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IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport
journal contribution
posted on 2019-10-18, 12:35 authored by Sameera
S. Abeyrathna, Nisansala S. Abeyrathna, Nathan Khoi Thai, Prithwijit Sarkar, Sheena D’Arcy, Gabriele MeloniIn intravacuolar
pathogens, iron is essential for growth and virulence.
In Legionella pneumophila, a putative transmembrane
protein inserted on the surface of the host pathogen-containing vacuole,
IroT/MavN, facilitates intravacuolar iron acquisition from the host
by an unknown mechanism, bypassing the problem of Fe(III) insolubility
and mobilization. We developed a platform for purification and reconstitution
of IroT in artificial lipid bilayer vesicles (proteoliposomes). By
encapsulating the fluorescent reporter probe Fluozin-3, we reveal,
by real-time metal transport assays, that IroT is a high-affinity
iron transporter selective for Fe(II) over other essential transition
metals. Mutational analysis reveals important residues in the transmembrane
helices, soluble domains, and loops important for substrate recognition
and translocation. The work establishes the substrate transport properties
in a novel transporter family important for iron acquisition at the
host–pathogen intravacuolar interface and provides chemical
tools for a comparative investigation of the translocation properties
in other iron transporter families.
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iron acquisitionhigh-affinity iron transporternovel transporter familylipid bilayer vesiclestransmembrane proteinVitro Real-Time Transportsubstrate transport propertiesmetal transport assayschemical toolssubstrate recognitionMutational analysisiron transporter familiestransition metalsFeMetal SelectivityTranslocation Kinetics Revealedintravacuolar pathogenstransmembrane helicestranslocation propertiesLegionella pneumophilaIroTintravacuolar iron acquisitionhost pathogen-containing vacuole
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