Abstract
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.
Highlights
The multidrug resistance protein, MRP1,1 is believed to function as an active exporter of many conjugated organic anions from cells [1,2,3,4,5,6,7]
To assess how applicable this model may be to other adenine nucleotide binding cassette (ABC) transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two nucleotide binding domains (NBDs) are much less similar than in P-glycoprotein
Photolabeling of MRP1 with N3[␣-32P]- or N3[␥-32P]ATP— MRP1 is an exporter of a variety of conjugated hydrophobic substrates [1,2,3,4,5,6,7] and has been shown to have ATPase activity that is stimulated by these substrates [11, 12]
Summary
The multidrug resistance protein, MRP1,1 is believed to function as an active exporter of many conjugated organic anions from cells [1,2,3,4,5,6,7]. The cell pellet was washed once with membrane preparation buffer (10 mM Tris-HCl, pH 7.5, 250 mM sucrose, and 0.2 mM MgCl2). The supernatant was overlaid on a 35% sucrose solution containing 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA and centrifuged at 16,000 ϫ g for 30 min. The interface was collected, diluted 5-fold with a solution containing 10 mM Tris-HCl (pH 7.5) and 250 mM sucrose, and centrifuged at 100,000 ϫ g for 45 min. Membrane vesicles for drug transport experiments were prepared exactly the same way except the pellet was resuspended in a solution containing 10 mM Tris-HCl, pH 7.5, 250 mM sucrose, and protease inhibitors and passed through a LiposofastTM vesicle extruder (200 nm filter, Avestin, Ottawa, Canada)
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