Coding

Part:BBa_K2271103

Designed by: Jan Maika and Philipp Rink   Group: iGEM17_Cologne-Duesseldorf   (2017-10-27)


pex15

Biology and Usage

Figure 1. The micrscopy showing a localisation of the fluorescence protein Venus with the typical peroxisomal shape. This indicates, an peroxisomal membrane licalisation of Venus-Pex15

This part contains a truncated version of Pex15 (aa315-383) from S. cerevisiea . [1]
Pex15 is a tail anchord peroxisomal membrane protein with its c-terminus directed to the peroxisomal lumen and its n-terminus directed to the cytosol. It is required for peroxisome biogenesis. In cells lacking Pex15 peroxisomal matrix proteins mislocalize to the cytosol while overexpression results in impaired peroxisome assembly. [2]

Experimental design

The functionality of this part was validated by microscopy. For the microscopy mVenus was fused to the C-Terminus of Pex15. We could observe a peroxisomal typical localisation in the cell.
Additionaly we tested this part as a membrane anchor for the v-SNARE Snc1. We co-expressed the Snc1-Pex15 construct with GUS-PTS1 to perform a GUS Assay. The secreted GUS in the supernatant was measured with the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU). The fluorescent 4-MU was measured with a plate reader (excitation: 365 nm, emission: 465 nm).

Figure 2. Relative fluorescence units per minute (RFU/min) measured for supernatants of different S. cerevisiae strains. The fluorescence was measured for 12 hours in intervals of 10 minutes with an excitation of 365 nm and an emission of 465 nm. For the strain BY4247 (wt) which was used as the background strain the fluorescence did not increase over measured period. The lysis controls (GUS-PTS1; ∆Pex11 GUS-PTS1) showing a lower activity than the samples of strain with peroxisomes decorated with Snc1. The highest activity could be measured in the strain using Pex15 with a linker as a membrane anchor (Pex15 L). The assay was performed in three technical replicates.

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]

References

[1] Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites.
André Halbach, Christiane Landgraf, Stephan Lorenzen, Katja Rosenkranz, Rudolf Volkmer-Engert, Ralf Erdmann, and Hanspeter Rottensteiner (2006)
Journal of Cell Science 119, 2508-2517 Published by The Company of Biologists 2006 doi:10.1242/jcs.02979

[2] Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.
Ype Elgersma, Liane Kwast, Marlene van den Berg, William B. Snyder, Ben Distel, Suresh Subramani, Henk F. Tabak(1997)
EMBO J 16(24):7326-41 DOI 10.1093/emboj/16.24.7326


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