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 ultimate goal of our project was to accomplish a fully orthogonal import into the peroxisomes − we wanted to provide a PEX5 variant with the following abilities:

• Recognition of our artificial PTS1* sequence and rejection of the natural PTS1 − rejection of our designed PTS1* by the wild type PEX5 receptor
• Full functionality − cargo release and receptor recycling should still work
• Correct protein folding

In order to design this synthetic receptor we utilized molecular dynamics simulation software. Using this software we planned different mutations of the binding pocket facilitating molecular interaction and recongition of distinguished PTS1 peptides.

Verification of our orthogonal protein import machinery was ensured using the frourescence protein mTurquoise combined with our artificial PTS1* sequence. Peroxisomal localization was proved by coexpression of a peroxisomal marker protein PEX13 fused to the fluorescence protein mRuby.

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Results

Figure 1:PEX5 knock out strain, transformed with the R19 variant, mTurqouise tagged with the PTS1 and the PEX13 membrane anchor fused to mRuby.

Figure 2:PEX5 knock out strain, transformed with the R19 variant, mTurqouise tagged with the PTS1 and the PEX13 membrane anchor fused to mRuby.

Figure 1 and 2 display our results that verify the function of our orthogonal protein import machinery. Coexpression of our receptor with mTurquoise tagged to PTS1* leads to import of the fluorescent reporter protein, indicated by localized fluorescence areas. The negative control consists of the wild type yeast strain carrying mTurquoise tagged with our designed PTS1* demonstrate the exact opposite: Fluorescence was detected in the whole cell, indicating that our receptor is not capable to recognize and import the modified peroxisomal targeting signal with its cargo. Peroxisomal localization was verified by coexpression of our import machinery with the peroxisomal marker protein PEX13 fused to the fluorescence marker protein mRuby.

Usage and Biology

This part should be used in combination with the corresponding PTS1* (https://parts.igem.org/Part:BBa_K2271016) in a PEX5 knockout strain to obtain an orthogonal import of any protein of interest. This mechanism enables different opportunities like relocating 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]