ABCMdb

PMID: 21199866

Fukuda Y, Aguilar-Bryan L, Vaxillaire M, Dechaume A, Wang Y, Dean M, Moitra K, Bryan J, Schuetz JD
Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8.
J Biol Chem. 2011 Mar 11;286(10):8481-92. Epub 2011 Jan 3., [PubMed]

Sentences

No. Mutations Sentence Comment

52 ABCC8 p.Cys26Ala K562 cells were transduced with MSCV-IRES-GFP containing no insert or inserts encoding ABCB6-FLAG, ABCB6-C26A-FLAG, or ABCB6-Walker A mutant-FLAG by retroviral gene transfer (13). Login to comment 53 ABCC8 p.Cys26Ala K562 cells were transduced with MSCV-IRES-GFP containing no insert or inserts encoding ABCB6-FLAG, ABCB6-C26A-FLAG, or ABCB6-Walker A mutant-FLAG by retroviral gene transfer (13). Login to comment 59 ABCB6 p.Cys26Ala ABCB6 p.Cys26His The pcDNA3.1- hABCB6-C8S/C26A-V5-His was generated by introducing a point mutation into pcDNA3.1-hABCB6-C8S-V5-His at Cys-26. Login to comment 61 ABCB6 p.Cys50Ala ABCB6 p.Cys50Ala ABCB6 p.Cys120Ala ABCB6 p.Cys120Ala The region encoding the amino-terminal 210 amino acids was then amplified with a FLAG tag at the carboxyl terminus from pcDNA3.1-hABCB6-C50A/C120A-V5 or pcDNA3.1-hABCB6-C8S/C50A/C120A-V5 by using primers (sense, 5Ј-GCCATGGTGACTGTGGGCAACTACTG- CGAGGCCG-3Ј; antisense, 5Ј-CCTACTTATCGTCGTCATC- CTTGTAATCACGAAGTCCAGGGGCCCAGAG-3Ј) under the following conditions: heating at 94 °C for 4 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and reaction at 68 °C for 1 min. Login to comment 63 ABCB6 p.Cys50Ala ABCB6 p.Cys50Ala The inserts were subcloned into pcDNA3 using HindIII/XbaI sites to obtain pcDNA3.1- hABCB6-C50A/C120AN1-210-FLAG or pcDNA3.1-hABCB6-C8S/C50A/C120AN1-210-FLAG constructs. Login to comment 65 ABCC1 p.Cys32Ala Using this wild-type construct as a template, we introduced a point mutation at Cys-32 to generate hMRP1-MSD0-C32A-GFP by using the QuikChange mutagenesis kit and the primers listed in the supplemental table. Login to comment 66 ABCC1 p.Cys32Ala Using this wild-type construct as a template, we introduced a point mutation at Cys-32 to generate hMRP1-MSD0-C32A-GFP by using the QuikChange mutagenesis kit and the primers listed in the supplemental table. Login to comment 88 ABCC1 p.Cys32Ala Indirect Immunofluorescence Microscopy-HEK293 cells were seeded on poly-L-lysine-coated coverslips 24 h before transfection with plasmid DNA (50 ng) encoding hMRP1-MSD0-GFP or hMRP1-MSD0-C32A-GFP by using Lipofectamine Plus according to the manufacturer`s protocols. Login to comment 89 ABCC1 p.Cys32Ala Indirect Immunofluorescence Microscopy-HEK293 cells were seeded on poly-L-lysine-coated coverslips 24 h before transfection with plasmid DNA (50 ng) encoding hMRP1-MSD0-GFP or hMRP1-MSD0-C32A-GFP by using Lipofectamine Plus according to the manufacturer`s protocols. Login to comment 142 ABCB6 p.Cys26Ala ABCB6 p.Cys26Ser ABCB6 p.Cys26Ser Therefore, we next determined whether ABCB6 engineered to contain free thiols in the ER was ER-retained by generating the ABCB6 mutants C26A and C26S as well the mutants lacking both cysteine residues, C8S/C26S. Login to comment 157 ABCB6 p.Cys26Ala To determine whether ABCB6 with cysteine substitutions at either position 8 or 26 was unstable, we performed pulse-chase experiments using K562 cells expressing either ABCB6, C8S, or C26A. Login to comment 160 ABCB6 p.Cys26Ala In contrast, both C8S-ABCB6 and C26A- ABCB6 had almost identical decay curves with an estimated half-life of 9.4 and 8.0 h, respectively (Fig. 3D). Login to comment 174 ABCB6 p.Cys26Ala ABCB6 was still glycosylated when Cys-8 was substituted with Ser to make a consensus N-glycosylation motif. B, K562 cells were transduced with plasmids containing IRES-GFP and wild-type ABCB6-, Walker A mutant (mt)-, C8S-, or C26A-FLAG. Login to comment 176 ABCB6 p.Cys26Ala Cysteine mutants C8S and C26A were expressed at low levels. Login to comment 180 ABCB6 p.Cys26Ala D, K562 cells stably expressing wild-type ABCB6, C8S, or C26A were labeled with [35 S]Met/Cys for 5 min, washed, and chased for the indicated times. Login to comment 200 ABCB6 p.Cys26Ser ABCB6 p.Cys26Ser The ER retention and