Molecular Genetic Analysis of Pdr5p, a Major Yeast Multidrug Transporter

项目摘要 多药转运蛋白的过表达导致广谱耐药性的增加是一种 病原体和恶性肿瘤治疗中的主要医学问题。我们使用生化和 研究Pdr5的遗传学方法，Pdr5是一种主要的酵母外排泵，它是一个大型 临床上重要的真菌ABC转运蛋白亚家族。 这项提案关注的是所有ABC出口商都面临的一个核心问题。一旦有毒药物被排出 细胞，如何防止它通过药物结合部位重新进入，这些结合部位现在面临着 胞外方向？亲和力降低10-30倍，这通常发生在 运输周期不足以排除回流。我们实验室的工作确定了Pdr5 传输是单向的，因此这种外排泵是一个分子二极管。我们还确认了 一种残基，Ser-1368，是二极管功能所必需的。产生S1368A突变体的替代 当执行一种新的二极管分析时，显示出相当大的回流。 目前的建议有两个目的：第一，结合使用突变抑制和定点定位 在突变过程中，我们将确定组成Pdr5二极管的更多残基。这些突变体将会是 使用几种生化分析方法进行了仔细的表征，其中包括一种专用于二极管功能的生化分析方法。 一个中心问题是这种转运体的两个部分是否都参与了二极管的形成，或者是否类似 其他几个生化过程的二极管功能是不对称的。这项建议的第二个目的是 对二极管进行生化表征。例如，我们将确定是否有任何底物 二极管操作的专属性。三磷酸腺苷水解酶的作用和已确定的Pdr5传递 将评估二极管功能中的界面。

Project Summary Overexpression of multidrug transporters leading to increased, broad-spectrum drug resistance is a major medical problem in the treatment of pathogens and malignant tumors. We use biochemical and genetic approaches to study Pdr5, a major yeast efflux pump that is the founding member of a large subfamily of clinically important fungal ABC transporters. This proposal focuses on a central issue common to all ABC exporters. Once a toxic drug is expelled from the cell, how is it prevented from reentry via the drug-binding sites which are now facing in an extracellular direction? A simple 10-30 fold reduction in affinity which is typically seen during the transport cycle is not sufficient to preclude reflux. Work in our laboratory established that Pdr5 transport is unidirectional and that this efflux pump is therefore a molecular diode. We also identified one residue, Ser-1368 that is essential for diode function. A substitution creating a S1368A mutant shows considerable reflux when a novel diode assay is performed. The present proposal has two aims: First, using a combination of mutant suppression and site-directed mutagenesis, we will identify additional residues that make up the Pdr5 diode. These mutants will be carefully characterized using several biochemical assays including one that is specific for diode function. A central issue is whether both halves of this transporter participate in diode formation or whether like several other biochemical processes diode function is asymmetric. The second aim of the proposal is to biochemically characterize the diode. For instance, we will determine whether there is any substrate specificity to diode operation. The role of ATP hydrolysis and the well-established Pdr5 transmission interface in diode function will be evaluated.