title: Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+exchanger.
abstract: Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe2+into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells. Cells that expressed PfVIT had decreased levels of total cellular iron compared with cells that did not express the protein. Qualitative transport assays performed on inverted vesicles enriched with PfVIT revealed that the transporter catalysed Fe2+/H+exchange driven by the proton electrochemical gradient. Furthermore, the PfVIT transport function in this system did not require the presence of any Plasmodium-specific factor such as post-translational phosphorylation. PfVIT purified as a monomer and, as measured by intrinsic protein fluorescence quenching, bound Fe2+in detergent solution with low micromolar affinity. This study of PfVIT provides material for future detailed biochemical, biophysical and structural studies to advance understanding of the vacuolar iron transporter family of membrane proteins from important human pathogens.
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Paola
authors.initials: P
authors.lastname: Labarbuta
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Katie
authors.initials: K
authors.lastname: Duckett
authors.affiliation: School of Biology, Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, United Kingdom.
authors.forename: Catherine H
authors.initials: CH
authors.lastname: Botting
authors.affiliation: Spectroscopy Group, Analytical Services, Almac, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, United Kingdom.
authors.forename: Osama
authors.initials: O
authors.lastname: Chahrour
authors.affiliation: Spectroscopy Group, Analytical Services, Almac, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Malone
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: John P
authors.initials: JP
authors.lastname: Dalton
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Law
mesh_terms: D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D005813:Genes, Synthetic; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D007501:Iron / Q000378:metabolism* / Q000494:pharmacology; D016778:Malaria, Falciparum / Q000469:parasitology; D050296:Microbial Viability; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D027682:Cation Transport Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006859:Hydrogen; D007501:Iron