Functional characterization of Candida albicans ABC transporter Cdr1p.
In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and DeltaF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (DeltaF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized DeltaF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).
Shukla
Suneet
S
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Saini
Preeti
P
Smriti
Jha
Sudhakar
S
Ambudkar
Suresh V
SV
Prasad
Rajendra
R
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002176:Candida albicans / Q000187:drug effects / Q000378:metabolism*; D017124:Conserved Sequence; D003513:Cycloheximide / Q000494:pharmacology; D026141:Drug Resistance, Multiple, Fungal; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012235:Rhodamines; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D012235:Rhodamines; C026188:rhodamine 6G; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D000251:Adenosine Triphosphatases