Trafficking of ABC proteins in yeast

描述（由申请人提供）：内质网“质量控制”（ERQC）是一个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内质网流出。ERQC包括两个连续的过程：1)未折叠蛋白反应（UPR），指的是使细胞能够应对错误折叠蛋白的伴侣等基因的转录上调，然后是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，基于其错误折叠病变的拓扑位置，膜蛋白有两类ERAD底物，即管腔(L)或细胞质(C)。在本项目中，我们提出了这一观点的扩展，即细胞采用两个机械上不同的内质网质量控制分支：ERQC-L（包括urel - l和errad - l）和ERQC-C（包括urel - c和errad - c），分别处理结构域为管腔或细胞质的底物。该项目的长期目标是鉴定和机械剖析酿酒酵母ERQC-C途径的组成和工作原理，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）转运体Ste6p和Ycflp的突变形式的研究，它们受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，包括定义了一个突出的ER区（ERAC）作为UPR-C的标记，定义了erd - c和erd - l之间的机制差异，并初步了解了UPR-C和UPR-L转录诱导谱的差异。这些调查结果为本建议奠定了基础。在这里，我们将运用传统的和高通量的酵母遗传、分子和细胞生物学方法来实现以下目标：1)通过定义uprc胁迫引起的关键调控因子、上调基因和细胞保护机制来阐明uprc信号通路的电路；2)通过探索E3泛素连接酶的底物特异性和鉴定新的ERAD成分，明确ERAD- c通路的机制、步骤和机制；3)进一步开发MRP蛋白作为模型ERQC底物，特别是对ERQC与ER退出之间的关系有新的认识。我们的研究有望揭示各种膜蛋白转运疾病，最好的例子是囊性纤维化，最常见的结果是内质网保留和CFTR-deltaF508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.