The Signaling Interface of the Yeast Multidrug Transporter Pdr5 Adopts a Cis Conformation, and There Are Functional Overlap and Equivalence of the Deviant and Canonical Q-Loop Residues

摘要

ABC transporters are polytopic proteins. ATP hydrolysis and substrate transport take place innseparate domains, and these activities must be coordinated through a signal interface. We previouslyncharacterized amutation (S558Y) in the yeastmultidrug transporter Pdr5 that uncouples ATP hydrolysis andndrug transport. To characterize the transmission interface, we used a genetic screen to isolate second-sitenmutations of S558Y that restore drug transport.We recovered suppressors that restore drug resistance; theirnlocations provide functional evidence for an interface in the cis rather than the trans configuration indicatednby structural and cross-linking studies of bacterial and eukaryotic efflux transporters. One mutation, E244G,ndefines the Q-loop of the deviant portion ofNBD1, which is the hallmark of this group of fungal transporters.nWhen moved to an otherwise wild-type background, this mutation and its counterpart in the canonical ATP-nbinding siteQ951Gshow a similar reduction in drug resistance and in the very high basal-levelATP hydrolysisncharacteristic of Pdr5. A double E244G, Q951G mutant is considerablymore drug sensitive than either of thensingle mutations. Surprisingly, then, the deviant and canonical Q-loop residues are functionally overlappingnand equivalent in a strikingly asymmetric ABC transporter.