Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants

Faraz Quazi,Robert S Molday

doi:10.1074/jbc.m113.508812

Abstract

ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.

Highlights

Lipid transport by the A-subfamily of ATP-binding cassette (ABC) transporters implicated in human disorders is poorly understood
Purification and Reconstitution of ABCA Proteins into Proteoliposomes—ABCA1, ABCA7, and ABCA4 and corresponding mutant proteins in which lysine residues in the Walker A motifs (GXXGXK(S/T)) of NBD1 and NBD2 were replaced with methionine (ABCA-MM), all containing a 9amino acid C-terminal 1D4 tag, were transiently expressed in HEK293 cells and purified from CHAPS-solubilized cell lysates on a Rho1D4 immunoaffinity matrix
We provide the first direct biochemical evidence that ABCA1 and ABCA7 transport or flip specific phospholipids from the cytoplasmic leaflet to the exocytoplasmic leaflet of the lipid bilayer, and we determine the phospholipid specificity for these ABCA transporters

Summary

Background

Lipid transport by the A-subfamily of ATP-binding cassette (ABC) transporters implicated in human disorders is poorly understood. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation. Lipid Transport Activity of ABCA Transporters phenotypes, analysis of knock-out mice, and cell-based studies suggest that many ABCA proteins play a crucial role in cellular lipid homeostasis [6, 7]. Despite the importance of ABCA transporters in various physiological and pathological processes, relatively little is known about the substrates and direction of transport by the various members of this subfamily This is largely due to the difficulty in isolating sufficient amounts of purified ABCA proteins for functional analysis and the lack of sensitive assays to measure substrate transport across membranes. As part of this study, we describe the effect of Tangier mutations in ABCA1 and corresponding Stargardt mutations in ABCA4 on their ATPase and phospholipid transport activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION