Iron-Dependent Metabolic Remodeling in S. Cerevisiae ⁎ Jerry Kaplan A, , Diane Mcvey Ward A, Robert J

Biochimica et Biophysica Acta 1763 (2006) 646–651 www.elsevier.com/locate/bbamcr

Review Iron-dependent metabolic remodeling in S. cerevisiae ⁎ Jerry Kaplan a, , Diane McVey Ward a, Robert J. Crisp a, Caroline C. Philpott b

a Department of Pathology, School of Medicine University of Utah, SLC, UT 84132-2501, USA b Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MA 20892, USA

Received 18 January 2006; received in revised form 28 March 2006; accepted 29 March 2006 Available online 5 April 2006

Abstract

All eukaryotes require iron although iron is not readily bioavailable. Organisms expend much effort in acquiring iron and in response have evolved multiple mechanisms to acquire iron. Because iron is essential, organisms prioritize the iron use when iron is limiting; iron-sparing enzymes or metabolic pathways are utilized at the expense of iron-rich enzymes. A large percentage of cellular iron containing proteins is devoted to oxygen binding or metabolism, therefore, changes in oxygen availability affect iron usage. Transcriptional and post-transcriptional mechanisms have been shown to affect the concentration of iron-containing proteins under iron or oxygen limiting conditions. In this review, we describe how the budding yeast Saccharomyces cerevisiae utilizes multiple mechanisms to optimize iron usage under iron limiting conditions. © 2006 Elsevier B.V. All rights reserved.

Keywords: Iron; Heme; Metabolism; Oxygen; Sterol; Yeast

1. Introduction Further, iron is not found uniformly on the earth and great expanses are iron-poor. Data exists suggesting that iron is the Iron is an essential element required by all eukaryotes and the rate-limiting element in growth of oceanic biomass, leading to vast majority of prokaryotes. The ability of iron to easily gain the hypothesis that seeding the ocean with iron may lead to and lose electrons, transitioning between two valence states has increased biomass through expansion of diatom populations made iron an essential component for a broad range of oxidation/ [1,2]. reduction reactions. In addition to acting as a prosthetic group on Many iron-containing proteins in eukaryotes participate in proteins, as oxo-diiron or as a Fe–S cluster, iron is an essential oxygen-dependent reactions. Such reactions include those that substrate for heme. Heme is the major oxygen-binding molecule involve heme (oxygen binding, respiration) and those that utilize of eukaryotes and also participates in a wide range of electron oxygen as a substrate. Examples of the latter include: porphyrin transfer reactions. As useful as iron is in enzymatic reactions, the synthesis, sterol metabolism, lipid desaturation and iron tran- acquisition of iron is problematic. While iron is the third most sport. While heme and oxo-diiron containing proteins almost abundant element on earth, it is generally present in forms that invariably participate in oxygen-dependent reactions, iron– are biologically unavailable. Ferric iron (Fe3+) reacts with sulfur containing proteins participate in a broad variety of re- oxygen leading to insoluble ferric oxides. Solubilization of iron actions some that are oxygen independent. Yeast can survive requires either the synthesis of high affinity ferric chelators, without heme if the products of heme-dependent enzymes are termed siderophores or the synthesis of heme containing pro- provided. They cannot, however, survive without iron–sulfur teins capable of reducing ferric iron to the more soluble ferrous cluster synthesis. form. Consequently, solubilization of iron from insoluble ferric The scarcity of iron, as well as iron's involvement in oxygen forms requires a significant investment of cellular components. metabolism, are the major factors that determine iron use in metabolic reactions. Species in all biological kingdoms have devised mechanisms to remodel metabolic activities in line with ⁎ Corresponding author. Tel.: +1 801 581 7427; fax: +1 801 581 6001. iron and oxygen availability. The availability of iron dramati- E-mail address: [email protected] (J. Kaplan). cally affects the use of metabolic pathways and enzymes. In the

