Assembly of the Peroxisomal Translocon

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The assembly of peroxisomal translocons for matrix proteins is an unknown multi-step process, which is initiated by the interaction between cytosolic receptors and fully folded cargo-proteins. Together with the membrane protein Pex14p, the receptor Pex5p and the co-receptor Pex18p become integral constituents of PTS1- or PTS2-specific import channels, respectively. It has been suggested that the assembly of translocons is mainly driven by interaction of receptors with peroxisomal phospholipids and Pex14p, either simultaneously or sequentially. Within the first funding period of this project, we focussed on the characterization of receptor interactions with various cargo-proteins, PEX14 and phospholipids. Moreover, we established methods which allow to dissect the single steps of translocon assembly in a spatial and temporal manner. In the next period, we will specifically address the following goals: A. Analysis of the role of phospholipid binding of PTS receptors for translocon assemblyB. Elucidation of the function of PEX14 for pore formationC. Role of PEX19 for translocon assemblyD. Investigation of a role of the peroxisomal PTS1 translocon in metabolite transport In the first part of the project, we will map phospholipid-interacting sites of the human PTS1 receptor and study how this affects its conformation, dynamics and molecular interactions by NMR. The contribution of these sites for the assembly of functional pores will be assessed by mutagenizing these sites and insertion into horizontal lipid bilayers (HLB) allowing electrophysiological pore measurements. The physiological role of the lipid interaction will also be validated by expressing PEX5 variants in PEX5-deficient human cells. For the second part of the project, a similar experimental strategy will be employed to study the role of all known PEX5-PEX14 interacting sites using site-directed mutagenesis in vitro and in vivo. This includes the WxxxF/Y motifs in the N-terminal region and the C-terminal binding site in PEX5, which have been identified in this funding period. PEX19, the receptor for peroxisomal membrane proteins, binds to PEX14 in a PEX5-competitive way. In the third part of the project, the in vitro effects of PEX19 on translocon assembly will be studied by combining HLB- and NMR-studies. These analyses could provide mechanistic insights into the convergence of import pathways for matrix and membrane proteins. The fourth part of the project will address the question whether the pores formed by PTS receptors can translocate other molecules than proteins and in this context remain associated with the folded enzymes. Although specific metabolite transporters in the peroxisomal membrane have been proven to exist, they could not be identified so far. A Pex5p subcomplex at the peroxisomal membrane of yeast is an ideal candidate to prove this assumption.