Trafficking of ABC proteins in yeast

描述(申请人提供)：ER“质量控制”(ERQC)是防止错误折叠的分泌物和膜蛋白从ER退出的基本和保守的细胞过程。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的蛋白质的伴侣等基因的转录上调；2)内质网相关降解(ERAD)，即错误折叠的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，根据其错误折叠损伤的拓扑位置，存在两种ERAD底物，鲁米娜(L)和胞质(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UPR-L和ERAD-L)和ERQC-C(包括UPR-C和ERAD-C)，分别处理结构域为腔结构域或胞质结构域的底物。该项目的长期目标是鉴定和机械剖析酿酒酵母中ERQC-C途径的组成和工作原理，并确定ERQC-C和ERQC-L的不同之处。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Ste6p和Ycflp的突变形式的研究，这两种转运蛋白受到ERQC的影响。利用这些作为模型ERQC-C底物，我们在这个项目中取得了重大进展，包括定义了一个显著的内质网隔室(ERAC)作为UPR-C的标记，定义了ERAD-C和ERAD-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。这些调查结果为目前的提议奠定了基础。在这里，我们将应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UPR-C应激诱导的关键调节因子、上调基因和细胞保护机制来阐明UPR-C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寻找新的ERAD成分来确定ERAD-C途径的机制、步骤和机制；以及3)进一步开发MRP蛋白作为模型ERQC底物，特别是对ERQC和ER Exit之间的关系有新的见解。我们的研究有望揭示一系列不同的膜蛋白转运疾病，最好的例证是囊性纤维化，这最常见的原因是CFTR-deltaF508的内质网滞留和降解。

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.