0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.03.008 J. Kaplan et al. / Biochimica et Biophysica Acta 1763 (2006) 646–651 647 face of iron scarcity, both iron-independent enzymes and iron- the high affinity iron transport system. Reduction of ferric to independent metabolic pathways are enhanced while iron- ferrous iron occurs at the cell surface through the action of the dependent pathways are minimized. For most iron-dependent heme containing metalloreductases, Fre1p and Fre2p [6,7].In reactions, changes in oxygen tension also affect selection of standard yeast media with glucose as a carbon source, the metabolic reactions. Decreased oxygen tension selects against amount of iron available is such that yeast can grow in the iron-based enzymes that use oxygen in favor of iron-indepen- absence of Fet3p/Ftr1p, through the activity of low affinity iron dent reactions that do not use oxygen. In this review, we discuss transport systems. In iron limited media the high affinity iron how changes in oxygen and or iron lead to metabolic remodeling transport system is required for growth [8]. Even in iron–replete in yeast. Iron- and oxygen-based remodeling occurs in all media, increased cellular demand for iron requires the activity eukaryotes and many prokaryotes, however, this review focuses of the high affinity iron transport system. Most commonly, on the budding yeast Saccharomyces cerevisiae for several increased iron demand is necessitated by mitochondrial respi- reasons. First, S. cerevisiae is a single cell organism and is con- ration, where iron is utilized for synthesis of mitochondrial sidered to be among the simplest of the eukaryotes. Second, S. respiratory proteins, which require both heme and iron–sulfur cerevisiae has tractable genetics, which has made it a model clusters. Depending on the yeast strain, a need for respiration organism for both genome wide genetic screens and transcript may lead to a two- to five-fold increase in cellular iron. Cyto- analysis. Third, as opposed to most eukaryotes, loss of respi- chromes involved in respiration require heme, and iron in the ration is not a lethal event in S. cerevisiae and the budding yeast form of iron–sulfur clusters. The Bc1 complex contains three can grow either aerobically without respiration or anaerobically. iron atoms as heme (two b-type hemes, one c-type heme) and S. cerevisiae has an enormous capacity for glycolysis and while two iron atoms in a [2Fe–2S] cluster. Many yeast strains with the amount of ATP per mole of glucose resulting from glycolysis mutations that lead to a defective high affinity iron transport is reduced compared to respiratory metabolism, the budding system result in an inability to grow on respiratory substrates yeast shows significant metabolic adaptations to non-respiratory while still permitting growth on fermentative substrates [9]. metabolism. These adaptations include multiple cell surface This “genetic experiment” formally demonstrates that in S. hexose transporters and many glycolytic enzymes that constitute cerevisiae respiration is not essential and that yeast prioritize the a significant amount of cellular mass. The ability to grow distribution of iron into essential metabolic events such as sterol aerobically and anaerobically makes S. cerevisiae exceedingly synthesis and heme synthesis for non-respiratory activities. adaptable, giving the organism a high degree of metabolic plas- Yeast naturally shift from fermentation to respiration (the ticity in response to changes in both