title: ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry.
abstract: In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.
authors.affiliation: Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Sudhakar
authors.initials: S
authors.lastname: Jha
authors.forename: Neelam
authors.initials: N
authors.lastname: Dabas
authors.forename: Neerja
authors.initials: N
authors.lastname: Karnani
authors.forename: Preeti
authors.initials: P
authors.lastname: Saini
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D000251:Adenosine Triphosphatases / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D015153:Blotting, Western; D002176:Candida albicans / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014170:Transformation, Genetic / Q000502:physiology
chemical_list: D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D000251:Adenosine Triphosphatases