Biochimica et Biophysica Acta 1763 (2006) 652 – 667
www.elsevier.com/locate/bbamcr
Review
Mechanisms of iron–sulfur protein maturation in mitochondria,
cytosol and nucleus of eukaryotes
Roland Lill ⁎, Rafal Dutkiewicz, Hans-Peter Elsässer, Anja Hausmann, Daili J.A. Netz,
Antonio J. Pierik, Oliver Stehling, Eugen Urzica, Ulrich Mühlenhoff
Institut für Zytobiologie, Philipps Universität Marburg, Robert-Koch-Str. 6, 35037 Marburg, Germany
Received 6 February 2006; received in revised form 26 April 2006; accepted 5 May 2006
Available online 23 May 2006
Abstract
Iron–sulfur (Fe/S) clusters are important cofactors of numerous proteins involved in electron transfer, metabolic and regulatory processes. In
eukaryotic cells, known Fe/S proteins are located within mitochondria, the nucleus and the cytosol. Over the past years the molecular basis of Fe/S
cluster synthesis and incorporation into apoproteins in a living cell has started to become elucidated. Biogenesis of these simple inorganic
cofactors is surprisingly complex and, in eukaryotes such as Saccharomyces cerevisiae, is accomplished by three distinct proteinaceous
machineries. The ‘iron–sulfur cluster (ISC) assembly machinery’ of mitochondria was inherited from the bacterial ancestor of mitochondria. ISC
components are conserved in eukaryotes from yeast to man. The key principle of biosynthesis is the assembly of the Fe/S cluster on a scaffold
protein before it is transferred to target apoproteins. Cytosolic and nuclear Fe/S protein maturation also requires the function of the mitochondrial
ISC assembly system. It is believed that mitochondria contribute a still unknown compound to biogenesis outside the organelle. This compound is
exported by the mitochondrial ‘ISC export machinery’ and utilised by the ‘cytosolic iron–sulfur protein assembly (CIA) machinery’. Components
of these two latter systems are also highly conserved in eukaryotes. Defects in the mitochondrial ISC assembly and export systems, but not in the
CIA machinery have a strong impact on cellular iron uptake and intracellular iron distribution showing that mitochondria are crucial for both
cellular Fe/S protein assembly and iron homeostasis.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Iron–sulfur cluster; Ferredoxin; ABC protein; Chaperone; P-loop ATPase; Glutathione
1. Introduction
Iron–sulfur (Fe/S) clusters came into the focus of biochemical
interest in the early 1960s when electron paramagnetic resonance
spectroscopy and chemical analyses gave evidence for these
inorganic cofactors in a number of proteins such as plant and
bacterial ferredoxins and complexes I, II and III of the respiratory chain including the prominent Rieske Fe/S protein
(reviewed in [1,2]. It soon became clear that Fe/S clusters are
ubiquitous cofactors of numerous bacterial and eukaryotic
proteins. Most Fe/S proteins contain the relatively simple rhombic
[2Fe–2S] or the cubic [4Fe–4S] clusters, but also [3Fe–4S] forms
with one iron missing at one edge of the cube are known. In the
⁎ Corresponding author. Tel.: +49 6421 286 6449; fax: +49 6421 286 6414.
E-mail address: [email protected] (R. Lill).
0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2006.05.011
late 1960s chemical reconstitution of simple protein-bound Fe/S
clusters was achieved leading to a fully re-activated Fe/S protein
[3]. Over the years more and more complex structures of Fe/S
clusters, often in contact with other metals such as molybdenum,
nickel or vanadium were discovered and characterised biochemically and biophysically [4–6]. Well-studied examples include
nitrogenase of various azototrophic bacteria and various types of
hydrogenases [7–11].
While the chemical re-constitution of simple Fe/S clusters had
suggested a spontaneous, non-catalysed assembly of these cofactors in proteins, it became clear through elegant genetic studies
around 1990 that in bacterial cells the nitrogenase Fe/S protein
required a plethora of genes assisting the assembly of its cofactors
and their insertion into the apoprotein [12]. It was not until 1998
that initial evidence became available that also simple Fe/S clusters depended on a complex set of biogenesis proteins [13,14].
This was the starting point for the rapid discovery of numerous
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
biogenesis components in both eubacteria and eukaryotes involved in the generation of Fe/S cofactors in vivo (see, e.g., [15–
18]. To date, research on the biogenesis of Fe/S proteins is an
intensive field concentrated on the functional characterisation of
participating proteins, the elucidation of the molecular mechanisms underlying the pathway, and the identification of novel biogenesis proteins. This review will summarise the currently known
principles and concepts of Fe/S protein biogenesis in (non-plant)
eukaryotes. The survey is intended to introduce non-specialists to
this fascinating new field of biochemistry and cell biology of
metals. More detailed or focused reviews have been published
recently elsewhere with particular emphasis on bacterial Fe/S
protein maturation [19–21] or on biogenesis in yeast, mammals or
plants [22–24].
1.1. A brief look at the structures and properties of Fe/S clusters
and Fe/S proteins
Fe/S clusters are formally composed of ferrous (Fe2+) and/or
ferric (Fe3+) iron, inorganic sulfide (S2−) ions, and in rare cases
additional metals or cofactors [25,26]. Their chemical behaviour,
however, cannot be described as that of a simple iron salt, but
rather is best explained by the ligand field theory of co-ordination
chemistry. The most simple form of an Fe/S center is the [2Fe–2S]
cluster. It is present in, e.g., ferredoxins, Rieske Fe/S protein, biotin
synthase and ferrochelatase (Table 1). Formally, the [4Fe–4S]
clusters can be viewed as a duplication of [2Fe–2S] clusters. They
consist of a cubic structure with Fe and S occupying alternating
corners of the cube which due to the shorter distance between the
Fe ions is usually distorted. This type of cluster is present in many
proteins such as aconitase and aconitase-like proteins, ferredoxins,
sulfite reductase, and DNA glycosylase (Table 1). The Fe ions
within the clusters have a tetrahedral co-ordination, as they additionally bind to nucleophilic ligands of the protein. The most
common co-ordination partner of the Fe ions is the sulfur atom of
cysteine residues. However, also histidine, and in rare cases arginine, serine, peptidyl-N and non-protein ligands (homocitrate, CO,
CN−) may be used. For example, in the [4Fe–4S] cluster of
aconitase-like proteins the fourth iron is not co-ordinated by a
protein-bound ligand, but binds to the substrate/product of the
enzyme (e.g., citrate, homocitrate, isopropyl malate) or to a water
molecule. Hence, the co-ordination sphere of the fourth iron in
aconitase-like proteins changes to octahedral.
Some proteins such as subunits of complexes I and II of the
respiratory chain contain [3Fe–4S] clusters in which one corner of
the cube is unoccupied (Table 1). There are numerous proteins
which contain more than one Fe/S cluster. The most extreme form
is complex I (NADH-ubiquinone oxidoreductase; not present in
Saccharomyces cerevisiae) of the respiratory chain which contains 8 (eukaryotes) to 9 (bacteria) Fe/S clusters [27]. Likewise,
complex II (succinate-ubiquinone oxidoreductase) contains
[2Fe–2S], [3Fe–4S] and [4Fe–4S] clusters [28]. Higher order
structures of Fe/S clusters with additional metal centers such as
those present in bacterial nitrogenase, CO dehydrogenase or
various hydrogenases have been studied extensively and many
3D structures have been resolved [4]. Some Fe/S proteins such as
xanthine dehydrogenase, plant aldehyde oxidase and plant nitrate
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reductase contain an additional molybdenum cofactor which
requires Fe/S proteins for its own synthesis [29]; see also article
by Mendel and Bittner [137] in this issue.
The electronic charge of the Fe/S center is concentrated at the
sulfur moiety. Depending on the overall oxidation state of the Fe/S
cluster the Fe ions may harbour unpaired electrons, and the resulting electron spin can be detected by electron paramagnetic
resonance (EPR) spectroscopy. This biophysical technique
provides valuable information mainly about the oxidation state,
the type and the electronic environment of the clusters [30]. Other
biophysical methods to study the structure and properties of the
Fe/S clusters are Mössbauer, EXAFS, ENDOR, NMR, and resonance Raman spectroscopies. They provide specific information
on the electronic and magnetic properties of the Fe/S clusters in
their particular environment. In UV/Vis spectrophotometric measurements Fe/S proteins exhibit typical absorption maxima around
420 nm ([4Fe–4S] clusters) and 320, 410 and 560 nm ([2Fe–2S]).
These spectral properties give rise to the dark brown colour of
purified Fe/S holoproteins.
Fe/S clusters and Fe/S proteins are fairly sensitive to damage by
oxidative conditions and may decompose readily in air or by the
exposure to NO or H2O2. This propensity has been exploited in
nature for using Fe/S proteins as sensors and regulatory switches
[31–33]; see also below).
Even though there is no single common consensus motif for
binding an Fe/S cluster, some primary structural features are reoccurring. These include the conserved positioning of cysteine residues in the protein. Examples include the CX4CX2CX≈30C motif
in plant and mammalian [2Fe–2S] ferredoxins. [4Fe–4S] clusters
are often co-ordinated by the consensus motif CX2CX2CX20–40C
which was originally defined in [4Fe–4S] ferredoxins, but seems
present in many other members of the [4Fe–4S] cluster type. It
appears that the spacing of the cysteine residues can also be
reversed. Usually, a conserved proline (or sometimes glycine)
flanks one of the cysteine residues. Care has to be taken not to
confuse the presence of conserved cysteine residues with similar
patterns of Zn-finger proteins or other metal binding motifs.
Despite the existence of these fairly defined Fe/S consensus
sequences there exist numerous Fe/S proteins that do not follow
these simple rules for binding the Fe/S cofactor. On the one
hand, this lack of consensus makes it rather hard to predict and
identify new Fe/S proteins based on primary sequence
information contained in genomes. On the other hand, this
shows that binding of Fe/S clusters seems to rely on a substantial
amount of flexibility of the protein partner during selection of the
co-ordination sites within the protein. In fact, mutational analyses
in some ferredoxins and other Fe/S proteins have shown that,
upon exchange of particular cysteine to serine or alanine residues,
the Fe/S proteins may be flexible enough to provide alternative coordination partners within the polypeptide sequence [30,34].
Hence, frequently only mutation of two cysteine residues leads to
the complete loss of the Fe/S center.
1.2. Diverse functions of Fe/S proteins
The biological functions of Fe/S proteins can be grouped into
three categories, namely electron transfer, enzyme catalysis and
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Table 1
Fe/S proteins in (non-plant) eukaryotes: Function, bound cluster types, and sub-cellular localisation
Fe/S protein
Yeast name
Localisation
Fe/S proteins involved in electron transport
Complex I
–
Mitochondrial
membrane
Complex II
Sdh2
Mitochondrial
membrane
Complex III
Rip1
Mitochondrial
membrane
Ferredoxin (adrenodoxin)
Yah1
Mitochondrial
ETFQO
Fe/S proteins in metabolism
Aconitase
Homoaconitase
Dihydroxy-acid
dehydratase
Lipoate synthase
Biotin synthase
Ferrochelatase
MOCS1A
Isopropylmalate isomerase
Sulfite reductase
Glutamate dehydrogenase
CMP-N-acetyl-neuraminic
acid hydroxylase
Dihydro-pyrimidine
dehydrogenase
Xanthine dehydrogenase
DNA glycosylase
Function
Cluster type
inner
Respiration (NADH ubiquinone oxidoreductase)
inner
Respiration (succinate dehydrogenase)
8 clusters of 2F,
3F, 4F
2F, 3F, 4F
inner
4F
Yor356w
Mitochondria
Respiration (ubiquinone cytochrome c oxidoreductase,
‘Rieske Fe/S protein’)
Maturation of Fe/S proteins, biosynthesis of heme A,
steroid biosynthesis in mammals
Catabolism of amino acids and choline, β-oxidation of fatty acids
Aco1
Lys4
Ilv3
Mitochondrial matrix
Mitochondrial matrix
Mitochondrial matrix
Citric acid cycle
Biosynthesis of lysine (aconitase-like)
Biosynthesis of branched-chain amino acids
4F
4F
4F
Lip5
Bio2
Hem15
matrix
matrix
inner
Biosynthesis of lipoic acid
Biosynthesis of biotin
Heme biosynthesis (no cluster in Saccharomyces cerevisiae)
2F ?, 4F
2F, 4F
2F
–
Leu1
Ecm17
Glt1
–
Mitochondrial
Mitochondrial
Mitochondrial
membrane
Mitochondrial
Cytosol
Cytosol
Cytosol
Cytosol
matrix
Biosynthesis of
Biosynthesis of
Biosynthesis of
Biosynthesis of
Biosynthesis of
3F ?, 4F
4F
4F
4F
2F
–
Cytosol
Degradation of pyrimidine nucleotides
4× 4F
–
Ntg2
Cytosol
Nucleus
Degradation of xanthine to urate, contains FAD and molybdopterin
DNA repair (Endonuclease III-like glycosylase 2)
2× 2F
4F
Post-transcriptional regulation of iron uptake, storage and
utilisation in mammals (‘cytosolic aconitase’)
Biogenesis of ribosomes, rRNA processing, translation initiation,
assembly of RNA complexes
Glutathione-dependent oxidoreductase (no cluster in
Saccharomyces cerevisiae)
Histone acetyltransferase subunit of the Elongator complex,
cluster binds S-adenosyl-methionine
Negative regulator of receptor tyrosine kinase signalling
4F
matrix
Fe/S protein involved in regulation and assembly
Iron regulatory protein 1
–
Cytosol
(IRP1)
ABC protein Rli1
Rli1
Cytosol and nucleus
(ABCE1)
Glutaredoxin 2
Grx2
Mitochondria
(mammals)
Elp3
Elp3
Nucleus
Sprouty (mammals)
–
Cytosol
Fe/S proteins involved in Fe/S protein biogenesis
Ferredoxin
Yah1
Mitochondrial matrix
P-loop ATPase Nbp35
Nbp35
Cytosol and nucleus
Iron-only hydrogenaseNar1
Cytosol and nucleus
like Nar1
Moco (molybdenum co-factor)
leucine (aconitase-like)
methionine, contains siroheme
glutamate
N-glycolyl neuraminic acid, Rieske-like protein
Maturation of mitochondrial Fe/S proteins, possibly reduction of sulfur
Maturation of cytosolic/nuclear Fe/S proteins, complex with Cfd1
Maturation of cytosolic/nuclear Fe/S proteins, binds to Nbp35 and Cia1
2F
2F
2× 4F ?
