Mechanism and Regulation of the Cellular Compartmentalization of Iron between Mitochondria and Cytosol.
Iron is a key trace element for virtually all organisms as it functions as an essential co-factor in central cellular processes. Although iron is highly abundant, its bioavailability is low due to its poor solubility under ambient conditions. Therefore all cells have developed efficient iron uptake systems to meet cellular iron demands (see for instance, Anderson and Vulpe, 2009; Kaplan and Kaplan, 2009; Muckenthaler et al., 2008). For microbial pathogens, acquisition of host iron is frequently crucial for virulence (Haas et al., 2008). Accordingly, mammals react to microbial infections by inducing iron withholding defence systems, in order to deprive pathogens of essential iron in body fluids (Weinberg, 2009).
Cellular iron levels must be delicately balanced, as intracellular iron is both a source and an amplifier of reactive oxygen species and thus toxic at higher concentrations. To maintain appropriate cellular iron levels and to avoid iron-loading, cells have developed sophisticated systems for assuring a balanced cellular iron homeostasis. At the cellular level, this balance is achieved through a strict coupling of cellular iron uptake at the plasma membrane to intracellular iron demands and a balanced intracellular distribution of iron between the cellular compartments involved in iron-utilization and storage. A tightly regulated iron metabolism is essential, and disruption or over-expression of iron-related molecules can have significant health consequences. Defects in mammalian proteins involved in iron transport, its regulation, or its utilization in mitochondria are frequently associated with recessive chronic degenerative disorders with either chronic anaemia or systemic iron overload (Anderson and Vulpe, 2009; Dunn et al., 2007; Lill and Mühlenhoff, 2008). In the latter case, the cytotoxic effects of elevated intracellular iron levels result in chronic progressive tissue damage and ultimately failure of the organs involved (Heeney and Andrews, 2004; Muckenthaler et al., 2008).
In eukaryotes, mitochondria are the major iron-utilizing cell organelles as they play a central role in the maturation of all cellular Fe/S proteins and are the unique site for heme synthesis (Lill and Mühlenhoff, 2008). Consistent with this central role in iron metabolism, mitochondria are also key players in the regulation of cellular iron homeostasis (see Fig. 1). Cells with defective mitochondrial Fe/S cluster (ISC) assembly and ISC export systems accumulate high levels of iron within mitochondria. This un-physiological event is observed in S. cerevisiae, mice and human tissue and is indicative for a deregulated cellular iron homeostasis. In S. cerevisiae, the disruption of the mitochondrial ISC assembly and export systems is associated with the constitutive activation the iron-responsive transcription factors Aft1 and Aft2 and, consequently, increased cellular iron uptake (Kaplan and Kaplan, 2009; Lill and Mühlenhoff, 2008). Since the impairment of mitochondrial ISC assembly is not associated with the depletion of cytosolic iron pools, Aft1 requires a signal molecule produced and exported by the mitochondrial ISC assembly systems for proper sensing of iron (Fig. 1 and Fig. 2). The functional role of mitochondria in the regulation of cellular iron homoestasis is functionally conserved in higher eukaryotes despite the fact that they use completely different systems for iron regulation (Fig. 1). Furthermore, defects in the ISC assembly systems are associated with heme deficiency caused by the inhibition of ferrochelatase, the last enzyme of heme biosynthesis (Lill and Mühlenhoff, 2008).
In the model organism S. cerevisiae, the iron-responsive transcription factors Aft1 and Aft2 play a central role in the regulation of cellular ion homeostasis (Kaplan and Kaplan, 2009). Upon iron deprivation, Aft1-Aft2 activate the transcription of the iron regulon, a set of ~ 40 genes encoding cell surface iron transporters and proteins involved in intracellular iron utilization (Fig. 2). Aft1 shuttles between the cytosol and nucleus in an iron-responsive manner and acts as transcriptional activator under iron-limiting conditions (Fig. 1 & 2). Nuclear export of Aft1 is promoted by an iron-dependent interaction with the nuclear export factor Msn5 (Kaplan and Kaplan, 2009). Sensing of intracellular iron by Aft1 involves the cytosolic-nuclear glutaredoxins Grx3 and Grx4 (Grx3/4), that play a central role in the passage of iron throughout the cytosol. This regulatory function of cytosolic glutaredoxins is functionally conserved in fungi that utilize iron-responsive transcription systems unrelated to those from S. cerevisiae (Haas et al., 2008; Kaplan and Kaplan, 2009).
