Part:BBa_K2271067

PEX5 variant R19

Brief introduction

Figure 1: TPR domains of the yeasts PEX5 protein.

Protein import into the peroxisome is mediated by two peroxins − PEX5 and PEX7. PEX5 is the protein that is responsible for most of the protein import into the peroxisomal. It recodnizes the very twelve amino acids at the C-terminus and mediates the import of the cargo protein. PEX5 of Saccharomyces cerevisiae contains 612 amino acid, that includes seven tetratricopeptide (TPR) regions which are specific interacting motifs of the receptor facilitating PTS1 binding (figure 1).
Figure 2 describes the whole import mechanisms of Pex5. Initially, Pex5 recognizes the PTS1 sequence of the espective protein enabling the translocation to the membrane. Subsequently, PEX5 interacts with PEX13, PEX14 and PEX17 leading to membrane integration and pore formation of PEX5. Afterwards, PEX5 binds to PEX8, which is combined with PEX2, PEX10 and PEX12, leading to the release of the cargo protein by competetive inhibition. Finally, ubiquitination of PEX5 leads either to receptor recycling or degradation, contolled by the degree of ubiquitination − while mono- or di-ubiquitination cause recycling, polyubiquitination causes degradation.

Figure 2: Import mechanism (Peroxisomal matrix protein import: the transient pore model, Erdmann et al. (2005))

Targeted mutagenesis

Figure 3: Workflow of our molecular dynamics approach

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

We wanted to achieve our aim with the help of molecular dynamics simulations. Therefore, we checked which amino acids of the receptor are interacting with the targeting signal and tried different mutations in this model.

This PEX5 variant was one of the promising candiates for experiments in the laboratory. We ordered the synthesis at IDT and started cloning once we got this part. Our plan for the validation of correct functionality was to tag a fluorescent protein − in this case mTurqouise − with the PTS1* and see if we have right localization.

Usage and Biology

This part should be used in combination with the corresponding PTS1* in a PEX5 knock out strain to obtain an orthogonal import of any protein of interest. With that, one is able to e.g. relocate metabolic pathways into the empty peroxisome. Thus, it prevents interferences, ensures a higher metabolite concentration due to the small volume and increases resistance to toxic substances because of the spatial isolation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1341
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1128
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1633
  
• 1000
  COMPATIBLE WITH RFC[1000]