2F
4F
2F
2F
4F ?
2 clusters of
complex type
Most Fe/S proteins contain clusters of the [2Fe–2S] (2F), [3Fe–4S] (3F), or [4Fe–4S] (4F) type. The chemical structure of some Fe/S clusters is currently unknown
(indicated by ‘?’) due to the lack of spectroscopic and 3D structural information.
regulation (Table 1). The most well-known function of Fe/S
proteins is in various redox processes. This function is based on the
property of the iron ions of the Fe/S clusters in switching between a
reduced (ferrous Fe2+) and oxidised (ferric Fe3+) state. Hence, the
Fe/S clusters can easily accept electrons from one partner and
donate them to other molecules. The redox potential range depends
mainly on the protein environment of the clusters. For instance, the
relatively high redox potential of Rieske-type Fe/S proteins is
caused by the two co-ordinating histidine ligands on one of the two
Fe ions with the result that it can be reduced at a considerably
higher potential than those Fe ions bound via cysteine residues.
Typical proteins involved in redox reactions are complexes I, II
and III of the respiratory chain, mitochondrial and plant ferredoxins and the plant photosystem I. A fascinating example of
step-wise electron transfer can be seen in respiratory complexes I
and II, where electrons are transferred from the substrates NADH
and succinate, respectively, via several Fe/S clusters and other cofactors to the electron acceptor ubiquinone in the bacterial or
mitochondrial inner membrane. The characteristic redox potentials
of Fe/S clusters range from −500 mV to +300 mV. The reducing
energy is typically a few hundred mV, but in some Fe/S proteins
such as nitrogenase or 2-hydroxy-glutaryl-CoA dehydratase the
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extra energy of ATP hydrolysis assures high reduction potentials of
greater than 1 V [8,35]. In these fascinating proteins the Fe/S
cluster is co-ordinated between two subunits of the protein, i.e. the
cofactor serves as a bridging unit. The additional energy is supplied by conformational changes that are induced by ATP binding
and hydrolysis thus allowing the redox interaction with the
substrates.
The classical example for a role of Fe/S clusters in enzyme
catalysis is aconitase of the citric acid cycle (Table 1). This
enzyme and related proteins such as homoaconitase (yeast Lys4
involved in lysine biosynthesis) and isopropylmalate dehydratase (yeast Leu1, leucine biosynthesis) catalyse the removal and
re-addition of water, resulting in an isomerisation of the
substrates. In these enzymes, the fourth Fe of the [4Fe–4S]
cluster serves as a Lewis acid to bind the substrate and stabilise
the transition state of the reaction. Other enzymatic reactions
supported by Fe/S clusters are the final steps of biotin and lipoic
acid synthesis. In these cases, the enzymes bind two clusters, one
of which is possibly used as a source of the sulfur atom to be
inserted into the precursors desthiobiotin and octanoic acid [36].
An interesting case of an Fe/S protein is ferrochelatase
catalysing the final step of heme biosynthesis [37]; see also
article by Ajioka et al. [138] in this issue. Since this enzyme is a
major consumer of iron in cells and relies on an Fe/S cluster for
its function, the two major iron metabolising pathways are
connected in this protein. It is important to note that some
ferrochelatases such as that of S. cerevisiae (Hem15; Table 1) do
not contain an Fe/S cluster. Further Fe/S proteins in eukaryotes
are involved the biosynthesis of methionine (sulfite reductase)
and the molybdenum cofactor (see article by Mendel and Bittner
[137] in this issue). For some of the mentioned enzymes it is still
unclear what exact role the cluster may play in catalysis. It is well
possible that in some cases the Fe/S cluster may simply serve a
structural role stabilising the rest of the polypeptide chain.
However, such a stabilising function was long suspected for Fe/S
proteins involved in repair of DNA damage (DNA glycosylases;
Table 1), yet recent investigations have provided ample evidence
to suggest a role of the Fe/S cofactor in DNA binding [38].
An intriguing function of Fe/S proteins lies in the regulation
of biological processes. The chemical lability of the Fe/S
cofactors and their pronounced sensitivity to oxidative damage
has been exploited by Nature to sense intracellular or
environmental conditions by Fe/S clusters and hence regulate
gene expression in eukaryotes or prokaryotes. While regulation
in bacteria by the transcription factors SoxR, IscR, and FNR
occurs on a transcriptional level, regulation in mammals by iron
regulatory protein 1 (IRP1) is on a post-transcriptional level
[31–33]. IRP1 appears to have a dual role in the cell. As an Fe/S
protein it functions as a cytosolic aconitase, while under
conditions leading to the loss of the Fe/S cluster the IRP1
apoprotein binds to RNA stem–loop structures designated iron
regulatory elements (IRE) of specific mRNAs encoding proteins
involved in iron uptake, utilisation, storage or export. As a result
of IRP1-IRE binding, some mRNAs are stabilised and thus
prevented from degradation resulting in enhanced protein
synthesis. In other mRNAs stem–loop structures at the 5′untranslated end block efficient translation by the ribosome
655
leading to lower protein abundance (for details of IRP1 function
see article by Wallander et al. [139] in this issue).
1.3. Overview on the biogenesis of Fe/S proteins in eukaryotes
In the late 1990s several research groups working with the yeast
S. cerevisiae almost simultaneously realised that the biogenesis of
mitochondrial Fe/S proteins depended on a number of conserved
proteins now known as members of the Fe/S cluster (ISC) assembly machinery (Figs. 1 and 2). Initially these groups were
interested in understanding the rather diverse processes of oxidative stress, cellular iron homeostasis, and the function of mitochondrial chaperones or ABC transporters [14,39–41]. The
unifying and surprising theme was that all these groups studied
processes or proteins that played a decisive role in the assembly of
Fe/S proteins. The mitochondrial ISC assembly system turned out
to be strikingly similar to that of bacteria, and in all likelihood has
been inherited from the bacterial endosymbiont that gave rise to
modern mitochondria [19,42]. It was realised early on that mitochondria perform a prime role in Fe/S protein biogenesis as they
were found to be responsible not only for maturation of Fe/S
proteins inside but also outside the organelle [41,43]. Since an
ABC transporter in the mitochondrial inner membrane was
required for efficient maturation of extra-mitochondrial Fe/S
proteins, this created the idea of a mitochondrial “export system” to
support the generation of Fe/S proteins in the cytosol and nucleus
(Fig. 1).
It was not until 2003 that the first component of the so-called
cytosolic Fe/S protein assembly (CIA) machinery was discovered by a genetic approach [44]. Thus, the current view of Fe/S
protein biogenesis in eukaryotes, as derived from studies in the
model organism S. cerevisiae, is that of an interplay of three
complex proteinaceous systems termed ISC assembly, ISC
export, and CIA machineries (Fig. 1). The ISC assembly
machinery is required for all cellular Fe/S proteins, whereas the
functions of the ISC export and the CIA machineries are
Fig. 1. Three distinct systems participate in the generation of Fe/S proteins in
eukaryotes. Fe/S proteins have been identified in mitochondria, cytosol and
nucleus of eukaryotes. The mitochondrial ISC assembly machinery is required for
maturation of virtually all cellular Fe/S proteins. The ISC export apparatus and the
CIA machinery are specifically involved in the formation of cytosolic and nuclear
Fe/S proteins. The Fe/S cluster in proteins is indicated by red/yellow circles.
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R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
the maturation of a [2Fe–2S] ferredoxin [48]. The technique
allows the distinction of the apo- and holoforms of this
ferredoxin, yet is restricted to isolated systems, e.g., isolated
mitochondria. Finally, the RNA binding of IRP1 (see above) has
been used in higher eukaryotes and in yeast to indirectly follow
its association with a [4Fe–4S] cluster [44,49–51]. The
increased binding of IRP1 to RNA is reciprocally taken as a
measure of diminished amounts of Fe/S cluster associated with
the protein.
1.4. Biogenesis of mitochondrial Fe/S proteins—the first steps
Fig. 2. A model for the mechanism of Fe/S protein biogenesis in mitochondria.
After membrane potential-dependent (pmf) import of ferrous iron (Fe2+) facilitated
by the carrier proteins Mrs3 and Mrs4 and other unknown proteins (X), iron is used
for heme synthesis from protoporphyrin IX (PPIX) by ferrochelatase (Hem15) and
for the biogenesis of Fe/S proteins. The latter process starts with the release of sulfur
from cysteine by the Nfs1/Isd11 cysteine desulfurase complex and the formation of
a transient Fe/S cluster on the Isu1/2 scaffold proteins. The electron (e−) transfer
chain NADH → ferredoxin reductase Arh1→ ferredoxin Yah1 and the putative
iron donor Yfh1 (frataxin) are needed for transient Fe/S cluster synthesis on Isu1/2.
ISC assembly proteins required later in biogenesis, i.e. after Fe/S cluster synthesis
on Isu1/2, are the dedicated chaperones Ssq1, Jac1 and Mge1 and the monothiol
glutaredoxin Grx5. Isa1 and Isa2 are specifically and additionally required for the
maturation of aconitase-type Fe/S proteins. The exact role of Nfu1 is unclear so far.
Nfs1 performs a second function as a cysteine desulfurase in thio-uridine modification of mitochondrial tRNA (tRNAS).
restricted to the maturation of cytosolic and nuclear Fe/S
proteins. The high conservation of the ISC assembly, ISC export
and CIA proteins in virtually all eukaryotes [22] and pioneering
functional studies in human cell culture and multi-cellular
organisms (see below) support the view that biogenesis in lower
and higher eukaryotes occurs along similar pathways.
Before we concentrate on the pathways of Fe/S protein maturation, we will briefly address the issue of how the process can
be followed experimentally in vivo or in cell extracts. The most
classical and widely used technique is to follow the activities of
particular Fe/S proteins, functioning as enzymes (aconitase,
isopropylmalate isomerase, sulfite reductase etc.) or in electron
transport (complexes II and III of the respiratory chain). This
approach usually requires verification that observed activity
changes are not caused by altered gene expression. Therefore,
protein levels have to be analysed in parallel by immunostaining.
The approach does not directly measure de novo assembly but
rather the steady state levels of the Fe/S proteins. Thus, it may be
difficult to distinguish long-time damage of Fe/S proteins (for
instance by oxidative stress) from an impairment of the maturation process. A more direct assay of biogenesis is the
incorporation of radiolabelled iron (or sulfur) into the nascent
Fe/S proteins [41,45–47]. Fe/S cluster synthesis and assembly
on the apoproteins is then measured by immunoprecipitating the
Fe/S proteins of interest with subsequent quantitative analysis by
scintillation counting. Since the times used for radiolabelling are
usually short, the assay gives a reasonable estimation of de novo
assembly. Native gel electrophoresis can be employed to follow
The requirements for the making of an Fe/S cluster are deceptively trivial: inorganic iron and sulfide. While both components may occur in cells in free form, they both are toxic to the cell,
and therefore the intracellular free concentrations have to be rather
low. This may preclude the spontaneous assembly of an Fe/S
cluster in cells, the more so as rather high concentrations of iron
and sulfide and anaerobic conditions are needed for chemical in
vitro reconstitution of model Fe/S proteins. Therefore, sophisticated systems have been developed in living cells to assemble the
clusters and to specifically insert them into proteins. In yeast
mitochondria the ISC assembly system encompasses 14 known
proteins of diverse functions (Fig. 2). The similarity to the bacterial
counterparts suggests shared mechanisms of biosynthesis in
bacteria and mitochondria. In fact, many functional observations
for the ISC proteins have first been made with bacterial proteins.
We summarise an emerging consensus mechanism of biogenesis
for the eukaryotic (yeast) ISC system making use of numerous
observations from the bacterial ISC system [19].
