Chemical Modification Identifies Two Populations of Glycerophospholipid Flippase in Rat Liver ER

Abstract

Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…Flippase Assay-Proteoliposomes containing NBD-lipids were assayed for flippase activity as described previously (17)(18)(19). Briefly, 50 l of proteoliposomes (ϳ4 mM phospholipid) was diluted to 2 ml in Buffer B in a cuvette.…”

“…Protein Modification-TE was treated with NEM and DEPC as described previously (18). Stock solutions of NEM (200 mM) and DEPC (200 mM) were prepared in Buffer B and added to TE at a final concentration of 40 mM.…”

“…We previously used a variety of assays to demonstrate ATPindependent phospholipid flippase activity in proteoliposomes reconstituted with ER membrane proteins (14,18,(33)(34)(35)(36). The most convenient of these assays (17,18) exploits the ability of sodium dithionite to eliminate selectively the fluorescence of NBD-lipids located on the outer leaflet of sealed vesicles.…”

“…The most convenient of these assays (17,18) exploits the ability of sodium dithionite to eliminate selectively the fluorescence of NBD-lipids located on the outer leaflet of sealed vesicles. Thus, when dithionite is added to large unilamellar vesicles containing NBD-lipids symmetrically distributed between the two leaflets of the membrane, fluorescence is expected to drop by 50% as NBD-lipids in the outer leaflet react.…”

“…Flippase Activity-Phospholipid flippase activity in the ER is due to two distinct pools of flippases that can be distinguished by their sensitivity to the protein modification reagents NEM and DEPC (18,19). Thus, as shown in Fig.…”