In vertebrates, iron is administered to tissue cells through the plasma iron transport protein transferrin (Tf) (Anderson and Vulpe, 2009; Dunn et al., 2007). Tf binds to transferrin receptor-1 (TfR1) on the cell membrane of iron-consuming cells and is internalized by receptor-mediated endocytosis. Iron released from Tf is reduced by endosomal ferric reductases and transported into the cytosol via the divalent metal transporter DMT1. Iron is then used for cellular processes, and excess iron is stored within the storage protein ferritin. In vertebrates, cellular iron levels are post-transcriptionally controlled by iron regulatory proteins (IRP)-1 and IRP2. In iron-deficient cells, IRP1 and IRP2 bind to iron-responsive elements (IRE) in the 3’- or 5’-untranslated regions of mRNA transcripts of molecules involved in iron metabolism, such as Tf-receptor (TfR1), ferritin H and L chains, or DMT1, stabilizing them against degradation or inhibiting translation, respectively (Anderson and Vulpe, 2009; Dunn et al., 2007; Kaplan and Kaplan, 2009; Muckenthaler et al., 2008) (See Fig. 1). This results in increased cellular iron uptake through TfR1 and decreased intracellular iron storage within ferritin. IRP1 is a cytosolic Fe/S protein with homology to mitochondrial aconitase. Upon iron deprivation, IPR1 is converted from an active Fe/S cluster enzyme to its iron-regulatory apo-form that binds to IRE (Muckenthaler et al., 2008).
During the current funding period, project A2 follows three major aims. (1) In the previous funding periods, we have worked out a portrayal of the mechanism underlying the global changes of the transcriptome to conditions of to defects in cellular Fe/S protein maturation in S. cerevisiae. This allowed us to understand the connection between mitochondria and iron-responsive gene expression in this organism. We will now study the transcriptional response to defects in cellular Fe/S protein maturation and to iron deprivation in human cells. We expect that the connection between mitochondria and the regulation of cellular iron homeostasis is likely a common theme for eukaryotes, regardless of whether these organisms regulate their iron metabolism by transcriptional or post-transcriptional mechanisms and that we will identify novel components involved in this process. (2) For efficient adjustment of cellular iron metabolism to ambient iron levels, changes of the transcriptome alone are not likely sufficient and must be bolstered by further appropriate post-transcriptional changes in the proteome. Factors involved in the iron-responsive modulation of the proteome are largely unknown. We will characterize cytoplasmic and mitochondrial factors that mediate this crucial adaptation in S. cerevisiae. (3) The transport of iron across mitochondrial membranes is a central aspect of intracellular iron metabolism in eukaryotes. Since mitochondrial ISC assembly is an essential process, iron uptake into mitochondria, in turn, is essential for viability as well. We will characterize novel components involved in this process.
Figure 1: Comparison of the impact of Fe/S protein biogenesis on iron homeostasis in yeast and mammalian cells. (Left) In yeast, the transcription factors Aft1 and Aft2 sense an Fe-dependent signal molecule that is provided by the mitochondrial iron-sulfur cluster (ISC) assembly and export systems and the Fe/S co-factor of the cytosolic glutaredoxins, Grx3/4. (Right) In mammalian cells, the Fe/S cluster on IRP1 serves as a binary switch, determining the binding capacity of IRP1 to iron-responsive elements (IRE) on mRNAs encoding proteins involved in iron uptake (e.g. Tf receptor) or storage (e.g. ferritin). In its apo-form, IRP1 binds to IREs, thereby stabilizing mRNAs with 3’-UTR IREs and blocking translation of mRNAs with 5’-UTR IREs. In contrast to yeast, iron regulation by mammalian cells involves the cytosolic Fe/S protein assembly system (CIA).
Figure 2: Intracellular iron trafficking and the regulation of cellular iron uptake in S. cerevisiae. Iron acquired at the plasma membrane by the high- and low-affinity iron uptake systems enters the cytosol, where it likely binds to diverse low molecular weight biological ligands. The essential mono-thiol Grx3/4 accept iron from this “labile iron pool”, in form of an Fe/S cluster (Muhlenhoff et al., 2010). Iron is donated to cytosolic iron-dependent enzymes, the CIA system involved in the maturation of cytosolic Fe/S proteins, and iron transporters in the mitochondrial, and likely, vacuolar membranes. In the absence of Grx3/4 or its bound Fe/S cluster, iron accumulates in the cytosol in a biologically unavailable form, eventually resulting in cell death. Grx3/4 (mammalian PICOT), the mitochondrial iron transporters Mrs3/4 (vertebrate Mitoferrin 2) and the vacuolar iron transporter Smf3 (mammalian DMT1) are conserved in higher eukaryotes. The vacuolar iron exporter Ccc1 is conserved in plants. Genes involved in iron uptake at the plasma membrane are members of the yeast iron regulon, a set of iron-responsive genes that is controlled by the iron-responsive transcription factors Aft1 and Aft2. Aft1 shuttles between the cytosol and nucleus in an iron-responsive manner, and acts as transcriptional activator under iron-limiting conditions. Aft1 responds to two iron-dependent intracellular signals: (1) Aft1 interacts with Grx3/4. The Grx3/4 bound Fe/S-cluster functions as sensor for the status of the cytosolic iron pool. (2) The mitochondrial ISC assembly system (ISC) and export systems produce and sequester a regulatory molecule (X) that signals the status of mitochondrial iron status to Aft1. In addition, X is utilized for the maturation of cytosolic Fe/S proteins by the CIA system. In the absence of X or the Fe/S cluster on Grx3/4, Aft1 activates the transcription of the iron regulon which results in increased cellular iron uptake and increased cytosolic iron pools.
Dr. Oliver Stehling, Post-doc
Bastian Hoffmann, PhD student
Nicole Rietzschel, PhD student
Martin Stümpfig, technician.
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