ABCG5 and ABCG8 Are Obligate Heterodimers for Protein Trafficking and Biliary Cholesterol Excretion

Abstract

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette (ABC) transporters that limit intestinal absorption and promote biliary excretion of neutral sterols. Mutations in either ABCG5 or ABCG8 result in an identical clinical phenotype, suggesting that these two half-transporters function as heterodimers. Expression of both G5 and G8 is required for either protein to be transported to the plasma membrane of cultured cells. In this paper we used immunofluorescence microscopy to confirm, in vivo, that G5 is localized to the apical membranes of mouse enterocytes and hepatocytes. Other ABC halftransporters function as homodimers or as heterodimers with other subfamily members. To determine whether G5 or G8 complex with other ABCG half-transporters, we co-expressed G1, G2, and G4 with either G5 or G8 in cultured cells. G1, G2, and G4 co-immunoprecipitated with G5, and G4 co-immunoprecipitated with G8, but the putative dimers were retained in the endoplasmic reticulum (ER). Adenovirus-mediated expression of either G5 or G8 in the liver of G5G8 null mice resulted in ER retention of the expressed proteins and no increase in biliary cholesterol. In contrast, co-expression of G5 and G8 resulted in transit of the proteins out of the ER and a 10-fold increase in biliary cholesterol concentration. Finally, adenoviral expression of G2 in the presence or absence of G5 or G8 failed to promote sterol excretion into bile. These experiments indicate that G5 and G8 function as obligate heterodimers to promote sterol excretion into bile. ABCG5 (G5)1 and ABCG8 (G8) are members of the large family of ATP-binding cassette (ABC) transporters that facilitate translocation of a wide variety of substrates across cellular membranes (1). Eukaryotic ABC transporters consist of two modules, a magnesium-dependent ATPase catalytic domain and a transmembrane domain containing 6 -12 membranespanning segments (2). ABC transporters are divided into halftransporters, which contain one ATPase domain and one membrane-spanning domain, and full-transporters, which contain paired modules in tandem (2). G5 and G8 are both half-transporters belonging to the G subfamily of ABC proteins. Like the other three members of the human G subfamily of ABC transporters (ABCG1, ABCG2, and ABCG4), the ATPase catalytic domains of G5 and G8 are located N-terminal to the transmembrane domain.Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an autosomal recessive disorder characterized by the accumulation of both plant-derived (primarily sitosterol) and animalderived (cholesterol) sterols in plasma and tissues (3-5). In mice, G5 and G8 limit the absorption of dietary sterols and promote the excretion of cholesterol into bile (6, 7). In sitosterolemia there is a generalized increase in the absorption of dietary neutral sterols and a defect in the efflux of these sterols into bile (8 -10). These changes in sterol trafficking result in deposition of neutral sterols in skin as xanthomas and in coronary arteries, resulting in premature atherosclerosis.G5 and G8 are both glycop...

The sterol transporting heterodimer ABCG5/ABCG8 requires bile salts to mediate cholesterol efflux

“…Since this cholesterol acceptor is very potent and may overrule the importance of Abcg5/g8 we have used limiting amounts of the acceptor and carried out kinetic studies. Similar results were obtained at lower concentrations 1-4), intracellular (lanes [5][6][7][8] or plasmamembrane (lanes 9-12) fraction was detected by immunoblotting with a rabbit polyclonal antibody against mouse Abcg5. The plasma membrane fraction was isolated after biotinylation of the cells as described in Section 2.…”

“…Abrogating the activity of both or just one of the half transporters not only influenced plant sterol transport but also strongly decreased secretion of cholesterol into the bile [3][4][5]. The transporters require co-expression and probably accessory proteins to invoke trafficking from their site of synthesis in the ER to the apical site of hepatocytes and only the heterodimer shows catalytic activity [6,7]. Recent studies making use of reconstituted ABCG5 and ABCG8 have provided crucial information about their function.…”

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

“…42 Biliary cholesterol excretion is mediated by ABCG5/ ABCG8 heterodimer, which is expressed in the canalicular membrane of the hepatocyte. 43 We find an approximate twofold increase in biliary cholesterol output in Atp8b1 G308V/G308V mutant mice when challenged with taurocholate, without a change in Abcg5 protein and mRNA expression levels. This suggests that the cholesterol output in mutant mice was enhanced by extraction from the canalicular membrane.…”

Similar serum plant sterol responses of human subjects heterozygous for a mutation causing sitosterolemia and controls to diets enriched in plant sterols or stanols

“…This is owing to the action of two half-transporters, ATP-binding cassette transporters (ABC) G5 and G8, which form a heterodimer located in the apical cell membranes of enterocytes that is capable of pumping plant sterols and stanols back into the intestinal lumen once they have been taken up . ABCG5 and ABCG8 are also expressed in hepatocytes (Graf et al, 2003), where they are involved in the excretion of sterols and stanols into the bile (Sudhop et al, 2002b). Owing to the high effectiveness of these transporters, plant sterols and stanols are both poorly absorbed and quickly removed from the human body (Sudhop et al, 2002b).…”