ABCMdb

PMID: 19556522

Singaraja RR, Kang MH, Vaid K, Sanders SS, Vilas GL, Arstikaitis P, Coutinho J, Drisdel RC, El-Husseini Ael D, Green WN, Berthiaume L, Hayden MR
Palmitoylation of ATP-binding cassette transporter A1 is essential for its trafficking and function.
Circ Res. 2009 Jul 17;105(2):138-47. Epub 2009 Jun 25., [PubMed]

Sentences

No. Mutations Sentence Comment

95 ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser Circulation Research July 17, 2009 C23S, and the double C3/C23S. Login to comment 100 ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser Circulation Research July 17, 2009 at C23S, and the double C3/C23S. Login to comment 103 ABCA1 p.Cys1111Ser Assessment of palmitoylation of these sites in the context of full-length ABCA1 showed that ABCA1 was still palmitoylated (Figure 3D, lane C1110/C1111S). Login to comment 104 ABCA1 p.Cys1111Ser However, generation of the quadruple mutant C3/23/1110/1111S in full-length ABCA1 resulted in a significant reduction in ABCA1 palmitoylation (Figure 3D, lane C3/C23/C1110/C1111S; and Figure 3E). Login to comment 108 ABCA1 p.Cys1111Ser Assessment of palmitoylation of these sites in the context of full-length ABCA1 showed that ABCA1 was still palmitoylated (Figure 3D, lane C1110/C1111S). Login to comment 109 ABCA1 p.Cys1111Ser However, generation of the quadruple mutant C3/23/1110/1111S in full-length ABCA1 resulted in a significant reduction in ABCA1 palmitoylation (Figure 3D, lane C3/C23/C1110/C1111S; and Figure 3E). Login to comment 120 ABCA1 p.Met1091Thr As a control, cells expressing the ABCA1 mutant M1091T were used. Login to comment 124 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser We generated stable cell lines harboring each of the C3S, C23S, C1110S, and C1111S mutants singly and in combination and determined the extent of both cholesterol and phospholipid efflux to ApoA-I facilitated by these cell lines. Login to comment 125 ABCA1 p.Met1091Thr As a control, cells expressing the ABCA1 mutant M1091T were used. Login to comment 129 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser We generated stable cell lines harboring each of the C3S, C23S, C1110S, and C1111S mutants singly and in combination and determined the extent of both cholesterol and phospholipid efflux to ApoA-I facilitated by these cell lines. Login to comment 130 ABCA1 p.Cys23Ser C23S .....ߪߪߪߪߪߪߪߪߪ.ߪߪ..ߪߪ...ߪSߪߪߪߪߪߪ..ߪߪߪߪߪߪߪߪߪߪߪߪ.ߪ. Login to comment 131 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser C3S/C23S .....S.ߪߪߪߪߪ.ߪߪߪ.ߪߪ.ߪ......Sߪߪߪߪߪߪߪ..ߪߪߪ..ߪ..ߪߪߪߪߪߪߪ C1110S/C1111S ....ߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪߪ..ߪߪߪߪߪߪ..ߪ..ߪ.SSߪߪߪ.ߪߪ. Login to comment 132 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser C3S/C23S/ C1110S/C1111S .....S.ߪߪߪߪߪߪߪߪߪ......ߪ......Sߪߪߪߪߪ..ߪ..ߪ..ߪ.....SSߪ..ߪߪߪߪ. Login to comment 133 ABCA1 p.Cys1111Ser ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser B C 3 / C 2 3 / C 1 1 1 0 / C 1 1 1 1 S C3/C23S C 100KDa W T C3S C23S IP: b1;-His WB: b1;-Myc 75KDa 100KDa 75KDa 3H fluorogram W T C 3 S C 2 3 S C 3 / C 2 3 S C 1 1 1 0 / C 1 1 1 1 S WB: b1;-ABCA1 3H fluorogram 250KDa 250KDa D COOH H2N B A B A C C C3S C23S C1110S C1111S WT C3/C23/C1110/C1111S Palmitoylation (Normalized to total protein) E 0 20 40 60 80 100 120 p=0.000002 W T C 3 / 2 3 / 1 1 1 0 / 1 1 1 1 S W T C 3 / 2 3 / 1 1 1 0 / 1 1 1 1 S W T C 3 / 2 3 / 1 1 1 0 / 1 1 1 1 S 3H fluorogram ABCA1 ABCA1 250KDa 250KDa WB: b1;-ABCA1 Figure 3. Login to comment 135 ABCA1 p.Cys23Ser C23S .....……………………….……..……...…S………………..……………………………….…. Login to comment 136 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser C3S/C23S .....S.…………….……….…….…......S…………………..………..…..………………… C1110S/C1111S ....……………………………………………………..………………..…..….SS……….……. Login to comment 137 ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser C3S/C23S/ C1110S/C1111S .....S.………………………......…......S……………..…..…..….....SS…..