Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8.
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.
Wang
Jin
J
Eugene McDermott Center for Human Growth and Development, Department of Internal Medicine, and the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8591, USA.
Zhang
Da-Wei
DW
Lei
Ying
Y
Xu
Fang
F
Cohen
Jonathan C
JC
Hobbs
Helen H
HH
Xie
Xiao-Song
XS
D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D019281:Dimerization; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D016415:Sequence Alignment; D013261:Sterols / Q000378:metabolism*
C462600:ABCG5 protein, mouse; C462601:ABCG8 protein, mouse; D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D008074:Lipoproteins; D013261:Sterols; D000251:Adenosine Triphosphatases