Targeting of Proteins for Export in Escherichia coli

描述(申请人提供)：大肠杆菌的SEC依赖的蛋白质输出途径负责将分泌蛋白质通过内膜转移到外膜或周质空间的最终目的地。SecA、SecE和SecY是转位酶的重要亚基，在其他真细菌、古生物、酵母和哺乳动物中也发现了SecE和SecY的同源物。此外，易位过程的机制似乎在物种之间是保守的。因此，从对大肠杆菌易位途径的持续研究中获得的信息将直接适用于更好地理解将蛋白质靶向内质网。分泌蛋白以前体形式合成，带有氨基末端信号序列，在穿过内膜时被切割。尽管信号序列起着中心作用，但已经证明，它既不必要也不足以直接输出分泌蛋白。这个实验室和其他实验室之前的结果表明，除了有助于SEC蛋白质识别的信号序列外，分泌蛋白质还包含信息。这一假设基于以下数据：信号序列的缺失显著减少了分泌蛋白的输出，但并不完全取消输出；向非分泌蛋白添加信号序列不一定足以针对该蛋白质进行输出；完全缺乏信号序列的突变分泌蛋白在PRL抑制菌株中被非常有效地输出。我们建议(1)通过基因筛选来研究折叠在蛋白质输出中的作用，这将识别在PRL抑制株中输出的慢折叠细胞质蛋白质，以及(2)通过去除靶向信息来选择分泌蛋白质的细胞质定位。拟议的研究将有助于对正常的出口过程的一般理解，并使我们能够定义针对其出口途径的分泌蛋白的特征。

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.