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Targeting of Proteins for Export in Escherichia coli

大肠杆菌中出口蛋白质的靶向

基本信息

批准号：
6555969
负责人：
ANN M FLOWER
金额：
$ 14.02万
依托单位：
UNIVERSITY OF NORTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2006-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6555969
关键词：
Escherichia coli cytoplasm endoplasmic reticulum genetic screening green fluorescent proteins membrane proteins molecular cloning polymerase chain reaction protein folding protein localization protein signal sequence protein transport secretory protein transport proteins western blottings

项目摘要

DESCRIPTION (provided by applicant): The Sec-dependent protein export pathway of Escherichia coli is responsible for translocation of secretory proteins across the inner membrane to final destinations in the outer membrane or periplasmic space. SecA, SecE, and SecY are the essential subunits of translocase, and homologs of SecE and SecY have been identified in other eubacteria, archae, yeast and mammals. Further, the mechanisms of the translocation process seem to be conserved across species. Therefore, information gained from continued studies of the E. coli translocation pathway will be directly applicable to a greater understanding of targeting of proteins to the endoplasmic reticulum. Secretory proteins are synthesized in precursor form with an amino terminal signal sequence that is cleaved upon traversal of the inner membrane. Despite the central role of the signal sequence, it has been shown that it is neither necessary nor sufficient to direct export of a secretory protein. Previous results in this lab and others demonstrate that secretory proteins contain information in addition to the signal sequence that contributes to recognition by the Sec proteins. This hypothesis is based on the following data: deletion of the signal sequence significantly reduces export of a secretory protein, but does not completely abolish export; addition of a signal sequence to a non-secretory protein is not necessarily sufficient to target that protein for export; and mutant secretory proteins that completely lack a signal sequence are exported quite efficiently in a Prl suppressor strain. We propose to (1) examine the role of folding in protein export through a genetic screen that will identify slow-folding cytoplasmic proteins that are exported in a Prl suppressor strain, and (2) select for cytoplasmic localization of a secreted protein by removal of targeting information. The proposed studies will contribute to the general understanding of the normal export process and allow us to define the characteristics of a secretory protein that target it to the export pathway.

描述（由申请方提供）：大肠杆菌的Sec依赖性蛋白输出途径负责分泌蛋白跨内膜转运至外膜或周质空间中的最终目的地。SecA、SecE和SecY是转位酶的重要亚基，并且在其他真细菌、古细菌、酵母和哺乳动物中已经鉴定出SecE和SecY的同源物。此外，易位过程的机制似乎是跨物种保守的。因此，继续研究E.大肠杆菌转运途径将直接适用于更好地理解蛋白质的内质网靶向。分泌蛋白以前体形式合成，其氨基末端信号序列在穿过内膜时被切割。尽管信号序列的中心作用，它已被证明，它既不是必要的，也不足以直接输出的分泌蛋白。本实验室和其他实验室的先前结果表明，分泌蛋白除了包含有助于Sec蛋白识别的信号序列外，还包含信息。该假设基于以下数据：信号序列的缺失显著减少分泌蛋白的输出，但不完全消除输出;向非分泌蛋白添加信号序列不一定足以靶向该蛋白用于输出;并且完全缺乏信号序列的突变分泌蛋白在Prl抑制菌株中相当有效地输出。我们建议（1）通过遗传筛选来检查折叠在蛋白质输出中的作用，该遗传筛选将鉴定在Prl抑制菌株中输出的缓慢折叠的细胞质蛋白，以及（2）通过去除靶向信息来选择分泌蛋白的细胞质定位。拟议的研究将有助于一般的理解正常的出口过程，并允许我们定义的分泌蛋白的特性，它的目标出口途径。