In mitochondrial Fe/S cluster assembly free cysteine is the
source of sulfur which is released by the cysteine desulfurase Nfs1
in a pyridoxal phosphate-dependent fashion (Fig. 2). Nfs1 possesses numerous well-studied relatives in bacteria such as NifS,
IscS, and SufS of the NIF, ISC and SUF systems (reviewed in
[52,53]. The purified Nfs1 enzyme produces sulfide in vitro [54].
However, the sulfide production by Nfs1 (and its bacterial counterparts) likely does not mimic the in vivo pathway of the sulfur
from cysteine into an Fe/S cluster. As discussed above cells have
to prevent an unregulated release of toxic sulfide. To circumvent
liberation of sulfide, the sulfur is first transiently bound to Nfs1
via a persulfide group at a conserved cysteine residue on the
enzyme before it is further transferred to the Isu scaffold proteins
(Fig. 2). These latter proteins may initially receive the sulfur also
as a covalently bound persulfide (see below).
It has long been thought that Nfs1 is sufficient to support this
transfer reaction. However, sulfur transfer to the Isu proteins
was recently found to involve an additional essential protein
designated Isd11 which forms a stable complex with Nfs1 but is
not conserved in the bacterial systems [55,56]. The precise role
of Isd11 is unclear so far, but it is interesting to compare the
situation with a similar complex in the bacterial or plastid SUF
systems [18,24], where the Nfs1 homologue SufS binds to
SufE. This small protein transiently receives the sulfur released
from cysteine by SufS, before sulfur is further transferred to
scaffold proteins (e.g., SufA). Sulfur binding to SufE occurs via a
persulfide group formed on a conserved cysteine residue. Since
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Isd11 does not contain any cysteine residue, the functional
mechanism of Isd11 must be different from that worked out for
SufE.
A central concept of Fe/S protein biogenesis is the de novo
synthesis of the Fe/S cluster on so-called scaffold proteins before
its transfer to apoproteins [57]. In vivo studies in yeast have shown
that Isu1 (and presumably Isu2) fulfil this scaffold function in
mitochondria [58]. Fe/S cluster association with Isu1 was followed
by radiolabelling cells with iron and immunoprecipitation of Isu1.
In these experiments, over-produced Isu1 was necessary to
overcome the detection limits. The specificity of Fe/S cluster
association with Isu1 is evident from its dependence on other ISC
proteins including Nfs1, Isd11, Yah1 and Yfh1 (see also below).
The source of iron for Fe/S cluster formation on the Isu
scaffold proteins is less clear. Iron is taken up by mitochondria
in reduced form (Fig. 2). Uptake requires an energised inner
membrane and the members of the mitochondrial carrier family
termed Mrs3 and Mrs4 [59–63]. It presently is unclear whether
these transporters are directly trafficking iron and in which form
(free or chelated) iron may be transported. Since the two yeast
carriers are not essential for iron transfer into the mitochondrial
matrix, additional transporters must exist to support this
reaction (‘X’ in Fig. 2). The form of iron in which it is stored
in the mitochondrial matrix before its use in Fe/S cluster
assembly is unknown.
After import into mitochondria iron may be delivered to the
Isu proteins by Yfh1, the yeast homologue of frataxin (Fig. 2; see
also below). This model is now favoured by many groups in the
field. The purified protein was found to bind and oxidise iron,
even though it is still debated whether this binding is specific or
unspecific [64–66]. Further, Yfh1 binds to Isu1 in an ironstimulated fashion in mitochondria [67] suggesting that complex
formation may facilitate iron transfer from frataxin to Isu1 for
Fe/S cluster formation. In keeping with this view, Yfh1 was
found to be necessary for transient Fe/S cluster assembly on Isu1
[58] reassuring the role of frataxin/Yfh1 in Fe/S protein
assembly.
Frataxin was claimed to play various additional roles in heme
synthesis, iron storage, and prevention of oxidative stress prevailing in frataxin-deficient yeast cells. Most of these effects seem to be
a secondary consequence of a defective Fe/S cluster assembly and
the accompanying iron accumulation in mitochondria (see below).
In particular, the role of frataxin in iron storage is a matter of debate.
This function requires the formation of a higher order structure
similar to that of ferritin. However, mutations preventing the formation of aggregates of Yfh1 display no detectable phenotype [65].
Frataxin/Yfh1 was proposed to deliver iron not only to Fe/S cluster
biosynthesis, but also to ferrochelatase for heme formation from
protoporphyrin IX (Fig. 2; [68]). However, no primary defects in
heme synthesis upon Yfh1 depletion were seen [45]. An interaction
between ferrochelatase and Yfh1 was detected in vitro by plasmon
resonance technique, but no complex formation was observed in
mitochondria [69]. Further, experiments with triple mutants in
YFH1, MRS3 and MRS4 genes suggested that the heme defect in
cells entirely lacking YFH1 may be indirect [62]. Finally, no
immediate ferrochelatase defect seems to be evident in human cells
depleted of frataxin [50,70]. In summary, it seems reasonable to
657
believe that Yfh1/frataxin plays a role as an iron donor in Fe/S
cluster assembly on the Isu scaffold proteins, yet the precise molecular function remains to be established.
The chemistry of cluster formation of the Isu proteins is far
from being understood (discussed in [18]. While experiments
with purified bacterial proteins have led to the conflicting
interpretation that either sulfur or iron initially bind to the Isu (or
bacterial IscU or NifU) proteins, it is clear that three conserved
cysteine residues are important for Fe/S cluster assembly and
transient binding. From a solution structure of the apoform of
IscU (determined by NMR) one might expect that bound Fe/S
clusters may be surface-exposed, a situation that may facilitate the
transfer of the pre-assembled cluster to apoproteins. It was shown
that the bacterial IscU or NifU can assemble both [2Fe–2S] and
[4Fe–4S] clusters, and recently it was demonstrated that these
clusters can be specifically transferred to appropriate apoproteins
[71,72]. Thus, it appears that the Isu/IscU/NifU scaffolds are
capable of facilitating the assembly of both types of clusters [73].
In addition to Nfs1, Isd11 and Yfh1 two other ISC components
are required early in biogenesis (Fig. 2; [43,58,74]). The ferredoxin
Yah1 and the ferredoxin reductase Arh1 form an electron transfer
chain that gains its electrons from NADH and, most likely, transfers
them to the sulfur moiety (S0) released from cysteine by the Nfs1/
Isd11 complex to generate the sulfide (S2−) present in the Fe/S
cluster. The precise mode of how the sulfur is reduced and at which
step of the reaction path this occurs has remained elusive. Interestingly, Yah1 carries a [2Fe–2S] cluster itself, and thus pre-existing
holo-Yah1 is presumably involved in the conversion of newly
imported apo-Yah1 to the holoprotein. While the role of Yah1/Arh1
in the reduction of sulfur is likely (yet not proven), it is not excluded
that the proteins are required at a second step of biogenesis, for
instance during Fe/S cluster transfer from the Isu scaffold proteins
to the acceptor apoproteins. Insight into this problem will require the in vitro reconstitution of the reactions. To date, no in vitro
evidence for the potential role of this electron chain has been
provided.
1.5. Components acting late in Fe/S protein maturation
A dedicated chaperone system consisting of the Hsp70 chaperone Ssq1, its cognate J-type co-chaperone Jac1, and the
nucleotide exchange factor Mge1 has been long recognised to be
required for Fe/S protein assembly in mitochondria [16,
40,48,75–77]. Recent in vivo and in vitro experiments have
demonstrated that the proteins are not required for de novo Fe/S
cluster assembly on the Isu scaffold proteins [58,78]. Rather, upon
depletion of these proteins up to fivefold more Fe/S cluster
accumulates on Isu1. This has been taken to propose a role for the
chaperone system late in biogenesis, for instance in a partial
reaction between the stage of transient Fe/S cluster binding to the
Isu scaffold and the incorporation of the pre-assembled Fe/S
cluster into target apoproteins (Fig. 2). One might assume that
these chaperones specifically interact with the apoform of Fe/S
proteins to maintain them in a conformation capable of accepting
the Fe/S cluster from the Isu scaffold proteins. However, no
specific interaction between these protein classes has been
reported hitherto.
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Ssq1 is specifically targeted to its substrate Isu1 by Jac1 [77,79].
Ssq1 binds to Isu1 via a highly conserved LPPVK motif present in
Isu/IscU proteins [80,81]. Binding is facilitated by the co-chaperone Jac1 which also directly binds to Isu1 in a J-domain independent fashion via its C-terminal domain [82]. Even though
complex formation is not specific for either the apo- or holoform of
Isu/IscU, it is clear that the chaperone system functions in close
contact to the scaffold proteins. Hence, it seems reasonable to
propose that the chaperones facilitate Fe/S cluster transfer, that they
stabilise the conformation of the scaffold proteins or that they
regenerate the scaffold proteins for the next round of cluster
assembly. A recently developed in vitro system to study Fe/S
cluster formation on Isu1 with purified components may be
extended to unravel the role of these (and other) components [78].
Another ISC component acting late in biogenesis is the monothiol glutaredoxin Grx5 (Fig. 2; [83]. Yeast contains two dithiol
(Grx1, Grx2) and three monothiol (Grx3, Grx4, Grx5) glutaredoxins, but only Grx5 seems to play a specific role in Fe/S protein
biogenesis. As seen with the chaperones, depletion of Grx5 in yeast
results in a general defect in Fe/S protein assembly, yet transiently
bound Fe/S clusters accumulate on Isu1 [58]. The exact function of
Grx5 remains unclear, but, in contrast to the role of the chaperones,
Grx5 is dispensable for this process, as deletion of its gene results
in a minor growth defect only. Recently, a zebrafish mutant with
hypochromic anemia was found to be impaired in Grx5 function
[84]. Zebrafish Grx5 could complement the respective yeast
deletion mutant. Loss of Fe/S cluster assembly in Grx5-deficient
animals activated IRP1 thus leading to decreased levels of δaminolaevulinate synthase 2 (ALAS2) and diminished heme
synthesis. These results not only show that Grx5 is important for
(cytosolic) Fe/S protein assembly in higher eukaryotes, but also
documented that heme formation in the differentiating red cell is
regulated by a member of the ISC assembly machinery (see also
[63].
1.6. A specific role of Isa proteins in the maturation of
aconitase-like proteins
The mitochondrial proteins Isa1 and Isa2 contain a fairly conserved sequence motif that is also present in IscA and SufA proteins
of bacteria. Most eukaryotes contain only close sequence relatives
of Isa1. The Isa2 protein is unique in that closest relatives are
present only in the genomes of a few yeasts. In S. cerevisiae the two
Isa proteins form a hetero-oligomer, and the deletion of their genes
results in similar phenotypic consequences, such as the failure to
grow on non-fermentable carbon sources, loss of mitochondrial
DNA and a general defect in respiration [85–87]. Initial experiments had indicated that yeast cells depleted in Isa1 and/or Isa2
show a defect in Fe/S proteins. A more detailed study indicated a
specialised requirement of the Isa proteins for aconitase-type Fe/S
proteins in mitochondria (Fig. 2; U. Mühlenhoff, unpublished). In
contrast, the de novo assembly of other Fe/S proteins such as the
ferredoxin Yah1, Rieske Fe/S protein and biotin synthase were
unaffected. In the bacterial ISC and SUF systems the IscA proteins
were shown in vitro to serve as alternative scaffold proteins assembling an Fe/S cluster, which is competent for transfer to target
apoproteins (for reviews see [18,19,21]). This made it appear likely
that those proteins are the specific scaffolds for aconitase-type Fe/S
proteins. Several observations refute this idea for yeast as a model.
First, maturation of aconitase-type Fe/S proteins required the functional involvement of the Isu proteins, in addition to the Isa proteins. Second, despite the possibility to assemble an Fe/S cluster on
purified Isa proteins in vitro, attempts to find an Isa-associated Fe/S
cluster in vivo failed (U. Mühlenhoff, unpublished). Instead, the
proteins isolated from cell extracts bind iron (but no sulfur). Finally,
the phenotypes of Isa-depleted yeast cells are fully explained by
the defects in aconitase (e.g., glutamate auxotrophy, mtDNA loss,
respiratory defect) and homoaconitase (lysine auxotrophy). The
yeast Isa proteins therefore perform a specific role in the maturation
of aconitase-like Fe/S proteins. This is in marked contrast to the role
of the Isu proteins which are responsible for maturation of virtually
all cellular Fe/S proteins. Further clarification of the molecular role
of the Isa proteins in Fe/S cluster assembly will require faithful in
vitro approaches for reconstitution of aconitase.
1.7. The role of mitochondria in the maturation of
extra-mitochondrial Fe/S proteins
The importance of mitochondria in Fe/S protein assembly was
recognised early on in the molecular dissection of this process [41].
Virtually the entire ISC assembly system is required for maturation
of extra-mitochondrial Fe/S proteins such as Leu1, Rli1, sulfite
reductase, Nbp35, Nar1, and Ntg2 (Fig. 3). In two cases, for Nfs1
and for the Isu proteins, experimental evidence has been provided
that these ISC proteins need to be located inside yeast mitochondria to be functional in extra-mitochondrial Fe/S protein assembly
[41,54,73]. The exclusive presence of a cytosolic/nuclear form of
Nfs1 or Isu1 did not lead to any Fe/S cluster incorporation into
cytosolic/nuclear Fe/S proteins. This suggests that both Nfs1 and
Isu1/Isu2 have to be located inside yeast mitochondria to be useful
in extra-mitochondrial Fe/S protein maturation.
