Identification of Intra- and Intermolecular Disulfide Bridges in the Multidrug Resistance Transporter ABCG2

Abstract

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 Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 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. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 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 Cys-592 or Cys-608 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, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter-and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations

“…Cysteine residues that are potentially disulfide bridged do not participate in the structure of putative MCT8 protomer interface(s) in oligomers (Visser et al 2009), as has been reported, for example, for GPCRs (Berthouze et al 2007) and other members of the MFS (Hastrup et al 2003, Henriksen et al 2005. Moreover, it has been demonstrated that the amino acids from positions 267 to 360 (putative TMH4-6) are not involved in MCT8 protomer contacts (Visser et al 2009).…”

ATP-binding cassette, subfamily G (ABCG family)

“…Dimerization is not essential for membrane localization and transport activity since treatment with mercaptoethanol, a reducing agent, did not affect the ATP-dependent transport of methotrexate by ABCG2 [54]. Furthermore, substitution of alanine for cysteine 603 did not affect membrane localization and resistance to mitoxantrone, although it abolished dimerization [24]. It should be noted that, recently, higher forms of oligomers (tetramer and dodecamer) of ABCG2 have been suggested, in which a homodimer linked by a disulfide bond is the minimum unit [84].…”

“…ABCG2 has three preserved cysteine residues (592, 603, and 608) in the large extracellular loop between putative transmembrane domain 5 and 6. Among these residues, cysteine 603 is involved in the formation of an intermolecular disulfide bond, whereas the others have been suggested to form an intramolecular disulfide bond [24]. Dimerization is not essential for membrane localization and transport activity since treatment with mercaptoethanol, a reducing agent, did not affect the ATP-dependent transport of methotrexate by ABCG2 [54].…”

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

“…For several other membrane proteins that are able to form multimers, oligomerization was shown to be a prerequisite for the export from the ER and for correct PM targeting (Gao and Dean, 2000;Liang et al, 2004;Salahpour et al, 2004). Intra-and intermolecular disulfide bridges together were shown to be important for the structure and functions of the human multidrug resistance transporter ABCG2 (Henriksen et al, 2005). In mitochondria, a disulfide relay system catalyzes the import of proteins into the intermembrane space by an oxidative folding mechanism (Mesecke et al, 2005), and for immunoglobulins free thiol groups play a role in the transport through the secretory pathway and the quality control (Alberini et al, 1990;Fra et al, 1993).…”