Investigations of Protein Translocation in Archaea

宾夕法尼亚大学的M.Pohlschroder蛋白质跨疏水膜转运是所有生物的一个基本过程。对细菌和真核生物系统的生化和遗传分析表明，许多蛋白质通过进化上保守的孔穿过这些膜。机制和能量学似乎是截然不同的，关于这一过程中涉及的组件的作用需要了解很多。序列比较表明，生命的第三个领域的生物，古生菌，含有细菌和真核生物蛋白质转运成分的同系物的组合，这表明这些生物通过细胞膜进行蛋白质转运的某些方面是独特的。然而，古生菌中的蛋白质易位研究一直局限于序列比较。本研究旨在研究一种古生物蛋白质易位系统。该项目的具体目标是：1.克隆和测序已知蛋白质易位成分的古生菌同源物，并为研究和验证实验系统提供必要的工具；以及2.使用生化方法和反向遗传学来纯化与古生菌易位相关的蛋白质。除了鉴定已知的细菌和真核生物蛋白质转运机制的H.Volcanii同源物外，这项研究还可能分离出古生菌特有的成分，或以前在细菌和真核生物中尚未鉴定的成分。了解古生菌的蛋白质转位可能有助于我们更好地了解这一保守的过程，包括生命、细菌、古生菌和真核生物的各个领域。例如，在真核生物中，分泌的抗体对于抵御病原体是必不可少的。另一方面，许多原核生物分泌毒素，这是它们致病的主要特征。因此，更好地了解蛋白质穿过细胞膜的机制可能会使我们能够调节致病化合物的分泌，并优化对它们的防御机制。原核生物和低等真核生物也分泌酶来清除细胞外分子。这些蛋白质中有许多具有有用的生物技术应用。了解蛋白质输出机制将有助于重组分泌型蛋白质的过度表达。古生菌与细菌和真核生物一起构成了生命的三个领域之一，在我们的蛋白质易位研究中代表着一个相对未被探索的领域。对这些生物体中蛋白质转运生物学的探索不仅将有助于揭示蛋白质穿过疏水古生菌膜的新过程，而且还将为以前发现的细菌和真核蛋白质转运系统的机制提供新的见解。

M. PohlschroderUniversity of Pennsylvania Protein translocation across hydrophobic membranes is an essential process in all living organisms. Biochemical and genetic analyses of the bacterial and eukaryotic systems have shown that many proteins cross these membranes via an evolutionarily conserved pore. The mechanisms and energetics seem to be distinct and much has to be learned about the roles of the components involved in this process. Sequence comparisons suggest that organisms of the third domain of life, the archaea, contain a combination of homologs of the bacterial and eukaryotic protein translocation components, suggesting that some aspects of protein translocation through the cytoplasmic membrane of these organisms are unique. However, protein translocation studies in archaea, have been limited to sequence comparisons. This research is aimed at studying an archaeal protein translocation system. The specific aims of the project are: 1. To clone and sequence archaeal homologs of known protein translocation components, and provide necessary tools to study and validate the experimental system; and 2. To use a biochemical approach and reverse genetics to purify proteins associated with the archaeal translocon. In addition to identifying H. volcanii homologs of the known bacterial and eukaryotic protein translocation machinery, this research may lead to the isolation of archaea-specific components, or components that have not been previously identified in bacteria and eukaryotes. Gaining insights into archaeal protein translocation may help us to obtain a better understanding of this conserved process all domains of life, bacteria, archaea, and eukaryotes.The ability to transport proteins across hydrophobic membranes is fundamental to the growth and survival of all cells. For example, in eukaryotes, secreted antibodies are essential for the defense against pathogens. On the other hand, many prokaryotes secrete toxins, which are central features of their pathogenicity. Thus, a better understanding of the mechanisms by which proteins traverse the membranes may allow us to modulate the secretion of pathogenic compounds as well as optimize defense mechanisms against them. Prokaryotes and lower eukaryotes also secrete enzymes to scavenge extracellular molecules. Many of these proteins have useful biotechnology applications. Understanding protein export mechanisms will help in the overexpression of recombinant secreted proteins. The archaea, which, along with bacteria and eukaryotes comprise one of the three domains of life, represent a relatively unexplored area in our study of protein translocation. An exploration of the biology of protein translocation in these organisms will not only shed light on novel processes by which proteins traverse hydrophobic archaeal membranes, but will also provide new insights into the mechanisms of previously identified bacterial and eukaryotic protein translocation systems.