Abstract
ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.
Highlights
Once an ATP-binding cassette (ABC) multidrug transporter efflux pump expels a substrate, its reentry must be prevented
The S1368A mutation presents a novel phenotype never before seen in any ABC transporter
S1368A exhibited profound, broad multidrug hypersensitivity, and yet its ATPase activity was indistinguishable from WT, as was the allosteric inhibition of this enzyme by clo
Summary
Once an ABC multidrug transporter efflux pump expels a substrate, its reentry must be prevented. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5 This substitution created broad, severe drug hypersensitivity, drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. A second exclusion mechanism is suggested from the observation that with both P-gp and Pdr, high concentrations of a subset of drug substrates cause allosteric (trans) inhibition of the ATPase activity (10 –14). One might think that drugs that do not cause trans inhibition of ATPase activity could slip back through the transporter Another alternative proposed by Gupta et al [15] is that ABC efflux pumps are molecular diodes or unidirectional gates that are remodeled after drug release and ATP hydrolysis so that back transport is eliminated even if some drug is bound to a transport site. Because of its unusual behavior in several kinds of transport and reflux assays with rhodamine 6G (R6G), we posit that S1368A prevents the reflux of drugs during transport
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