Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Abstract

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2,5-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2,5-dihydroxychalcone (2,5-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 M), indicating competitive inhibition. In contrast, 2,5-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.Glutathione (GSH) is a tripeptide (␥-glutamyl-cysteinyl-glycine) that is maintained at millimolar concentrations within the cell. It is important in many signaling processes, including cell proliferation, post-translational modification of proteins, immune responses, and apoptosis (1, 2). The importance of GSH in normal cell function as well as its role in disease states such as cancer, cystic fibrosis, Parkinson disease, AIDS, liver dysfunction, and a host of other maladies has been well documented (3-8). For the purpose of detoxification and to respond to extracellular oxidative stress, cells express membrane proteins capable of transporting GSH, glutathione disulfide (GSSG), and GSH conjugates (GS-X).2 Currently, there are only a handful of proteins identified as capable of transporting GSH across membranes.The first human protein identified as a GS-X and/or GSSG transporter was the ATP-binding cassette (ABC) protein ABCC1, first called the GSH S-conjugate pump and later identified as the multidrug resistance protein MRP1 (9 -11). Since the discovery of ABCC1 as a GSH transporter, five other ABC family members have been shown to transport GSH and/or GS-X, including ABCC2, ABCC3, ABCC4, ABCC5, and ABCC7 (12-17). GSH transport has also been linked to the organic anion transporting polypeptide protein family, a subfamily of the solute carrying (SLC) protein superfamily. The SLC superfamily also contains mitochondria-specific membrane proteins (SLC25) capable of transporting GSH (18).Numerous compounds stimulate GSH, GSSG, and GS-X efflux through the ABC protei...

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