ABCB6 p.Cys26Ala
| Predicted by SNAP2: | A: D (75%), D: D (91%), E: D (91%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, D: D, E: D, F: D, G: 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Conserved intramolecular disulfide bond is critica... J Biol Chem. 2011 Mar 11;286(10):8481-92. Epub 2011 Jan 3. 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., [PMID:21199866]
Abstract [show]
The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.
Comments [show]
None has been submitted yet.
No. Sentence Comment
59 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.
X
ABCB6 p.Cys26Ala 21199866:59:25
status: NEW142 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.
X
ABCB6 p.Cys26Ala 21199866:142:136
status: NEW157 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.
X
ABCB6 p.Cys26Ala 21199866:157:183
status: NEW160 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).
X
ABCB6 p.Cys26Ala 21199866:160:32
status: NEW174 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.
X
ABCB6 p.Cys26Ala 21199866:174:226
status: NEW176 Cysteine mutants C8S and C26A were expressed at low levels.
X
ABCB6 p.Cys26Ala 21199866:176:25
status: NEW180 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.
X
ABCB6 p.Cys26Ala 21199866:180:57
status: NEW229 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.
X
ABCB6 p.Cys26Ala 21199866:229:229
status: NEW231 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.
X
ABCB6 p.Cys26Ala 21199866:231:57
status: NEW262 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.
X
ABCB6 p.Cys26Ala 21199866:262:188
status: NEW