Analysis of proteins that bind promoter of the PDR5 multidrug transporter gene in Saccharomyces cerevisiae using minichromosome isolation and LC-MS/MS

The Pleiotropic Drug Resistance (PDR) network is central to the drug response in fungi, and its overactivation is associated with drug resistance. However, gene regulation of the PDR network is not well understood. Here, we established a method to identify proteins that bind promoter of the PDR5 multidrug transporter gene in Saccharomyces cerevisiae using minichromosome isolation and SILAC-based quantitative proteomics, and identified the SWI/SNF chromatin remodelling complex as a PDR5 promoter-binding complex. We also purified the SWI/SNF complex from S. cerevisiae by immunoprecipitating Flag-tagged Snf6, a subunit of SWI/SNF, and identified the subunits of SWI/SNF and its binding proteins by LC-MS/MS.

多药耐药（Pleiotropic Drug Resistance, PDR）网络是真菌药物应答过程中的核心调控网络，其过度激活与药物耐药性密切相关。然而，目前学界对PDR网络的基因调控机制尚未得到充分阐明。本研究建立了一种通过微染色体分离与基于稳定同位素标记氨基酸培养（SILAC）的定量蛋白质组学技术，鉴定酿酒酵母（Saccharomyces cerevisiae）中多药转运蛋白基因PDR5的启动子结合蛋白的方法，并成功鉴定出SWI/SNF染色质重塑复合物为PDR5的启动子结合复合物。此外，我们通过免疫沉淀带Flag标签的SWI/SNF复合物亚基Snf6，从酿酒酵母中纯化得到SWI/SNF复合物，并借助液相色谱-串联质谱联用（LC-MS/MS）技术鉴定了该复合物的亚基及其结合蛋白。