title: Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2.
abstract: The malaria parasite Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2. Among them, EXP2 is thought to constitute the transmembrane pore, whereas the other components seem to play a role in unfolding the luggage proteins or providing a driving force. However, detailed functional and structural characterizations of PTEX proteins have not been performed. In this study, we expressed and characterized the membrane-associated component EXP2. Because expression of EXP2 is lethal to E. coli, EXP2 was expressed as a fusion protein with GST, and the recombinant EXP2 was obtained by protease digestion. The recombinant EXP2 formed pores in bilayer lipid membranes. The inner diameter of the pore was estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, we determined that the pore comprises approximately 10-12 EXP2 subunits. However, there is a possibility that the pore structure is different in the PTEX complex. These results provide important insights in the protein transport mechanism of PTEX, which will aid in developing new drugs targeting PTEX.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Hakamada
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Kawano
authors.affiliation: Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Noguchi
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address: yohda@cc.tuat.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D003062:Codon; D004912:Erythrocytes / Q000469:parasitology*; D004926:Escherichia coli / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D010963:Plasmodium falciparum / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D003062:Codon; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Bilayer lipid membrane; Channel; Malaria; Plasmodium; Pore-forming protein; Translocon