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、转位和成熟的调节机制。内质网有一个复杂的大分子组装，称为转运子，是底物进入分泌途径的蛋白质传导通道。转位蛋白参与多种细胞活动，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细胞质进行降解。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中发生的位置的问题。所有这些转运子相关的活动是否均匀地分布在内质网中，或者它们是在空间和时间维度上组织和调节的，以满足细胞不断变化的需求？目前对这个问题的了解很少，很大程度上是因为目前理解蛋白质易位的方法利用的是失去空间关系的生化系统。 与Jennifer Lippincott-Schwartz的实验室合作，我们正在利用生物物理技术，如荧光共振能量转移(FRET)，原位探索易位机制组件的分子组织。在最初的研究中，ER蛋白转运子的主要成分Sec61p复合体亚单位之间的FRET分析被用来直接监测转运子在细胞中的组装状态。我们的研究表明，虽然转运子可以根据生化系统中蛋白质转运的配体进行组装，但它不会在体内连续几轮运输之间拆解和重新组装。相反，活跃参与的移位子与静止的移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体的形成与生化活动联系起来，我们努力直接显示内质网的功能分离和组织，并监测它在细胞代谢、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.