title: 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.
authors.affiliation: From the Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105,; Interdisciplinary Program, College of Graduate Health Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Pacific Northwest Research Institute, Seattle, Washington 98122.
authors.forename: Lydia
authors.initials: L
authors.lastname: Aguilar-Bryan
authors.affiliation: CNRS UMR 8090 Unit, Institute of Biology and Pasteur Institute, Lille 59019, France, and.
authors.forename: Martine
authors.initials: M
authors.lastname: Vaxillaire
authors.affiliation: CNRS UMR 8090 Unit, Institute of Biology and Pasteur Institute, Lille 59019, France, and.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Dechaume
authors.affiliation: From the Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Laboratory of Experimental Immunology, NCI, National Institutes of Health, Frederick, Maryland 21702.
authors.forename: Michael
authors.initials: M
authors.lastname: Dean
authors.affiliation: Laboratory of Experimental Immunology, NCI, National Institutes of Health, Frederick, Maryland 21702.
authors.forename: Karobi
authors.initials: K
authors.lastname: Moitra
authors.affiliation: Pacific Northwest Research Institute, Seattle, Washington 98122.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bryan
authors.affiliation: From the Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105,. Electronic address: John.Schuetz@stjude.org.
authors.forename: John D
authors.initials: JD
authors.lastname: Schuetz
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D002239:Carbohydrate Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006946:Hyperinsulinism / Q000235:genetics / Q000378:metabolism; D007003:Hypoglycemia / Q000235:genetics / Q000378:metabolism; D020014:K562 Cells; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D064233:Sulfonylurea Receptors
chemical_list: C107991:ABCB6 protein, human; C577507:ABCC8 protein, human; D018528:ATP-Binding Cassette Transporters; C514489:Abcb6 protein, mouse; C577508:Abcc8 protein, mouse; D015853:Cysteine Proteinase Inhibitors; D004220:Disulfides; D007976:Leupeptins; D024101:Mitochondrial Proteins; D024661:Potassium Channels, Inwardly Rectifying; D061988:Proteasome Inhibitors; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde