Part:BBa_K2271105
PEX5 variant R15
Brief introduction
Protein import into the peroxisome is mediated by two so called peroxins − PEX5 and PEX7. PEX5 is the protein that is responsible for most of the protein import into the peroxisomal membrane. It detects the very twelve amino acids at the C-terminus and then mediates the import of the protein attached to it. The PEX5 of Saccharomyces cerevisiae is a 612 amino acid long proteins, that contains seven tetratricopeptide (TPR) regions which are interacting motifs of the receptor. These predicted TPR domains can be seen in figure 1 − this structure was used for the targeted mutagenesis based on educated guesses (see part design).
Figure 2 shows all steps of the import mechanisms. It starts with the binding of the PTS1, then the transport to the membrane, where PEX5 interacts with PEX13, PEX14 and PEX17 which leads to membrane integration and pore formation of PEX5. Then the interaction with PEX8, which is bound to PEX2, PEX10 and PEX12, causes cargo release into the matrix. Subsequently ubiquitination of PEX5 lead either to receptor recycling or degradation. This depends on the degree of ubiquitination − while mono- or di-ubiquitination cause recycling, polyubiquitination causes degradation.
Targeted mutagenesis
The achievement of an orthogonal import into the peroxisomes was our teams most important objective − we wanted to offer a PEX5 variant with the following features: * Interaction with a new PTS1* signal and no interaction with the natural PTS1 − by implication this means that the wildtype does not interact with the PTS1* * Full functionality − cargo release and receptor recycling should still work * Protein missfolding should not happen
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 409
- 1000COMPATIBLE WITH RFC[1000]
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