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M502937200v1
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Papers In Press, published online ahead of print August 17, 2005
J. Biol. Chem, 10.1074/jbc.M502937200
Submitted on March 17, 2005
Revised on August 16, 2005
Accepted on August 17, 2005

Identification of intra-and intermolecular disulfide-bridges in the multidrug resistance transporter ABCG2

Ulla Henriksen, Jacob U. Fog, Thomas Litman, and Ulrik Gether

Department of Pharmacology, University of Copenhagen, Copenhagen DK 2200 N

Corresponding Author: gether{at}neuropharm.ku.dk

ABCG2 is an ATP binding cassette (ABC) half transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide-bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys592 or Cys608 to alanine (C592A and C608A) ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Since the transporter migrated as a dimer in SDS-PAGE when only Cys603 was present (C592A-C608A), the data suggest that Cys603 forms a symmetrical intermolecular disulfide-bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys592 or Cys608 were present (C592A-C603A and C603A-C608A) the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A-C608A) partially restored plasma membrane expression; however, whereas transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports that Cys592 and Cys608 form an intramolecular disulfide-bridge in ABCG2 that is critical for substrate specificity. Finally, we mutated all three cysteines simultaneously resulting in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an intermolecular and an intramolecular disulfide-bridge together are of fundamental importance for the structural and functional integrity of ABCG2.


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