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

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. ABCG5 (G5) 1 and ABCG8 (G8) are ABC half-transporters that limit the accumulation of neutral sterols in the body (1). The two proteins are expressed primarily in enterocytes and hepatocytes, where they heterodimerize in the endoplasmic reticulum prior to being transported to apical membranes (1-3). Inactivating mutations in either G5 or G8 cause sitosterolemia, a recessive disorder characterized by hypercholesterolemia, phytosterolemia, and premature coronary artery disease (3,4). The role of G5 and G8 in sterol trafficking in vivo has been † This work was supported by Grant HL72304 from the National Institutes of Health (NIH). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript examined in detail using genetically modified mice, in which G5 and G8 are either overexpressed or inactivated (5-10). In the intestine, the G5G8 heterodimer limits the absorption of dietary sterols, especially plant-derived sterols (5). G5G8 synthesized in the liver is located in the bile canalicular membrane and is required for efficient secretion of neutral sterols into bile (5).Previously, we developed an in vitro assay using recombinant G5G8 expressed in Sf9 cells to elucidate the mechanism by which G5G8 promotes translocation of sterols across membranes (11). Membrane vesicles prepared from Sf9 cells expressing wild-type G5 and G8 supported the transfer of cholesterol from donor vesicles. G5G8-mediated transfer was stereoselective and specific for neutral sterols. Introduction of mutations predicted to disrupt ATP hydrolysis abolished G5G8-mediated sterol transfer. The recombinant G5G8 transporter was purified to near homogeneity using affinity chromatography, and the purified heterodimer retained ATPdependent sterol transfer activity when incorporated into proteoliposomes (11). These studies provided the first direct evidence that neutral sterols are the primary transport substrate f...

Intestinal Farnesoid X Receptor Controls Transintestinal Cholesterol Excretion in Mice

“…Single ABCG8 knock-out mice phenocopy ABCG5/G8 double knock-outs because the proteins need to dimerize to allow trafficking of the transporter to the apical membrane of hepatocytes and enterocytes. 32 We challenged ABCG8 KO mice and WT littermates with PX either or not in combination with ezetimibe and assessed the effects on body cholesterol fluxes. Severely blunted inductions of FNS excretion ( Figure 5B, left panel) and TICE ( Figure 5C, left panel) in response to treatment with PX or PX/ezetimibe clearly demonstrate that ABCG5/G8 is mediating the vast majority of cholesterol that is secreted via the TICE pathway under these stimulated conditions.…”

Phytosterols and Lack of Occurrence of Cholestasis in Rats Nourished Parenterally or Orally

“…When mutations affect the heterodimers transporter complex, sitosterolemia and hypercholesterolemia occur and atherosclerosis may develop. This process was highlighted in homologous animal proteins (10,11).…”

The ABCs of sterol transport

“…(46) and references therein) demonstrated that ABCG5 and ABCG8 proteins are glycosylated and that heterodimerization is required for mobilization to the cell surface. Mutation studies identified the Walker A and Walker B motifs of ABCG5 and the Signature motif of ABCG8 as being impor-tant for sterol transport, whereas mutations of Walker A or B motifs of ABCG8 or the Signature motif of ABCG5 had a small effect (46).…”

“…(46) and references therein) demonstrated that ABCG5 and ABCG8 proteins are glycosylated and that heterodimerization is required for mobilization to the cell surface. Mutation studies identified the Walker A and Walker B motifs of ABCG5 and the Signature motif of ABCG8 as being impor-tant for sterol transport, whereas mutations of Walker A or B motifs of ABCG8 or the Signature motif of ABCG5 had a small effect (46). Studies using recombinant protein or endogenous ABCG5/ABCG8 purified from mouse livers showed that the heterodimer transported both cholesterol and sitosterol (46), consistent with the accumulation of both sterols in Abcg5 2/2 and Abcg8 2/2 mice and patients with sitosterolemia.…”

“…Mutation studies identified the Walker A and Walker B motifs of ABCG5 and the Signature motif of ABCG8 as being impor-tant for sterol transport, whereas mutations of Walker A or B motifs of ABCG8 or the Signature motif of ABCG5 had a small effect (46). Studies using recombinant protein or endogenous ABCG5/ABCG8 purified from mouse livers showed that the heterodimer transported both cholesterol and sitosterol (46), consistent with the accumulation of both sterols in Abcg5 2/2 and Abcg8 2/2 mice and patients with sitosterolemia. The finding that ATP hydrolysis by ABCG5/ABCG8 was stimulated in vitro by various androgen hormones and analogs, but not by cholesterol or sitosterol (47), is perplexing as loss of ABCG5/ABCG8 is not linked to changes in androgens in vivo.…”