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

Abstract

ABC transporters are polytopic proteins. ATP hydrolysis and substrate transport take place in separate domains, and these activities must be coordinated through a signal interface. We previously characterized a mutation (S558Y) in the yeast multidrug transporter Pdr5 that uncouples ATP hydrolysis and drug transport. To characterize the transmission interface, we used a genetic screen to isolate second-site mutations of S558Y that restore drug transport. We recovered suppressors that restore drug resistance; their locations provide functional evidence for an interface in the cis rather than the trans configuration indicated by structural and crosslinking studies of bacterial and eukaryotic efflux transporters. One mutation, E244G, defines the Q-loop of the deviant portion of NBD1, which is the hallmark of this group of fungal transporters. When moved to an otherwise wild-type background, this mutation and its counterpart in the canonical ATP-binding site Q951G show a similar reduction in drug resistance and in the very high basal-level ATP hydrolysis characteristic of Pdr5. A double E244G, Q951G mutant is considerably more drug sensitive than either of the single mutations. Surprisingly, then, the deviant and canonical Q-loop residues are functionally overlapping and equivalent in a strikingly asymmetric ABC transporter.

Evidence for a Molecular Diode-based Mechanism in a Multispecific ATP-binding cassette (ABC) Exporter

“…We determined the cell density after 24-h rather than 48-h growth. Therefore, our IC 50 values were somewhat different from those reported previously (14,18,20).…”

“…We confirmed that the transformant we used had a mutant and a WT allele by plating cells on 5-fluororotic acid medium (17,18) and qualitatively testing the resulting segregants for their relative clo resistance by replica plating. We recovered both WT and S1368A mutant alleles.…”

“…Most show at least some reduction in ATPase activity (14,17,18,20). V656A (intracellular loop-2) and S558Y (TMH2), which exhibited severe, broad drug hypersensitivity much like S1368A, also had a Pdr5-specific ATPase that was more resistant to allosteric inhibition by clo.…”

“…This strain offers numerous other advantages for genetic and biochemical analyses, which are described in detail elsewhere (17,18). We cultured the strains at 30°C.…”

“…Consistently good amplification requires relatively pure DNA (260/ 280, ϳ2.0; 260/230, ϳ2.2) at a concentration of 60 -120 ng. We performed 40 rounds of amplification as described previously (18). We sent the PCR product of ϳ4.5 kbp to SeqWright (Houston, TX) for purification and sequencing.…”