Evaluation of the Role of Phosphatidylserine Translocase Activity in ABCA1-mediated Lipid Efflux

Jonathan D. Smith,Ping Zheng,Christine Waelde,Andrew Horwitz

doi:10.1074/jbc.m201594200

Jonathan D. Smith, Ping Zheng + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m201594200

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Journal: Journal of Biological Chemistry	Publication Date: May 1, 2002
Citations: 110	License type: cc-by

Affiliation: Rockefeller University

Abstract

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.

Highlights

The ATP binding cassette (ABC)1 family of proteins serves to pump a diverse set of molecules out of cells
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
ABCA1 Expression or Apoptosis Increases Cell Surface PS and ApoAI Binding—We have demonstrated previously that 8-Br-cAMP treatment of RAW267.4 cells leads to ϳ50-fold induction of ABCA1 mRNA, which in turn mediates cholesterol and phospholipid efflux to extracellular lipid-free apoAI or apoE [10]

Summary

EXPERIMENTAL PROCEDURES

Fluorescent Annexin V and ApoAI Binding and Uptake Studies— RAW264.7 cells were trypsinized and plated in high glucose DMEM containing 10% fetal bovine serum on Permanox Lab-Tek 4-chamber tissue culture plastic slides (Nunc) at a density of 50,000 cells/chamber. Cells were incubated for 24 h in serum-free DMEM containing 0.2% BSA in the presence or absence of 0.1– 0.3 mM 8-Br-cAMP (Sigma), used to induce ABCA1. For Cy5-apoAI uptake studies, this ligand was incubated at 37 °C for 5 or 60 min with cells in serum-free medium containing 0.2% BSA. On the following day the cells were treated for 24 h in DMEM, 0.2% BSA with 50 ␮g/ml acetylated low density lipoprotein, 0.3 mM 8-Br-cAMP, and (for some dishes) 0.05– 0.2 mM NBD-PC Prior to FRAP, the NBD-PC-containing medium was aspirated, and the cells were washed once and preincubated at 37 °C for 10 min in serum-free DMEM. Statistical Analyses—Two-tailed Student’s t tests, analysis of variance with Newman-Keuls post-test, and non-linear regression analysis were performed using Prism software (GraphPad)

RESULTS

The ability of apoAI and annexin V to compete with each

DISCUSSION