SYNTHESIS AND FUNCTION OF THE YEAST ABC TRANSPORTER STE6

描述（来自申请人的摘要）适当的细胞内折叠和 膜蛋白的运输，包括转运蛋白、通道和 受体是促成其功能的关键事件。步骤6p，交配 酵母中的信息素a因子转运体，被用作 剖析多跨膜蛋白的运输步骤。STE 6P是一个 ATP结合盒（ABC）超家族的成员，包括囊性 纤维化蛋白CFTR、多药耐药转运蛋白和相关蛋白 对人类健康和疾病至关重要。本文提出的研究重点是两个 代表血浆关键调控点的Step 6p运输方面 膜蛋白：1）ER质量控制，与ER出口交织的过程， （2）内吞作用。申请人列举了先前的几项进展， 项目期间：a）确定不同类别的ER保留ste 6 突变体，其在ER质量控制中显示出相当大的复杂性; B） 发现了一个突出的新结构，ER相关体（ERAB）， 在某些ste 6突变体的反应中形成，并可能提供关于ER的线索。 质量控制; c）证明泛素在ER质量中的作用 控制和内吞作用。这些发现为目前的研究奠定了基础 识别参与“决策”方面的细胞成分的目标 内质网的退出和内吞。更新列出了以下目标：1）隔离 ER保留的ste 6突变体的染色体抑制子和增强子，以鉴定 参与ER质量控制和/或ER退出的细胞组分。（二） 鉴定ERAB中富集的或在其诱导下诱导的细胞组分。 形成，通过生化，显微镜和微阵列分析。3)建立 通过突变体研究，有序的事件途径（磷酸化-> 泛素化->内吞作用（PUE途径）是Ste 6p内化所需的， 并从遗传学上鉴定新的Ste 6p内吞特异性因子。4)比较 导致ER相关降解（ERAD）的亚基化要求 与WT Ste 6p的内吞作用相比。人膜蛋白 “贩运疾病”很多。申请人希望这些努力能够 确定参与贩运的细胞成分将为 为这些疾病和相关疾病设计合理的药物。

DESCRIPTION (from applicant's Abstract) The proper intracellular folding and trafficking of membrane proteins, including transporters, channels, and receptors are key events that contribuute to their function. Ste6p, the mating pheromone a-factor transporter in yeast, is being used as a model protein for dissecting the trafficking steps of mulitspanning membrane proteins. Ste6p is a member of the ATP binding cassette (ABC) superfamily that includes the cystic fibrosis protein CFTR, multidrug resistance transporters, and related proteins important in human health and disease. Studies proposed here focus on two aspects of Ste6p trafficking that represent key regulatory points for a plasma membrane protein: 1) ER quality control, a process intertwined with ER exit, and 2) endocytosis. The applicant cites several advances in the previous project period: a) The identification of distinct classes of ER-retained ste6 mutants, which revealed considerable complexity in ER quality control; b) Discovery of a prominent novel structure, the ER-associated body (ERAB) that is formed in response to certain ste6 mutants and may provide clues about ER quality control; c) Demonstration of a role for ubiquitin both in ER quality control and endocytosis of Ste6p. These findings set the stage for the present goal of identifying cellular components involved in "decision making" aspects of ER exit and endocytosis. The renewal lists the following aims: 1) Isolate chromosomal suppressors and enhancers of ER-retained ste6 mutants to identify cellular components that participate in ER quality control and/or ER exit. 2) Identify cellular components enriched within ERABs or induced upon their formation, by biochemical, microscopic, and microarray analysis. 3) Establish by mutant studies an ordered pathway of events (phosphorylation -> ubiquitination -> endocytosis (PUE pathway) required for Ste6p internalization, and genetically identify novel Ste6p endocytosis specficity factors. 4) Compare the requirements for ubqiuitination leading to ER-associated degradation (ERAD) of mutant Ste6p versus endocytosis of WT Ste6p. Human membrane protein "trafficking diseases" are numerous. The applicant hopes that these efforts to identify cellular components involved in trafficking will provide targets for rational drug design for these and related diseases.