Understanding Pdr5 function through cycloheximide ultra resistant mutations

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 bindin