Insights into iron trafficking and handling in eukaryotic cells
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Iron is essential for growth and replication of eukaryotic cells and must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast - a multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene - were shown to mediate high-affinity iron uptake. Fe 3+ generated by Fet3p is ligand for the iron permease, Ftr1p. To work as a permease, Ftr1p must have amino acid residues critical for iron transport. We identify iron-binding motifs conserved in fungal permeases and essential to Ftr1, for iron transport in Saccharomyces cerevisiae. The DASE motif in extracellular Loop 6 plays an important role in channeling the Fe 3+ from Fet3p to Ftr1p. The REXLE motifs of transmembrane (TM) 1 and TM 4 were shown to have distinct roles in the membrane permeation of iron. The arginine and glutamic acids in the RECLE motif of TM 1 and the glutamic acids in the REGLE motif of TM 4 are absolutely essential. Once it has passed through the channel, the iron associates with the carboxyl terminus of Ftr1p, which was shown to be essential in iron uptake. The accepted view is that upon entering the eukaryotic cell, the increased cellular iron levels appear initially in a transitional pool, the labile iron pool, LIP. This pool is accessed by several proteins, involved in cell and/or organismal iron homeostasis. Ribonucleotide reductase, RR, is the only de novo route to deoxyribonucleotides required for efficient replication. It contains an essential iron prosthetic group presumably obtained from the LIP and is produced by all vertebrates and many eukaryotic large DNA viruses. We tested if manipulation of host iron status can lead to a modulation of the proliferation and virulence of vaccinia, a large DNA pox virus. We manipulated the labile iron pool of eukaryotic cells by stably over-expressing ferritin. We show that both ferritin and the viral R2 obtain iron from the same source i.e. the newly arrived iron in the labile iron pool. The over-production of Ft H chain leads to 2-fold increase in iron responsive protein (IRP) binding activity, helping us to determine the status of the pool of cell iron to which this iron sensor responds. However, over-production of Ft H chain does not have any significant effect on blocking or suppressing viral pathogenicity. In multicellular organisms, iron must be distributed from the site of absorption to cells that require it. In mammals, iron uptake into the intestinal epithelial cells is followed by its subsequent release into the plasma where it binds transferrin to be transported to other organs for transferrin receptor-mediated iron uptake. Ceruloplasmin (Cp), a multicopper ferroxidase, plays a critical role in the iron cycle by establishing a rate of iron oxidation sufficient for iron release from cells. Cp catalyses the formation of Fe 3+ species for incorporation into apo transferrin. Loss-of-function mutations in the ceruloplasmin gene results in aceruloplasminemia (AC), a neurodegenerative disease. We have developed a mammalian cell system for the rapid and robust production of recombinant human ceruloplasmin. We optimized the necessary conditions which includes expression vector, cell line, culture medium and transient transfection process of suspension-growing cells, to obtain a high yield of r-hCp. Under these conditions, 8 mg of purified hCp could be obtained from a 1L culture following purification steps standardized for Cp. We also show that certain mutant forms of Cp found in AC patients, get synthesized and secreted using this system. Recombinant forms of WT hCp and mutant hCp proteins will be useful in biomedical research for the determination of structure-function relationships that will elucidate the molecular basis as well as a possible treatment of the human disease aceruloplasminemia.