Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+ exchanger
MetadataShow full item record
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.
Labarbuta , P , Duckett , K , Botting , C H , Chahrour , O , Malone , J , Dalton , J & Law , C 2017 , ' Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe 2+ /H + exchanger ' Scientific Reports , vol 7 , 42850 . DOI: 10.1038/srep42850
© The Author(s) 2017. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
This work was funded in part by a Royal Society Wolfson Research Merit Award (to J.P.D). It was also supported by the Wellcome Trust (grant number WT079272AIA) which funded the MALDI TOF-TOF analyser at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews. P.L was supported by a Northern Ireland Department of Employment and Learning (DEL) postgraduate studentship.
Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.