…………. Login to comment 138 ABCA1 p.Cys1111Ser ABCA1 p.Cys1111Ser ABCA1 p.Cys1111Ser ABCA1 p.Cys1111Ser ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser ABCA1 p.Cys23Ser ABCA1 p.Cys1110Ser B C3/C23/C1110/C1111S C3/C23SC 100KDa W T C3S C23S IP: α-His WB: α-Myc 75KDa 100KDa 75KDa 3H fluorogram W T C3S C23S C3/C23S C1110/C1111S WB: α-ABCA1 3H fluorogram 250KDa 250KDa D COOHH2N B A B A C C C3S C23S C1110S C1111S WT C3/C23/C1110/C1111S Palmitoylation (Normalizedtototalprotein) E 0 20 40 60 80 100 120 p=0.000002 W T C3/23/1110/1111SW T C3/23/1110/1111SW T C3/23/1110/1111S 3H fluorogram ABCA1 ABCA1 250KDa 250KDa WB: α-ABCA1 Figure 3. Login to comment 142 ABCA1 p.Met1091Thr Circulation Research July 17, 2009 harboring the naturally occurring ABCA1 mutation M1091T that was previously characterized as severe loss-of-function mutation8 was reduced by b07;80%. Login to comment 147 ABCA1 p.Met1091Thr Circulation Research July 17, 2009 at harboring the naturally occurring ABCA1 mutation M1091T that was previously characterized as severe loss-of-function mutation8 was reduced by Ϸ80%. Login to comment 151 ABCA1 p.Met1091Thr The severe loss-of-function patient mutation M1091T, previously shown to be absent from the plasma membrane, was used as a positive control. Login to comment 156 ABCA1 p.Met1091Thr The severe loss-of-function patient mutation M1091T, previously shown to be absent from the plasma membrane, was used as a positive control. Login to comment 164 ABCA1 p.Cys1111Ser A D p=0.0007 p=0.04 p=0.002 p=0.0005 p=0.0002 p<0.0001 0 20 40 60 80 100 120 W T C 3 S C 2 3 S C 3 S / C 2 3 S C 1 1 1 0 S / C 1 1 1 1 S C 3 / C 2 3 / C 1 1 1 0 / C 1 1 1 1 S M 1 0 9 1 T % cholesterol efflux (normalized to wt) p=0.003 p=0.009p=0.01 p=0.03 p=0.03 p<0.0001 0 20 40 60 80 100 120 W T C 3 S C 2 3 S C 3 S / C 2 3 S C 1 1 1 0 S / C 1 1 1 1 S C 3 / C 2 3 / C 1 1 1 0 / C 1 1 1 1 S M 1 0 9 1 T %phosphocholine efflux (normalized to wt) Empty Vector WT- No Biotin 150 100 250 75 50 37 Input IP Input IP Input IP WT+Biotin C3/C23/ C1110/C111S+Biotin ABCA1 GAPDH Controls WB: b1;ABCA1 WB: b1;GAPDH C3/C23/C1110/C1111S 0 20 40 60 80 100 120 p=0.00003 W T Biotin labeled ABCA1 (normalized to WT) Input IP B C Figure 5. Login to comment 169 ABCA1 p.Cys1111Ser A D p=0.0007 p=0.04 p=0.002 p=0.0005 p=0.0002 p<0.0001 0 20 40 60 80 100 120 W T C 3S C 23SC 3S/C 23S C 1110S/C 1111S C 3/C 23/C 1110/C 1111S M 1091T %cholesterolefflux(normalizedtowt) p=0.003 p=0.009p=0.01 p=0.03 p=0.03 p<0.0001 0 20 40 60 80 100 120 W T C 3S C 23SC 3S/C 23S C 1110S/C 1111S C 3/C 23/C 1110/C 1111S M 1091T %phosphocholineefflux(normalizedtowt) Empty Vector WT- No Biotin 150 100 250 75 50 37 Input IP Input IP Input IP WT+Biotin C3/C23/ C1110/C111S+Biotin ABCA1 GAPDH Controls WB: αABCA1 WB: αGAPDH C3/C23/C1110/C1111S 0 20 40 60 80 100 120 p=0.00003 W T BiotinlabeledABCA1(normalizedtoWT) Input IP B C Figure 5. Login to comment 172 ABCA1 p.Met1091Thr As a control, efflux of lipids was reduced by b07;80% in a cell line harboring M1091T, a naturally occurring loss-of-function patient mutation. Login to comment 177 ABCA1 p.Met1091Thr As a control, efflux of lipids was reduced by Ϸ80% in a cell line harboring M1091T, a naturally occurring loss-of-function patient mutation. Login to comment 204 ABCA1 p.Met1091Thr As controls for the efflux assay, we used a cell line harboring M1091T, a naturally occurring patient mutation that was previously characterized as severe loss-of-function mutation8 and showed b0e;80% reduction in efflux, likely representing the absence of ABCA1 both at the plasma membrane and at intracellular sites of function. Login to comment 209 ABCA1 p.Met1091Thr As controls for the efflux assay, we used a cell line harboring M1091T, a naturally occurring patient mutation that was previously characterized as severe loss-of-function mutation8 and showed Ͼ80% reduction in efflux, likely representing the absence of ABCA1 both at the plasma membrane and at intracellular sites of function. Login to comment