Investigation of YG-Rich Regions of Trypanosoma Brucei Peroxins 13.1 and 13.2

Anne Crosswell, Emily Knight, Meredith Morris

Abstract

Kinetoplastids are unique to other Eukaryotes in that they have two Peroxin 13 (Pex13) proteins, Pex13.1 and Pex13.2. All other Eukaryotes only contain one Pex13 gene. We are investigating the evolutionary benefit of possessing both isoforms. Both Pex13.1 and 13.2 have an N-terminal YG-rich region and are involved in glycosome biogenesis. In aligning the amino acid sequences, it was found that the Pex13.1 YG region has conserved serine residues where Pex13.2 has conserved leucine residues. This difference may cause differing binding affinities between Pex13.1 and 13.2 for peroxisome import receptors, leading to an increased peroxisomal import efficiency. We will use Immunoprecipitation to determine the binding affinities that the receptors, Pex5 and Pex7, have for both the Pex13.1 and 13.2 YG-rich regions.

Introduction

Trypanosoma brucei is a species of parasite responsible for causing African Sleeping Sickness. It is transmitted to humans and animals by the tsetse fly and can be fatal. Trypanosomes exist in both a procyclic form and a bloodstream form. When in the bloodstream form, trypanosomes rely on compartmentalization of glycolytic processes into “glycosomes” in order to produce ATP. Glycosomes are homologous to peroxisomes. They sequester glycolytic and other metabolic enzymes from the rest of the cell and are essential for viability. [1]

Pex5 and Pex7 are receptors that recognize PTS1 (Peroxisome Targeting Sequence) and PTS2-containing proteins [2]. Pex5 and Pex7 bind these glycosome targeted proteins and bring them to the glycosomal surface [2]. Pex13 and Pex14 form a complex creating a pore in the membrane for translocation of PTS1/PTS2 proteins [D]. Pex5 and Pex7 are then recycled [2]. This process is illustrated in Figure 1. 

Kinetoplastids are the only Eukaryotes that produce two isoforms of Peroxin 13. The two forms of the Pex13 gene likely arose from a gene duplication event. Duplications of genes are typically are only conserved if there is a division of function, making both genes necessary.  [4].

Both isoforms have two transmembrane regions and a YG rich N terminus [2]. Pex13.1 contains a SH3 region at the C terminus [2]. (Figure 2)

Hypothesis

The fact that both Pex13.1 and 13.2 are duplicate genes and have been conserved in Kinetoplastids, there is likely an evolutionary advantage to producing both. Pex13.1 and 13.2 have slightly different structures (including different conserved sequences in the YG-rich region)  which may cause to differing affinities for Pex5 and Pex7. This may result in increased glycosomal import efficiency.

Materials and Methods

The following proteins will be expressed using cloning techniques in E. Coli:

  • Pex13.1 YG-rich region
  • Pex13.2 YG-rich region
  • Pex13.1 SH3 region
  • Pex5
  • Pex7

Steps of Immunoprecipitation Assay to determine if there is a difference in binding affinity between Pex13.1 and 13.2 for Pex7 and Pex5

Immunoprecipitation techniques will be used in order to establish if there is any difference in the binding affinities for Pex5/ Pex7 between Pex13.1 and 13.2. (Figure 3). 

Figures

  • Figure 1.

    Process of protein import into glycosome. PTS1 containing sequences are recognized by Pex5, and PTS2 by Pex7 receptors. Pex5 and Pex7 then bind the Pex13/Pex14 glycosomal membrane complex. Translocation occurs and the receptor is recycled.

  • Figure 2.

    Kinetoplastids contain two isoforms of Pex13. Pex13.1 and 13.2 both have two transmembrane regions and an N-terminal YG-rich region. Pex 13.1 contains a C-terminal SH3 region where Pex13.2 does not.

  • Figure 3.

    Immunoprecipitation techniques will be used in order to establish if there is any difference in the binding affinities for Pex5/ Pex7 between Pex13.1 and 13.2.

Results