Analysis of the High-Affinity Iron Uptake Proteins, FOX1 and FTR1, in Chlamydomonas reinhardtii

Multicopper oxidase (MCO) proteins play a vital role in iron metabolism in bacteria, fungi, algae and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of the MCO protein, Fet3p, to Fe3+ transport into the cell by the ferric iron permease, Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas reinhardtii FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to yeast since Chlamydomonas expresses a ferric iron permease homolog, FTR1. In this thesis, FOX1 was shown to support high-affinity iron uptake in Chlamydomonas by demonstrating that a reduction in high-affinity iron uptake correlated with a loss of FOX1 protein. FOX1 was classified as a multicopper oxidase with specificity toward ferrous iron by analysis of a FOX1 structural model and purified recombinant FOX1 protein. Furthermore, FOX1 was shown to be localized to the Chlamydomonas plasma membrane with its MCO domain in the extracellular milieu. The role of FTR1 was determined by demonstrating that it supports ferroxidase-dependent iron uptake in S. cerevisiae. The substrate for this uptake was shown to be Fe2+, not Fe3+, suggesting Fe3+-transport by FTR1 is coupled to ferroxidase activity. Additionally, the topology and localization of FTR1 confirmed its resemblance to the fungal Ftr proteins. Finally, the kinetic inhibition of high-affinity iron uptake in Chlamydomonas using a series of ferric iron chelators is consistent with a kinetic Fe 3+ channeling mechanism in which iron permeation is coupled to Fe 2+ oxidation by FOX1.