Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution.
ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP >> CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.
Wang
Jin
J
Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, 75390-8591, USA.
Sun
Fang
F
Zhang
Da-wei
DW
Ma
Yongming
Y
Xu
Fang
F
Belani
Jitendra D
JD
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 / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism*; D019281:Dimerization; D008074:Lipoproteins / Q000737:chemistry / Q000502:physiology*; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera; D014638:Vanadates / Q000494:pharmacology
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; D008074:Lipoproteins; D008081:Liposomes; D011994:Recombinant Proteins; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002784:Cholesterol