SYNTHESIS AND FUNCTION OF THE YEAST ABC TRANSPORTER STE6

描述(摘自申请人摘要)正确的细胞内折叠和 膜蛋白的运输，包括转运体、通道和 受体是影响其功能的关键事件。第六步，交配 酵母菌中的信息素a因子转运体正被用作 剖析了多跨膜蛋白的转运步骤。步骤6P是一个 三磷酸腺苷结合盒(ABC)超家族的成员，包括囊状 纤维化蛋白cftr、多药耐药转运蛋白及相关蛋白 对人类健康和疾病很重要。这里提出的研究集中在两个方面 代表血浆关键调节点的Ste6P贩运的各个方面 膜蛋白：1)ER质控，一个与ER出口交织在一起的过程， (2)内吞作用。申请人引用了前一年中的几个预付款 项目期间：a)确定保留ER的不同类别的Step 6 突变体，揭示了ER质量控制的相当复杂；b) 发现了一种突出的新结构，内质网相关小体(ERAB)，即 在对某些ste6突变体的反应中形成，可能提供有关ER的线索 质量控制；c)展示泛素在ER质量中的作用 Ste6p的控制和内吞作用。这些发现为目前的研究奠定了基础 确定“决策”方面涉及的元胞组件的目标 内质网退出和内吞作用。更新列出了以下目标：1)隔离 ER保留的ste6突变体的染色体抑制子和增强子鉴定 参与ER质量控制和/或ER退出的蜂窝组件。2) 识别在ERAB中丰富的细胞成分或根据其诱导的细胞成分 形成，通过生化、显微和微阵列分析。3)建立 通过突变研究事件的有序途径(磷酸化-&GT； 泛素化--Ste6p内化所需的内吞作用(PUE途径) 并从基因上鉴定新的Ste6p内吞特异性因子。4)比较 导致内质网相关降解(ERAD)的子化要求 突变的Ste6p与WT Ste6p的内吞作用。人膜蛋白 “贩卖疾病”不计其数。申请人希望这些努力能够 识别参与贩运的蜂窝组件将为以下目标提供目标 针对这些疾病及相关疾病的合理药物设计。

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.