The amino acid residues of transmembrane helix 5 of multidrug resistance protein CaCdr1p of Candida albicans are involved in substrate specificity and drug transport.
In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida albicans in azole resistance, we have characterized its ability to efflux variety of substrates by subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant variants of putative 21 amino acids of TMS 5 and native CaCdr1p were over expressed as a GFP-tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility pattern, the mutant variants could be grouped into two categories. The variants belonging to first category were susceptible to all the tested drugs, as compared to those belonging to second category which exhibited resistance to selective drugs. The mutant variants of both the categories were analyzed for their ATP catalysis and drug efflux properties. Irrespective of the categories, most of the mutant variants of TMS 5 showed an uncoupling between ATP hydrolysis and drug efflux. The mutant variants such as M667A, F673A, I675A and P678A were an exception since they reflected a sharp reduction in both K(m) and V(max) values of ATPase activity when compared with WT CaCdr1p-GFP. Based on the competition experiments, we could identify TMS 5 residues which are specific to interact with select drugs. TMS 5 residues of CaCdr1p thus not only impart substrate specificity but also selectively act as a communication link between ATP hydrolysis and drug transport.
Puri
Nidhi
N
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Gaur
Manisha
M
Sharma
Monika
M
Shukla
Suneet
S
Ambudkar
Suresh V
SV
Prasad
Rajendra
R
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D015725:Fluconazole / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017964:Itraconazole / Q000378:metabolism; D007654:Ketoconazole / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008825:Miconazole / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism; D021381:Protein Transport; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity
D001386:Azides; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012235:Rhodamines; C026188:rhodamine 6G; D017964:Itraconazole; D008825:Miconazole; D015725:Fluconazole; C041552:azidoprazosin; D000251:Adenosine Triphosphatases; D007654:Ketoconazole; D011224:Prazosin