was detected by adding a membrane-impermeable quenching compound called dithionite to the medium surrounding the vesicles. Dithionite reacts with the fluorescent head group, destroying its ability to strate that the vesicles containing ABCB4 exhibited ATP-dependent some flippase activity, whereas those without ABCB4 did not. The structures and mechanism of action of some flippases are covered in Chapter 7.

Flip-flopping between leaflets, lateral diffusion, and membrane fusion and fission are not the only dynamic processes of phospholipids in membranes. Their fatty acyl chains and, in some cases, their head groups are subject to ongoing cova-lent remodeling (e.g., hydrolysis of fatty esters by phospholi-pases and resynthesis by acyl transferases). Another key fluoresce (gray). In the presence of the quencher, only labeled phospholipid in the protected environment on 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. Step 4: Addition of detergent to the vesicles generates micelles and makes all fluorescent lipids accessible to the quenching agent and lowers the fluorescence to baseline values. [Adapted from S. Ruetz and P Gros, 1994, Cell 77:1071.]

External labeled lipids: unprotected, quenched\

Add fluorescent phospholipids

Isolated secretory vesicles containing ABCB4 protein

ATPase

External labeled lipids: unprotected, quenched\

Add fluorescent phospholipids

Detergent

+ Quencher + Light

ATPase

ABCB4 (flippase)

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