It is still unknown what mitochondria provide for cytosolic/
nuclear Fe/S protein assembly. The current consensus is that
the mitochondrial ISC assembly machinery produces a substrate that is exported from mitochondria to serve in Fe/S
cluster formation and incorporation in the cytosol. Components involved in this step are summarised as ISC export
machinery [88]. A central component is the mitochondrial
ABC transporter Atm1 which is involved in mitochondria to
cytosol trafficking (Fig. 3; [41]). The protein is located in the
inner membrane with its ABC domains facing the matrix.
Deletion of Atm1 is associated with severe effects including a
drastic growth defect of yeast cells, loss of cytochromes and
respiration and an accumulation of iron in mitochondria.
Gradual depletion of Atm1 in a regulated yeast mutant
revealed primary functions of Atm1 in Fe/S protein maturation
in the cytosol and nucleus, and in iron accumulation in
mitochondria (see below). In an attempt to biochemically characterise Atm1, the membrane protein was purified and reconstituted into proteoliposomes [89]. Its ATPase activity is specifically
stimulated by compounds containing free sulfhydryl (-SH) groups,
in particular by peptides with multiple cysteine residues. It is thus
conceivable that the substrate of Atm1 contains free thiol groups,
possibly in a peptidic environment.
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
659
dithiothreitol [93]. As noted in the case of defects in Atm1 or Erv1,
GSH-depleted cells were hampered in cytosolic and nuclear, but
not in mitochondrial Fe/S protein biogenesis. Further insights into
the molecular mechanisms underlying the function of these three
components of the ISC export machinery will depend on the
identification of the substrate exported by Atm1.
1.8. The cytosolic Fe/S protein assembly (CIA) machinery
Fig. 3. An emerging model for cytosolic Fe/S protein maturation and the
mitochondrial influence on cellular iron homeostasis in yeast cells. The
mitochondrial ISC assembly machinery (Fig. 2) is required for maturation of
cytosolic and nuclear Fe/S proteins. The hypothetical model proposes that the ISC
assembly machinery generates a compound (designated by ?) that is exported to the
cytosol by the ISC export machinery comprised of the inner membrane ABC
transporter Atm1, the sulfhydryl oxidase Erv1 of the intermembrane space and the
tripeptide glutathione (GSH). The maturation of Fe/S proteins further requires the
P-loop NTPases Cfd1 and Nbp35, the iron-only hydrogenase-like protein Nar1 and
the seven-bladed WD40 repeat protein Cia1. Note that the CIA proteins Nbp35 and
Nar1 are Fe/S proteins themselves. Whether ATP or GTP is used by the P-loop
NTPases remains to be resolved. The indicated protein interactions have been
established in vivo. The two ISC systems are also responsible for regulation of the
yeast iron uptake system via the Aft1/2 transcription factors. In the absence of
functional ISC systems, Aft1/2 translocate into the nucleus and activate genes of
the iron regulon encoding proteins involved in iron uptake and utilisation. The
precise molecular role of the mitochondrial ISC machineries in both cytosolic/
nuclear Fe/S protein maturation and in iron regulation mediated by Aft1/2 is
thought to be understood. Small amounts of Nfs1 in the nucleus are not yet
involved in thio-uridine modification of some cytosolic tRNAs (tRNAS).
Conspicuously, the two other components of the ISC export
machinery, Erv1 and the tripeptide glutathione (GSH), deal with
sulfhydryl chemistry (Fig. 3). Erv1 is a component of the intermembrane space and has an in vitro activity as a sulfhydryl oxidase
catalysing the reaction
R1 SH þ R2 SH þ O2 →R1 S S R2 þ H2 O2 [90].
The electron acceptor in vivo is not known yet. Depletion of the
protein has virtually the same phenotype as Atm1 defects suggesting its involvement in cytosolic/nuclear Fe/S protein maturation and in mitochondrial iron homeostasis [91]. In addition, the
protein performs a crucial role in the import of preproteins into the
intermembrane space [92]. The protein forms mixed disulfides
with Mia40, a protein involved in the import small TIM proteins
into the intermembrane space and their oxidative folding. Thus,
Erv1 appears to function as an enzyme required for the oxidation of
sulfhydryl groups in the mitochondrial intermembrane space. It
will be crucial, yet challenging to identify the specific substrate of
Atm1 and Erv1 responsible for Fe/S protein maturation in the
cytosol/nucleus.
Glutathione is known to be the most important redox regulator
of yeast cells. In addition, a specific requirement of this reagent
in cytosolic Fe/S protein assembly was shown by depleting this
tripeptide while the redox level was maintained by the addition of
The past 3 years have seen the identification of four components of the CIA machinery involved in cytosolic/nuclear Fe/S
protein maturation (Fig. 3). The first cytosolic component for
which such a function has been described is the P-loop NTPase
Cfd1 [44]. A sophisticated genetic screen aimed at the reconstitution of Fe/S cluster assembly on the human IRP1 in yeast cells
lacking mitochondrial aconitase identified Cfd1 as a prime candidate for Fe/S protein maturation. Mutations in Cfd1 are associated with defects in virtually all cytosolic and nuclear, but not
mitochondrial Fe/S proteins ([44]; D.J.A. Netz, unpublished). The
protein possesses, in addition to its ATP/GTPase domain, a stretch
of four conserved cysteine residues. Two of those were found
important for function and cell viability, but, presently it is unclear
what role these residues might play in biogenesis.
Another P-loop NTPase protein of the CIA machinery is
Nbp35 which shows sequence similarity to Cfd1 in the central
and C-terminal parts [94]. In addition, Nbp35 contains a
stretch of ∼50 residues at its N-terminus which co-ordinates an
Fe/S cluster via four conserved cysteine residues (Fig. 3).
Removal of the N-terminus of Nbp35 or mutation of the
cysteine residues impaired the ability of the protein to bind an
Fe/S cluster and destroyed its function suggesting an important
role of the N-terminal Fe/S cluster [94]; D.J.A. Netz,
unpublished). The protein forms a stoichiometric complex
with Cfd1 and therefore a hetero-oligomeric form of Cfd1 and
Nbp35 may be the functional form that plays an essential role in
Fe/S protein assembly (Fig. 3). Interestingly, assembly of the Fe/S
clusters on Nbp35 and Nar1 (see below) depend on the
mitochondrial ISC assembly and export systems. This observation
and the fact that these CIA proteins are required for their own
maturation emphasise that the process is indispensable for cells. It
should be noted that bacteria contain a similar P-loop protein
termed ApbC which appears to perform a role in Fe/S protein
maturation [95]. Likewise, a plant-specific protein (Hcf101) was
proposed to be involved in Fe/S cluster incorporation into
photosystem I [96]. These proteins, however, differ in the number
and location of conserved cysteine residues in the C-terminal part of
the protein. Nevertheless, it is possible that this class of P-loop
NTPases plays a general role in Fe/S protein assembly.
The CIA protein Nar1 exhibits some similarity to iron-only
hydrogenases of bacteria and algae, even though the protein does
not function as a hydrogenase [10,11,97]. Similarly to true hydrogenases, Nar1 is an Fe/S protein with two electronically and
magnetically interacting Fe/S clusters (Fig. 3; [98]). However, it is
evident from EPR spectra of isolated Nar1 that the Fe/S clusters of
Nar1 differ characteristically from those defined in the 3D structures of iron-only hydrogenases. The clusters are likely co-ordinated by eight conserved cysteine residues, four of which are
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concentrated at the N-terminus and the remainder of which are
scattered in the middle and C-terminal parts. Assembly of the Fe/S
clusters of Nar1 requires the mitochondrial ISC assembly and
export systems, and additionally Cfd1 and Nbp35 [94]. Apparently, a pre-existing functional CIA machinery is necessary for the
assembly of newly assembled components of this system. This
chicken and egg situation is reminiscent of the assembly of Yah1
in the mitochondrial matrix and of the TOM complex in mitochondrial outer membrane which depend on pre-existing components for their own de novo assembly.
The most recently discovered CIA member termed Cia1 was
originally identified on the basis of a fusion gene with CFD1 in
Schizosaccharomyces pombe making it likely that the fused
proteins perform a function in the same process [99]. Depletion of
yeast Cia1 elicits a somewhat different phenotype as compared to
defects in Cfd1, Nbp35 and Nar1. While Cia1 deficiency is
accompanied by an impairment in the assembly of true Fe/S target
proteins such as Leu1, Rli1 and Ntg2, Cia1 is not required for the
Fe/S cluster-containing CIA components Nar1 and Nbp35. On the
first glimpse, this result is counterintuitive, as also Nar1 and Nbp35
should initially behave as Fe/S targets before conversion into
functional proteins. However, a closer inspection shows that the
situation may be reminiscent of the observations made for depletion of the matrix chaperone system Ssq1/Jac1/Mge1 or of Grx5
with respect to Fe/S cluster accumulation on either the Isu1 scaffold protein or on target proteins (see above). Consequently, one
might expect a role of Cia1 late in biogenesis, i.e., after the function
of Nbp35 and Nar1. A further possibility arising from these
observations is that Nbp35 and/or Nar1 might serve as scaffold
proteins binding an Fe/S cluster in a transient fashion. This hypothesis can now be tested following the lead of the studies with Isu1
[58].
Cia1 is homologous to human Ciao1 which was proposed to
play a role in transcription regulation of the Wilms tumour suppressor protein, a protein not present in yeast. This suggests that
the proteins may perform functions in both Fe/S protein biogenesis and transcription regulation. This expectation is further
supported by the sub-cellular localisation of Cia1 that differs
from that of the other three CIA proteins [99]. Cia1 is mainly
located in the nucleus with minor amounts in the cytosol, while
the other three CIA proteins show the opposite sub-cellular
partitioning. In addition, the cellular concentration of Cia1 is
larger than that of the other CIA proteins. Therefore, a role of
Cia1 in transcription similar to human Ciao1 is not excluded.
Cia1 is well conserved in eukaryotes and belongs to a large
protein family characterised by the WD40 repeat domains [100].
Even though more than 50 WD40 repeat proteins with close
similarity to Cia1 are present in yeast, the protein is unique and
can be discriminated from other members with distinct
functions.
At the moment, we can only speculate about the molecular
mechanism underlying Fe/S protein assembly in the cytosol and
nucleus of yeast. Future functional insights can make use of
several distinct protein interactions between the CIA components that have been defined by in vivo and in vitro experiments
(Fig. 3; [99]). As mentioned above, Cfd1 and Nbp35 form a tight
complex, while Nbp35 interacts with Nar1. The latter protein
serves as a specific docking partner of Cia1. The predicted
doughnut shape of Cia1 typical for WD40 proteins may be
ideally suited to serve as a platform for the binding of other
proteins during the assembly pathway of Fe/S proteins.
Surprisingly, no direct contact has been reported yet for Cfd1
and Cia1, despite the existence of the above mentioned fusion
gene in S. pombe. Nevertheless, this genetic situation strongly
suggests a functional co-operation of the two proteins in the CIA
machinery-supported pathway. It is tempting to predict that
future research will see the identification of new components of
this apparently complex machinery. This will facilitate the
elucidation of the molecular mechanisms leading to the
maturation of cytosolic/nuclear Fe/S proteins.
1.9. Fe/S protein assembly in higher eukaryotes
The ISC assembly, ISC export and CIA machineries are highly
conserved in eukaryotes from yeast to man [22,23]. Thus, one
might expect similar pathways and mechanisms of Fe/S protein
assembly in lower and higher eukaryotes. To date only a few
studies exist to put this expectation to the test. The thorough
investigation of mammalian Fe/S protein assembly is an important
topic for several reasons. First, pilot experiments have to examine
the degree of similarity or divergence in the yeast and mammalian
Fe/S protein maturation mechanisms. Second, components such as
Atm1 are duplicated in mammals (ABCB6 and ABCB7; [101])
posing the question of which particular function the individual
proteins may perform. Third, diseases are associated with mutations in frataxin (Friedreich's ataxia) and ABCB7 (X-linked
sideroblastic anemia and ataxia, XLSA/A). Detailed information
on molecular issues of these disorders can be found in other
comprehensive reviews (see, e.g., [102–105]). Fourth and finally,
the close connection of Fe/S protein assembly and (intra-)cellular
iron homeostasis in yeast (see below) raises the question of whether
such links exist in mammals. It has become clear over the past
decade that the iron metabolism in yeast and mammals is governed
by radically different mechanisms.
