ABCG8 p.Gly216Asp
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (91%), E: D (85%), F: D (85%), H: D (85%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (80%), P: D (91%), Q: D (85%), R: D (91%), S: D (66%), T: D (80%), V: D (80%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Functional asymmetry of nucleotide-binding domains... J Biol Chem. 2006 Feb 17;281(7):4507-16. Epub 2005 Dec 12. Zhang DW, Graf GA, Gerard RD, Cohen JC, Hobbs HH
Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8.
J Biol Chem. 2006 Feb 17;281(7):4507-16. Epub 2005 Dec 12., 2006-02-17 [PMID:16352607]
Abstract [show]
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.
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No. Sentence Comment
179 We therefore substituted aspartate for glycine in the signature motif of G5 and G8 (G5-G197D and G8-G216D) to investigate the possible role of this motif in protein function.
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ABCG8 p.Gly216Asp 16352607:179:100
status: VERIFIED183 Thus, G5-G197D and G8-G216D did not affect significantly the ATP labeling and ADP trapping in G5 and G8.
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ABCG8 p.Gly216Asp 16352607:183:22
status: VERIFIED185 In contrast to these results, co-expression of G8-G216D with wild-type G5 did not result in any increase in biliary cholesterol level and produced only a modest increase in the level of biliary plant sterols (Fig. 6B).
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ABCG8 p.Gly216Asp 16352607:185:50
status: VERIFIED[hide] Sterol transfer by ABCG5 and ABCG8: in vitro assay... J Biol Chem. 2006 Sep 22;281(38):27894-904. Epub 2006 Jul 25. Wang J, Sun F, Zhang DW, Ma Y, Xu F, Belani JD, Cohen JC, Hobbs HH, Xie XS
Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution.
J Biol Chem. 2006 Sep 22;281(38):27894-904. Epub 2006 Jul 25., 2006-09-22 [PMID:16867993]
Abstract [show]
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.
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No. Sentence Comment
37 Mutations predicted to inactivate the ATPases were introduced into the Walker A motif of G5 (K93M) and into the signature motif of G8 (G216D) using the QuickChange site-directed mutagenesis kit (Stratagene) (13, 14).
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ABCG8 p.Gly216Asp 16867993:37:135
status: VERIFIED108 A missense mutation in a highly conserved residue in the signature motif was introduced into G8 (G216D); substitution of the corresponding residue in two other ABC transporters impairs the ATPase and transport activity of both transporters (14, 23).
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ABCG8 p.Gly216Asp 16867993:108:97
status: VERIFIED138 Recombinant wild type and mutant G5 (K93M) and G8 (G216D) were expressed in Sf9 cells.
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ABCG8 p.Gly216Asp 16867993:138:51
status: VERIFIED38 Mutations predicted to inactivate the ATPases were introduced into the Walker A motif of G5 (K93M) and into the signature motif of G8 (G216D) using the QuickChange site-directed mutagenesis kit (Stratagene) (13, 14).
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ABCG8 p.Gly216Asp 16867993:38:135
status: NEW109 A missense mutation in a highly conserved residue in the signature motif was introduced into G8 (G216D); substitution of the corresponding residue in two other ABC transporters impairs the ATPase and transport activity of both transporters (14, 23).
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ABCG8 p.Gly216Asp 16867993:109:97
status: NEW139 Recombinant wild type and mutant G5 (K93M) and G8 (G216D) were expressed in Sf9 cells.
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ABCG8 p.Gly216Asp 16867993:139:51
status: NEW39 Mutations predicted to inactivate the ATPases were introduced into the Walker A motif of G5 (K93M) and into the signature motif of G8 (G216D) using the QuickChange site-directed mutagenesis kit (Stratagene) (13, 14).
X
ABCG8 p.Gly216Asp 16867993:39:135
status: NEW110 A missense mutation in a highly conserved residue in the signature motif was introduced into G8 (G216D); substitution of the corresponding residue in two other ABC transporters impairs the ATPase and transport activity of both transporters (14, 23).
X
ABCG8 p.Gly216Asp 16867993:110:97
status: NEW140 Recombinant wild type and mutant G5 (K93M) and G8 (G216D) were expressed in Sf9 cells.
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ABCG8 p.Gly216Asp 16867993:140:51
status: NEW[hide] Purification and reconstitution of sterol transfer... Biochemistry. 2008 May 6;47(18):5194-204. Epub 2008 Apr 11. Wang J, Zhang DW, Lei Y, Xu F, Cohen JC, Hobbs HH, Xie XS
Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8.
Biochemistry. 2008 May 6;47(18):5194-204. Epub 2008 Apr 11., 2008-05-06 [PMID:18402465]
Abstract [show]
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.
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No. Sentence Comment
77 The knockout mice were infected with adenoviruses (5 × 1012 particles/kg injected into the tail vein) expressing either wild-type G5 and G8 or mutant forms of the protein that were previously demonstrated to lack ATPase activity: G5 (K93M) and G8 (G216D) (12).
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ABCG8 p.Gly216Asp 18402465:77:254
status: VERIFIED