Academic journal article Genetics

Genome-Wide Screen for Genes with Effects on Distinct Iron Uptake Activities in Saccharomyces Cerevisiae

Academic journal article Genetics

Genome-Wide Screen for Genes with Effects on Distinct Iron Uptake Activities in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.

RECENT advances have made possible genome-wide approaches to the biology of eukaryotes. Many genes and proteins implicated in iron metabolism have been identified in the past 10 years using classical genetic approaches of mutagenesis, phenotype screening, complementation, and homology searching. More recently, global transcriptome analysis has become possible, and the effects of iron manipulations on gene expression have been studied. Using these methods, a number of iron transporters and regulators have been discovered (KOSMAN 2003). However, many aspects are still missing from a complete picture of cellular iron metabolism. Mediators of intracellular iron trafficking, distribution, organelle transport, and processing must exist, although few have been identified. Regulators coordinating use of iron in cofactors such as heme and Fe-S also must exist and the cofactors must be made, trafficked, and delivered to target apoproteins. Most of these key components are yet to be found. Finally, essential processes such as transcription, translation, protein trafficking, organelle biogenesis, and secretion are likely to influence iron metabolism to different degrees and in different ways. A new tool, the haploid knockout collection of Saccharomyces cerevisiae, has recently become available. Each strain carries a complete deletion of a single ORF, and the collection can be manipulated and examined in microtiter well format. We wondered whether we could use this collection to discover novel genes involved in iron metabolism.

The idea underlying this screen is that yeast expresses two independent pathways by which iron can enter cells: siderophore and reductive (LESUISSE and LABBE 1989; DANOIS et al. 1990). Both pathways are regulated and responsive to iron availability (YUN et al. 2000a). Therefore, random or targeted mutations in the genome that alter basic processes involved in cellular iron metabolism will affect activities for both pathways. By contrast, mutations selectively impacting one or the other system will be due to altered expression, localization, or function of pathway-specific components.

The siderophore pathway mediates iron uptake from siderophores (YUN et al. 2000b; LESUISSE et al. 2001). Siderophores are small molecules that bind, solubilize, and chelate ferric iron in the environment with tremendous affinity. They are synthesized by a nonribosomal enzymatic process and secreted by bacteria and fungi, although not by S. cerevisiae. This yeast, however, has evolved a means for acquiring iron from siderophores made by other organisms. …

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