Almost half of all enzymes must associate with a particular metal to function. An ambition is to understand why each metal-protein partnership arose and how it is maintained. Metal availability provides part of the explanation, and has changed over geological time and varies between habitats but is held within vital limits in cells. Such homeostasis needs metal sensors, and there is an ongoing search to discover the metal-sensing mechanisms. For metalloproteins to acquire the right metals, metal sensors must correctly distinguish between the inorganic elements.

All living organisms use numerous signal-transduction systems to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we review recent progress in our understanding of how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental cues.

The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.

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The transcription factors Aft1p and Aft2p from Saccharomyces cerevisiae regulate the expression of genes that are involved in iron homeostasis. In vitro studies have shown that both transcription factors bind to an iron-responsive element (FeRE) that is present in the upstream region of genes in the iron regulon. We have used DNA microarrays to distinguish the genes that are activated by Aft1p and Aft2p and to establish for the first time that each factor gives rise to a unique transcriptional profile due to the differential expression of individual iron-regulated genes. We also show that both Aft1p and Aft2p mediate the in vivo expression of FET3 and FIT3 through a consensus FeRE. In addition, both proteins regulate MRS4 via a variant FeRE with Aft2p being the stronger activator from this particular element. Like other paralogous pairs of transcription factors within S. cerevisiae, Aft1p and Aft2p are able to interact with the same promoter elements while maintaining specificity of gene activation.

Aging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did not include oxidative stress genes. Our findings suggest how the interplay between ROS, antioxidants, and efficiency of energy production regulates the lifespan. The data also suggest a model wherein factors implicated in aging (for example, ROS) may influence the lifespan yet not be the cause of aging.A ging is typically viewed as the accumulation of damage that results in death. This damage is thought to be caused by reactive oxygen species (ROS). Indeed, ROS can damage biomolecules, accelerate aging, and shorten lifespan (1-3), whereas antioxidant enzymes alleviate these effects, providing support for a free radical theory of aging (4). The contrasting effects of ROS and antioxidants, as well as correlation between accumulation of oxidative damage in cellular components and the lifespan of an organism, have been the main arguments in favor of this theory.Proteins become oxidatively damaged by oxidation of their amino acid residues and cofactors. Methionine residues in proteins are particularly susceptible to oxidation by ROS, resulting in methionine-S-sulfoxides (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO) (5). Oxidized methionines can be repaired by antioxidant enzymes Met-S-SO reductase (MsrA) and Met-R-SO reductase (MsrB) (6). Recent studies revealed that methionine sulfoxide reduction provides lifespan extension in animals: deletion of the MsrA gene in mice reduced lifespan by Ϸ40% (7), whereas MsrA overexpression, predominantly in the nervous system, extended fruit fly lifespan by Ϸ70% (8). The correlation between methionine sulfoxide reductase activity and lifespan has been attributed to antioxidant function of MsrA. The role of MsrB in lifespan regulation has not been previously addressed.Most known modulators of the rate of aging are conserved across species, suggesting that common, conserved processes regulate and cause aging in diverse organisms. For example, caloric restriction (CR) is a dietary regimen that is known to extend lifespan in organisms from yeast to mammals (3, 9, 10). Saccharomyces cerevisiae has been extensively used as a model organism in studies on the mechanisms of aging (9, 10). Yeast express methionine sulfoxide reductases and can grow both aerobically and anaerobically, and in the present study, we used these features to examine the casual role of ROS-dependent processes in aging. Materials and MethodsYeas...

Iron, copper, and zinc are all essential nutrients. The electron transfer properties of iron and copper are fundamental to processes such as respiration and photosynthesis. Zinc forms the catalytic center in numerous enzymes and has an important structural role in a wide range of proteins. However, all these metals can be toxic if their levels and distribution are not carefully regulated, as their inappropriate binding may compromise cellular function. The uncontrolled redox activity of iron and copper can also lead to the generation of damaging oxygen radicals. Therefore, organisms maintain cytoplasmic metal concentrations at a nontoxic level that is sufficient for growth. A variety of homeostatic mechanisms have been identified, which include the control of translation and RNA stability by iron-regulatory proteins and the metal-dependent trafficking or degradation of metal transporters (39,109,138). This review focuses on the role that metal-responsive transcription factors have in regulating trace metal metabolism. These factors are able to sense changes in metal concentrations and coordinate the expression of genes that are involved in the acquisition, distribution, sequestration, and use of metals. Consequently, the ability to mediate metal-responsive gene expression is an important aspect of metal homeostasis in those organisms that contain these factors.

Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1⌬aft2⌬ deletion was generated. Growth assays established that the single aft2⌬ strain exhibited no iron-dependent phenotype. However, the double-mutant aft1⌬aft2⌬ strain was more sensitive to lowiron growth conditions than the single-mutant aft1⌬ strain. A mutant allele of AFT2 (AFT2-1 up ), or overexpression of the wildtype AFT2 gene, led to partial complementation of the respiratorydeficient phenotype of the aft1⌬ strain. The AFT2-1 up allele also increased the uptake of 59 Fe in an aft1⌬ strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1 up -dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1 up allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles. metalloregulation ͉ iron homeostasis