iron - sulfur ( fe - s ) clusters are important prosthetic groups with unusual chemical properties that enable the proteins that contain them ( fe - s proteins ) to function in pathways ranging from metabolism to dna repair .
they are evolutionarily ancient and are present in essentially all organisms , including archaea , bacteria , plants and animals .
the high level of evolutionary conservation is consistent with the possibility that fe - s clusters contributed to the success of early life forms . in the anaerobic atmosphere of ancient earth ,
fe - s inorganic metal compounds were probably already present in hydrothermal vents ( martin et al . , 2008 ) .
evolving primitive organisms might have used these versatile fe - s compounds and incorporated them into early metabolic pathways ; in fact , fe - s cluster activities might have contributed to the origin of life itself .
once primitive organisms evolved , they could use the reducing power of their metabolic pathways to generate organic molecules and to spread to less protected environments ( russell and martin , 2004 ) .
however , as plants oxygenated the earth s atmosphere , fe - s clusters were no longer as readily assembled or as stable owing to the oxidation of iron and of fe - s clusters themselves ( imlay , 2006 ) . despite their inherent vulnerability to oxidation and degradation , fe - s clusters are crucial for facilitating enzyme activities in all kingdoms of life because they can bind electron - rich enzymatic substrates , accept or donate single electrons and stabilize specific protein conformations that are important to the activities of numerous proteins .
their importance for normal mammalian physiology is illustrated by the fact that mutations in proteins involved in fe - s cluster biogenesis cause at least five distinctive human diseases ( table 1 ) , including friedreich s ataxia ( frda ) , iscu myopathy and a specific sideroblastic anemia , all of which seem to be caused by mutations in proteins in the main fe - s cluster biogenesis pathway ( reviewed by ye and rouault , 2010 ) .
in addition , mutations in nubpl ( calvo et al . , 2010 ) , also known as ind1 , a protein involved in transferring fe - s clusters to complex i ( bych et al . , 2008 ; sheftel et al . , 2009 ) , were recently discovered to cause deficiency of respiratory chain complex i , resulting in mitochondrial encephalomyopathy .
finally , two forms of a disease called multiple mitochondrial dysfunctions syndrome , caused by mutations in either nfu1 or bola3 , indicate that some fe - s proteins might acquire their clusters from a specific and essential subset of donor proteins ( cameron et al .
careful phenotyping of humans with problems caused by defects in fe - s cluster biogenesis should help to clarify aspects of the pathway , particularly regarding how target fe - s proteins are identified , that have remained elusive in studies of bacteria and yeast model systems .
determining why these diseases affect some tissues , but not others , is an important challenge that will probably be resolved by detailed studies of molecular pathophysiology .
diseases caused by defects in fe - s cluster biogenesis insights into the pathophysiology of these diseases depends on gaining a better understanding of fe - s cluster biogenesis and its regulation in eukaryotic cells , particularly in the mitochondria , which must synthesize fe - s clusters for incorporation into the citric acid cycle enzymes aconitase and succinate dehydrogenase , and into respiratory chain complexes i  iii .
many studies have been performed to investigate fe - s cluster biogenesis in the model organism saccharomyces cerevisiae ; however , there are apparently differences between aspects of mammalian and yeast fe - s cluster biogenesis , including multicellularity , the role of frataxin ( an important component of the initial fe - s biogenesis complex ; see below ) ( bridwell - rabb et al . , 2011 ; schmucker et al . , 2011 ) and differences in the cytosolic fe - s cluster biogenesis machinery .
notably , in mammalian cells , accumulating evidence suggests that several key fe - s cluster biogenesis proteins are present not only in mitochondria but also in cytosolic and/or nuclear compartments , whereas their yeast counterparts are alleged to reside only in mitochondria [ with the exception of cysteine desulfurase ( biederbick et al . , 2006 ) ] .
thus , there is a need to clarify the situation in the mammalian system to obtain a better understanding of the role of the fe - s synthetic machinery in normal and diseased physiology .
this commentary focuses on how advances in understanding the steps of basic fe - s cluster biogenesis can enhance our understanding of the pathogenesis of a relatively new class of human diseases .
in addition , it highlights important features of the compartmentalization of the fe - s biogenesis machinery in mammalian cells , which seem to differ from those of the lower eukaryote s. cerevisiae .
fe - s clusters are cofactors that are generally ligated to cysteine residues of proteins , where they facilitate numerous types of reactions . composed of iron and inorganic sulfur ,
they are most frequently found in a cubane form that contains four iron and four inorganic sulfur atoms ( meyer , 2008 ) .
fe - s clusters are highly chemically versatile because both iron and sulfur can readily donate or accept multiple electrons ( beinert , 2000 ) , and these chemical features can synergize so that the affinity of an fe - s cluster for electrons can be fine - tuned across an extremely broad electrochemical range by its surrounding protein residues . for example , in mitochondrial complex i , seven fe - s clusters with gradually increasing reduction potentials align to form a wire - like pathway along which electrons ascend .
