ABCMdb

ABCC8 p.Cys26Ser

Predicted by SNAP2:	A: D (71%), D: D (85%), E: D (85%), F: D (85%), G: D (75%), H: D (85%), I: D (85%), K: D (85%), L: D (85%), M: D (85%), N: D (80%), P: D (91%), Q: D (85%), R: D (85%), S: D (75%), T: D (63%), V: D (80%), W: D (85%), Y: D (85%),
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,

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[hide] Role of Derlin-1 protein in proteostasis regulatio... J Biol Chem. 2012 Mar 23;287(13):10482-93. Epub 2012 Feb 6. Wang F, Olson EM, Shyng SL
Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels.
J Biol Chem. 2012 Mar 23;287(13):10482-93. Epub 2012 Feb 6., [PMID:22311976]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of beta-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.

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171 We therefore studied three SUR1 mutations, C26S, A116P and F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on current topology model of SUR1 (Supplemental Figure 3) (43). Login to comment
228 Curiously, while a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite that A116P had overall lower protein levels than C26S and F1388. Login to comment
188 We therefore studied three SUR1 mutations, C26S, A116P, and èc;F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on the current topology model of SUR1 (supplemental Fig. 3) (43). Login to comment
227 A, HEK293 cells were transfected with Kir6.2 and WT, C26S, A116P, or èc;F1388 SUR1 along with Derlin-1 shRNA or the scramble control. Login to comment
317 Curiously, although a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite the fact that A116P had overall lower protein levels than C26S and èc;F1388. Login to comment

[hide] Transmembrane topology of the sulfonylurea recepto... J Biol Chem. 2001 Nov 2;276(44):41270-8. Epub 2001 Aug 23. Conti LR, Radeke CM, Shyng SL, Vandenberg CA
Transmembrane topology of the sulfonylurea receptor SUR1.
J Biol Chem. 2001 Nov 2;276(44):41270-8. Epub 2001 Aug 23., [PMID:11546780]

Abstract [show]
Sulfonylurea receptors (SURx) are multi-spanning transmembrane proteins of the ATP-binding cassette (ABC) family, which associate with Kir6.x to form ATP-sensitive potassium channels. Two models, with 13-17 transmembrane segments, have been proposed for SURx topologies. Recently, we demonstrated that the amino-terminal region of SUR1 contains 5 transmembrane segments, supporting the 17-transmembrane model. To investigate the topology of the complete full-length SUR1, two strategies were employed. Topology was probed by accessibility of introduced cysteines to a membrane-impermeable biotinylating reagent, biotin maleimide. Amino acid positions 6/26, 99, 159, 337, 567, 1051, and 1274 were accessible, therefore extracellular, whereas many endogenous and some introduced cysteines were inaccessible, thus likely cytoplasmic or intramembrane. These sites correspond to extracellular loops 1-3, 5-6, and 8 and the NH2 terminus, and intracellular loops 3-8 and COOH terminus in the 17-transmembrane model. Immunofluorescence was used to determine accessibility of epitope-tagged SUR1 in intact and permeabilized cells. Epitopes at positions 337 and 1050 (putative external loops 3 and 6) were labeled in intact cells, therefore external, whereas positions 485 and 1119 (putative internal loops 5 and 7) only were accessible after permeabilization and therefore internal. These results are compatible with the 17-transmembrane model with two pairs of transmembrane segments as possible reentrant loops.

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57 The resulting SUR1AAA construct containing C6S, C26S, C170A, C1051S, and C1057S was referred to as NEC (no external cysteines). Login to comment
55 The resulting SUR1AAA construct containing C6S, C26S, C170A, C1051S, and C1057S was referred to as NEC (no external cysteines). Login to comment

[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.

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266 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 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