The first functional in vivo study on a specific component of
Fe/S protein biogenesis in the human system has been performed
on frataxin. Depletion of this protein by the RNAi technology
resulted in a growth defect of HeLa cells and a specific depletion
of the activities of both mitochondrial and cytosolic Fe/S proteins [50]. Hence, this study confirmed findings made in yeast
for Yfh1, the yeast frataxin homologue, suggesting a function of
the human protein in the biogenesis of Fe/S proteins. Since the
defects in Fe/S proteins accompany the depletion of frataxin, the
Fe/S protein deficiencies previously observed in Friedreich's
ataxia patients appear to be specific rather than secondary effects, e.g., by damage of Fe/S proteins [106]. Conspicuously, the
frataxin-depleted HeLa cells were not compromised in heme
biosynthesis catalysed by ferrochelatase suggesting that frataxin
depletion in these cells was not severe enough to affect this Fe/S
cluster-dependent enzyme. A similar effect was seen in cultured
cells from Friedreich's ataxia patients [70]. Other alterations of
heme metabolites (conversion of heme b to heme a, downregulation of the heme synthesis pathway) may be explained by a
defect in the Fe/S cluster-containing adrenodoxin, a homologue
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of Yah1 required for heme a synthesis [107], and by IRP1
function regulating ALAS2 expression (see above). In fact,
cytosolic IRP1 was not efficiently converted into aconitase in
frataxin-depleted cell culture suggesting that frataxin plays a
crucial role in Fe/S protein assembly in the cytosol [50]. An
earlier study had provided initial, yet indirect evidence for the
participation of energised mitochondria in the conversion of
IRP1 to cytosolic aconitase [49]. Recent RNAi-mediated depletion
studies in Drosophila have provided strong in vivo evidence for a
role of frataxin in Fe/S protein biogenesis [108], while its knockdown in nematodes resulted in a pleiotropic phenotype with the
possibility of secondary effects [109]. Interestingly, an extra-mitochondrial localisation of frataxin in overexpressing cells has been
reported recently that is important for cell survival and prevention
of mitochondrial damage [110,111]. The precise function of the
cytosolic form of frataxin remains to be determined.
The cysteine desulfurase Nfs1 in human cells is located
predominantly in mitochondria, while minor amounts are present
in the cytosol and nucleus [112]. The protein shows activity as a
cysteine desulfurase, and, together with human Isu1, can assemble
IRP1 holoprotein in vitro [113]. The differential targeting of human
Nfs1 results from alternative translation initiation [23]. The
subcellular distribution of human Nfs1 is reminiscent to that of
yeast Nfs1 where both the mitochondrial and nuclear versions are
essential for cell viability [114]. Cell biological studies using RNAi
technology to deplete human Nfs1 have now shown that the
mitochondrial version is indispensable for Fe/S protein maturation both inside and outside mitochondria [115]. The human
protein could be replaced effectively by its mouse homologue,
demonstrating the specificity of the RNAi approach. Removal of
the mitochondrial targeting sequence from the murine Nfs1
resulted in its cytosolic/nuclear localisation. This form does not
complement the defects in cell growth and mitochondrial/
cytosolic Fe/S protein activities of human Nfs1-depleted cells.
Hence, the results demonstrate the importance of human
mitochondrial Nfs1 for biogenesis of both mitochondrial and
cytosolic Fe/S proteins, and do not identify an independent role of
the cytosolic/nuclear version of this protein. The situation
therefore is strikingly similar to yeast [54]. One should mention,
however, that these data do not exclude a participation of extramitochondrial Nfs1 in Fe/S protein maturation, but this function
would be in addition to mitochondrial Nfs1.
Genetic studies in yeast had suggested an essential task of Nfs1
outside mitochondria [114]. What might be the function of Nfs1 in
the cytosol/nucleus? Pioneering studies on IscS, the bacterial
homolog of Nfs1, have shown its requirement as a sulfur donor for
thiouridine modification of tRNAs [116]. The same function has
recently been shown for yeast mitochondrial and cytosolic/nuclear
Nfs1 ([54,117]; Figs. 2 and 3). Thus, thiouridine modification of
tRNAs in the nucleus seems to be a second indispensable function
of yeast Nfs1. It is likely, but has not been experimentally
confirmed yet, that the nuclear fraction of Nfs1 is responsible for
thiouridine modification. It seems possible that the human protein
may play a similar role in the nucleus/cytosol as its yeast
counterpart.
Small amounts of human Isu1 and Nfu1 have been found
outside mitochondria, while the majority is located inside these
661
organelles [118,119]. Isu1 in human cells is targeted to the cytosol
by alternative splicing of a single mRNA. Simultaneous depletion
of both forms of human Isu1 by RNAi results in a strong defect in
both mitochondrial and cytosolic aconitase activities documenting the importance of human Isu1 proteins for cellular Fe/S protein
biogenesis [51]. In addition, both aconitases could not be reactivated following treatment with H2O2 or an iron chelator which
results in damage or loss of the Fe/S cluster on aconitases. This
shows that regeneration of cellular Fe/S proteins after cluster loss
requires functional Isu1. Selective depletion of either mitochondrial
or cytosolic Isu1 isoforms by RNAi was inefficient and did not
significantly impact on the function of cellular Fe/S proteins precluding conclusions on the role of the individual Isu1 isoforms
under normal growth conditions. In cells containing diminished
levels of cytosolic Isu1 the activities of both aconitases fully
recovered over time after treatment with H2O2 or iron chelator. The
slightly slower reactivation of cytosolic compared to mitochondrial
aconitase was proposed to indicate a function of cytosolic Isu1 in
the regeneration of that Fe/S protein. Reduction of the mitochondrial Isu1 level by RNAi led to impaired recovery of both
mitochondrial and cytosolic aconitases after iron chelator treatment
demonstrating that this isoform is important for Fe/S protein
regeneration in both compartments. Taken together, these data
indicate that human Isu1 performs a crucial task in cellular Fe/S
protein biogenesis. While mitochondrial Isu1 plays the major role
in Fe/S cluster biogenesis in human cells, cytosolic Isu1 may
enhance regeneration/repair of cytosolic Fe/S proteins during
recovery from iron depletion or oxygen stress. It will be interesting
to learn more about this repair process, as nothing is known so far,
neither in yeast nor in human cells.
An extensive in vivo study has been performed on murine
ABCB7, the homologue of yeast Atm1 [120]. Ablation of the Xchromosome-linked ABCB7 gene is embryonic lethal indicating
the important function of this protein in mammals. Liver is the
only tissue in which a viable gene knockout was obtained by
conditional gene inactivation. Biochemical analyses of ABCB7depleted liver tissue showed diminished activities of cytosolic
Fe/S proteins including IRP1, yet the function of mitochondrial
Fe/S proteins was unaltered. This phenotype is strikingly similar
to the observations made for yeast Atm1 (see above), and
convincingly show that mitochondria play a crucial role in
cytosolic Fe/S protein biogenesis in mice. Together with the
above mentioned studies on zebrafish Grx5 and Mrs3 [63,84] the
available body of data suggests that there is a high degree of
similarity in the mechanisms of Fe/S protein biogenesis in yeast
and in higher eukaryotes. What appears to be strikingly different
from yeast in higher eukaryotes is the functional connection of
Fe/S protein biogenesis to iron uptake regulation and heme
synthesis. This is certainly, but likely not exclusively due to the
presence of the cytosolic Fe/S protein IRP1 in higher eukaryotes
but not in yeast. Maturation of IRP1 strongly depends on
mitochondrial ISC assembly and export components
[51,63,115,120]. Since the presence or absence of an Fe/S
cluster on IRP1 is crucial for the regulation of the synthesis of
key components of iron homeostasis and also of heme metabolism in erythroid cells (see article by Wallander et al. [139] in
this issue), these two processes respond to the activity of
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mitochondria in Fe/S protein biogenesis. While the critical role
of mitochondria in cellular iron homeostasis seems to be
conserved, the mode of regulation of iron homeostasis appears to
be entirely different in higher and lower eukaryotes.
1.10. The intimate link between Fe/S protein biogenesis and
iron homeostasis
Depletion of virtually any component of the yeast ISC assembly and export systems not only results in Fe/S protein
defects, but also has severe consequences for (intra-)cellular iron
homeostasis [15]. For instance, depletion of Nfs1, Isu1/2 or Atm1
results in a mitochondrial iron overload [39–41]. At the same time
yeast cells respond with an increased iron uptake into the cell
which on first sight seems somewhat counterintuitive because
also the total cell iron is increased. Initially, it was believed that a
satisfactory explanation was the depletion of cytosolic iron as a
result of the more efficient transport into mitochondria, thereby
resulting in an activation of the iron-regulated transcription
factors Aft1/Aft2 (Fig. 3). However, clever experiments using the
iron-dependent enzyme Erg25 of the ergosterol biosynthesis
pathway as an indicator for cytosolic iron levels argued against
major changes of the cytosolic iron concentration [121]. It was
shown in this and later studies that Aft1/Aft2 do not sense
cytosolic iron but rather the iron availability inside mitochondria
via a product generated by the mitochondrial ISC assembly
machinery and exported by Atm1 (Fig. 3; [122]). Thus, the ISC
assembly and export components are responsible for the
signalling of the iron status to Aft1/Aft2. Whether the compound
exported by Atm1 is identical, similar or entirely different from
the substance exported from mitochondria to facilitate Fe/S
protein assembly is unknown. At any rate, the export reaction
leads to an inactivation of Aft1/Aft2 transcription factors in the
cytosol, while conversely the CIA system is converted to a
functional biogenesis system (Fig. 3). It is clear from these and
other studies that mitochondria, as the major “consumers” of
intracellular iron, are critical regulators of iron homeostasis in
yeast. Surprisingly, changes in the activity of the CIA machinery
have no striking effects on iron uptake and storage in yeast cells.
Depletion of these proteins neither leads to mitochondrial iron
accumulation nor to an increased transcription of iron uptake
genes by activation of Aft1/Aft2. Therefore, iron regulation in
yeast is unlikely to be mediated by a canonical cytosolic Fe/S
protein which is matured with the help of the CIA proteins
distinguishing this regulatory step from the function of IRP1 in
mammals. Rather, the molecule exported by Atm1 directly or
indirectly leads to the regulation of the Aft1/Aft2 transcriptional
activity. It is likely that, in addition to Aft1/Aft2, further proteins
are involved in this regulatory path.
1.11. Fe/S protein biogenesis is essential. Why?
The process of Fe/S proteins biogenesis in mitochondria has
been discovered after some 50 years of intense research on these
organelles. One might ask how central this mitochondrial function
is for life. More than half of the components of the ISC assembly,
ISC export and CIA machineries are encoded by essential genes in
yeast [22]. The only other group of proteins fitting this criterion
are the constituents of the mitochondrial biogenesis apparatus, i.e.
the TOM and TIM proteins supporting preprotein import, mitochondrial chaperones required for proper protein import and
folding, the presequence peptidase and some other import-associated factors. Thus, together with the synthesis of heme (see
article by Ajioka et al. [138] in this issue), the pathway of Fe/S
protein biogenesis seems to be a key task of mitochondria in yeast
and higher eukaryotes, and organisms cannot survive without it.
In contrast, respiration is dispensable when yeast cells are grown
in the presence of fermentable carbon sources such as glucose.
Even human cells in culture can survive the loss of respiration
when fed with high amounts of glucose [123].
These considerations raise the immediate question of which
process makes Fe/S protein biogenesis in eukaryotes so central to
life [124]. The issue comes down to the question of which cellular
Fe/S proteins play an indispensable role for cell viability and what
is their function. To date, four genes are known in yeast that code
for essential Fe/S proteins, namely Yah1, Nbp35, Nar1 and Rli1.
As detailed above, the first three components are central components of Fe/S protein biogenesis (Table 1), and therefore the
‘chicken and egg’ situation does not provide a reasonable
explanation for essentiality of the process. This leaves us with
Rli1. The protein contains a bipartite, ferredoxin-like motif at its
N-terminus which associates with Fe/S clusters (Fig. 4; [125]). At
the C-terminus there are two typical ABC domains, giving Rli1 its
alternative name ABCE1 [126]. A 3D crystal structure has been
obtained for archaeal versions of Rli1 lacking the Fe/S cluster
motif [127]. Mutation of critical cysteine residues of the Fe/S
cluster motif to alanine resulted in the loss of cell viability showing
the essential character of these Fe/S clusters [125]. As found for
other cytosolic/nuclear Fe/S proteins, maturation of Rli1 was
dependent on the mitochondrial ISC assembly and export
machineries, as well as on all four known CIA components
providing a logical explanation why Yah1, Nbp35, and Nar1 are
essential.
