Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2

Area of Science:

• Molecular Biology
• Parasitology
• Biophysics

Background:

• The malaria parasite Plasmodium falciparum relies on the Plasmodium translocon of exported proteins (PTEX) for proliferation in human red blood cells.
• PTEX facilitates the export of hundreds of parasite proteins into the host cell cytosol.
• PTEX is a complex of five proteins: EXP2, PTEX150, PTEX88, Hsp101, and TRX2, with EXP2 hypothesized to form the transmembrane pore.

Purpose of the Study:

• To functionally and structurally characterize EXP2, a key membrane-associated component of the PTEX complex.
• To elucidate the role of EXP2 in protein transport across the parasite membrane.

Main Methods:

• Recombinant EXP2 was expressed as a GST fusion protein in E. coli due to its toxicity.
• Protease digestion was used to obtain purified recombinant EXP2.
• Pore formation was assessed in bilayer lipid membranes.
• Pore diameter was determined using electron microscopy and channel current analysis.
• Subunit stoichiometry was estimated via size exclusion chromatography and blue native PAGE.

Main Results:

• Recombinant EXP2 successfully formed pores in artificial lipid bilayers.
• Electron microscopy and channel current measurements indicated an inner pore diameter of approximately 3.5 nm.
• Size exclusion chromatography and blue native PAGE suggested the pore is composed of approximately 10-12 EXP2 subunits.
• The precise structure within the native PTEX complex may differ.

Conclusions:

• EXP2 forms functional pores with specific dimensions, providing critical insights into the PTEX protein export machinery.
• Understanding EXP2's structure and function is vital for developing novel anti-malarial drugs targeting the PTEX complex.