Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
Biochem Biophys Res Commun. 2011 Jan 7;404(1):357-63. doi: 10.1016/j.bbrc.2010.11.123. Epub 2010 Dec 4.
We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.
我们利用匹配的唑类敏感（AS）和唑类耐药（AR）临床分离株的天然环境，表达了其主要的 ABC 多药转运蛋白 CgCdr1p，用于结构和功能分析。这是通过在 CgCDR1 的 ORF 下游标记绿色荧光蛋白（GFP），并将所得融合蛋白整合到 AS 和 AR 背景下的其天然染色体位置来完成的。表征证实，与 AS 分离株相比，由于其超活性天然启动子，CgCdr1p-GFP 在 AR 分离株中过表达，并且 GFP 标签在两种构建体中均不影响其功能。我们观察到，除了 Rhodamine 6 G（R6G）和氟康唑（FLC）外，最近鉴定的多药转运蛋白荧光底物尼罗红（NR）也可以被 CgCdr1p 排出。与这些底物的竞争测定显示，CgCdr1p 中存在重叠的多个药物结合位点。点突变利用定点诱变证实，对 ATP 催化和 ABC 药物转运蛋白定位至关重要的独特氨基酸残基的作用在 C. glabrata 中与其他酵母一样得到很好的保守。这项研究展示了第一个体内新型系统，其中 GFP 标记的 MDR 转运蛋白的过表达可以由 AR 分离株的自身超活性启动子驱动。总之，这个体内系统可以用于 CgCdr1p 和类似蛋白的结构和功能分析，其中在使用异源系统时遇到的人为问题完全排除。
