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

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.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations

“…G70E and G111R have previously been reported to compromise channel biogen- esis and trafficking (25), which are in agreement with our results. Cys-6 in SUR1 has been shown previously to form a disulfide bond with Cys-26, and mutation of Cys-6 to an alanine prevents maturation of the SUR1 band in cells co-expressing Kir6.2 (28). Our observation that C6G also disrupts SUR1 processing is consistent with the importance of this cysteine residue in channel biogenesis.…”

Missense mutations in the ABCB6 transporter cause dominant familialpseudohyperkalemia

“…The purple circle is the mutated amino acid in the three families studied here. Green circles are consensus N-glycosylation sites, only one of which (branched glycan symbol at residue 6) is experimentally validated by Fukuda [47]. C. Evolutionary conservation of mutated residue Arg 375 (red box), across the species indicated at right (with Uniprot codes).…”

“…B. Schematic representation of the two-dimensional structure of ABCB6 in the RBC membrane, based on the experimentally supported structure of Fukuda [47]. Circles represent individual amino acid residues.…”

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

“…Hydrophobicity and sequence homology analysis suggests that the TMD contains six transmembrane helices with the N and C termini located in the cytoplasm. The minimal functional unit has been suggestedto be a homodimer residing in the outer mitochondrial membrane (11,12). ABCB6 has been characterized as a mitochondrial transporter involved in the translocation of coproporphyrinogen III (CPIII) from the cytoplasm into the mitochondria (11,13).…”