Krishnamurthy S, Chatterjee U, Gupta V, Prasad R, Das P, Snehlata P, Hasnain S E, Prasad R
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Yeast. 1998 Apr 30;14(6):535-50. doi: 10.1002/(SICI)1097-0061(19980430)14:6<535::AID-YEA254>3.0.CO;2-5.
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.
Cdr1p是一种来自致病性酵母白色念珠菌的ATP结合盒转运蛋白，它能使细胞对几种不相关的药物产生抗性，包括抗念珠菌药物（Prasad等人，1995b）。我们证明，从CDR1的3'末端缺失237 bp（79个氨基酸）（其中包含假定转运蛋白的跨膜结构域（TM）12）并不会导致其排出细胞毒性药物的能力完全丧失。虽然酵母中deltaCDR1的表达导致对环己酰亚胺、茴香霉素、甲磺隆和抗真菌药物制霉菌素等药物的敏感性受损，但其赋予抗性的能力对邻菲罗啉、4-硝基喹啉-N-氧化物、浅蓝菌素、唑类、寡霉素、红霉素和苯菌灵等药物保持不变。与人类MDR1p相似，Cdr1p可能在TM 12中也有定位的药物结合位点，但并非所有药物都是如此。TM 12的缺失也没有导致NTPase活性的任何显著损害。完整的Cdr1p和deltaCdr1p的ATPase和UTPase活性均未显著改变，它们排出罗丹明123和类固醇激素如[3H]-β-雌二醇的能力也是如此。为了进一步剖析Cdr1p的功能，其截短版本在杆状病毒-昆虫细胞表达系统中过表达。Sf9细胞中deltaCdr1p的合成作为杆状病毒多角体蛋白基因启动子的函数受到时间调控。Sf9细胞衍生的deltaCdr1p约为130 kDa，低于预期大小，这可能是由于糖基化的差异。然而，这并不影响deltaCdr1p的功能。TM 12的缺失不影响该蛋白的靶向定位，通过免疫荧光检测发现deltaCdr1p仅定位于Sf9细胞的质膜中。杆状病毒-昆虫表达系统中deltaCdr1p的表达产生了高药物刺激的质膜结合ATPase活性，而当deltaCdr1p在酵母中表达时则未表现出这种活性。
