Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8.
The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion.
Zhang
Da-Wei
DW
McDermott Center for Human Growth and Development, the Department of Molecular Genetics, University of Texas Southwestern Medical Center, 75390, USA.
Graf
Gregory A
GA
Gerard
Robert D
RD
Cohen
Jonathan C
JC
Hobbs
Helen H
HH
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; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D008074:Lipoproteins / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D013329:Structure-Activity Relationship
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; D001386:Azides; D008074:Lipoproteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate