Regulation of Saccharomyces cerevisiae FET4 by Oxygen and Iron

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Abstract

Saccharomyces cerevisiae expresses two distinct iron transport systems under aerobic and anaerobic conditions. The high affinity transporters, Ftr1p and Fet3p, are primarily expressed in oxygenated cultures, whereas anaerobic conditions induce the low affinity iron transporter, Fet4p. The oxygen regulation of FET4 was found to involve the Rox1p transcriptional repressor. The physiological significance of this control by Rox1p is twofold. First, FET4 repression by Rox1p under oxygenated conditions helps minimize metal toxicity. Sensitivity towards cadmium was high in either anaerobically grown wild-type yeast or in oxygenated rox1Δ strains, and in both cases cadmium toxicity was reversed by FET4 mutations. Secondly, the loss of Rox1p repression under anaerobic conditions serves to induce FET4 and facilitate continual accumulation of iron. We noted that fet4 mutants accumulate lower levels of iron under anaerobic conditions. Regulation of FET4 was examined using FET4-lacZ reporters. We found that FET4 contains a complex promoter regulated both by oxygen and iron status. The region surrounding ≈−960 to −490 contains two consensus Rox1p binding sites and mediates Rox1p, but not iron control of FET4. Sequences downstream of −490 harbor a consensus binding site for the iron regulatory factor Aft1p that is essential for iron regulation in wild-type strains. In addition, a secondary mode of iron regulation becomes evident in strains lacking AFT1. The induction by iron limitation in conjunction with low oxygen is more than additive, suggesting that these activities are synergistic. Fet4p is not the only metal transporter that is negatively regulated by oxygen; we find that Rox1p also represses S. cerevisiae SMF3, proposed to function in vacuolar iron transport. This oxygen control of iron transporter gene expression is part of an adaptation response to changes in the redox state of transition metals.

Introduction

The appearance of oxygen in the biosphere ≈two billion years ago had a major impact on the utility of metals in biological systems. For example, oxidation of copper to cupric Cu(II) increased the solubility of this ion and enhanced its bio-availability for living organisms.1 By contrast, the oxidation of iron to ferric Fe(III) rendered this metal largely insoluble, making it potentially unavailable for living cells.1 The bakers yeast Saccharomyces cerevisiae has evolved with at least two iron uptake systems that circumvent the solubility problem of environmental ferric iron. First, iron can be taken up in the form of low molecular mass siderophores that bind ferric iron with high affinity. However, S. cerevisiae does not synthesize these molecules and therefore relies on neighboring microbes for the production of environmental siderophores.2., 3. As a more self-sufficient mechanism of iron uptake, S. cerevisiae ordinarily obtains the metal through a high affinity iron transport system that works in conjunction with ferric reductases.

High affinity iron uptake requires the activities of several proteins including the metalloreductases Fre1p and Fre2p, 4., 5. the high affinity iron permease Ftr1p,6 and a multicopper oxidase Fet3p.6., 7. Iron is initially reduced from Fe(III) to Fe(II) by the metalloreductase activity of either Fre1p or Fre2p.4., 5. The reduced iron is thought to be the substrate for Fet3p, which catalyzes the oxidation of Fe(II) to Fe(III) using molecular oxygen as a co-substrate8., 9. followed by transport into the cell via Ftr1p.6 This entire system is regulated at the level of gene transcription by iron sensing transcription factors, Aft1p or Aft2p. Under iron starvation conditions, Aft1p/Aft2p activates genes for high affinity uptake of iron and for iron–siderophore transport. Under iron replete conditions, Aft1p and Aft2p fail to activate transcription and both uptake systems are repressed.10., 11., 12., 13., 14., 15.

In addition to these aerobic iron uptake pathways, S. cerevisiae also needs to acquire iron under anaerobic conditions and a change in expression of metal uptake genes may assist the cell in obtaining required nutrients. In recent analyses of anaerobically grown yeast, the expression of S. cerevisiae FET3 and FTR1 was found to be dramatically reduced.16., 17. This anaerobic repression of high affinity transport was quite logical because the Fet3p reaction requires molecular oxygen. But then how does yeast acquire iron under anaerobic conditions?

S. cerevisiae also expresses a low affinity iron uptake pathway encoded by FET4. Fet4p is a cell surface divalent metal transporter that can transport metals other than iron, including toxic cadmium ions.18., 19. FET4 gene transcription is repressed by iron,19., 20., 21. but it has been suggested that iron regulation of FET4 does not require Aft1p.19 Moreover, unlike the high affinity transport system, metal transport by Fet4p does not require oxygen.17 Genome-wide transcriptional analysis of aerobic and anaerobic cultures using DNA microarrays revealed that the down-regulation of FET3 and FTR1 in low oxygen is accompanied by a large induction of FET4.16 This suggests a role for Fet4p in iron uptake in anaerobic conditions. Yet the mechanisms by which FET4 is down regulated by oxygen and by iron are not understood.

Here, we demonstrate that the strong oxygen repression of FET4 involves the action of a S. cerevisiae repressor known as Rox1p.22., 23. This aerobic repression of FET4 by Rox1p serves to protect aerobic cells from Fet4p-mediated metal toxicity. Rox1p appears to regulate only a strict sub-set of iron homeostasis genes. FET3 involved in high affinity iron uptake is not regulated by Rox1p, however, Rox1p and oxygen do repress expression of S. cerevisiae SMF3, encoding a putative vacuolar transporter for iron. In addition to regulation by oxygen, FET4 is also regulated by the iron status of the cell through mechanisms that are independent of Rox1p.

Section snippets

Increased cadmium sensitivity in anaerobically grown cultures of S. cerevisiae

Earlier genome wide analysis of oxygen-regulated genes demonstrated that S. cerevisiae FET4 is repressed under aerobic conditions.16 Since Fet4p has the capacity to transport toxic metals such as cadmium,18., 21. it was possible that the oxygen repression of Fet4p helps guard the cell against toxicity from unwanted metals. We therefore examined cadmium sensitivity in wild-type versus fet4Δ yeast that were grown either in air or in a CO2-enriched, oxygen-depleted environment. As seen in Figure

Discussion

Micro-organisms that thrive under both aerobic and anaerobic conditions are faced with the challenge of acquiring essential metals with variable redox states. In response to a change in oxygen status, the bakers yeast S. cerevisiae expresses two distinct iron uptake pathways. Under aerobic conditions, a high affinity iron transport system that employs molecular oxygen is the primary route of ferric iron acquisition. However, when oxygen is limiting, the genes for high affinity uptake are turned

Yeast strains and culture conditions

The yeast strains in this study were derived from BY4741 (MATa leu2Δ0 met15Δ0 ura3Δ0 his3Δ1). Strains 1090 (aft2Δ::kanMX4) and 6461 (fet4Δ::kanMX4) were obtained from Research Genetics, Inc. The rox1Δ deletion strains LJ198 (rox1Δ::LEU2) and LJ230 (rox1Δ::LEU2 fet4Δ::kanMX4) were created by disrupting the ROX1 gene of BY4741 and 6461 using a rox1::LEU2 deletion plasmid as described.36 Strains LJ194 (aft1Δ::LEU2) and MP127 (aft1Δ::LEU2 aft2Δ::kanMX4) have been described.25 Yeast transformations

Acknowledgements

We thank Dennis Winge for vectors pAR1 and AFT1-lup-313 and for invaluable discussions. This work was supported by the JHU NIEHS center and by NIH grant RO1 ES 08996 awarded to V.C.C.

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