Deletion of transmembrane domain 12 of CDR1, a multidrug transporter from Candida albicans, leads to altered drug specificity: expression of a yeast multidrug transporter in baculovirus expression system.
Cdr1p, an ATP-binding cassette transporter from the pathogenic yeast Candida albicans, confers resistance to several unrelated drugs including anti-Candida drugs (Prasad et al., 1995b). We demonstrate that the deletion of 237 bp (79 aa) from the 3' end of CDR1 (which encompasses the transmembrane domain (TM) 12 of the putative transporter) did not result in the total loss of its ability to efflux cytotoxic agents. While the expression of deltaCDR1 in yeast resulted in impaired sensitivity to drugs like cycloheximide, anisomycin, sulfomethuron methyl and antifungal nystatin, its ability to confer resistance remained unaltered to drugs such as o-phenanthroline, 4-nitroquinoline-N-oxide, cerulenin, azoles, oligomycin, erythromycin, and benomyl. Similar to human MDR1p. Cdr1p might also have localized drug binding sites in TM 12, but that might not be the case for all the drugs. The TM 12 deletion also did not lead to any significant impairment in NTPase activities. Both ATPase and UTPase activities of complete Cdr1p and deltaCdr1p were not significantly altered, as was the case with respect to their ability to efflux Rh123 and steroid hormone like [3H]-beta-estradiol. To further dissect the functionality of Cdr1p, its truncated version was overexpressed in a baculovirus-insect cell expression system. The synthesis of deltaCdr1p in Sf9 cells was temporally regulated as a function of the baculovirus polyhedrin gene promoter. The Sf9 derived deltaCdr1p was approximately 130 kDa, which was lower than the expected size, probably due to the differences in glycosylation. This, however, did not affect the functionality of deltaCdr1p. The deletion of TM 12 did not affect the targeting of the protein and deltaCdr1p was exclusively localized in plasma membrane of Sf9 cells as detected by immunofluorescence. The expression of deltaCdr1p in the baculovirus-insect expression system generated a high drug-stimulated plasma membrane-bound ATPase activity which was not demonstrable when deltaCdr1p was expressed in yeast.
Krishnamurthy
S
S
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Chatterjee
U
U
Gupta
V
V
Prasad
R
R
Das
P
P
Snehlata
P
P
Hasnain
S E
SE
Prasad
R
R
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000187:drug effects / Q000235:genetics*; D001692:Biological Transport; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D004958:Estradiol / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D017384:Sequence Deletion; D018411:Spodoptera
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D012235:Rhodamines; D020112:Rhodamine 123; D004958:Estradiol; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C023278:uridine triphosphatase