iron and oxygen levels. diauxic shift) during growth in glucose-containing medium as The transition metals iron and copper are required for respi- the glucose is depleted and the ethanol produced during fer- ratory growth and the first evidence of metabolic remodeling mentation is metabolized through respiration. This shift to occurs when these essential transition metals become limiting. respiration is accompanied by an increase in iron uptake that is The importance of iron in S. cerevisiae is emphasized by the fact mediated by both high-affinity transport systems and requires that there are multiple transport systems that translocate iron the major iron-dependent transcription factor, Aft1p, and the across the cell surface (for review see [3]). There are two low Snf1p/Snf4p kinase pathway [10]. affinity transport systems, products of the FET4 and SMF1 genes that are not specific for iron but can also transport other transition 2. Oxygen-dependent remodeling metals. In addition to the low affinity iron transport systems there are two high affinity transport systems. One system, com- The distribution of iron from non-essential to essential pro- prised of four different gene products, affects the transport of cesses results from changing patterns of transcription. In the siderophore–iron complexes. Siderophores are small organic case of oxygen, heme has a central role in yeast metabolism, as molecules produced by bacteria and fungi that bind iron with it is the sensor for aerobic/anaerobic metabolism. Heme binds very high affinity. While S. cerevisiae does not make side- oxygen and the synthesis of porphyrins is oxygen dependent. rophores it can accumulate siderophores synthesized by other There are two steps in porphyrin synthesis that require oxygen: organisms [4,5]. These siderophore transporters permit S. cere- the oxidation of coproporphyrinogen to protoporphyrinogen visiae to accumulate a large variety of chemically disparate mediated by coproporphyrinogen oxidase (Hem13p) and oxi- siderophores. The four siderophore transporters are highly ho- dation of protoporphyrinogen to protoporphyrin IX mediated by mologous and are thought to arise from an ancestral gene. protoporphyrinogen oxidase (Hem14p). In both yeast and man, The high affinity elemental iron transport system is com- these two enzymes are localized to the inner matrix of the prised of two cell surface proteins, Fet3p a multicopper oxidase mitochondria, as is the last step in heme synthesis: the insertion and Ftr1p a transmembrane permease. Fet3p oxidizes ferrous of iron into protoporphyrin IX mediated by ferrochelatase iron to ferric iron, which is then transported across the plasma (Hem15p). membrane by Ftr1p. The electrons abstracted from ferrous iron As oxygen decreases there is a reduction in the rate of heme are then used to reduce molecular oxygen to water. Most envi- synthesis. Decreased amounts of heme lead to dramatic changes ronmental iron is found as ferric iron, however, the substrate for in metabolism including lowered rates of synthesis of respiratory Fet3p is ferrous iron. Similarly, the substrate for the low affinity proteins such as cytochromes (Fig. 1). The decreased synthesis iron transport systems, either Fet4p or Smf1p is also ferrous of respiratory proteins results from reduced transcription activity iron. Ferric iron must be reduced before it can be transported by due to the loss of a heme binding transcriptional activator 648 J. Kaplan et al. / Biochimica et Biophysica Acta 1763 (2006) 646–651

