Genetic Requirements for Protein Degradation at the Endoplasmic Reticulum Translocon

项目总结 大多数内膜系统和分泌的真核蛋白穿过内质网(ER) 在合成过程中或合成后不久发生移位。强调维持功能转运蛋白的关键性质， 真核生物具有多种促进降解的保守转位质量控制(TQC)机制 通道堵塞蛋白。与其他ER蛋白质质量控制相比，TQC仍然知之甚少 小路。高度保守的酵母锌金属蛋白酶Ste24(哺乳动物中的ZMPSte24)催化 转运子堵塞蛋白的降解。初步数据表明，内质网菱形蛋白水解酶家族 Dfm1(与哺乳动物Derlin蛋白同源)可能通过Ste24机制参与TQC。 与国家普通医学研究所的使命一致，拟议的 工作是使用酵母作为模型，提高对TQC的理解，TQC是细胞生物学的一个重要医学方面 系统。Ste24促进健康衰老，并凭借其在TQC中的功能，可能起到保护作用 抑制糖尿病的发展。此外，至少有两种对代谢具有深远意义的蛋白质 生理学一直在与变性人打交道。拟议的实验将检验Dfm1和Dfm1的假设 它的辅因子CDC48 ATPase部分地从转运子中提取泛素化的堵塞蛋白，以使 被Ste24切割，胞浆片段被蛋白酶体和管腔片段降解 通过内膜系统运输到液泡进行降解。这个项目的具体目标是 目的是(1)确定STE24在TQC中的功能；(2)确定Dfm1和CDC48在TQC中的作用。致信地址 这些目标，以及STE24和Dfm1/CDC48在TQC中作用的几个机制方面将被探索。这个 将确定Ste24和Dfm1/CDC48 TQC基板的宽度。实验将解决Ste24是否 TQC底物在识别之前被泛素化，如果TQC底物在依赖于Ste24的 方式，以及Ste24 TQC底物最终如何降解(即由蛋白酶体或液泡)。DFM1 将评估与转运子和TQC底物的相关性，以及Dfm1和Dfm1的贡献。 CDC48转Ste24介导和Ste24非依赖性TQC。保守的Dfm1元件在TQC中的作用是 下定决心。最后，缺乏表达TQC底物的DFM1的酵母表现出一个严重的适应性缺陷，即 迅速而稳定地被抑制。抑制的遗传决定因素将被识别出来。本科生和 硕士研究生将参与这个项目的各个方面，包括实验设计、数据收集和 结果的沟通。这些实验将产生对保守的、生物医学的新的机械学见解 全面质量控制的相关机制。一旦完成，拟议的工作将极有可能向 针对多种人类疾病，包括胆固醇升高，开发改进的治疗策略 还有糖尿病。

PROJECT SUMMARY Most endomembrane system and secreted eukaryotic proteins traverse the endoplasmic reticulum (ER) translocon during or shortly after synthesis. Underscoring the critical nature of maintaining functional translocons, eukaryotes possess multiple conserved translocon quality control (TQC) mechanisms that promote degradation of channel-clogging proteins. TQC remains poorly understood relative to other ER protein quality control pathways. The highly conserved yeast zinc metalloprotease Ste24 (ZMPSte24 in mammals) catalyzes degradation of translocon-clogging proteins. Preliminary data indicate that ER rhomboid-family pseudoprotease Dfm1 (homologous to mammalian derlin proteins) contributes to TQC, likely via the Ste24 mechanism. Consistent with the mission of the National Institute of General Medical Sciences, the objective of the proposed work is an improved understanding of TQC, a medically important facet of cell biology, using yeast as a model system. Ste24 promotes healthy aging and, by virtue of its function in TQC, is likely to play a protective role against the progression of diabetes. Further, at least two proteins with profound significance for metabolic physiology persistently engage translocons. The proposed experiments will test the hypothesis that Dfm1 and its cofactor, the Cdc48 ATPase, partially extract ubiquitylated clogging proteins from the translocon to enable cleavage by Ste24, whereupon the cytosolic fragment is degraded by the proteasome and the luminal fragment is trafficked through the endomembrane system to the vacuole for degradation. The specific aims of this project are to (1) characterize STE24 function in TQC and (2) determine the role of Dfm1 and Cdc48 in TQC. To address these objectives, several mechanistic aspects of Ste24 and Dfm1/Cdc48 function in TQC will be explored. The breadth of Ste24 and Dfm1/Cdc48 TQC substrates will be determined. Experiments will address whether Ste24 TQC substrates are ubiquitylated prior to recognition, if TQC substrates are cleaved in a Ste24-dependent manner, and how Ste24 TQC substrates are ultimately degraded (i.e. by the proteasome or vacuole). Dfm1 association with the translocon and TQC substrates will be assessed, as will the contributions of Dfm1 and Cdc48 to Ste24-mediated and Ste24-independent TQC. The role of conserved Dfm1 elements in TQC will be determined. Finally, yeast lacking DFM1 expressing TQC substrates exhibit a profound fitness defect that is rapidly and stably suppressed. The genetic determinants of suppression will be identified. Undergraduate and master’s students will participate in all aspects of this project, including experiment design, data collection, and communication of results. These experiments will yield novel mechanistic insights into conserved, biomedically relevant mechanisms of TQC. Upon completion, the proposed work has the strong potential to inform the development of improved therapeutic strategies for multiple human conditions, including elevated cholesterol and diabetes.