Conserved Intramolecular Disulfide Bond Is Critical to Trafficking and Fate of ATP-binding Cassette (ABC) Transporters ABCB6 and Sulfonylurea Receptor 1 (SUR1)/ABCC8

Yu Fukuda,Martine Vaxillaire,Lydia Aguilar-Bryan,Aurelie Dechaume,Yao Wang,John D. Schuetz,Karobi Moitra,Michael Dean,Joseph Bryan

doi:10.1074/jbc.m110.174516

Abstract

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.

Highlights

ATP-binding cassette (ABC)2 transporters utilize ATP to facilitate the transmembrane movement of a variety of biologically important molecules [1]
These NXC glycosylation sites are rare in the glycoproteome [18], this endoplasmic reticulum (ER) luminal cysteine (Cys-8) in ABCB6 is not required for glycosylation; it is required for ABCB6 stability because it forms a disulfide bond with another conserved luminal cysteine (Cys26), which is required for ABCB6 stability
ABCB6 Is Glycosylated at Atypical NXC Site—We have previously demonstrated that ABCB6 expression increases during erythroid differentiation and that overexpression of ABCB6 increases porphyrin synthesis [13]

Summary

Introduction

ATP-binding cassette (ABC)2 transporters utilize ATP to facilitate the transmembrane movement of a variety of biologically important molecules [1]. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. Their mobility was identical to that of the PNGase F-treated wild-type protein and was unchanged by either PNGase F or Endo H treatment (Fig. 2C), indicating that this atypical site is the sole glycosylation site in ABCB6.

Results

Conclusion