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Title: Protein interactions in the peroxisomal docking complex of Arabidopsis thaliana
Author: Lanyon-Hogg, Thomas
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Peroxisomes are metabolic organelles found in almost all eukaryotic organisms. As peroxisomes possess no DNA, the matrix protein content required for function is post-translationally imported from the cytosol via one of two peroxisomal targeting signals (PTS). The PTSl and PTS2 are recognised by the cytosolic receptors PEX5 and PEX7, respectively, PEX5 also functions as a coreceptor for PTS2 import. The cytosolic import complex then docks at PEX14 and PEX13 at the peroxisomal membrane for import. To gain insight into the interactions involved in formation of the docking complex representative soluble protein constructs of PTS 1 and PTS2 cargo, PEX5, PEX7 and PEX14 were expressed and purified. Additionally, synthetic peptides representative of the key interaction motifs were obtained. These tools were utilised in biochemical and biophysical assays to generate both quantitative and qualitative data for modelling of interaction steps. A new approach for labelling of binding partners in solution through Enzyme Mediated Activation of Radical Sources was also investigated. A low affinity interaction was detected between PEX14 and peptide motifs from PEX5, and it was shown that binding of PTSl cargo to PEX5 was not a prerequisite for interaction with PEX14. The PEX5 receptor was also shown to be capable of forming a complex with both PTS 1 and PEX7 -PTS2 cargos simultaneously, thus representing a point of convergence of the two pathways in A. thafiana. Recent studies have implicated PEX14 in release of catalase, an atypical PTS 1 cargo, from PEX5. In a range of biophysical and biochemical assays, PEX14 dependent PTSl cargo release was not observed. However, a new function of PEX14, PEX14 dependent release of PTS2 cargo from the PEX7- PEX5 complex was observed. This demonstrates the docking complex to be the point of PTS2 cargo-unloading, and opens the possibility that interplay with PEX13 may be required for PTSl cargo-unloading.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available