instability of ABCB6-C8S, -C26S, and -C8S/C26S suggests that these residues have a crucial role in the folding of the ABCB6 amino terminus, a domain that appears to be important in determining the stability of ABCB6. Login to comment 217 ABCB6 p.Cys50Ala To rule out the contribution of the two additional cysteines (Cys-120 and Cys-150) present in this segment, we then developed a chimeric expression plasmid encoding the amino-terminal 210 amino acids of ABCB6 fused to a carboxyl-terminal FLAG epitope where both Cys-120 and Cys-150 were substituted with Ala (ABCB6-C50A/C120AN1-210-FLAG). Login to comment 220 ABCB6 p.Cys50Ala ABCB6 p.Cys50Ala We next compared the electrophoretic migration of ABCB6-C50A/C120AN1-210-FLAG and the cysteine mutant ABCB6-C8S/C50A/C120AN1-210-FLAG in the presence versus absence of the reducing agent DTT (Fig. 6B). Login to comment 223 ABCB6 p.Cys50Ala ABCB6-C50A/ C120AN1-210-FLAG exhibited three faster migrating bands in the absence of DTT (Fig. 6B). Login to comment 229 ABCB6 p.Cys26Ala ABCB6 p.Lys629Gly As ABCB6 is expressed almost exclusively in the mitochondria of K562 cells (13, 37), these cells were transduced with retroviruses expressing ABCB6-FLAG, ABCB6- K629G-FLAG (a non-functional Walker A lysine mutant (13)), or ABCB6-C26A/S-FLAG by using plasmids containing IRES-GFP. Login to comment 231 ABCB6 p.Cys26Ala The mean PPIX fluorescence in cells expressing the ABCB6-C26A mutant or nonfunctional ABCB6 was reduced in cells expressing ABCB6 (Fig. 6C and supplemental Fig. 4), indicating a loss of ability to stimulate porphyrin synthesis. Login to comment 238 ABCA1 p.Cys23Ala When the cysteines at ABCA1 residues 3 and 23 were substituted with alanine, confocal microscopic analysis showed that the mutant C3A/C23A-ABCA1 localized to both the plasma membrane and intracellular compartments (supplemental Fig. 5A). Login to comment 257 ABCB6 p.Cys50Ala ABCB6 p.Cys50Ala B, ABCB6-C50A/C120AN1-210-FLAG and ABCB6-C8S/C50A/C120AN1-210-FLAG were expressed in NIH3T3 cells, treated with N-ethylmaleimide to prevent spontaneous disulfide bonds, separated by SDS-PAGE with or without DTT, and analyzed by immunoblotting. Login to comment 262 ABCB6 p.Cys26Ala Representative images from two separate experiments are shown. C, intracellular PPIX content in K562 cells transduced with GFP and with ABCB6-FLAG, the Walker A mutant (mt)-FLAG, or ABCB6-C26A-FLAG was measured by flow cytometric analysis of GFP-positive cells. Login to comment 266 ABCC8 p.Cys26Ser This analysis enabled us to identify a role for the previously unrecognized defective SUR1/ABCC8 allele in a patient with hyperinsulinemic hypoglycemia, a recessive genetic disease where a point mutation results in a Cys-26 to serine substitution (Fig. 7A). Login to comment 267 ABCC8 p.Cys26Ser ABCC8 p.Cys26Ser The patient was a compound heterozygote: one mutant ABCC8/SUR1 allele was a previously reported trunca- Conserved Disulfide Bond in ABC Transporters 8490 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 10•MARCH tion (19), whereas the other had only a C26S substitution. Login to comment 278 ABCC1 p.Cys32Ala HEK293 cells were transfected with a plasmid encoding an MRP1-MSD0-GFP chimera containing either wild-type MSD0 or MSD0 with a C32A substitution. Login to comment 282 ABCC1 p.Cys32Ala In contrast, the construct with the C32A substitution was Endo H-sensitive. Login to comment 301 ABCC8 p.Cys6Ser A, a point mutation resulting in Cys-6 to Ser substitution in SUR1/ABCC8 was identified in one allele of a hyperinsulinemic patient as shown in the electropherogram. Login to comment 305 ABCC1 p.Cys32Ala D, MRP1-MSD0-GFP containing either wild-type MRP1 or a C32A-MRP1 mutant was transiently expressed in HEK293 cells. Login to comment 306 ABCC1 p.Cys32Ala Merged indirect immunofluorescence microscopy images (left) show the wild-type protein (green) on the cell surface, whereas the C32A mutant protein remains in the ER where it co-localizes with the ER protein protein-disulfide isomerase (PDI) (red, which is shown as yellow in the co-localized cell). Login to comment