Genetic Requirements for Protein Degradation at the Endoplasmic Reticulum Translocon

项目摘要 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，强调维持功能性translocons的关键性质， 真核生物具有多种保守的促进降解的易位子质量控制（TQC）机制 通道堵塞蛋白质。TQC相对于其他ER蛋白质质量控制仍然知之甚少 途径。高度保守的酵母锌金属蛋白酶Ste 24（哺乳动物中的ZMPSte 24）催化 translocon堵塞蛋白质的降解。初步数据表明ER菱形家族假蛋白酶 Dfm 1（与哺乳动物derlin蛋白同源）可能通过Ste 24机制参与TQC。 根据国家普通医学科学研究所的使命，拟议的 工作是提高对全面质量管理的理解，这是细胞生物学的一个医学重要方面，使用酵母作为模型 系统Ste 24促进健康老龄化，并凭借其在TQC中的功能，可能起到保护作用 对抗糖尿病的发展此外，至少两种对代谢具有深远意义的蛋白质， 生理学持续地与translocons结合。所提出的实验将检验Dfm 1和 它的辅因子Cdc 48 ATP酶从易位子中部分提取泛素化的堵塞蛋白， 被Ste 24切割，于是胞质片段被蛋白酶体降解，管腔片段 通过内膜系统运输到液泡进行降解。该项目的具体目标 目的是（1）确定STE 24在TQC中的功能;（2）确定Dfm 1和Cdc 48在TQC中的作用。解决 为了达到这些目的，我们将探讨Ste 24和Dfm 1/Cdc 48在TQC中的作用机制。的 确定Ste 24和Dfm 1/Cdc 48 TQC底物的宽度。实验将解决Ste 24是否 如果TQC底物在Ste 24-依赖性蛋白酶中被切割，则TQC底物在识别之前被泛素化。 方式，以及Ste 24 TQC底物最终如何降解（即通过蛋白酶体或液泡）。DFM 1 将评估与易位子和TQC底物的结合，以及Dfm 1和 Cdc 48对Ste 24介导的和Ste 24非依赖性的TQC。保守的Dfm 1元件在TQC中的作用将是 测定最后，缺乏表达TQC底物的DFM 1的酵母表现出严重的适应性缺陷， 迅速而稳定地抑制。将确定抑制的遗传决定因素。本科和 硕士生将参与该项目的各个方面，包括实验设计，数据收集， 沟通结果。这些实验将产生新的机制见解保守，生物医学 TQC的相关机制。完成后，拟议的工作有很大的潜力， 为多种人类疾病（包括胆固醇升高）开发改进的治疗策略 和糖尿病

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.