The function of Rli1 has remained enigmatic for a long time,
but recent studies have provided first clues as to what processes
Rli1 might support. Initial studies had suggested roles of human
Rli1 in virus-infected cells, yet these functions do not comprise
the normal cellular function, as Rli1 is encoded in all Eukarya and
Archaea [128,129]. Depletion of Rli1 in yeast resulted in defects
in translation initiation and an accumulation of ribosomal subunits
in the nucleus [125,130,131]. This nuclear export defect of
ribosomal subunits is generally seen in all mutants with defects in
components of the CIA machinery. Concomitantly, defects in the
processing of rRNA were observed. Collectively, these data
suggest roles of Rli1 in protein translation initiation and in the
maturation of ribosomal subunits in the nucleus. Only a minor
fraction of Rli1 is present in the nucleus, but the protein can be
accumulated in this compartment using nuclear protein export
mutants suggesting that the protein is shuttling between cytosol
and nucleus. The consensus behind these findings appears to be a
function of Rli1 centered around protein synthesis on cytosolic
80S ribosomes. Consistent with these ideas, Rli1-depleted cells
exhibit a protein translation defect, yet this develops after the
occurrence of the nuclear ribosome export failure. Nevertheless, it
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
663
Fig. 4. The essential Fe/S-cluster containing ABC protein Rli1. The bottom scheme shows the functional domains of Rli1. The highly conserved bipartite Fe/S cluster
binding motif at the N-terminus possibly co-ordinates two Fe/S clusters of unknown type (blue). The two ABC domains in the center and at the C-terminus are each
followed by a short hinge region (green). The numbers indicate the amino acid residues of the protein from S. cerevisiae. The top part shows a hypothetical sketch of
the 3D domain structure of Rli1. The design was inspired by the 3D crystal structures of N-terminally truncated versions of Rli1 from Archaea lacking the Fe/S
cluster domain [127]. As typical for ABC proteins, the ATP binding site and the ABC signature motif (Sig) of the two ABC domains mutually interact in the solved
structures.
seems unlikely that the translational defect upon Rli1 depletion is
only a secondary consequence of the nuclear ribosome export
defect, as Rli1 was found to directly interact with major
translation initiation factors in both yeast [130] and human cells
[126]. These specific complex formations suggest a specific
functional role of Rli1 in protein translation initiation. Collectively, Rli1 might perform multiple roles in both normal and virusinfected cells. As a consensus model the protein may function in
the assembly of RNA–protein complexes such as ribosomal
particles, translation initiation complexes, and virus particles.
Archaeal Rli1 proteins may prove useful to decipher the basic
function(s) of this interesting protein.
In summary, the functional studies on Rli1 have pointed to
crucial link between two central and ancient processes in cells,
Fe/S protein biogenesis and protein synthesis. The biogenesis of
Fe/S proteins might have been a critical evolutionary step arising
from the catalytic phase of the “iron–sulfur” world proposed by
Wächtershäuser [132]. Protein synthesis facilitated by ribosomes is central for the development of a living cell.
These contemplations provide logical explanation why mitochondria (and Rli1) cannot be lost in Eukarya, not even in cases
where respiration is dispensable. Such a situation is prevalent in
parasitic pathogens which live (mostly) inside their host and feed
on the host cytosol. Due to the ample availability of metabolites and
energy inside the host cell, many of these parasitic organisms have
lost most of their mitochondrial functions such as heme
biosynthesis, citric acid cycle, and most prominently respiration.
While these organisms are commonly known as “amitochondriates” because they lack classical mitochondria with cristae membranes, some of them recently were shown to contain mitochondrial
remnant organelles (for recent reviews on these organisms and
organelles see [42,133,134]. The most well-known forms of these
rudimentary organelles are the hydrogenosomes and mitosomes. It
is noteworthy in the context of this review that both types of
organelles were shown to harbour basic components of the ISC
assembly machinery (Isu1 and Nfs1) and an Hsp70, and thus may
be capable of producing Fe/S clusters [135,136]. In line with the
discussion on Rli1 (see above), the essentiality of this biosynthetic
pathway may be the reason why the remnant organelles have not
been lost in these organisms. A closer inspection of the genomes of
several amitochondriates indeed reveals that they contain sequence
relatives of the ISC assembly and CIA machineries, and of Rli1.
Interestingly, counterparts of the ISC export machinery are lacking,
raising the interesting question whether hydrogenosomes and
mitosomes may also play a role in extra-organellar Fe/S protein
biogenesis as expected from the presence of Rli1.
2. Conclusions and perspectives
The past 8 years have seen the identification of 21 components
with a direct function in the assembly of Fe/S proteins in yeast as a
paradigm for a eukaryotic cell. While initially the identification of
mitochondrial ISC components and their functional characterisation has largely been influenced from findings made in the related
ISC system of bacteria, work on the Atm1-linked export reaction
and the identification and characterisation of novel components of
the CIA machinery is unique for eukaryotes. Despite the considerable progress that has been made in our understanding of
cellular Fe/S protein biogenesis, many challenges lie ahead
including the elucidation of the molecular mechanisms underlying
Fe/S cluster assembly and incorporation into apoproteins in mitochondria, the definition of the exact function of the ISC components, the (bio)chemical mechanism of Fe/S cluster synthesis on
the Isu scaffold proteins, the identification of the nature of the
compound(s) exported by Atm1, the mechanism of the export
reaction, the analysis of the source of iron and sulfur for cytosolic/
nuclear Fe/S protein assembly, the definition of potential scaffold
proteins in the cytosol/nucleus, the molecular mechanism of Fe/S
protein assembly in the cytosol/nucleus, and finally the intriguing
connection of this process to iron homeostasis. While we hope this
review has left the impression that basic components and concepts
of Fe/S protein maturation in eukaryotes have been identified, we
foresee many exciting years of discovery and surprise for researchers involved in this field. Certainly, the elucidation of the
molecular foundations of Fe/S protein biogenesis has moved into a
more mature stage.
Acknowledgements
We thank Dr. W. Walden (Chicago) for discussion. Work in
our laboratory is supported by grants from Sonderforschungsbereiche 593 and TR1, Deutsche Forschungsgemeinschaft
664
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(Gottfried-Wilhelm Leibniz program), the European Commission (MitEURO), German-Israeli foundation GIF, and Fonds der
chemischen Industrie. We thank B. Niggemeyer, N. Richhardt,
and R. Rösser for excellent technical assistance.
References
[1] H. Beinert, R.H. Holm, E. Münck, Iron–sulfur clusters: nature's modular,
multipurpose structures, Science 277 (1997) 653–659.
[2] H. Beinert, Iron–sulfur proteins: ancient structures, still full of surprises,
J. Bioinorg. Chem. 5 (2000) 2–15.
[3] R. Malkin, J.C. Rabinowitz, The reconstitution of clostridial ferredoxin,
Biochem. Biophys. Res. Commun. 23 (1966) 822–827.
[4] D.C. Rees, Great metalloclusters in enzymology, Ann. Rev. Biochem. 71
(2002) 221–246.
[5] D.C. Rees, J.B. Howard, The interface between the biological and inorganic
worlds: iron–sulfur metalloclusters, Science 300 (2003) 929–931.
[6] S. Merchant, B.W. Dreyfuss, Post-translational assembly of photosynthetic metalloproteins, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49
(1998) 25–51.
[7] P.C. Dos Santos, D.R. Dean, Y. Hu, M.W. Ribbe, Formation and insertion
of the nitrogenase iron–molybdenum cofactor, Chem. Rev. 104 (2004)
1159–1173.
[8] D.C. Rees, J.B. Howard, Nitrogenase: standing at the crossroads, Curr.
Opin. Chem. Biol. 4 (2000) 559–566.
[9] F.A. Armstrong, Hydrogenases: active site puzzles and progress, Curr.
Opin. Chem. Biol. 8 (2004) 133–140.
[10] Y. Nicolet, C. Cavazza, J.C. Fontecilla-Camps, Fe-only hydrogenases:
structure, function and evolution, J. Inorg. Biochem. 91 (2002) 1–8.
[11] D.S. Horner, B. Heil, T. Happe, T.M. Embley, Iron hydrogenases-ancient
enzymes in modern eukaryotes, Trends Biochem. Sci. 27 (2002) 148–153.
[12] D.R. Dean, J.T. Bolin, L. Zheng, Nitrogenase metalloclusters: structures,
organization, and synthesis, J. Bacteriol. 175 (1993) 6737–6744.
[13] L. Zheng, V.L. Cash, D.H. Flint, D.R. Dean, Assembly of iron–sulfur
clusters. Identification of an iscSUA-hscBA-fdx gene cluster from
Azotobacter vinelandii, J. Biol. Chem. 273 (1998) 13264–13272.
[14] J. Strain, C.R. Lorenz, J. Bode, S. Garland, G.A. Smolen, D.T. Ta, L.E.
Vickery, V.C. Culotta, Suppressors of superoxide dismutase (SOD1)
deficiency in Saccharomyces cerevisiae. Identification of proteins predicted to mediate iron–sulfur cluster assembly, J. Biol. Chem. 273 (1998)
31138–31144.
[15] R. Lill, G. Kispal, Maturation of cellular Fe/S proteins: the essential
function of mitochondria, Trends Biochem. Sci. 25 (2000) 352–356.
[16] E.A. Craig, J. Marszalek, A specialized mitochondrial molecular
chaperone system: a role in formation of Fe/S centers, Cell. Mol. Life
Sci. 59 (2002) 1658–1665.
[17] J. Frazzon, D.R. Dean, Formation of iron–sulfur clusters in bacteria—An
emerging field in bioinorganic chemistry, Curr. Opin. Chem. Biol. 7
(2003) 166–173.
[18] M. Fontecave, S.O. Choudens, B. Py, F. Barras, Mechanisms of iron–
sulfur cluster assembly: the SUF machinery, J. Biol. Inorg. Chem. 10
(2005) 713–721.
[19] D.C. Johnson, D.R. Dean, A.D. Smith, M.K. Johnson, Structure, function
and formation of biological iron–sulfur clusters, Ann. Rev. Biochem. 74
(2005) 247–281.
[20] F. Barras, L. Loiseau, B. Py, How Escherichia coli and Saccharomyces
cerevisiae build Fe/S proteins, Adv. Microb. Physiol. 50 (2005) 41–101.
[21] S.S. Mansy, J.A. Cowan, Iron–sulfur cluster biosynthesis: toward an
understanding of cellular machinery and molecular mechanism, Acc.
Chem. Res. 37 (2004) 719–725.
[22] R. Lill, U. Mühlenhoff, Iron–sulfur protein biogenesis in eukaryotes,
Trends Biochem. Sci. 30 (2005) 133–141.
[23] T.A. Rouault, W.H. Tong, Iron–sulphur cluster biogenesis and mitochondrial iron homeostasis, Nat. Rev., Mol. Cell. Biol. 6 (2005) 345–351.
[24] J. Balk, S. Lobreaux, Biogenesis of iron–sulfur proteins in plants, Trends
Plant Sci. 10 (2005) 324–331.
[25] P.V. Rao, R.H. Hom, Synthetic analogues of the active sites of iron–sulfur
proteins, Chem. Rev. 104 (2004) 527–559.
[26] H. Beinert, J. Meyer, R. Lill, in: W.J. Lennarz, M.D. Lane (Eds.),
Encyclopedia of Biological Chemistry, Iron–Sulfur Proteins, vol. 2,
Academic Press, 2004, pp. 482–489.
[27] P. Hinchliffe, L.A. Sazanov, Organization of iron–sulfur clusters in
respiratory complex I, Science 309 (2005) 771–774.
[28] F. Sun, X. Huo, Y. Zhai, A. Wang, J. Xu, D. Su, M. Bartlam, Z. Rao,
Crystal structure of mitochondrial respiratory membrane protein complex
II, Cell 121 (2005) 1043–1057.
[29] R.R. Mendel, R. Hansch, Molybdoenzymes and molybdenum cofactor in
plants, J. Exp. Bot. 53 (2002) 1689–1698.
[30] J.M. Moulis, V. Davasse, M.P. Golinelli, J. Meyer, I. Quinkal, The
coordination sphere of iron–sulfur clusters: lessons from site-directed
mutagenesis experiments, J. Bioinorg. Chem. 1 (1996) 2–14.
[31] P.J. Kiley, H. Beinert, The role of Fe–S proteins in sensing and regulation
in bacteria, Curr. Opin. Microbiol. 6 (2003) 181–185.
[32] M.W. Hentze, M.U. Muckenthaler, N.C. Andrews, Balancing acts:
molecular control of mammalian iron metabolism, Cell 117 (2004)
285–297.
[33] K. Pantopoulos, Iron metabolism and the IRE/IRP regulatory system: an
update, Ann. N. Y. Acad. Sci. 1012 (2004) 1–13.
[34] H. Matsubara, K. Saeki, in: R. Cammack (Ed.), Iron–Sulfur Proteins,
Structural and Functional Diversity of Ferredoxins and Related Proteins,
vol. 38, Academic Press, San Diego, 1992, pp. 223–280.
[35] W. Buckel, M. Hetzel, J. Kim, ATP-driven electron transfer in enzymatic
radical reactions, Curr. Opin. Chem. Biol. 8 (2004) 462–467.
[36] J.T. Jarrett, The novel structure and chemistry of iron–sulfur clusters in
the adenosylmethionine-dependent radical enzyme biotin synthase, Arch.
Biochem. Biophys. 433 (2005) 312–321.
[37] H.A. Dailey, Terminal steps of haem biosynthesis, Biochem. Soc. Trans.
30 (2002) 590–595.
[38] O.A. Lukianova, S.S. David, A role for iron–sulfur clusters in DNA
repair, Curr. Opin. Chem. Biol. 9 (2005) 145–151.
[39] J. Li, M. Kogan, S.A. Knight, D. Pain, A. Dancis, Yeast mitochondrial
protein Nfs1p coordinately regulates iron–sulfur cluster proteins, cellular
iron uptake, and iron distribution, J. Biol. Chem. 274 (1999)
33025–33034.
