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

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 Fe^2+/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. [ABSTRACT FROM AUTHOR]