Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux.
The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37 degrees C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.
Smith
Jonathan D
JD
Rockefeller University, New York, New York 10021, USA. smithj@rockefeller.edu
Waelde
Christine
C
Horwitz
Andrew
A
Zheng
Ping
P
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D016632:Apolipoprotein A-I / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D019311:Staurosporine / Q000494:pharmacology
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D017304:Annexin A5; D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D008565:Membrane Proteins; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D002784:Cholesterol; D019311:Staurosporine