[40] B. Schilke, C. Voisine, H. Beinert, E. Craig, Evidence for a conserved
system for iron metabolism in the mitochondria of Saccharomyces
cerevisiae, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 10206–10211.
[41] G. Kispal, P. Csere, C. Prohl, R. Lill, The mitochondrial proteins Atm1p
and Nfs1p are required for biogenesis of cytosolic Fe/S proteins, EMBO
J. 18 (1999) 3981–3989.
[42] M. van der Giezen, J. Tovar, Degenerate mitochondria, EMBO Rep. 6
(2005) 525–530.
[43] H. Lange, G. Kispal, A. Kaut, R. Lill, A mitochondrial ferredoxin is
essential for biogenesis of intra- and extra-mitochondrial Fe/S proteins,
Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 1050–1055.
[44] A. Roy, N. Solodovnikova, T. Nicholson, W. Antholine, W.E. Walden, A
novel eukaryotic factor for cytosolic Fe–S cluster assembly, EMBO J. 22
(2003) 4826–4835.
[45] U. Mühlenhoff, N. Richhardt, M. Ristow, G. Kispal, R. Lill, The yeast
frataxin homologue Yfh1p plays a specific role in the maturation of
cellular Fe/S proteins, Hum. Mol. Genet. 11 (2002) 2025–2036.
[46] U. Mühlenhoff, N. Richhardt, J. Gerber, R. Lill, Characterization of iron–
sulfur protein assembly in isolated mitochondria: A requirement for ATP,
NADH and reduced iron, J. Biol. Chem. 277 (2002) 29810–29816.
[47] O. Stehling, P.M. Smith, A. Biederbick, J. Balk, R. Lill, U. Mühlenhoff,
Investigation of iron–sulfur protein maturation in eukaryotes, Methods
Cell Biol. (in press).
[48] T. Lutz, B. Westermann, W. Neupert, J.M. Herrmann, The mitochondrial
proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in
mitochondria, J. Mol. Biol. 307 (2001) 815–825.
[49] C. Bouton, M.J. Chauveau, S. Lazereg, J.C. Drapier, Recycling of RNA
binding iron regulatory protein 1 into an aconitase after nitric oxide
removal depends on mitochondrial ATP, J. Biol. Chem. 277 (2002)
31220–31227.
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
[50] O. Stehling, H.P. Elsässer, B. Brückel, U. Mühlenhoff, R. Lill, Iron–
sulfur protein maturation in human cells: evidence for a function of
frataxin, Hum. Mol. Genet. 13 (2004) 3007–3015.
[51] W.H. Tong, T.A. Rouault, Functions of mitochondrial ISCU and cytosolic
ISCU in mammalian iron–sulfur cluster biogenesis and iron homeostasis,
Cell. Metab. 3 (2006) 199–210.
[52] H. Mihara, N. Esaki, Bacterial cysteine desulfurases: their function and
mechanisms, Appl. Microbiol. Biotechnol. 60 (2002) 12–23.
[53] D. Kessler, Enzymatic activation of sulfur for incorporation in
biomolecules, FEMS Microbiol Lett. (in press).
[54] U. Mühlenhoff, J. Balk, N. Richhardt, J.T. Kaiser, K. Sipos, G. Kispal, R.
Lill, Functional characterization of the eukaryotic cysteine desulfurase
Nfs1p from Saccharomyces cerevisiae, J. Biol. Chem. 279 (2004)
36906–36915.
[55] N. Wiedemann, E. Urzica, B. Guiard, H. Müller, C. Lohaus, H.E. Meyer,
M.T. Ryan, C. Meisinger, U. Mühlenhoff, R. Lill, N. Pfanner, Essential
role of Isd11 in iron–sulfur cluster synthesis on Isu scaffold proteins,
EMBO J. 25 (2006) 184–195.
[56] A.C. Adam, C. Bornhövd, H. Prokisch, W. Neupert, K. Hell, The Nfs1
interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in
mitochondria, EMBO J. 25 (2006) 174–183.
[57] P. Yuvaniyama, J.N. Agar, V.L. Cash, M.K. Johnson, D.R. Dean, NifSdirected assembly of a transient [2Fe–2S] cluster within the NifU protein,
Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 599–604.
[58] U. Mühlenhoff, J. Gerber, N. Richhardt, R. Lill, Components involved in
assembly and dislocation of iron–sulfur clusters on the scaffold protein
Isu1p, EMBO J. 22 (2003) 4815–4825.
[59] F. Foury, T. Roganti, Deletion of the mitochondrial carrier genes MRS3
and MRS4 suppresses mitochondrial iron accumulation in a yeast
frataxin-deficient strain, J. Biol. Chem. 277 (2002) 24475–24483.
[60] U. Mühlenhoff, J. Stadler, N. Richhardt, A. Seubert, T. Eickhorst, R.J.
Schweyen, R. Lill, G. Wiesenberger, A specific role of the yeast
mitochondrial carriers Mrs3/4p in mitochondrial iron acquisition under
iron-limiting conditions, J. Biol. Chem. 278 (2003) 40612–40620.
[61] L. Li, J. Kaplan, A mitochondrial-vacuolar signaling pathway in yeast that
affects iron and copper metabolism, J. Biol. Chem. 279 (2004) 33653–33661.
[62] Y. Zhang, E.R. Lyver, S.A. Knight, E. Lesuisse, A. Dancis, Frataxin and
mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing
iron for heme synthesis, J. Biol. Chem. 280 (2005) 19794–19807.
[63] G.C. Shaw, J.J. Cope, L. Li, K. Corson, C. Hersey, G.E. Ackermann, B.
Gwynn, A.J. Lambert, R.A. Wingert, D. Traver, N.S. Trede, B.A. Barut,
Y. Zhou, E. Minet, A. Donovan, A. Brownlie, R. Balzan, M.J. Weiss, L.L.
Peters, J. Kaplan, L.I. Zon, B.H. Paw, Mitoferrin is essential for erythroid
iron assimilation, Nature 440 (2006) 96–100.
[64] T. Yoon, J.A. Cowan, Iron–sulfur cluster biosynthesis. Characterization
of frataxin as an iron donor for assembly of [2Fe–2S] clusters in ISU-type
proteins, J. Am. Chem. Soc. 125 (2003) 6078–6084.
[65] K. Aloria, B. Schilke, A. Andrew, E.A. Craig, Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo, EMBO
Rep. 5 (2004) 1096–1101.
[66] M. Nair, S. Adinolfi, C. Pastore, G. Kelly, P. Temussi, A. Pastore,
Solution structure of the bacterial frataxin ortholog, CyaY: mapping the
iron binding sites, Structure 12 (2004) 2037–2048.
[67] J. Gerber, U. Mühlenhoff, R. Lill, An interaction between frataxin and
Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1, EMBO Rep. 4
(2003) 906–911.
[68] E. Lesuisse, R. Santos, B.F. Matzanke, S.A. Knight, J.M. Camadro, A.
Dancis, Iron use for haeme synthesis is under control of the yeast frataxin
homologue (Yfh1), Hum. Mol. Genet. 12 (2003) 879–889.
[69] H. Lange, U. Mühlenhoff, M. Denzel, G. Kispal, R. Lill, The heme
synthesis defect of mutants impaired in mitochondrial iron–sulfur protein
biogenesis is caused by reversible inhibition of ferrochelatase, J. Biol.
Chem. 279 (2004) 29101–29108.
[70] R.A. Schoenfeld, E. Napoli, A. Wong, S. Zhan, L. Reutenauer, D. Morin,
A.R. Buckpitt, F. Taroni, B. Lonnerdal, M. Ristow, H. Puccio, G.A.
Cortopassi, Frataxin deficiency alters heme pathway transcripts and
decreases mitochondrial heme metabolites in mammalian cells, Hum.
Mol. Genet. 14 (2005) 3787–3799.
665
[71] J.N. Agar, C. Krebs, J. Frazzon, B.H. Huynh, D.R. Dean, M.K. Johnson, IscU
as a scaffold for iron–sulfur cluster biosynthesis: sequential assembly of [2Fe–
2S] and [4Fe–4S] clusters in IscU, Biochemistry 39 (2000) 7856–7862.
[72] A.D. Smith, G.N. Jameson, P.C. Dos Santos, J.N. Agar, S. Naik, C. Krebs, J.
Frazzon, D.R. Dean, B.H. Huynh, M.K. Johnson, NifS-mediated assembly
of [4Fe–4S] clusters in the N- and C-terminal domains of the NifU scaffold
protein, Biochemistry 44 (2005) 12955–12969.
[73] J. Gerber, K. Neumann, C. Prohl, U. Mühlenhoff, R. Lill, The yeast scaffold
proteins Isu1p and Isu2p are required inside mitochondria for maturation of
cytosolic Fe/S proteins, Mol. Cell. Biol. 24 (2004) 4848–4857.
[74] J. Li, S. Saxena, D. Pain, A. Dancis, Adrenodoxin reductase homolog
(Arh1p) of yeast mitochondria required for iron homeostasis, J. Biol.
Chem. 276 (2001) 1503–1509.
[75] R. Kim, S. Saxena, D.M. Gordon, D. Pain, A. Dancis, J-domain protein,
Jac1p, of yeast mitochondria required for iron homeostasis and activity of
Fe–S cluster proteins, J. Biol. Chem. 276 (2001) 17524–17532.
[76] C. Voisine, Y.C. Cheng, M. Ohlson, B. Schilke, K. Hoff, H. Beinert, J.
Marszalek, E.A. Craig, Jac1, a mitochondrial J-type chaperone, is
involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae,
Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1483–1488.
[77] R. Dutkiewicz, B. Schilke, H. Kneiszner, W. Walter, E.A. Craig, J.
Marszalek, Ssq1, a mitochondrial Hsp70 involved in iron–sulfur (Fe/S)
center biogenesis: Similarities to and differences from its bacterial
counterpart, J. Biol. Chem. 278 (2003) 29719–29727.
[78] R. Dutkiewicz, J. Marszalek, B. Schilke, E.A. Craig, R. Lill, U. Mühlenhoff,
The Hsp70 chaperone Ssq1p is dispensable for iron–sulfur cluster formation
on the scaffold protein Isu1p, J. Biol. Chem. 281 (2006) 7801–7808.
[79] K.G. Hoff, J.J. Silberg, L.E. Vickery, Interaction of the iron–sulfur cluster
assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of
Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7790–7795.
[80] K.G. Hoff, J.R. Cupp-Vickery, L.E. Vickery, Contributions of the LPPVK
motif of the iron–sulfur template protein IscU to interactions with the Hsc66–
Hsc20 chaperone system, J. Biol. Chem. 278 (2003) 37582–37589.
[81] R. Dutkiewicz, B. Schilke, S. Cheng, H. Knieszner, E.A. Craig, J.
Marszalek, Sequence-specific interaction between mitochondrial Fe–S
scaffold protein Isu and Hsp70 Ssq1 is essential for their in vivo function,
J. Biol. Chem. 279 (2004) 29167–29174.
[82] A.J. Andrew, R. Dutkiewicz, H. Knieszner, E.A. Craig, J. Marszalek,
Characterization of the interaction between the J-protein Jac1 and Isu1,
the scaffold for Fe–S cluster biogenesis, J. Biol. Chem. 281 (21) (2006)
14580–14587.
[83] M.T. Rodriguez-Manzaneque, J. Tamarit, G. Belli, J. Ros, E. Herrero,
Grx5 is a mitochondrial glutaredoxin required for the activity of iron/
sulfur enzymes, Mol. Biol. Cell 13 (2002) 1109–1121.
[84] R.A. Wingert, J.L. Galloway, B. Barut, H. Foott, P. Fraenkel, J.L. Axe,
G.J. Weber, K. Dooley, A.J. Davidson, B. Schmid, B.H. Paw, G.C. Shaw,
P. Kingsley, J. Palis, H. Schubert, O. Chen, J. Kaplan, L.I. Zon, Deficiency of glutaredoxin 5 reveals Fe–S clusters are required for vertebrate
haem synthesis, Nature 436 (2005) 1035–1039.
[85] L.T. Jensen, V.C. Culotta, Role of Saccharomyces cerevisiae ISA1 and
ISA2 in iron homeostasis, Mol. Cell. Biol. 20 (2000) 3918–3927.
[86] A. Kaut, H. Lange, K. Diekert, G. Kispal, R. Lill, Isa1p is a component of
the mitochondrial machinery for maturation of cellular iron–sulfur
proteins and requires conserved cysteine residues for function, J. Biol.
Chem. 275 (2000) 15955–15961.
[87] W. Pelzer, U. Mühlenhoff, K. Diekert, K. Siegmund, G. Kispal, R. Lill,
Mitochondrial Isa2p plays a crucial role in the maturation of cellular
iron–sulfur proteins, FEBS Lett. 476 (2000) 134–139.
[88] J. Balk, R. Lill, The cell's cookbook for iron–sulfur clusters: recipes for
fool's gold? ChemBioChem 5 (2004) 1044–1049.
