title: The transmission interface of the Saccharomyces cerevisiae multidrug transporter Pdr5: Val-656 located in intracellular loop 2 plays a major role in drug resistance.
abstract: Pdr5 is a major ATP-binding cassette (ABC) multidrug transporter regarded as the founding member of a fungal subfamily of clinically significant efflux pumps. When these proteins are overexpressed, they confer broad-spectrum ultraresistance. To better understand the evolution of these proteins under selective pressure, we exposed a Saccharomyces cerevisiae yeast strain already overexpressing Pdr5 to a lethal concentration of cycloheximide. This approach gave mutations that confer greater resistance to a subset of transport substrates. One of these mutations, V656L, is located in intracellular loop 2 (ICL2), a region predicted by structural studies with several other ABC transporters to play a critical role in the transmission interface between the ATP hydrolysis and drug transport domains. We show that this mutation increases drug resistance, possibly by altering the efficiency with which the energy from ATP hydrolysis is used for transport. Val-656 is a conserved residue, and an alanine substitution creates a nearly null phenotype for drug transport as well as reduced ATPase activity. We posit that despite its unusually small size, ICL2 is part of the transmission interface, and that alterations in this pathway can increase or decrease resistance to a broad spectrum of drugs.
authors.affiliation: Department of Biology, The Catholic University of America, Washington, District of Columbia, USA.
authors.forename: Marianne T
authors.initials: MT
authors.lastname: Downes
authors.forename: Jitender
authors.initials: J
authors.lastname: Mehla
authors.forename: Neeti
authors.initials: N
authors.lastname: Ananthaswamy
authors.forename: Adina
authors.initials: A
authors.lastname: Wakschlag
authors.forename: Micheala
authors.initials: M
authors.lastname: Lamonde
authors.forename: Elliot
authors.initials: E
authors.lastname: Dine
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.forename: John
authors.initials: J
authors.lastname: Golin
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000235:genetics; D003513:Cycloheximide / Q000378:metabolism* / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D026901:Membrane Transport Proteins; C092438:PDR5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide