Journal of Molecular Biology
Regulation of Saccharomyces cerevisiae FET4 by Oxygen and Iron
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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