[89] G. Kuhnke, K. Neumann, U. Mühlenhoff, R. Lill, Stimulation of the
ATPase activity of the yeast mitochondrial ABC transporter Atm1p by
thiol compounds, Mol. Membr. Biol. 23 (2) (2006) 173–184.
[90] J.E. Lee, G. Hofhaus, T. Lisowsky, Erv1p from Saccharomyces cerevisiae
is a FAD-linked sulfhydryl oxidase, FEBS Lett. 477 (2000) 62–66.
[91] H. Lange, T. Lisowsky, J. Gerber, U. Mühlenhoff, G. Kispal, R. Lill, An
essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in
the maturation of cytosolic Fe/S proteins, EMBO Rep. 2 (2001) 715–720.
666
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
[92] N. Mesecke, N. Terziyska, C. Kozany, F. Baumann, W. Neupert, K. Hell,
J.M. Herrmann, A disulfide relay system in the intermembrane space of
mitochondria that mediates protein import, Cell 121 (2005) 1059–1070.
[93] K. Sipos, H. Lange, Z. Fekete, P. Ullmann, R. Lill, G. Kispal, Maturation
of cytosolic iron–sulfur proteins requires glutathione, J. Biol. Chem. 277
(2002) 26944–26949.
[94] A. Hausmann, D.J. Aguilar Netz, J. Balk, A.J. Pierik, U. Mühlenhoff, R.
Lill, The eukaryotic P-loop NTPase Nbp35: an essential component of
the cytosolic and nuclear iron–sulfur protein assembly machinery, Proc.
Natl. Acad. Sci. U. S. A. 102 (2005) 3266–3271.
[95] E. Skovran, D.M. Downs, Lack of the ApbC or ApbE protein results in a
defect in Fe–S cluster metabolism in Salmonella enterica serovar
Typhimurium, J. Bacteriol. 185 (2003) 98–106.
[96] L. Lezhneva, K. Amann, J. Meurer, The universally conserved HCF101
protein is involved in assembly of [4Fe–4S]-cluster-containing complexes in Arabidopsis thaliana chloroplasts, Plant J. 37 (2004) 174–185.
[97] J. Balk, A.J. Pierik, D. Aguilar Netz, U. Mühlenhoff, R. Lill, The
hydrogenase-like Nar1p is essential for maturation of cytosolic and
nuclear iron–sulphur proteins, EMBO J. 23 (2004) 2105–2115.
[98] J. Balk, A.J. Pierik, D.J. Aguilar Netz, U. Muhlenhoff, R. Lill, Nar1p, a
conserved eukaryotic protein with similarity to Fe-only hydrogenases,
functions in cytosolic iron–sulphur protein biogenesis, Biochem. Soc.
Trans. 33 (2005) 86–89.
[99] J. Balk, D.J. Aguilar Netz, K. Tepper, A.J. Pierik, R. Lill, The essential
WD40 protein cia1 is involved in a late step of cytosolic and nuclear
iron–sulfur protein assembly, Mol. Cell. Biol. 25 (2005) 10833–10841.
[100] T.F. Smith, C. Gaitatzes, K. Saxena, E.J. Neer, The WD repeat: a common
architecture for diverse functions, Trends Biochem. Sci. 24 (1999) 181–185.
[101] R. Lill, G. Kispal, in: B.I. Holland, S.P.C. Cole, K. Kuchler, C.F. Higgins
(Eds.), ABC Proteins: From Bacteria to Man, ABC Transporters in
Mitochondria, Academic Press, Amsterdam, 2003, pp. 515–531.
[102] J. Gerber, R. Lill, Biogenesis of iron–sulfur proteins in eukaryotes:
components, mechanism and pathology, Mitochondrion 2 (2002) 71–86.
[103] H. Puccio, M. Koenig, Friedreich ataxia: a paradigm for mitochondrial
diseases, Curr. Opin. Genet. Dev. 12 (2002) 272–277.
[104] R.B. Wilson, Frataxin and frataxin deficiency in Friedreich's ataxia,
J. Neurol. Sci. 207 (2003) 103–105.
[105] I. Napier, P. Ponka, D.R. Richardson, Iron trafficking in the mitochondrion:
novel pathways revealed by disease, Blood 105 (2005) 1867–1874.
[106] A. Rötig, P. de Lonlay, D. Chretien, F. Foury, M. Koenig, D. Sidi, A.
Munnich, P. Rustin, Aconitase and mitochondrial iron–sulphur protein
deficiency in Friedreich ataxia, Nat. Genet. 17 (1997) 215–217.
[107] M.H. Barros, C.G. Carlson, D.M. Glerum, A. Tzagoloff, Involvement of
mitochondrial ferredoxin and Cox15p in hydroxylation of heme O, FEBS
Lett. 492 (2001) 133–138.
[108] P.R. Anderson, K. Kirby, A.J. Hilliker, J.P. Phillips, RNAi-mediated
suppression of the mitochondrial iron chaperone, frataxin, in Drosophila,
Hum. Mol. Genet. 14 (2005) 3397–3405.
[109] R.P. Vazquez-Manrique, P. Gonzalez-Cabo, S. Ros, H. Aziz, H.A. Baylis, F.
Palau, Reduction of Caenorhabditis elegans frataxin increases sensitivity to
oxidative stress, reduces lifespan, and causes lethality in a mitochondrial
complex II mutant, FASEB J. 20 (2006) 172–174.
[110] F. Acquaviva, I. De Biase, L. Nezi, G. Ruggiero, F. Tatangelo, C. Pisano,
A. Monticelli, C. Garbi, A.M. Acquaviva, S. Cocozza, Extra-mitochondrial localisation of frataxin and its association with IscU1 during
enterocyte-like differentiation of the human colon adenocarcinoma cell
line Caco-2, J. Cell Sci. 118 (2005) 3917–3924.
[111] I. Condo, N. Ventura, F. Malisan, B. Tomassini, R. Testi, A pool of
extramitochondrial frataxin that promotes cell survival, J. Biol. Chem.
(in press).
[112] T. Land, T.A. Rouault, Targeting of a human iron–sulfur cluster assembly
enzyme, nifs, to different subcellular compartments is regulated through
alternative AUG utilization, Mol. Cell 2 (1998) 807–815.
[113] K. Li, W.H. Tong, R.M. Hughes, T.A. Rouault, Roles of the mammalian
cytosolic cysteine desulfurase, ISCS, and scaffold protein, ISCU in iron–
sulfur cluster assembly, J. Biol. Chem. 281 (18) (2006) 12344–12351.
[114] Y. Nakai, M. Nakai, H. Hayashi, H. Kagamiyama, Nuclear localization of yeast
Nfs1p Is required for cell survival, J. Biol. Chem. 276 (2001) 8314–8320.
[115] A. Biederbick, O. Stehling, R. Rösser, B. Niggemeyer, Y. Nakai, H.P.
Elsässer, R. Lill, Role of human mitochondrial Nfs1 in cytosolic iron–
sulfur protein biogenesis and iron regulation, Mol. Cell. Biol. (in press).
[116] T. Kambampati, C.T. Lauhon, IscS is a sulfurtransferase for the in vitro
biosynthesis of 4-thiouridine in Escherichia coli tRNA, Biochemistry 38
(2006) 16561–16568.
[117] Y. Nakai, N. Umeda, T. Suzuki, M. Nakai, H. Hayashi, K. Watanabe, H.
Kagamiyama, Yeast Nfs1p is involved in thio-modification of both
mitochondrial and cytoplasmic tRNAs, J. Biol. Chem. 279 (2004)
12363–12368.
[118] W.H. Tong, T. Rouault, Distinct iron–sulfur cluster assembly complexes
exist in the cytosol and mitochondria of human cells, EMBO J. 19 (2000)
5692–5700.
[119] W.H. Tong, G.N. Jameson, B.H. Huynh, T.A. Rouault, Subcellular
compartmentalization of human Nfu, an iron–sulfur cluster scaffold
protein, and its ability to assemble a [4Fe–4S] cluster, Proc. Natl. Acad.
Sci. U. S. A. 100 (2003) 9762–9767.
[120] C. Pondarre, B.B. Antiochos, D.R. Campagna, S.L. Clarke, E.L. Greer,
K.M. Deck, A. McDonald, A.P. Han, A. Medlock, S.A. Anderson, R.S.
Eisenstein, M.D. Fleming, The mitochondrial ATP-binding cassette
transporter Abcb7 is essential in mice and participates in cytosolic iron–
sulphur cluster biogenesis, Hum. Mol. Genet. 15 (6) (2006) 953–964.
[121] O.S. Chen, R.J. Crisp, M. Valachovic, M. Bard, D.R. Winge, J. Kaplan,
Transcription of the yeast iron regulon responds not directly to iron but
rather to iron–sulfur cluster biosynthesis, J. Biol. Chem. 279 (2004)
29513–29518.
[122] J.C. Rutherford, L. Ojeda, J. Balk, U. Mühlenhoff, R. Lill, D.R. Winge,
Activation of the iron-regulon by the yeast Aft1/Aft2 transcription factors
depends on mitochondrial, but not cytosolic iron–sulfur protein biogenesis,
J. Biol. Chem. 280 (2005) 10135–10140.
[123] M.P. King, G. Attardi, Human cells lacking mtDNA: repopulation with
exogenous mitochondria by complementation, Science 246 (1989) 500–503.
[124] R. Lill, Z. Fekete, K. Sipos, C. Rotte, Is there an answer? Why are
mitochondria essential for life? IUBMB Life 57 (2005) 701–703.
[125] G. Kispal, K. Sipos, H. Lange, Z. Fekete, T. Bedekovics, T. Janaky, J.
Bassler, D.J. Aguilar Netz, J. Balk, C. Rotte, R. Lill, Biogenesis of
cytosolic ribosomes requires the essential iron–sulphur protein Rli1p and
mitochondria, EMBO J. 24 (2005) 589–598.
[126] Z.Q. Chen, J. Dong, A. Ishimura, I. Daar, A. Hinnebusch, M. Dean, The
essential vertebrate ABCE1 protein interacts with eukaryotic initiation
factors, J. Biol. Chem. 281 (11) (2006) 7452–7457.
[127] A. Karcher, K. Buttner, B. Martens, R.P. Jansen, K.P. Hopfner, X-ray
structure of RLI, an essential twin cassette ABC ATPase involved in
ribosome biogenesis and HIV capsid assembly, Structure (Camb) 13
(2005) 649–659.
[128] C. Bisbal, C. Martinand, M. Silhol, B. Lebleu, T. Salehzada, Cloning and
characterization of a RNAse L inhibitor. A new component of the
interferon-regulated 2–5A pathway, J. Biol. Chem. 270 (1995)
13308–13317.
[129] C. Zimmerman, K.C. Klein, P.K. Kiser, A.R. Singh, B.L. Firestein, S.C.
Riba, J.R. Lingappa, Identification of a host protein essential for
assembly of immature HIV-1 capsids, Nature 415 (2002) 88–92.
[130] J. Dong, R. Lai, K. Nielsen, C.A. Fekete, H. Qiu, A.G. Hinnebusch, The
essential ATP-binding cassette protein RLI1 functions in translation by
promoting preinitiation complex assembly, J. Biol. Chem. 279 (2004)
42157–42168.
[131] A. Yarunin, V. Panse, E. Petfalski, D. Tollervey, E. Hurt, Functional link
between ribosome formation and biogenesis of iron–sulfur proteins,
EMBO J. 24 (2005) 580–588.
[132] C. Huber, G. Wachtershauser, Peptides by activation of amino acids with
CO on (Ni,Fe)S surfaces: implications for the origin of life, Science 281
(1998) 670–672.
[133] S.D. Dyall, M.T. Brown, P.J. Johnson, Ancient invasions: from
endosymbionts to organelles, Science 304 (2004) 253–257.
[134] W. Martin, The missing link between hydrogenosomes and mitochondria,
Trends Microbiol. 13 (2005) 457–459.
[135] J. Tovar, G. Leon-Avila, L.B. Sanchez, R. Sutak, J. Tachezy, M. Van Der
Giezen, M. Hernandez, M. Muller, J.M. Lucocq, Mitochondrial remnant
R. Lill et al. / Biochimica et Biophysica Acta 1763 (2006) 652–667
organelles of Giardia function in iron–sulphur protein maturation, Nature
426 (2003) 172–176.
[136] R. Sutak, P. Dolezal, H.L. Fiumera, I. Hrdy, A. Dancis, M. DelgadilloCorrea, P.J. Johnson, M. Muller, J. Tachezy, Mitochondrial-type assembly
of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis, Proc. Natl. Acad. Sci. U. S. A. 101 (2004)
10368–10373.
667
[137] R.R. Mendel, F. Bittner, Cell biology of molybdenum, Biochim. Biophys.
Acta 1763 (2006) 621–635.
[138] R.S. Ajioka, J.D. Phillips, J.P. Kushner, Biosynthesis of heme in
mammals, Biochim. Biophys. Acta 1763 (2006) 722–735.
[139] M.L. Wallander, E.A. Leibold, R.S. Eisenstein, Molecular control of
vertebrate iron homeostasis by iron regulatory proteins, Biochim. Biophys.
Acta 1763 (2006) 668–689.