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

Abstract

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 ΔCDR1 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 ΔCdr1p were not significantly altered, as was the case with respect to their ability to efflux Rh123 and steroid hormone like [³H]-β-estradiol. To further dissect the functionality of Cdr1p, its truncated version was overexpressed in a baculovirus-insect cell expression system. The synthesis of ΔCdr1p in Sƒ9 cells was temporally regulated as a function of the baculovirus polyhedrin gene promoter. The Sƒ9 derived ΔCdr1p was ~130 kDa, which was lower than the expected size, probably due to the differences in glycosylation. This, however, did not affect the functionality of ΔCdr1p. The deletion of TM 12 did not affect the targeting of the protein and ΔCdr1p was exclusively localized in plasma membrane of Sƒ9 cells as detected by immunofluorescence. The expression of ΔCdr1p in the baculovirus-insect expression system generated a high drug-stimulated plasma membrane-bound ATPase activity which was not demonstrable when ΔCdr1p was expressed in yeast.