Purification and Reconstitution of Sterol Transfer by Native Mouse ABCG5 and ABCG8

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2008 Apr 12

PMID 18402465

Citations 18

Authors

Jin Wang

Da-Wei Zhang

Ying Lei

Fang Xu

Jonathan C Cohen

Helen H Hobbs

Xiao-Song Xie

Affiliations

Soon will be listed here.

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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