| ATP-binding cassette (ABC) transporters are present in all known organisms and comprise one of the largest families of integral membrane proteins. Clinically, ABC transporters are associated with numerous diseases, cause multidrug resistance in various types of cancer, and cause antifungal resistance in pathogenic yeasts. Pdr5 is a plasma membrane bound multidrug transporter and the founding member of the Pdr subfamily of ABC transporters.;In ABC transporters, the nucleotide binding domains dimerize to form two nucleotide binding sites, which are composed of highly conserved motifs. The deviant ATP-binding site, a unique feature of the Pdr subfamily, has been found to transmit signals between the various domains of Pdr5, specifically through the Q- loop (Ananthaswamy et al. 2010) and D-loop (Furman et al. 2013) motifs, and to facilitate ATP hydrolysis at its canonical ATP-binding site (Gupta et al. 2014). A Pdr5 deviant signature mutant, E1013A has reduced drug resistance, drug transport capability, and ATPase activity. In order to elucidate the role of the deviant signature motif, we isolated suppressors of E1013A. Two suppressors, SUP5 and SUP6, exhibited dominant gain-of-function phenotypes that significantly restored E1013A drug resistance independent of overexpressing Pdr5. Sup5 suppression fully restored the ATPase activity of Pdr5, while Sup6 suppression occurred via a different mechanism. Significantly, Sup5 and Sup6 were unable to rescue the loss-of-function phenotype present in the canonical signature mutant, G312A, suggesting that both are extragenic suppressors that interact specifically with the deviant ATP-binding site of Pdr5.;We verified and characterized the drug resistance phenotype, ATPase activity, and plasma membrane abundance of Pdr5 for SUP5 and SUP6. Several genetic mapping and screening methods were utilized to identify SUP5 and SUP6, but a successful Yep24 overexpression screen identified SKS1, a putative serine/threonine kinase. The overexpression of SKS1 in the E1013A mutant strain mediated multidrug resistance to clotrimazole and cerulenin, possibly by phosphorylating the linker 2 region of Pdr5 (Johnson et al. 2014). Sks1-mediated phosphorylation also conferred hyper-resistance to WT Pdr5 in a manner that did not increase the ATPase activity or increase the abundance of Pdr5 in the plasma membrane. A Sks1 K39I mutation was unable to suppress the E1013A mutant. Despite common expectations, knocking out SKS1 in WT had no decrease in drug resistance, which suggests that Sks1 is not essential for Pdr5-mediated drug resistance.;We proposed a mechanism for in vivo regulation of Pdr5 in low glucose and potential drug conditions based on evidence that positions Sks1 in the cAMP-PKA pathway, a pathway that controls cell division and stress responses. Sks1 has a homolog, SHA3, in C. albicans which functions in the cAMP-PKA pathway and is required for hyphal development and virulence. An SKS1 homolog, SNFK , was also found in humans, thus providing greater relevance for elucidating the Sks1 signaling network. |
