ABCMdb

PMID: 25732853

Sharpe LJ, Rao G, Jones PM, Glancey E, Aleidi SM, George AM, Brown AJ, Gelissen IC
Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain.
Biochim Biophys Acta. 2015 Jul;1851(7):956-64. doi: 10.1016/j.bbalip.2015.02.016. Epub 2015 Feb 27., [PubMed]

Sentences

No. Mutations Sentence Comment

107 ABCG1 p.Tyr667Leu Cholesterol docking studies were performed for the Y667 containing CRAC motifs as well as the Y667L ABCG1 mutant. Login to comment 109 ABCG1 p.Tyr667Leu Y667 in this model was replaced by leucine to simulate effects of the Y667L mutation on the interaction between this ABCG1 CRAC motif and cholesterol. Login to comment 147 ABCG1 p.Tyr667Leu ABCG1 p.Tyr649Leu ABCG1 p.Tyr672Leu Individual point mutations of Y649L, Y667L and Y672L were introduced into ABCG1 and stable cell lines overexpressing the individual mutants generated. Login to comment 148 ABCG1 p.Tyr667Leu ABCG1 p.Tyr649Leu ABCG1 p.Tyr672Leu Fig. 3A shows that the activity of both the Y649L and Y667L mutants was totally abolished, while the Y672L mutant had reduced cholesterol export activity (this latter difference was not statistically significant). Login to comment 149 ABCG1 p.Tyr667Leu Upon investigation of the protein stability in response to cholesterol starvation/enrichment, the Y667L mutant ABCG1 protein was not stabilised by addition of cholesterol (Fig. 3B). Login to comment 150 ABCG1 p.Tyr667Leu Some experiments suggested that the degradation of the Y667L mutant upon starvation was more pronounced than the wild type, however this was not consistent. Login to comment 151 ABCG1 p.Tyr649Leu ABCG1 p.Tyr672Leu The Y649L and Y672L mutant proteins however, were consistently stabilised by the addition of cholesterol, suggesting that their cholesterol "sensing" ability was still intact, despite having reduced or negligible cholesterol export capacity. Login to comment 153 ABCG1 p.Tyr667Leu Y667L is degraded via the ubiquitin-proteasomal system As mentioned, we showed previously that ABCG1 is ubiquitinated and degraded by the proteasome when cholesterol levels drop in the cell [5]. Login to comment 154 ABCG1 p.Tyr667Leu Investigation of the route of degradation of the Y667L mutant showed that this protein was also degraded via the ubiquitin-proteasomal system as was previously shown for the wild type ABCG1 (Fig. 3C). Login to comment 155 ABCG1 p.Tyr667Leu Overexposure of the Western blot after incubation with a proteasomal inhibitor (MG132) revealed a laddering and smearing pattern, which may indicate that Y667L protein is also ubiquitinated (Fig. 3C). Login to comment 156 ABCG1 p.Tyr667Leu Addition of lysosomal inhibitors, chloroquine and ammonium chloride, did not increase the Y667L protein levels, indicating that this pathway of degradation was not quantitatively important. Login to comment 158 ABCG1 p.Tyr667Leu Staining patterns were the same for the wild type and Y667L mutant, displaying a largely perinuclear distribution, which is identical to what we have shown previously for these cells (Supplementary Fig. 5; [12]). Login to comment 163 ABCG1 p.Tyr649Leu This may explain why the Y649L mutation inhibited the cholesterol transport activity of ABCG1 without affecting its stabilisation by cholesterol. Login to comment 174 ABCG1 p.Tyr667Leu ABCG1 p.Tyr649Leu ABCG1 p.Tyr672Leu 2A) Cholesterol export of parental CHOK1 cells (CHOK1), CHOK1 cells overexpressing wild-type ABCG1 (ABCG1), or two independent clones overexpressing triple Y649L, Y667L and Y672L mutants (Triple Y/L). Login to comment 181 ABCG1 p.Tyr667Leu In view of the above, and the marked effect of the Y667L mutation on both cholesterol-mediated rescue of ABCG1, as well as cholesterol efflux, we aimed to investigate the interaction of cholesterol with the Y667 CARC motif, by docking a cholesterol molecule to the structural homology model of the area containing residues F652 to R678. Login to comment 193 ABCG1 p.Tyr667Leu ABCG1 p.Tyr667Leu ABCG1 p.Tyr667Leu Docking of cholesterol to the Y667L mutant Considering that the Y667L mutant had no transport capacity and had lost its cholesterol-mediated stability, we performed an identical simulation by docking cholesterol to this mutant (Fig. 5; Y667L). Login to comment 203 ABCG1 p.Tyr667Leu ABCG1 p.Tyr649Leu ABCG1 p.Tyr672Leu 3A) Cholesterol export of CHOK1 cells overexpressing ABCG1 with a single mutation (Y649L, Y667L or Y672L). Login to comment 205 ABCG1 p.Tyr667Leu ABCG1 p.Tyr649Leu Statistically significant differences in HDL-mediated export (via 2-way ANOVA; p b 0.05) are indicated between cells overexpressing ABCG1 vs CHOK1, Y649L and Y667L mutants. Login to comment 206 ABCG1 p.Tyr672Leu Differences between ABCG1 vs Y672L were non-significant (N.S.). Login to comment 211 ABCG1 p.Tyr667Leu 3C) Left side: ABCG1 Y667L protein levels at T = 0 or after 8 h of treatment with BSA alone (0.1% in F12 media) or with addition of MG132 (20 bc;M), ammonium chloride (20 bc;'c;) or chloroquine (CQ, 200 bc;M). Login to comment 225 ABCG1 p.Tyr667Leu Time course of ligand binding to the wild type and Y667L mutant ABCG1 TM6 area. Login to comment 231 ABCG1 p.Tyr667Leu ABCG1 p.Tyr667Leu (Y667L) Y667L mutant. Login to comment 239 ABCG1 p.Tyr667Leu This model was further supported by an identical analysis of the Y667L mutant, where the binding locus of the cholesterol was altered due to the changed position of the R671 side chain. Login to comment 251 ABCG1 p.Tyr667Leu In addition to rendering ABCG1 inactive, the Y667L mutation also altered the post-translational regulation of the transporter protein by cholesterol. Login to comment 253 ABCG1 p.Tyr667Leu Considering that the Y667L protein was not stabilised by addition of cholesterol, this implies that binding of cholesterol to this site is necessary for "rescue" from degradation to occur. Login to comment