Analysis of the localization and interaction between proteins of the fungal heme uptake system

Fungal pathogens of the Candida clade have evolved a system that enables them to take environmental heme up as heme or iron source. The fungal heme uptake system consists of a family of differentially localized extracellular CFEM hemophores such as Rbt5, Pga7 and Csa2 that can capture heme from hemoglobin and other host proteins, and transfer it across the cell envelope to the cell membrane. At the plasma membrane, heme is thought to be endocytosed into the cell via Frp1 and Frp2, transmembrane proteins related to ferric reductases. To better understand the interactions between these proteins, we have tagged the CFEM hemophores Rbt5 and Pga7, as well as FRP1, with GFP and its derivatives mScarlet and mCherry. The tagged proteins were tested for activity and cellular localization under different conditions. Interaction between FRP proteins and CFEM hemophores is monitored in vivo by co-localization of fluorescent protein-tagged proteins. Mobility of the membrane-bound proteins is monitored by FRAP (Fluorescence Recovery after Photobleaching). Expression of recombinant CFEM hemophores, which were shown to exchange heme in vitro, as FP fusions enables to test their interactions by FRET (Forster Resonance Energy Transfer).