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 C andida albicans, confers resistance to several unrelated drugs includi ng anti-Candida drugs (Prasad ct nl., 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. Wh ile the expression of Delta CDR1 in yeast resulted in impaired sensiti vity to drugs like cycloheximide, anisomycin, sulfomethuron methyl and antifungal nystatin, its ability to confer resistance remained unalte red to drugs such as o-phenanthroline, 4-nitroquinoline-N-oxide: cerul enin, azoles, oligomycin, erythromycin, and benomyl. Similar to human MDR1p, Cdr1p might also have localized drug binding sites in TM 12, bu t that might not be the case for all the drugs. The TM 12 deletion als o did not lead to any significant impairment in NTPase activities. Bot h ATPase and UTPase activities of complete Cdr1p and Delta Cdr1p were nor significantly altered, as was the case with respect to their abili ty to efflux Rh123 and steroid hormone like [H-3]-beta-estradiol. To f urther dissect the functionality of Cdr1p, its truncated version was o verexpressed in a baculovirus-insect cell expression system. The synth esis of Delta Cdr1p in Sf9 cells was temporally regulated as a functio n of the baculovirus polyhedrin gene promoter. The Sf9 derived Delta C dr1p was similar to 130 kDa, which was lower than the expected size, p robably due to the differences in glycosylation. This, however, did no t affect the functionality of Delta Cdr1p. The deletion of TM 12 did n ot affect the targeting of the protein and Delta Cdr1p was exclusively localized in plasma membrane of Sf9 cells as detected by immunofluore scence. The expression of Delta Cdr1p in the baculovirus-insect expres sion system generated a high drug-stimulated plasma membrane-bound ATP ase activity which was not demonstrable when Delta Cdr1p was expressed in yeast. (C) 1998 John Wiley & Sons, Ltd.