| The ATP-binding cassette (ABC) superfamily of transporters is one of the largest classes of proteins and is expressed in organisms from bacteria to mammals. ABC proteins have many functions including maintaining membrane lipid homeostasis, facilitating nutrient uptake, mediating cellular detoxification and enabling drug resistance. Multidrug resistance remains a major obstacle in the successful treatment of human cancers as well as parasitic, bacterial and fungal infections. This problem is due, in part, to the overexpression of ABC multidrug efflux pumps. Pleiotropic Drug Response 5 gene ( PDR5) in Saccharomyces cerevisiae defines a subclass of fungal ABC efflux pumps. Overexpression of Pdr5 leads to hyper resistance to all tested substrates. We show that it is possible to select for naturally occurring mutations that further increase resistance, ultra resistance, by exposing these already hyper resistant strains to inhibitory concentrations of drugs. We were able to identify five PDR5 point mutations that confer ultra resistance to cycloheximide.;Full length ABC transporters are organized into two nucleotide binding domains and two transmembrane domains. Each transmembrane domain is composed of six membrane spanning alpha-helices connected by intracellular and extracellular loops. The energy from ATP binding and/or hydrolysis at the nucleotide binding domains is transmitted through a signal interface to effect conformational changes in the transmembrane domains allowing for drug efflux. The ICLs are part of this signal interface. Two of the cycloheximide ultra resistant mutations map to conserved residues of the ICLs. Surprisingly, these ultra resistant strains do not confer broad spectrum antifungal resistance. It is of great interest to understand how mutations in the signal interface can confer increased resistance to a single or subset of drug substrates, as these mechanisms may lead to better understanding of how the signal interface functions. One mutant, P596L, maps to ICL1 and increases resistance to only cycloheximide. A second mutant, V656L, maps to ICL2 and confers ultra resistance to a subset of Pdr5 substrates; however, there is also an increase in sensitivity to ATPase activity inhibition by certain drugs in this mutant strain. I propose several models to explain these observed changes in Pdr5 resistance phenotypes. |
