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Title: Re-design of a receptor-targeting signal interaction to create a new peroxisomal trafficking pathway
Author: Cross, Laura Louise
ISNI:       0000 0004 5991 7534
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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Peroxisomes are crucial for cell survival but do not possess a genome, so protein import is essential for correct functioning of the organelle. The PTS1 (peroxisomal targeting signal 1) pathway is the major, and in some organisms the only, transport pathway for proteins to enter the peroxisome. PTS1 is a recognition sequence at the C-terminus of peroxisomal cargo proteins, which allows their binding to the receptor protein PEX5 (peroxin 5). PEX5 then acts as the vehicle for transporting PTS1-cargo proteins into peroxisomes. This work concerned the disruption of the natural PEX5:PTS1 interaction and the generation of an interaction between a mutated form of PEX5 (PEX5*) and a peptide representing a non-natural PTS1 (PTS1*). An in vitro protein-peptide binding screen was developed to test Arabidopsis thaliana PEX5-C (AtPEX5-C), and variants, with a library of peptides to identify corresponding binding peptides. For wild-type AtPEX5-C, peptides identified were consistent with bioinformatics predictions for plant PTS1 sequences, confirming the validity of the screen. Fluorescence anisotropy was then used to validate binding peptides, revealing that two variants of AtPEX5-C, D505H and N601A, exhibited reduced affinity to a representative native PTS1 (YQSKL-CO2H), yet increased affinity to a non-PTS1 sequence (YQSYY-CO2H). When these two mutations were combined, the affinity for YQSKL was further reduced and the affinity for YQSYY was further enhanced. This effect was amplified when an additional mutation, D507T, was incorporated into the AtPEX5-C double variant. The resulting triple variant was termed PEX5*. Various 9-amino acid sequences were added upstream of YQSYY, and the optimal resulting 14-amino acid sequence was termed PTS1*. The in vitro-validated binding of PEX5* to PTS1* was tested in vivo (in the moss Physcomitrella patens; by R. Paudyal) to investigate whether expression of PEX5* would allow the import of a fluorescent protein with a C-terminal PTS1* tag into peroxisomes.
Supervisor: Berry, Alan ; Warriner, Stuart L. ; Baker, Alison Sponsor: Leverhulme Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available