Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER.
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.
Chang
Qing-long
QL
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, Wisconsin 53706-1569, USA.
Gummadi
Sathyanarayana N
SN
Menon
Anant K
AK
D000596:Amino Acids; D000818:Animals; D002352:Carrier Proteins / Q000145:classification* / Q000302:isolation & purification / Q000378:metabolism; D004047:Diethyl Pyrocarbonate; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005033:Ethylmaleimide; D020404:Glycerophospholipids / Q000378:metabolism*; D008099:Liver / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000201:enzymology; D008565:Membrane Proteins / Q000145:classification* / Q000302:isolation & purification / Q000378:metabolism; D015335:Molecular Probes; D017830:Octoxynol / Q000494:pharmacology; D049294:Phospholipid Transfer Proteins; D051381:Rats
D000596:Amino Acids; D002352:Carrier Proteins; D020404:Glycerophospholipids; D008565:Membrane Proteins; D015335:Molecular Probes; D049294:Phospholipid Transfer Proteins; D017830:Octoxynol; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide