ABCA1 is a newly described transporter that functions as a HDL receptor in the apoA-I mediated efflux of cellular sterol and phospholipids. A structural defect in the ABCA1 transporter has been identified as the genetic defect in Tangier disease. To gain insight into the cellular distribution and potential cellular site(s) of function of ABCA1 we have expressed a chimeric ABCA1-GFP protein in human fibroblasts and CHO cells. The chimeric protein was constructed by fusing EGFP in frame to the 3' end of the full-length human ABCA1 cDNA that was first modified by deleting its stop codon. The cDNA for the chimeric protein was utilized to make plasmids for transfection studies. Following transient transfection, cells were imaged by laser scanning confocal microscopy. The ABCA1-GFP fusion protein was functional since cells over-expressing the chimeric protein became depleted of intracellular cholesterol. ABCA1-GFP expressed in CHO cells localized at the cell surface, similar to the distribution of GFP, a marker for the plasma membrane. In addition, a significant fraction of the ABCA1-GFP was also localized to intracellular distributed vesicles. ABCA1-GFP co-localized in these vesicles with lpg-B, a marker for late endosomes and lysosomes in CHO cells. Decreasing the level of expression of the fusion protein with a regulated promoter did not alter the cellular distribution of ABCA1-GFP. ABCA1-GFP expressed in Tangier fibroblasts was also identified in the plasma membrane, late endocytic compartments, as well as the Golgi. In summary: 1) A functional ABCA1-GFP linked transporter has been developed which has permitted the analysis of the cellular location and function of ABCA1-GFP. 2) ABCA1 is present in both the plasma membrane and intracellular late endocytic vesicles. 3) Based on these results the efflux of cholesterol mediated by ABCA1 may involve the cycling of ABCA1 from the cell membrane to the Golgi through specific endocytic vesicles.
