Leishmania multidrug resistance (MDR) proteins of ATP-binding cassette (ABC) transporters : subcellular localization of and the role in drug resistance

最近对三磷酸腺苷结合盒(ABC)转运蛋白的研究进展表明，ABC转运蛋白在包括原生动物在内的各种生物的耐药性中发挥着重要作用。我们先前在亚马逊利什曼原虫中分离到了ABC蛋白的两个不同的多药耐药(MDR)亚家族基因LaMDR1和LaMDR2。我们发现LaMDR1是哺乳动物MDR1的同源基因，而LaMDR2是一个新的MDR基因。LaMDR2的过表达使其对5-氟尿嘧啶(5-FU)产生耐药性，药物摄取实验表明，LaMDR2可减少5-FU在转染体中的累积。在本研究中，我们检测了mdr2是定位于细胞膜并将5-FU排出细胞外，还是定位于细胞内细胞膜并将药物隔离在细胞器内。用抗LaMDR2多肽抗体进行Western印迹分析表明，在对数相中，LaMDR2在转染体中的表达明显高于稳定期。用RT-PCR在野生型细胞中检测到相应的蛋白。间接免疫荧光研究表明，这些抗体染色的是未知的细胞质结构，而不是质膜。荧光和激光共聚焦扫描显微镜研究表明，标记了绿色荧光蛋白(GFP)的LaMDR2存在于细胞内的管状、管状或圆形结构中。GFP信号在对数时相细胞中最强，但在不同的转染体中信号模式不同。GFP荧光的分布与溶酶体标记物基本重叠，但与线粒体标记物不重叠。这些发现表明，LaMDR2位于溶酶体的膜上，将5-FU转运到管腔内，DUG通过随后的胞吐作用被挤出细胞外。

Recent progress in research for ATP-binding cassette (ABC) transporters have shown their important roles in drug resistance in various organisms, including protozoan parasites. We previously isolated two different multidrug resistance (MDR) subfamily genes of the ABC proteins, LaMDR1 and LaMDR2, in Leishmania amazonensis. We found that the LaMDR1 was a homologue to mammalian MDR1, but the LaMDR2 was a novel MDR gene. Overexpression of the LaMDR2 conferred resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used as an anticancer drug, and drug uptake experiments demonstrated a decrease accumulation of 5-FU in transfectants with the LaMDR2. In the present study, we examined whether MDR2 is located in the plasma membrane and extrudes 5-FU out of the cell, or it is localized in the membrane of intracellular organelle and sequesters the drug inside the organelle. Western blot analysis using anti-LaMDR2 peptide antibodies indicated that the expression of LaMDR2 was much higher in the transfectants in the log-phase than the stationary phase. The corresponding protein was detected by RT-PCR in wild-type cells. Indirect immunofluorescence studies showed that the antibodies stained unknown cytoplasmic structures, but not the plasma membrane. Fluorescent and confocal scanning laser microscopic studies revealed that LaMDR2 tagged with green fluorescent protein (GFP) was found in the intracellular tubular, canal or round-shape structure. The GFP signal was most intense in the log-phase cells, but the signal patters varied in individual transfectants. The distribution of the GFP fluorescence was mostly overlapped with that of a lysosome marker, but not with that of a mitochondria marker. These findings suggest that LaMDR2 is located in the membrane of the lysosome and transports 5-FU into the lumen, and that the dug is extruded out of cell by subsequent exocytosis.