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

描述(申请人提供)：在真核生物中，大约三分之一的新合成的蛋白质进入内质网(ER)，在内质网中存在专门的机制来支持多肽的翻译后修饰和促进蛋白质折叠。然而，许多分泌的蛋白质中有相当大一部分折叠效率低下。这个问题对于整膜蛋白来说尤其明显，因为这些拓扑复杂的物种的天然构象必须在内质网管腔、内质网膜和细胞质中实现。在折叠延迟或中止的情况下，产生的多肽可以被选择，然后被细胞质蛋白酶体靶向降解。这一过程被称为内质网相关降解(ERAD)，可细分为以下几个步骤：底物识别、逆转位或错位(输送到细胞质)、泛素结合和降解。由于许多膜蛋白对细胞和生物的动态平衡至关重要，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，设计了模型底物来检验特定的假设，并开发了新的体外检测方法，其中可以检查降解途径中的每一步。这些方法是通过使用从酿酒酵母中制备的试剂来实现的，这种试剂允许使用从野生型或突变菌株中分离出来的成分。因此，在ERAD过程中催化每一步的因子可以在互补的体外和体内系统中分离、表征和测试。在本申请中提出的问题包括：需要ATP的“引擎”如何帮助从细胞膜中提取ERAD底物，受相关因素的调节？当膜锚定的疏水性改变时，或者当结构域通过脂质连接到膜上时，细胞质中错误折叠的结构域是如何识别、反向移位和破坏的？当结构域位于内质网上时，不同的因素是否作用于ERAD底物？还有，底物逆转移位后跨膜区是如何保留在溶液中的？对这些问题的回答将促进申请人的长期目标，即调节ERAD途径，以抵消ERAD相关疾病的灾难性后果。

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.