Characterization of the translocation pore of the peroxisomal protein import machinery

过氧化物酶体基质蛋白在胞质溶胶中合成，具有两种定义的过氧化物酶体靶向信号之一（PTS 1或PTS 2）。这些蛋白质被胞质输入受体识别和结合，其将货物分子引导至过氧化物酶体膜。这里，受体-货物复合物与对接复合物的结合触发易位复合物的组装。货物分子穿过过氧化物酶体膜通过对两类货物蛋白中的任一种具有特异性的充满水的易位孔。在此过程中，输入受体插入过氧化物酶体膜，成为转运机制的一个组成部分。在货物易位之后，受体从膜中提取并再循环回到胞质溶胶。这一机制产生了一个瞬时孔的想法，形成的需求和货物translocation.With这一建议，我们的目标是采取决定性的一步，对过氧化物酶体的蛋白质转运机制的分子理解。我们将具体解决以下目标：A）鉴定和表征的人过氧化物酶体易位孔B）功能分析的核心组件的过氧化物酶体易位孔C）表征的替代PTS 1-translocon在本项目的第一部分，我们继续成功的电生理研究的人过氧化物酶体易位。在参与小组的共同努力下，已建立的纯化和重建方案的易位复合物从人类细胞系将用于详细的生化，结构和电生理分析，这种复合物，以阐明的分子特征的translocon。为实现第二个目标，我们将研究对接复合物蛋白、PTS 1输入受体和货物蛋白之间的分子相互作用。将使用纯化的组分和各种模型膜系统进行分析。将通过体内实验彻底验证体外结果。本项目的重要部分是阐明和表征PTS 1货物分子的最小成孔单位。最后，本项目涉及最近发现的替代PTS 1易位子的结构-功能分析。将研究新型输入受体Pex 9 p对孔形成的重要性。我们将筛选新的Pex 9 p依赖性货物分子，并最终表征Pex 9 p与对接复合物之间的相互作用。

Peroxisomal matrix proteins are synthesized in the cytosol with one of two defined peroxisomal targeting signals (PTS1 or PTS2). These proteins are recognized and bound by cytosolic import receptors, which direct the cargo molecules to the peroxisomal membrane. Here, binding of the receptor-cargo complex to the docking complex triggers the assembly the translocation complex. Cargo molecules pass the peroxisomal membrane through water-filled translocation pores specific for either of the two classes of cargo proteins. During this process, the import receptors insert into the peroxisomal membrane and become an integral part of the translocation machinery. Subsequent to cargo translocation, receptors are extracted from the membrane and recycled back to the cytosol. This mechanism gave rise to the idea of a transient pore that forms on demand and is disassembled after cargo translocation.With this proposal, we aim to take a decisive step towards a molecular understanding of the protein translocation machineries of peroxisomes. We will specifically address the following goals:A) Identification and characterization of the human peroxisomal translocation poreB) Functional analysis of the core-components of the peroxisomal translocation poreC) Characterization of the alternative PTS1-transloconIn the first part of this project, we continue the successful electrophysiological studies of the human peroxisomal translocon. In a joint effort by participating groups, the established purification and reconstitution protocols for the translocation complex from human cell line will be used for a detailed biochemical, structural, and electrophysiological analysis of this complex to elucidate the molecular characteristics of the translocon. This work will be contribute to our understanding of peroxisomal diseases.To achieve the second goal of our proposal, we will investigate the molecular interactions between the docking complex proteins, the PTS1 import receptor and cargo proteins. Analyses will be carried out with purified components and various model membrane systems. In vitro results will be thoroughly verified by in vivo experiments. Important for this part of the project is to elucidate and characterize the minimal pore-forming unit for PTS1 cargo molecules.Finally, this project addresses the structure-function analysis of the recently identified alternative PTS1 translocon. The importance of the novel import receptor Pex9p for pore formation will be investigated. We will screen for new Pex9p-dependent cargo molecules and finally the interaction between Pex9p and the docking complex will characterized.