Yibmantasiri Ploi, Bircham Peter W, Maass David R, Bellows David S, Atkinson Paul H
School of Biological Sciences, Victoria University of Wellington, Room 321, MacDiarmid Building, Kelburn, Wellington, 6012, New Zealand.
Mol Biosyst. 2014 Jan;10(1):128-37. doi: 10.1039/c3mb70351g.
The pleiotropic drug response (PDR) or multidrug resistance (MDR) are cellular defence mechanisms present in all species to deal with potential toxicity from environmental small molecule toxins or bioactives. The rapid induction of MDR by xenobiotics in mammalian cells and PDR in budding yeast (S. cerevisiae) has been well studied but how pathway specificity is achieved across different structural classes of xenobiotics is not well understood. As a novel approach to this problem we investigated the genome-wide network of genes modulating the yeast PDR. Fluorescently-tagged ABC pumps Pdr5p-GFP and Yor1p-GFP were used as real-time reporters for the Pdr1p/Pdr3p controlled response. Using the yeast non-essential gene deletion set fifty-four gene deletions that suppressed up-regulation of reporter fluorescence to the cell surface in the presence of atorvastatin were identified by high content confocal automated microscopy. Secondary validation using spot dilution assays to known PDR substrates and Western blot assays of Pdr5p expression confirmed 26 genes able to modulate the PDR phenotype. By analysis of network connectivity, an additional 10 genes that fell below the primary screen cut-off were predicted to be involved in PDR and confirmed as above. The PDR modulating genes taken together were enriched in signalling (Rho-GTPase, MAPK), Mediator complexes, and chromatin modification (subunits of ADA and SAGA complexes). Many of the gene deletions cause extra sensitivity in Δpdr1Δpdr3 strains strongly suggesting that there are alternative pathways to upregulate PDR, independently of Pdr1p/Pdr3p. We present here the first high-content microscopy screening for PDR modulators, and identify genes that are previously unsuspected regulators of PDR apparently contributing via network interactions.
多效药物反应（PDR）或多药耐药性（MDR）是所有物种中存在的细胞防御机制，用于应对环境小分子毒素或生物活性物质的潜在毒性。哺乳动物细胞中异生素对MDR的快速诱导以及芽殖酵母（酿酒酵母）中PDR的快速诱导已得到充分研究，但对于不同结构类别的异生素如何实现途径特异性尚不清楚。作为解决这个问题的一种新方法，我们研究了调节酵母PDR的全基因组基因网络。荧光标记的ABC转运蛋白Pdr5p-GFP和Yor1p-GFP被用作Pdr1p/Pdr3p控制反应的实时报告基因。使用酵母非必需基因缺失集，通过高内涵共聚焦自动显微镜鉴定出54个基因缺失，这些缺失在阿托伐他汀存在下抑制了报告荧光向细胞表面的上调。使用斑点稀释试验对已知PDR底物进行二次验证以及对Pdr5p表达进行蛋白质印迹分析，证实了26个能够调节PDR表型的基因。通过网络连通性分析，预测另外10个低于初次筛选阈值的基因参与PDR，并如上所述得到证实。共同作用的PDR调节基因在信号传导（Rho-GTPase、MAPK）、中介复合物和染色质修饰（ADA和SAGA复合物的亚基）中富集。许多基因缺失在Δpdr1Δpdr3菌株中导致额外的敏感性，强烈表明存在独立于Pdr1p/Pdr3p上调PDR的替代途径。我们在此展示了首次针对PDR调节剂的高内涵显微镜筛选，并鉴定出以前未被怀疑的PDR调节基因，这些基因显然通过网络相互作用发挥作用。