Fig. 1. Heme-regulated gene expression. (A) Normal oxygen levels result in Hap1p-regulated expression of aerobic genes and Rox1p repression of anaerobic genes. (B) As oxygen levels decrease, Hap1p-regulated genes show decreased expression resulting in a reduction in the demand for iron; preserving iron and heme for essential functions such as sterol synthesis and ferrireductase (FRE1, FRE2). (C) As oxygen levels decrease to the point that heme synthesis is inhibited, transcription of genes that utilize or transport iron aerobically ceases. Iron and copper demand is minimal under anaerobic conditions and uptake of these elements can be accomplished by low affinity transition metal transporters, e.g., Fet4p, whose expression is increased in the absence of Rox1p. (D) Cells with a deletion in HEM1 were transformed with plasmids that contained lacZ reporter constructs for FET3, HMG2, and CYC1. In the presence of aminolevulinic acid (ALA, the product of Hem1p) cells show normal heme synthesis and express Hap1p-regulated genes (CYC1-lacZ), Aft1p-regulated genes (FET3-lacZ) but do not express Rox1p repressed genes (HMG2-lacZ). Decreased levels of ALA first lead to decreased expression of CYC1-lacZ, followed by decreased expression of FET3-lacZ and increased expression of HMG2-lacZ.

Hap1p. Hap1p is considered to be the master regulator of transport of iron by Fet3p is oxygen dependent with a Km for iron aerobic-based transcription. In the absence of heme, a large transport of 0.15 μM [8]. Studies of Fet3p in vitro show a number of genes are not transcribed [11]. Conversely, one of the measured Km for iron of 4.8 μM and a Km for oxygen of 1.3 μM genes regulated by Hap1p is the repressor Rox1p, which pre- [13]. Further, many iron containing proteins, such as those vents transcription of anaerobic genes. When Hap1p losses its involved in sterol and lipid synthesis that utilize oxygen as a transcriptional activity in the absence of heme, Rox1p is no substrate, are also not needed under anaerobic conditions [14]. longer synthesized and genes required for anaerobic growth are Consequently, the demand for transition metals such as iron and now transcribed, while genes required for aerobic growth are no copper is dramatically reduced. In the absence of heme, cells no longer made. longer transcribe the genes that encode for the high affinity iron Heme-containing respiratory proteins are not made in the and copper transport systems [12]. The lack of expression of absence of Hap1p, however, other heme containing non- these transport systems is due to the presence of a transcriptional respiratory proteins as well as iron–sulfur proteins continue to repressor, in which repression is relieved by heme [15]. Deletion be made. The ferrireductases, Fre1p/Fre2p and the high affinity of the heme transcriptional activators (HAP1, HAP2, HAP5), as iron transport system, Fet3p/Ftr1p, are not regulated by the well as deletion of the known transcriptional repressors (ROX1, Hap1p system and continue to be made in the absence of MOT3), does not affect expression of the iron regulon. Phy- mitochondrial respiration [12]. It is only when heme levels siological experiments confirm the observation that the Hap decrease dramatically that transcription of the ferrireductases system(s) is not involved in the regulation of iron and copper and the high affinity iron transport system are affected. The accumulation. When heme levels are reduced, decreased J. Kaplan et al. / Biochimica et Biophysica Acta 1763 (2006) 646–651 649 expression of Hap1p-regulated genes occurs before decreases in ABC-transporter family, as the putative exporter of iron–sulfur the expression of the iron transport genes (J. K. unpublished). clusters, although the nature of the metabolite transported is This result demonstrates that in the face of a declining resource unknown [26]. Three cytosolic proteins, Nar1p [27], Cfd1p [28] (substrate or oxygen) cells prioritize their metal needs; heme- and Nbp35p [29], are required to assemble iron–sulfur clusters based proteins that are not essential are not made while iron on cytosolic proteins. Deletion of genes that encode these containing proteins that are essential continue to be made. proteins, however, does not affect Aft1p-regulated transcription [30]. While mitochondrial iron–sulfur cluster synthesis is 3. Iron-dependent remodeling critical for regulation of the iron regulon, the nature of how the mitochondrial iron–sulfur clusters affect Aft1p remains Under iron sufficient conditions most of the iron transported elusive. One possibility that might explain the connection into the cells occurs by the action of low affinity transporters, between mitochondrial iron and Aft1p-regulated transcription is Fet4p and Smf1p. There is, however, a significant (although that iron–sulfur clusters could assemble on Aft1p or associated never precisely quantified) amount of iron that can enter the cell proteins without the cytosolic iron–sulfur assembly complex. by fluid phase endocytosis. This iron winds up in the vacuole, Another possibility is that Aft1p (or an associated protein) which is the S. cerevisiae site for iron storage [16]. Iron from the senses the metabolite transported by Atm1p. It is important