Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+exchanger

Paola Labarbuta,Osama Chahrour,John P. Dalton,Christopher J. Law,Catherine H. Botting,Katie Duckett,John Malone

doi:10.1038/srep42850

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.

Highlights

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
Our results demonstrate that heterologous overexpression of PfVIT in E. coli confers increased resistance to iron-mediated cell death to the bacterial cells and that the PfVIT transport mechanism is Fe2+/H+ antiport driven by the proton electrochemical gradient
Vacuolar iron transport proteins play a vital role in compartmentalisation of labile Fe2+ in non-animal cells and thereby contribute to iron homeostasis in those systems

Summary

Introduction

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. The same redox properties of iron that have been exploited for beneficial purposes make it potentially cytotoxic; oxidation of excess ferrous iron (Fe2+) to the ferric state (Fe3+) by the Fenton reaction in the cell cytoplasm results in production of reactive free hydroxyl radicals that cause oxidative damage to nucleic acids, lipids and proteins[6] To prevent such damage but at the same time ensure adequate supply of essential iron, cells have evolved integrated mechanisms for maintenance of iron homeostasis via tight and coordinated regulation of the systems that control iron provision and storage[4]. Bioinformatics studies performed by Martin et al.[15,16] suggested a 273 amino acid, ~31 kDa VIT family homologue termed PfVIT (PlasmoDB ID: PF3D7_1223700; Supplementary Fig. S2a) was localised to the digestive vacuole (DV) membrane of P. falciparum from where it transports ferrous iron into the acidic vacuole interior via an antiport reaction. Our results demonstrate that heterologous overexpression of PfVIT in E. coli confers increased resistance to iron-mediated cell death to the bacterial cells and that the PfVIT transport mechanism is Fe2+/H+ antiport driven by the proton electrochemical gradient

Methods

Results

Conclusion