Purification and ATP Hydrolysis of the Putative Cholesterol Transporters ABCG5 and ABCG8

Abstract

Mutations in the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 lead to sitosterolemia, a disorder characterized by sterol accumulation and premature atherosclerosis. ABCG5 and ABCG8 are both half-size transporters that have been proposed to function as heterodimers in vivo. We have expressed the recombinant human ABCG5 and ABCG8 genes in the yeast Pichia pastoris and purified the proteins to near homogeneity. Purified ABCG5 and ABCG8 had very low ATPase activities (<5 nmol min(-)(1) mg(-)(1)), suggesting that expression of ABCG5 or ABCG8 alone yielded nonfunctional transporters. Coexpression of the two genes in P. pastoris greatly increased the yield of pure proteins, indicating that the two transporters stabilize each other during expression and purification. Copurified ABCG5/G8 displayed low but significant ATPase activity with a V(max) of approximately 15 nmol min(-)(1) mg(-)(1). The ATPase activity was not stimulated by sterols. The catalytic activity of copurified ABCG5/G8 was characterized in detail, demonstrating low affinity for MgATP, a preference for Mg as a metal cofactor and ATP as a hydrolyzed substrate, and a pH optimum near 8.0. AlFx and BeFx inhibited MgATP hydrolysis by specific trapping of nucleotides in the ABCG5/G8 proteins. Furthermore, ABCG5/G8 eluted as a dimer on gel filtration columns. The data suggest that the hetero-dimer is the catalytically active species, and likely the active species in vivo.

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

“…4C ), even though both are phosphate analogs; this selectivity is similar to that of ABCG5/8 and ABCA1 ( 22,26 ). In contrast, many ABC transporters, including MDR1, are inhibited by both analogs.…”

“…The geometry around the ATP binding site may be slightly different in each ABC transporter. The ATPase activity of ABCG1 showed a narrower optimal pH range compared with other ABC transporters ( 26,28,29 ), suggesting a difference in the environment of the ATP binding site.…”

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

“…Both plasmids (50 g) were linearized with PmeI and cotransformed by electroporation into P. pastoris strain SMD1163 (his4 pep4 prb1) following standard procedures (18). Transformants were plated onto YPDS agar plates containing 0.1, 0.5, 1.0, or 2.0 mg/ml Zeocin to select for multicopy integrations.…”

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

“…Only one group was able to show stimulation of ATPase activity by sterols [9]. The Km of ATP seems to be relatively high indicating that the proteins may be sensitive to the energy state of the cell [8][9][10]. Although it is now clear that the heterodimer interacts with sterols and shows strong specificity for the configuration of the sterol ring, the molecular mechanism by means of which ABCG5/G8 translocates cholesterol remains to be elucidated.…”