to vacuole can be recovered by either high affinity iron tran- reemphasize that iron limitation, which affects the essential sporters Fet5p/Fth1p [17], which are orthologues of the cell process of iron–sulfur cluster synthesis, is responsible for surface iron transporters Fet3p/Ftr1p or by a lower affinity iron bringing iron into cells. transporter Smf3p [18], which is an orthologue of the cell sur- The Aft1p iron regulon consists of about twenty different face Smf1p. Extensive changes in iron metabolism occur under genes [31]. Some of the genes that regulated directly by Aft1p oxygen sufficient conditions when iron becomes limiting. When include genes that encode the siderophore transporters (ARN1, iron is limiting, neither the low affinity iron transporters nor ARN2, ARN3, ARN4) high affinity elemental iron transporter endocytosis can provide enough iron for sustained cell growth. system (FET3/FTR1) and genes that encode proteins that are Consequently, there is increased transcription of genes that required for copper loading of Fet3p (CCC2, ATX1) and genes encode the high affinity iron transporters, both the siderophore that encode the iron-reduction ferrireductase (FRE1–FRE6). The transport system and the elemental iron transport system [5]. most highly expressed genes regulated by Aft1p encode cell wall Increased expression of these genes can occur under iron- proteins (FIT1, FIT2, FIT3), which are thought to play a role in limiting conditions in the absence of any effect on cell growth, siderophore binding [32]. As might be expected, genes that suggesting that the increased expression of these transporters encode proteins that export iron from vacuole to cytosol (SMF3, can provide enough iron to maintain cell growth. FET5, FTH1) are also regulated by Aft1p. Expression of a The induction of expression of iron transporters results from putative heme oxygenase is also part of the iron regulon [33]. the increased activity of the iron-sensing transcription factor The increased expression of all of these genes gives rise to a Aft1p [19]. A paralogue to Aft1p, Aft2p is also expressed in similar end result, that is, increased cytosolic iron. The expres- yeast. Aft2p can bind to the promoter elements of genes that sion of Hmx1p, the putative heme oxygenase, also results in a encode cell surface iron transporters. However, deletion of AFT2 decrease in regulatory pools of heme and a decrease in the has minor effects on cell surface iron transport activity [20,21]. transcription of heme-responsive genes, such as the respiratory Aft2p may play a role in regulating genes involved in cytochromes [33]. intracellular iron metabolism. When cells are iron-replete In addition to genes required for iron transport, the iron– Aft1p is cytosolic, when cells are iron-limited Aft1p relocalizes regulon also contains genes that encode for proteins that affect to the nucleus where it then activates the promoters on metabolic activities in dramatic ways. The first insight into iron- approximately 20 genes, collectively referred to as the iron– related metabolism comes from analysis of an Aft1p-regulated regulon [22]. Recently, studies have suggested that Aft1p may gene-VTH1, which encodes a biotin transporter [31]. S. cere- not directly sense iron but rather Aft1p may sense the product of visiae requires biotin for growth and obtains it in two different mitochondrial iron metabolism. This conclusion was based on ways. First, it can synthesize biotin from a precursor 7-keto, the observation that deletion of genes responsible for mitochon- 8 amino-pelargonic acid through the activities encoded by BIO3, drial iron–sulfur cluster synthesis results in activation of the BIO4, BIO2 (Fig. 2). Under low iron conditions, transcripts for iron–regulon even though cells are iron replete [23]. It is known the mRNA of these three enzymes are reduced dramatically, that mitochondrial iron–sulfur cluster synthesis, as measured by resulting in a decrease in the ability to synthesize biotin. Simul- aconitase, is sensitive to cytosolic iron. Aconitase activity is taneously, there is an increase in the expression of the cell inversely related to activation of the iron–regulon, regardless of surface biotin transporter (Vth1p), which is directly activated by whether aconitase activity is reduced by the lack of iron or by the Aft1p. This reciprocal change in biotin synthesis and transport is lack of sulfide [24]. These observations suggest that iron–sulfur not an indirect result of iron limitation but rather is a direct clusters may have some function in signaling iron-regulated consequence of Aft1p activation, as it occurs in iron-sufficient transcription. In Saccharomyces, iron–sulfur clusters are assem- cells bearing a constitutive allele of AFT1 (AFT1-1up). By accu- bled in the mitochondria and are either used in the mitochondria mulating biotin from external sources, cells can dispense with or are exported for use in the cytosol or nucleus [25]. Lill and biosynthetic enzymes. It is thought that one of the reasons cells colleagues identified Atm1p, a mitochondrial member of the wish to switch from biotin synthesis to biotin transport is that 650 J. Kaplan et al. / Biochimica et Biophysica Acta 1763 (2006) 646–651

