Understanding and Exploiting Peroxisomal Import Mechanisms

The peroxisome is a near-ubiquitous organelle in eukaryotes, which must import proteins to carry out its functions. Most peroxisomal matrix proteins are targeted by a peroxisome targeting signal (PTS1) at the C-terminus. This tripeptide is recognised by the import receptor protein PEX5, which shuttles the matrix protein across the peroxisomal membrane. This work reports the design and synthesis of bifunctional chemical probes, which can bind to PEX5 via a PTS1 peptide and bind a second protein, which represents a cargo protein. Labelling a protein with a PTS1 in this way is a completely novel concept. These probes are used to investigate two different aspects of peroxisomal matrix protein import. Chapter 2 addresses the question of whether a protein can be labelled with a PTS1 via the probes and subsequently be imported into the peroxisome by PEX5. The development of such a system could be used to trigger peroxisomal import of protein not native to the peroxisome as a tool to study protein-protein interactions and investigate the effects of protein knockdown. In vitro pulldown assays successfully demonstrated the interaction of probe-labelled proteins with PEX5 proteins. Cell-based assays investigated if using these probes can result in the labelled protein becoming resident in the peroxisomal matrix. Further optimisation is required to validate their function in vivo. Chapter 3 develops a method to create a form of PEX5, which is permanently bound to a cargo protein. This would be a useful tool to elucidate the structure of peroxisomal matrix protein import complex and investigate the mechanisms of cargo unloading across the peroxisomal membrane. The probes interact with PEX5 via a PTS1, and subsequently become covalently bound to a cysteine on PEX5 to create a permanent PEX5-cargo complex. The probes were extended to include a cargo protein-binding motif and a protein complex of PEX5 with a covalently bound cargo protein was formed. Together these investigations show the utility of chemical probes and how they can be used to investigate and manipulate peroxisome import mechanisms.