Fluorescence-Based Proteoliposome Methods to Monitor Redox-Active Transition Metal Transmembrane Translocation by Metal Transporters.

Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.