IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport

Abstract

In 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 realtime 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.

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

“…47 IroT topology is characterized by 8 transmembrane (TM) helices and a long C-terminal domain. 48,49 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 49 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival.…”

“…48,49 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 49 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival. 48 The second protein selected is a copper P1B-type ATPase from E. coli (CopA), a transmembrane primary-active pump, and part of the P-type ATPase superfamily, that utilize energy generated by ATP hydrolysis to drive Cu + transport across biological membranes against electrochemical gradients.…”

Plastic recognition and electrogenic uniport translocation of 1st-, 2nd-, and 3rd-row transition and post-transition metals by primary-active transmembrane P1B-2-type ATPase pumps

“…The CmDZntA 166-794 orientation distribution cannot be controlled in the reconstitution procedure. 45,46 Thus, for all CmDZntA 166-794 activity determinations in proteoliposomes a 1 : 1 orientation distribution was assumed.…”

“…25,26,29,46 Thus, unique modalities of Cu + and Zn 2+ transport in P 1B -type ATPase pumps have been proposed, suggesting that substrate release mechanism can vary depending on the metal translocated and/or the subfamily considered. We thus expanded our "multi-channel" strategy with probes responsive to diverse stimuli (uorescent metal turn-on probes, pH probes, and membrane potential probes) 45,46 to investigate the mechanism of Zn(II)-pump translocation with all metal substrates that stimulate ATP hydrolysis (Zn 2+ , Cd 2+ , Pb 2+ , and Hg 2+ ). Considering the different coordination modes of these substrates possessing different ligand nuclearities (e.g., tetrahedral vs. trigonal pyramidal), different modalities during translocation could be envisaged.…”

Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport

“…This bacterium infects macrophage cells and relies on the formation of an intracellular compartment named the Legionella -containing vacuole (LCV). To meet the iron requirements of the L. pneumophila , IroT is recruited to the LCV membrane. , IroT is predicted to contain eight transmembrane α-helices and has also been shown to transport a wide array of divalent metals in addition to Fe 2+ , including Mn 2+ , Co 2+ , and Zn 2+ , with equivalent proficiency at least in vitro ; however, IroT is suggested to function only for Fe 2+ transport in vivo . While the exact mechanism of IroT-mediated metal transport has not been elucidated, several mutagenesis experiments display the importance of key amino acids for metal translocation.…”