Iron Metabolism in the Budding Yeast Saccharomyces cerevisiae

The experiments that confirmed the role of multicopper oxidases in iron metabolism in vertebrate systems came from studies on iron transport in yeast. Yeast, particularly the budding yeast Saccharo-myces cerevisiae is perhaps the simplest of the eucaryotes, as they grow as single cells and can be both haploid and diploid The haploid status of yeast permits the selection of yeast mutants. Once a phenotype has been selected, genes responsible for modifying a specific phenotype can be identified by complementation using a genomic library. Because most yeast genes do not have introns, a yeast genomic library is functionally equivalent, although much easier to prepare than a cDNA library. Because all yeast genes have been cloned, only a small amount of sequence and subcloning is required to identify which gene on a genomic plasmid is responsible for modifying the mutant phenotype. A gene can modify a phenotype because it is allelic to the mutant gene or because it is a suppressor and metabolically bypasses the defective step. These two alternatives can easily be discriminated by deleting specific genes and examining the resulting phenotype. As yeast readily undergo homologous recombination, it is relatively easy to inactivate specific genes. The ability to inactivate specific genes permits a rigorous analysis of gene function; indeed, homologous recombination extends

Fig. 1. Iron transport across the plasma membrane in the budding yeast S. cerevisiae. See text for details.

Koch's postulates to genetics. If the deletion strain is genetically and phenotypically identical to the mutant strain (i.e., a diploid between the two shows the mutant phenotype) and is phenotypically identical, then the identified gene is most probably the normal allele of the mutant gene.

Studies on yeast iron metabolism identified the existence of two different transport systems for elemental iron and a transport system for iron-siderophore complexes (9) (Fig. 1). Both transport systems for elemental iron recognize Fe(II) as a substrate. This was demonstrated by showing that Fe(II) chelators inhibited iron uptake, whereas Fe(III) chelators were unable to inhibit iron transport. Further, there was biochemical evidence for a cell surface reductase activity that converted Fe(III) to Fe(II) (10). The most compelling work resulted from the cloning of the reductase gene. Dancis et al. identified a yeast mutant unable to grow on Fe(III) because of the lack of ferrireductase activity (11). The gene for the ferrireductase was cloned by complementation analysis and was termed FRE1. Later studies identified a second cell-surface ferrireductase termed FRE2 (12).

These reductases provide the substrate for the two elemental iron-transport systems. The low-affinity transport system encoded by the FET4 gene is not specific for iron and can accumulate other transition metals (13). The other system, comprised of the products of the FET3 and FTR1 genes, is a high-affinity transport system that is specific for iron (Fig. 2). The two transport systems are regulated differently. The high-affinity iron-transport system is induced when cells are grown on low-iron media. The low-affinity system is also negatively regulated by media iron, but the concentration required to turn off the system is usually not seen in most media formulations. The two systems are genetically separable, as they are the products of different genes. That the two systems are different both genetically and physiologically permitted a genetic approach to identifying the systems. Cells can be mutagenized and mutants in the high-affinity transport system can be identified by their inability to grow on low-iron media. These mutants were "rescued" by the addition of iron, as they could still obtain iron through the low-affinity transport system.

Fig. 2. Proposed model for high-affinity iron transport. See text for details.

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Fig. 2. Proposed model for high-affinity iron transport. See text for details.

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