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. 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 have determined 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. The trafficking of the Arn1p transporter in response to ferrichrome (FC) suggested the presence of a high-affinity receptor for FC. Whether this receptor was a separate gene product or part of the Arn1p transporter was unknown. The ARN transporters contain a unique carboxyl-terminal domain consisting of two predicted transmembrane domains, an extracytosolic loop, and a cytosolic tail, which are not present in other MFS transporters. We have conducted a structure-function analysis of this domain to evaluate its role in FC binding and trafficking. Mutations in the extracytosolic loop indicate that it functions as a receptor domain, and the binding of FC to this domain controls the sorting of the transporter. Thus, for this simple eukaryote, FC receptor and transporter functions have been combined in a single gene product. 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. To identify the transcription factor that controls the iron-dependent activation of GLT1, we will screen for genes that stimulate GLT1 expression under conditions of iron deprivation, and these genes will be evaluated as potential iron-regulated transcription factors. A single protein of the Aft1p regulon remains uncharacterized. Tis11p is a Zn-finger protein of the CCCH family that has representatives in all eukaryotic organisms. We have determined that Tis11p is a peripheral membrane protein that localizes to the late Golgi/trans-Golgi network. We are conducting a screen to identify proteins that interact with Tis11p in its native context of Golgi membranes using the newly developed """"""""split ubiquitin"""""""" approach. This technique is designed to identify protein-protein interactions between integral membrane proteins without the need for translocation to the nucleus, as is required in a traditional two-hybrid screen. Very little is known about heme transport in eukaryotes and no fungal heme transporters have been identified. We have identified a gene from C. albicans that, when overexpressed in S. cerevisiae, facilitates the uptake of heme. This gene, tentatively termed HUF1 for heme utilization factor 1, is part of a fungal gene family with three homologues in C. albicans and, surprisingly, three orthologues in S. cerevisiae. These genes have ten predicted transmembrane domains and may function as transporters. Members of this family of genes serve an essential function in yeast, as deletion of two members of this family is lethal in S. cerevisiae. We have constructed a double deletion strain in S. cerevisiae that expresses Huf1p from a regulatable promoter, and we are phenotypically characterizing this strain. Localization studies with an epitope-tagged Huf1p and a screen for genes that rescue the growth defects of the double deletion mutant are underway.
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