Spatial Organization Of Endoplasmic Reticulum Functions

蛋白质生物发生单元研究哺乳动物内质网（ER）分泌和膜蛋白的合成，转运和成熟的调节机制。在ER的复杂大分子组装，称为translocon，作为一个蛋白质传导通道，底物进入分泌途径。易位子参与多种细胞活动，其范围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，到错误折叠的底物从ER到胞质溶胶的输出以进行降解。ER功能的一个很大程度上未被探索的方面是ER中这些不同事件发生的位置的问题。所有这些translocon相关的活动均匀地分布在整个ER中，还是在空间和时间维度上组织和调节以满足细胞不断变化的需求？目前很少或根本没有洞察到这个问题，主要是因为目前的方法来理解蛋白质易位利用生化系统中的空间关系丢失。 与Jennifer Lippincott-Schwartz实验室合作，我们正在利用生物物理技术，如荧光共振能量转移（FRET）来原位探测易位机制组件的分子组织。在初步研究中，对ER蛋白易位子的主要成分Sec 61 p复合物亚基之间的FRET分析用于直接监测细胞中易位子的组装状态。我们的研究表明，虽然在生化系统中，转位子可以从其组分中组装出来，以响应蛋白质转位的配体，但它在体内连续几轮运输之间不会分解和重新组装。相反，一个积极参与的转座区分从一个静止的转座的构象变化，可以直接检测到FRET的差异。通过将特定蛋白质复合物的形成与生物化学活性相关联，我们奋进直接可视化ER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中的潜在变化。

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.