IDENTIFICATION OF COTRANSIN-SENSITIVE PROTEINS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 新生多肽通过Sec 61易位子的共翻译易位是分泌性膜蛋白加工中的重要步骤。 为了翻译蛋白质以进入ER腔，易位子必须首先识别蛋白质的信号序列。 以前，我们的实验室已经确定了某些环状七缩酚肽，称为cotransins，作为第一个小分子共翻译易位抑制剂。 值得注意的是，共转蛋白以信号序列依赖性方式抑制易位。 迄今为止，仅测试了约40个信号序列的共转蛋白敏感性，导致对抗性敏感性基础的了解很少。 因此，有必要鉴定更多敏感或抗性的靶信号序列，以允许更多的全局比较。 我们打算通过使用无偏的蛋白质组学方法来确定确定共转运蛋白敏感性的信号序列特征。 通过耦合膜蛋白纯化技术和稳定同位素标记在细胞培养（SILAC），我们将量化cotransin依赖的蛋白质表达的变化，以确定膜蛋白信号序列的子集是敏感的cotransin。 通过比较敏感的信号序列与不敏感的信号序列，我们将开始了解cotransin敏感性的序列要求，并揭示这些分子区分单个信号序列的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cotranslational translocation of nascent polypeptides through the Sec61 translocon is an essential step in the processing of secreted membrane proteins. For translating proteins to gain access to the ER lumen, the translocon must first recognize a protein's signal sequence. Previously, our lab has identified certain cyclic heptadepsipeptides, called cotransins, as the first small molecule inhibitors of cotranslational translocation. remarkably, cotransins inhibit translocation in a signal sequence-dependent manner. To date, only ~40 signal sequences have been tested for cotransin sensitivity resulting in few insights into the basis of sensitivity of resistance. Therefore, it is necessary to identify more target signal sequences that are sensitive or resistant to allow for more global comparisons. We intend to identify signal sequence characteristics that determine cotransin sensitivity by using an unbiased proteomics approach. By coupling membrane protein purification techniques and stable isotope labeling in cell culture (SILAC), we will quantify cotransin dependent changes in protein expression to determine the subset of membrane protein signal sequences that are sensitive to cotransin. By comparing sensitive signal sequences to insensitive ones, we will begin to understand the sequence requirements for cotransin sensitivity and uncover the mechanism by which these molecules distinguish between individual signal sequences.