Iron in the form of heme and oxo-diiron is required for sterol synthesis. Sterol synthesis can occur in the absence of Hap1 activation, suggesting that sterol synthesis (and lipid desaturation) is more essential than respiration [14]. Transcription of genes that encode sterol synthetic enzymes only decrease when heme synthesis is severely diminished. Similar to the situation with biotin, a decrease in the ability to synthesize sterols is compensated by an increased ability to take up exogenous sterols. The ability to take up exogenous sterols is controlled by two transcriptional activators, Upc2p and Sut1p [35], which regulate Fig. 2. The yeast biotin synthesis pathway. The genes that encode enzymes in the the sterol transporters Aus1p and Pdr11p [36]. In the absence of biotin biosynthetic pathway are highlighted in purple. The genes that encode the heme synthesis, transcription of sterol transporters occurs, lead- transporters of biotin or biotin precursors are highlighted in green. Bio2p is a ing to uptake of exogenous sterols. At the same time, decreased 4Fe–4S cluster protein. (For interpretation of the references to colour in this transcription of ERG genes relieves the demand for heme and figure legend, the reader is referred to the web version of this article.) non-heme iron, again permits iron to be used for the most essential activities. Iron limitation, however, leads to increased tran- Bio2p is a Fe–S containing enzyme. Preventing the synthesis of scription of ERG genes [37]. The increased transcription results this enzyme would then free up iron for other enzymes, which from a decrease in sterols due to decreased activity of iron- might be more essential. Bio5p transports the precursors for containing (i.e., methyl sterol oxidase, Erg25p) enzymes [38]. biotin synthesis, and this gene is also expressed under low iron In addition to regulation at the transcriptional level, there is conditions under the control of Aft1p [34]. The lack of biotin extensive regulation of iron-related mRNA at the post-tran- synthesis under low iron conditions suggests that these pre- scriptional level through control of mRNA stability. The Aft1p- cursors may be stored by the cell, and incorporated into biotin regulated gene CTH2 (also referred to as TIS11) encodes a when cells become iron sufficient. protein that binds to specific AU rich elements in the 3′ This line of logic, iron limitation leading to the expression of untranslated region of a wide variety of mRNAs that encode iron-sparing metabolism, has been extended to other metabolic proteins involved in either oxygen or iron-dependent activities processes. For example, S. cerevisiae can synthesize nitrogenous [37]. Binding of Cth2p to the mRNA tails results in a decrease in compounds from ammonium, by incorporating the ammonium mRNA stability resulting in decreased expression. Microarray ion into amino groups of glutamate and glutamine. Glutamine analysis of cells grown in iron-limited media showed decreased synthesis occurs by different routes (Fig. 3). In the first route two levels for many mRNA that encode enzymes that participate in a enzymes, Gdh1p and Gdh3p, form glutamate from ammonia and wide variety of metabolic reactions. Deletion of CTH2 resulted α-ketoglutarate. In the second route ammonia is added to gluta- in an increase in those mRNAs. Many of the mRNAs that are mate by the enzyme Gln1p and the glutamine then formed can be affected by Cth2p are those that encode iron-binding proteins used to generate glutamate from α-ketoglutarate through the (e.g., cytochrome subunits, iron–sulfur cluster containing en- action of the enzyme glutamate synthase (Glt1p), an iron–sulfur zymes) or enzymes that are in synthetic pathways that use iron containing enzyme. In iron-limited media the activity and GLT1 (e.g., heme and iron–sulfur cluster synthesis). Cth2p expression transcript decreased dramatically, while the activities and is inversely correlated with cellular iron content, that is, through transcripts of the iron-independent enzymes Gdh1p and Gdh3p the activation of Aft1p. The changes in mRNA levels as a result were relatively unchanged [31]. Thus, under iron-limitation, of deletion of CTH2 are not great, approximately a two-fold cells shift their metabolism from an iron-dependent to an iron- change in mRNA levels. However, Cth2p-mediated mRNA independent mechanism. It has been suggested that regulation of half-life combined with iron-dependent transcription permits GLT1 mRNA results from an iron-dependent transcription cells to exquisitely regulate mRNA levels. factor, although the transcription factor has not been identified. Diverse mechanisms respond to iron or oxygen restriction, As iron availability becomes limited, the theme of decreased including message destabilization, transcriptional activation expression of iron-containing activities in favor of iron-independent activities is seen throughout the genome. Transcript analysis of iron-limited cells showed decreased expression of genes that encode enzymes in the tricarboxylic acid and respiratory pathways and for enzymes required for the synthesis of unsaturated fatty acids. The decline in many of these enzymes reflects a decrease in heme synthesis and/or accelerated heme degradation, as heme is required for both respiratory and non-respiratory cytochromes. Based on studies of oxygen limitation described above, decreased synthesis of heme is expected to lead to decreased expression of Hap-activated genes. A decrease in Hap-activated genes, which Fig. 3. Glutamate/Glutamine biosynthesis. 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