Part:BBa_K2271016

PTS1 variant P*

The peroxisomal targeting signal 1 (PTS1) is a protein localization signal which is recognized by the Pex5 receptor protein. Cargo proteins which contain this C-terminal PTS1 tag were imported into the peroxisomes in dependence to Pex5 interaction. The native PTS1 sequence contains three amino acids (SKL motive; Serine-Lysine-Leucin).
In our project, we designed an artificial PTS1 peptide sequence which is recognized by our modified Pex5 (BBa_K2271067) in order to provide a fully orthogonal peroxisomal protein import machinery in yeast strains deficient for the natural Pex5 gene.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]