Trafficking of ABC proteins in yeast

描述（由申请人提供）：ER“质量控制”（ERQC）是一种基本和保守的细胞过程，可防止错误折叠的分泌蛋白和膜蛋白从ER中流出。ERQC由两个连续过程组成：1）未折叠蛋白反应（UPR），指的是基因（如伴侣蛋白）的转录上调，使细胞能够科普错误折叠的蛋白质，其次是2）ER相关降解（ERAD），其中错误折叠的ER保留蛋白被泛素-蛋白酶体系统降解。最近的研究表明，对于膜蛋白，基于其错误折叠损伤的拓扑位置，存在两类ERAD底物，管腔（L）或胞质（C）。在本项目中，我们提出了这一观点的扩展，即细胞采用两个机制不同的分支ER质量控制：ERQC-L（包括UPR-L和ERAD-L）和ERQC-C（包括UPR-C和ERAD-C），以科普基板的结构域是管腔或胞质，分别。该项目的长期目标是识别和机械解剖酿酒酵母中ERQC-C途径的组成和工作，并确定ERQC-C与ERQC-L的区别。ERQC不同分支的证据是基于我们对酵母ATP结合盒（ABC）转运蛋白Ste 6p和Ycflp突变形式的研究，这些突变形式受ERQC影响。使用这些作为模型ERQC-C底物，我们在该项目中取得了重大进展，包括定义一个突出的ER隔室（ERAC）作为UPR-C的标记，定义ERAD-C和ERAD-L之间的机械差异，并初步了解UPR-C和UPR-L的转录诱导谱的差异。这些调查结果为本提案奠定了基础。本论文将应用传统的酵母遗传学、分子生物学和细胞生物学方法，通过对UPR-C信号通路的研究，阐明UPR-C信号通路中的关键调控因子、上调基因以及UPR-C胁迫诱导的细胞保护机制; 2）通过探索E3泛素连接酶的底物特异性和鉴定新的ERAD组分来确定ERAD-C途径的机制、步骤和机制; 3）进一步开发MRP蛋白作为ERQC模型底物，特别是对ERQC和ER退出之间的关系有新的认识。我们的研究有望揭示多种膜蛋白运输疾病，最好的例子是囊性纤维化，其最常见的结果是ER保留和CFTR-deltaF 508的降解。

DESCRIPTION (provided by applicant): ER "quality control" (ERQC) is a fundamental and conserved cellular process that prevents the exit of misfolded secretory and membrane proteins from the ER. ERQC consists of two sequential processes: 1) the unfolded protein response (UPR), which refers to the transcriptional upregulation of genes such as chaperones that enable the cell to cope with misfolded proteins, followed by 2) ER-associated degradation (ERAD), whereby misfolded ER-retained proteins are degraded by the ubiquitin-proteasome system. Recent studies suggest that for membrane proteins there are two classes of ERAD substrates, based on the topological location of their misfolded lesion, either luminal (L) or cytosolic (C). In the present project, we propose an extension of this view, namely that cells employ two mechanistically distinct branches of ER quality control: ERQC-L (comprising UPR-L and ERAD-L) and ERQC-C (comprising UPR-C and ERAD-C), to cope with substrates whose domains are luminal or cytosolic, respectively. The long-term goal of this project is to identify and mechanistically dissect the components and workings of the ERQC-C pathway in Saccharomyces cerevisiae, and determine how ERQC-C differs from ERQC-L. Evidence for distinct branches of ERQC is based on our studies of mutant forms of the yeast ATP-binding cassette (ABC) transporters Ste6p and Ycflp, that are subject to ERQC. Using these as model ERQC-C substrates, we have made significant advances in this project that include defining a prominent ER compartment (the ERAC) as a marker for UPR-C, defining differences in machinery between the ERAD-C and ERAD-L, and gaining an initial glimpse into differences in the transcriptional induction profiles of UPR-C and UPR-L. These findings set the stage for the present proposal. Here, we will apply traditional and high-throughput yeast genetic, molecular, and cell biological methodologies to accomplish the following aims: 1) To elucidate the circuitry of the UPR-C signaling pathway by defining the key regulators, upregulated genes, and cytoprotective mechanisms evoked by a UPR-C stress; 2) To define the machinery, steps, and mechanism of the ERAD-C pathway by probing the substrate specificity of E3 ubiquitin ligases and identifying novel ERAD components; and 3) To further develop MRP proteins as model ERQC substrates, in particular to gain new insights into the relationship between ERQC and ER exit. Our studies are expected to shed light on a diverse array of membrane protein trafficking diseases, best exemplified by cystic fibrosis, which most commonly results from the ER-retention and degradation of CFTR-deltaF508.