Spatial Organization Of Endoplasmic Reticulum Functions

内质网功能的空间组织

• 批准号: 6672673
• 负责人: Ramanujan S Hegde
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6672673
• 关键词: cell migration; cell proliferation; cellular pathology; conformation; endoplasmic reticulum; fluorescence resonance energy transfer; protein protein interaction; protein structure function; protein transport

The Unit on Protein Biogenesis studies the mechanisms regulating the synthesis, translocation and maturation of secretory and membrane proteins at the mammalian endoplasmic reticulum (ER). A complex macromolecular assembly at the ER, termed the translocon, serves as a protein-conducting channel where substrates enter the secretory pathway. The translocon participates in diverse cellular activities that range from the import of secretory proteins, to topogenesis and assembly of complex multi-spanning membrane proteins, to the export of misfolded substrates from the ER to the cytosol for degradation. A largely unexplored aspect of ER function is the question of where within the ER these various events occur. Are all of these translocon-associated activities homogeneously distributed throughout the ER, or are they organized and regulated in the spatial and temporal dimensions to meet the changing needs of the cell? There is presently little or no insight into this question, largely because the current approaches to understanding protein translocation utilize biochemical systems in which spatial relationships are lost. In collaboration with the laboratory of Jennifer Lippincott-Schwartz, we are utilizing biophysical techniques such as fluorescence resonance energy transfer (FRET) to probe, in situ, the molecular organization of the components of the translocation machinery. In initial studies, analysis of FRET between subunits of the Sec61p complex, a principal component of the ER protein translocon, was used to directly monitor the assembly state of the translocon in cells. Our studies have revealed that while the translocon can be assembled from its components in response to ligands for protein translocation in biochemical systems, it does not disassemble and reassemble between successive rounds of transport in vivo. Instead, an actively engaged translocon is distinguished from a quiescent translocon by conformational changes that can be directly detected by differences in FRET. By correlating the formation of particular protein complexes with biochemical activities, we endeavor to directly visualize the functional segregation and organization of the ER, and to monitor potential changes in it during cellular metabolism, development, or disease pathogenesis.

蛋白质生物发生单元研究调节哺乳动物内质网 (ER) 分泌蛋白和膜蛋白的合成、易位和成熟的机制。内质网的一个复杂的大分子组装体，称为易位子，充当蛋白质传导通道，底物在此进入分泌途径。易位子参与多种细胞活动，从分泌蛋白的输入，到复杂的多跨膜蛋白的拓扑发生和组装，再到将错误折叠的底物从内质网输出到胞质溶胶进行降解。 ER 功能的一个很大程度上未被探索的方面是这些不同事件在 ER 内发生的位置的问题。所有这些与易位子相关的活动是否均匀分布在整个内质网中，或者它们是否在空间和时间维度上进行组织和调节，以满足细胞不断变化的需求？目前对这个问题的了解很少或根本没有，主要是因为当前理解蛋白质易位的方法利用了丢失空间关系的生化系统。 我们与 Jennifer Lippincott-Schwartz 实验室合作，利用荧光共振能量转移 (FRET) 等生物物理技术来原位探测易位机制组件的分子组织。在最初的研究中，对 Sec61p 复合物（ER 蛋白易位子的主要成分）亚基之间的 FRET 进行分析，用于直接监测细胞内易位子的组装状态。我们的研究表明，虽然易位子可以响应生化系统中蛋白质易位的配体而由其组件组装而成，但它不会在体内连续几轮运输之间分解和重新组装。相反，主动参与的易位子与静止易位子的区别在于构象变化，这些变化可以通过 FRET 的差异直接检测到。通过将特定蛋白质复合物的形成与生化活性联系起来，我们努力直接可视化内质网的功能分离和组织，并监测其在细胞代谢、发育或疾病发病机制过程中的潜在变化。