The Cellular Response To Iron Starvation And Intoxicatio

Diabetes, Digestive, Kidney Diseases

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Abstract

Iron Metabolism in the Model Eukaryote Saccharomyces cerevisiae Iron is an essential nutrient for virtually every organism, yet it can also be a potent cellular toxin. Dysregulated iron metabolism and iron overload are features of a growing number of human diseases. Some genes involved in cellular iron uptake and export have been identified, yet very little is known about inter- and intracellular iron transport, intracellular iron utilization, and the regulation of these processes. A combination of genetic, biochemical, and cell biological approaches is needed to understand iron metabolism and the role of iron in human disease. These approaches can be combined in the simple eukaryote, Saccharomyces cerevisiae. Studies of metal metabolism in budding yeast have yielded important insights into iron, copper, and zinc metabolism in both humans and pathogenic microorganisms. Genetic studies of iron metabolism in a simple eukaryote will allow us to discover new genes involved in iron homeostasis as well as to determine the cellular response to iron overload and iron deprivation. We have used cDNA microarrays representing the entire yeast genome to identify genes that are regulated according to the availability of iron and the activity of Aft1p, the major iron-dependent transcription factor. Using available genome and protein databases, we have grouped these newly identified genes into families and have begun their functional evaluation. Three genes that are predicted to encode GPI-anchored proteins of the yeast cell wall are new members of the Aft1 regulon. The corresponding mRNA transcripts show over a 50-fold induction under conditions of iron deprivation and Aft1-1up expression and the upstream control regions of the genes contain multiple Aft1p consensus sites. We confirmed that these proteins were incorporated into the cell wall by immunofluorescence. Strains bearing deletions of these genes were constructed and they exhibit defects in the retention of iron in the cell wall. Additionally, these strains exhibit reduced rates of siderophore iron uptake and evidence of altered intracellular iron sensing, indicating that cell wall mannoproteins contribute to iron uptake at the plasma membrane. We have identified and genetically characterized a novel system of eukaryotic iron uptake. Four homologous genes regulated as part of the Aft1-regulon (ARN1-4) were found to facilitate the transport of siderophores. We are investigating how the Arn proteins facilitate the uptake of siderophore when they do not appear to be expressed on the plasma membrane. To track the path of Arn proteins within the cell, we have examined siderophore uptake and Arn protein localization in a series of well-characterized yeast mutants that exhibit temperature-sensitive defects in protein sorting. These experiments have revealed that, in the absence of transport substrate, Arn1p is sorted directly from the Golgi to the late endosome/pre-vacuolar compartment and does not cycle to the plasma membrane. Upon the addition of low concentrations of specific substrate, however, Arn1p stably relocalizes to the plasma membrane. Higher concentrations of siderophore induce cycling between the plasma membrane and endosomal compartments. Our data suggest that siderophore binding at the plasma membrane and siderophore transport across the endosomal membrane may be separate and necessary steps in siderophore uptake. In order to understand control of the intracellular trafficking of Arn1p, we have performed site-directed mutagenesis of Arn1p and constructed a series of plasmids that encode proteins with mutations in the predicted extracellular domains. We have determined that Arn1p exhibits two ferrichrome binding sites of differing affinities, and that mutations in the extracellular domains result in loss of substrate binding activity, loss of uptake activity, and altered intracellular trafficking. We have also identified sequences in the carboxyl-terminal tail of Arn1p that control intracellular trafficking. Because siderophore iron uptake has been shown to be important for virulence in pathogenic fungi, we entered into collaboration with the laboratory of Dr. Jerry Kaplan to identify siderophore transporters in Candida albicans. Using strains of S. cerevisiae developed in our laboratory, the C. albicans ferrichrome transporter was cloned and functionally characterized. We are functionally characterizing the gene HMX1, a new member of the Aft1p regulon. HMX1 encodes a protein that is similar to human and bacterial heme oxygenases. We have determined that HMX1 is localized to the endoplasmic reticulum, binds heme, and can catalyze the enzymatic degradation of heme. Our microarray analysis unexpectedly revealed that other metabolic pathways are regulated in response to iron availability. VHT1 is a newly identified member of the Aft1p regulon and encodes the plasma membrane biotin transporter. The expression of the biotin transporter and the biotin biosynthetic pathway are inversely regulated by iron. Furthermore, we determined that the biotin biosynthetic pathway is transcriptionally down-regulated and inactive when cells are grown in limiting concentrations of iron. These observations are likely due to the existence of an iron-sulfur cluster protein in the biosynthetic pathway, indicating that yeast shift from iron-dependent to iron-independent systems under conditions of iron deprivation. The iron-dependent enzyme glutamate synthetase is also transcriptionally down-regulated in response to iron deprivation, and we have characterized the transcriptional control regions of this gene to identify the iron-responsive sequences. We intend to identify the transcription factor(s) that contribute to this iron-regulated pattern of gene expression. We plan to continue our functional analysis of new members of the Aft1 regulon and to investigate the cellular response to iron deprivation and iron overload. Our aim is to extend this work to examine analogous processes in human cells.