Abstract
Phospholipid translocation (flip-flop) across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters. These assays have been used in previous work to show that phospholipid translocation in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum is facilitated by specific membrane proteins (flippases). To extend these studies to natural phospholipids while providing a framework to guide the purification of a flippase, we now describe an assay to measure the transbilayer translocation of dipalmitoylphosphatidylcholine, a membrane-embedded phospholipid, in proteoliposomes generated from detergent-solubilized rat liver endoplasmic reticulum. Translocation was assayed using phospholipase A(2) under conditions where the vesicles were determined to be intact. Phospholipase A(2) rapidly hydrolyzed phospholipids in the outer leaflet of liposomes and proteoliposomes with a half-time of approximately 0.1 min. However, for flippase-containing proteoliposomes, the initial rapid hydrolysis phase was followed by a slower phase reflecting flippase-mediated translocation of phospholipids from the inner to the outer leaflet. The amplitude of the slow phase was decreased in trypsin-treated proteoliposomes. The kinetic characteristics of the slow phase were used to assess the rate of transbilayer equilibration of phospholipids. For 250-nm diameter vesicles containing a single flippase, the half-time was 3.3 min. Proportionate reductions in equilibration half-time were observed for preparations with a higher average number of flippases/vesicle. Preliminary purification steps indicated that flippase activity could be enriched approximately 15-fold by sequential adsorption of the detergent extract onto anion and cation exchange resins.
Highlights
Phospholipid translocation across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters
In the absence of any transbilayer transport as in liposomes, all phospholipids in the outer leaflet are expected to be converted to lysophospholipids and fatty acids by phospholipase A2 (PLA2), whereas phospholipids in the inner leaflet will be protected from hydrolysis
If the vesicles contain a flippase capable of facilitating bidirectional translocation of phospholipids, the extent of hydrolysis is expected to increase to 100% as inner leaflet lipids are flipped to the outer leaflet and become accessible to PLA2 (outer leaflet lysophospholipids ϩ fatty acid complexes are presumed to be flipped to the inner leaflet to compensate [24])
Summary
Phospholipid translocation (flip-flop) across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters These assays have been used in previous work to show that phospholipid translocation in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum is facilitated by specific membrane proteins (flippases). A recent publication from our laboratory [17] described a reconstitution system in which a detergent extract of ER membranes was used to prepare proteoliposomes capable of transporting dibutyroyl-PC, a watersoluble analog of phosphatidylcholine This reconstitution system was used to show that dibutyroyl-PC translocation required a specific membrane protein(s) whose abundance was estimated at ϳ0.2% by weight of ER membrane proteins. We conclude by using our assay procedures to show that flippase activity can be readily enriched by biochemical fractionation of the ER detergent extract, laying the groundwork for eventual purification of the transporter
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