Endoplasmic Reticulum (ER) Associated Degradation of Membrane Proteins in Yeast

描述（由申请人提供）：真核生物中所有新合成的蛋白质中约有三分之一进入内质网（ER），内质网是一个隔室，其中存在专门的机制以支持多肽的翻译后修饰并促进蛋白质折叠。然而，许多分泌蛋白质的显著比例折叠效率低下。这个问题是特别明显的整合膜蛋白，考虑到这些拓扑复杂的物种的天然构象必须实现在ER腔，ER膜内，和在细胞质中。在折叠被延迟或中止的情况下，可以选择所得多肽，然后靶向通过细胞质蛋白酶体降解。该过程被称为ER相关降解（ERAD），并且可以细分为以下步骤：底物识别、反向易位或移位（递送至细胞质）、泛素缀合和降解。由于许多膜蛋白对于细胞和生物体内平衡是必不可少的，因此越来越多的ERAD底物与人类疾病有关也就不足为奇了。为了确定ERAD底物最终被破坏的途径，设计了模型底物以测试特定假设，并开发了新型体外试验，其中可以检查降解途径中的每个步骤。这些方法通过使用由酵母S制备的试剂而得到加强。酿酒酵母，其允许使用从野生型或突变菌株分离的组分。因此，在ERAD过程中催化每个步骤的因子可以在互补的体外和体内系统中分离、表征和测试。在这个应用程序中提出的问题包括：如何是一个ATP需要的“引擎”，这有助于提取ERAD底物从膜调节相关因素？当膜锚的疏水性改变时，或者当结构域通过脂质连接到膜时，细胞质的错误折叠结构域是如何被识别、反向易位和破坏的？当ERAD底物位于内质网腔和细胞质中时，是否有不同的因子作用于ERAD底物？以及，跨膜结构域如何在底物反向易位后保留在溶液中？对这些问题的回答将促进申请人调节ERAD途径以抵消ERAD相关疾病的灾难性后果的长期目标。

DESCRIPTION (provided by applicant): Approximately one-third of all newly synthesized proteins in eukaryotes enter the endoplasmic reticulum (ER), a compartment in which specialized machinery exists to support the post-translation modification of polypeptides and to facilitate protein folding. Nevertheless, a significant proportion of many secreted proteins fold inefficiently. This problem is particularly evident for integral membrane proteins, given that the native conformations of these topologically complex species must be achieved in the ER lumen, within the ER membrane, and in the cytoplasm. In the event that folding is delayed or aborted, the resulting polypeptide may be selected and then targeted for degradation by the cytoplasmic proteasome. This process has been termed ER associated degradation (ERAD) and can be sub-divided into the following steps: substrate recognition, retro-translocation or dislocation (delivery to the cytoplasm), ubiquitin conjugation, and degradation. Because many membrane proteins are essential for cellular and organismal homeostasis, it is not surprising that a growing number of ERAD substrates have been linked to human disease. In order to define the pathway by which ERAD substrates are ultimately destroyed, model substrates were designed to test specific hypotheses and novel in vitro assays were developed in which each step during the degradation pathway can be examined. These approaches have been empowered by the use of reagents prepared from the yeast S. cerevisiae, which permits the use of components isolated from wild type or mutant strains. Therefore, factors that catalyze each step during ERAD can be isolated, characterized, and tested in complementary in vitro and in vivo systems. The questions asked in this application include: How is an ATP-requiring "engine", which helps extract ERAD substrates from the membrane regulated by associated factors? How is a cytoplasmic, misfolded domain recognized, retro-translocated, and destroyed when the hydrophobicity of the membrane anchor is altered, or when the domain is linked to the membrane by a lipid? Do different factors act on an ERAD substrate when the domain is positioned in the ER lumen versus cytoplasm? And, how are transmembrane domains retained in solution after substrate retro-translocation? Answers to these questions will further the applicant's long-term goal to modulate the ERAD pathway in order to off-set the catastrophic consequences of ERAD-associated diseases.