Characterization of the translocation pore of the peroxisomal protein import machinery

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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.