SYNTHESIS & FUNCTION OF THE YEAST ABC TRANSPORTER, STE6

细胞生理学的一个关键方面是选择性地运输 离子、营养物质、蛋白质和信号分子在细胞和 细胞膜，由膜转运蛋白介导。这个 酿酒酵母STE6蛋白是三磷酸腺苷结合蛋白的一员 盒式磁带(ABC)超家族，包括大量的膜 在真核生物中调节运输和通道功能的蛋白质和 原核生物。ABC家族临床上重要的成员包括MDR， 哺乳动物的多药耐药蛋白和CFTR蛋白 囊性纤维化患者存在缺陷。酵母中的STE6转运蛋白 调节脂肽交配信息素a因子的输出，因此 酵母交配所必需的。我们将使用生化，遗传和 STE6和TO结构和功能的分子研究进展 确定其折叠和转运所涉及的细胞成分 薄膜。 这里提出的工作的一个主要焦点是用分子术语来定义 如何实现α-因子的跨膜转运并确定 STE6是如何识别其a因子底物的。与此相关的具体目标 目标包括：1)建立STE6介导的体外系统 将允许核苷酸检查的a因子的移位 STE6的水解性和底物专一性，2)鉴定 STE6中对底物识别至关重要的残基 分离识别改变的a因子的ste6抑制突变体， 3)将多药耐药基因从药物转运体转化为优化的基因载体。 因子转运体；4)STE6功能结构域的鉴定 “显性阴性”突变体的分离。这些突变的蛋白质将是 体内STE6转运生化剖析的强效试剂 第一个目标是建立体外培养系统。 膜蛋白的功能取决于其正确的折叠和 膜插入。人们对这些因素知之甚少。 ABC等多跨膜蛋白的研究进展 传送者。该提案的第二个主要关注点是确定 参与STE6折叠和组装的细胞组件：5) 快速致病功能丧失的ste6突变体的分离 退化，以及6)随后分离恢复的抑制子 对不稳定的STE6突变蛋白的稳定性；这些抑制物是 预计识别执行的分子伴侣和折叠酶 STE6的折叠、组装和胞内转运。 这些实验将提供该函数的高分辨率视图， STE6的折叠和贩运，这也将与其他ABC相关 蛋白质。这些信息可以为预防多药耐药提供洞察力 耐药株对化疗药物耐药，并进入囊性治疗 纤维化，这是由于CFTR折叠错误或功能不正常造成的。

A critical aspect of cellular physiology is the selective transport of ions, nutrients, proteins, and signaling molecules across cellular and organellar membranes, mediated by membrane transporter proteins. The Saccharomyces cerevisiae STE6 protein is a member of the ATP binding cassette (ABC) superfamily which includes a large number of membrane proteins that mediate transport and channel functions in eukaryotes and prokaryotes. Clinically important members of the ABC family include MDR, the mammalian multidrug resistance protein, and CFTR, the protein defective in patients with cystic fibrosis. The STE6 transporter in yeast mediates export of the lipopeptide mating pheromone a-factor, .and thus is required for mating by yeast. We will use biochemical, genetic, and molecular approaches to dissect the structure and function of STE6 and to identify cellular components involved in its folding and transit to the membrane. One major focus of the work proposed here is to define in molecular terms how transport of a-factor across the membrane is achieved and to determine how STE6 recognizes its a-factor substrate. Specific aims related to this goal include: 1) development of an in vitro system for STE6-mediated translocation of a-factor that will allow examination of nucleotide hydrolysis and substrate specificity of STE6, 2) identification of residues within STE6 that are critical for substrate recognition by isolation of ste6 suppressor mutants that recognize an altered a-factor, 3) genetic conversion of MDR from a drug transporter into an optimized a- factor transporter, and 4) identification of functional domains of STE6 by isolation of "dominant negative" mutants. These mutant proteins will be powerful reagents for biochemical dissection of STE6 transport in the in vitro system developed in the first aim. The function of a membrane protein depends upon its proper folding and membrane insertion. Little is known about factors that assist these processes for multispanning membrane proteins such as the ABC transporters. A second major focus of this proposal is to identify cellular components involved in the folding and assembly of STE6 by: 5) isolation of ste6 mutants whose loss of function is due to rapid degradation, and 6) subsequent isolation of suppressors which restore stability to an unstable STE6 mutant protein; these suppressors are expected to identify molecular chaperones and foldases which execute folding, assembly, and intracellular transport of STE6. These experiments will provide a high resolution view of the function, folding, and trafficking of STE6, that will also be relevant to other ABC proteins. This information can provide insight into preventing multi-drug resistance to chemotherapeutic agents by MDR, and into treating cystic fibrosis, which results from misfolded or misfunctional CFTR.