Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8.

Biochemistry
Jin WangXiao-Song Xie

Abstract

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette half-transporters that limit intestinal uptake and promote biliary secretion of neutral sterols. Here, we describe the purification of endogenous G5G8 from mouse liver to near homogeneity. We incorporated the native proteins into membrane vesicles and reconstituted sterol transfer. Native gel electrophoresis, density-gradient ultracentrifugation, and chemical cross-linking studies indicated that the functional native complex is a heterodimer. No higher order oligomeric forms were observed at any stage in the catalytic cycle. Sterol transfer activity by purified native G5G8 was stable, stereospecific, and selective. We also report that G5 but not G8 is S-palmitoylated and that palmitoylation is not essential for dimerization, trafficking, or biliary sterol secretion. Both G5 and G8 have short but highly conserved cytoplasmic tails. The functional roles of these C-terminal regions were examined using an in vivo functional assay.

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Citations

Jan 7, 2011·The Journal of Biological Chemistry·Jin WangXiao-Song Xie
Jan 24, 2013·Current Opinion in Lipidology·Elizabeth J Tarling
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