thus , the ability of fe - s clusters to maintain low reduction potentials ( i.e. low affinity for electrons ) facilitates efficient capture of chemical energy from nadh as electrons move progressively through respiratory chain complexes ( hirst , 2010 ) .
fe - s clusters are versatile in other ways , because they can directly facilitate chemical reactions by binding to an fe - s protein s substrate , as in the enzyme aconitase , which binds and interconverts citrate and isocitrate in the citric acid cycle .
in addition , fe - s proteins have been found to function as sensors , as in the bacterial fnr and iscr proteins ( kiley and beinert , 2003 ) , and in mammalian irp1 , which regulates cytosolic iron metabolism in mammalian cells ( rouault , 2006 ; muckenthaler et al . , 2008 ) .
the highly conserved general fe - s cluster biogenesis pathway has been the subject of intense study in numerous species of bacteria , plants , yeast and mammals since it was first described in bacteria ( reviewed by frazzon and dean , 2003 ) .
many of the general steps of the pathway are common to all kingdoms of life , but it seems that the situation is more complicated in eukaryotes , in which fe - s proteins are functional and necessary in multiple subcellular compartments , including mitochondria , plastids , cytosol and nucleus .
the initial stage of fe - s cluster biogenesis is accomplished by a multimeric protein complex in which a dimer of a cysteine desulfurase [ called iscs ( escherichia coli ) , nfs1 ( s. cerevisiae ) or nfs1 ( mammals ) ] forms a core to which two monomers of a dedicated scaffold protein ( iscu in bacteria , isu1 or isu2 in yeast and iscu in humans ) bind at either end ( shi et al . , 2010 )
pyridoxal phosphate , nfs1 supplies sulfur by removing it from cysteine residues , and iscu provides the backbone structure and the cysteine ligands upon which a new cluster consisting of covalently bound iron and inorganic sulfur is synthesized ( raulfs et al . , 2008 ;
py and barras , 2010 ; bandyopadhyay et al . , 2008 ; lill , 2009 ) .
the source of the iron in the nascent fe - s cluster has not been clearly identified , but suggested sources include iron that is bound to acidic patches on frataxin ( see below ) ( stemmler et al . , 2010 ) , or iron that is donated from a complex of glutathione and glutaredoxin that tethers an fe - s cluster ( qi and cowan , 2011 ) . in eukaryotes ,
the stability of the cysteine desulfurase depends on its binding to a small partner protein called isd11 ( wiedemann et al . , 2006 )
( adam et al . , 2006 ) , which is found in the mitochondrial matrix of s. cerevisiae , but has been detected in both the mitochondrial matrix and the cytosolic and/or nuclear compartments of mammalian cells ( shi et al . ,
isd11 apparently became an indispensable binding partner for the eukaryotic cysteine desulfurase early in the evolution of eukaryotes ( richards and van der giezen , 2006 ) .
the discovery that the isd11 protein is present in the mammalian nucleus supported previous reports that human nfs1 ( land and rouault , 1998 ) , iscu ( tong and rouault , 2000 ) and an alternative scaffold protein called nfu1 ( tong et al . , 2003 )
are present and active in the cytosolic and/or nuclear compartments of mammalian cells ( tong and rouault , 2006 ) .
structural modeling suggests that the protein frataxin ( yfh1 in yeast ) then binds in a pocket - like region between nfs1 and iscu ( fig .
1 ) , where it might either repress , stabilize or enhance activity of the fe - s cluster biogenesis core complex ( prischi et al . , 2010 ; schmucker et al . , 2011 ; tsai and barondeau , 2010 ) . structural data demonstrating the inclusion of frataxin in the initial multimeric
complex detracts somewhat from previous hypotheses that the role of frataxin was to deliver bound iron to the complex ( cook et al . ,
rather , it now seems that frataxin binding to the nfs1-iscu complex drives a useful conformational change that enhances initial cluster formation ( bridwell - rabb et al . , 2011 ) .
assembly of the nascent clusters from the iron and sulfur building blocks available in cells relies on a source of electrons to achieve the electronic configurations that are observed in fe - s clusters .
candidates for the provision of electrons to nascent fe - s clusters include glutaredoxin 5 ( glrx5 ) ( lillig et al . , 2008 ) and ferredoxin ( ewen et al . , 2011 ; shi et al . , 2011 ) ; ferredoxin has been shown to facilitate reductive coupling of two distinct [ 2fe-2s ] clusters into a single [ 4fe-4s ] cluster on bacterial iscu in vitro ( chandramouli et al . , 2007 ) .
glutathione , which reduces glutaredoxins , might also serve as a previously unrecognized iron source ( hider and kong , 2011 ) .
notably , a mutation in human mitochondrial glrx5 causes sideroblastic anemia and impairs fe - s cluster biogenesis , but the function of glrx5 is still not clear .
glrx5 can dimerize and ligate a single bridging fe - s cluster in vitro , using a cysteine from each glrx5 monomer and two glutathiones as the ligands of a [ 2fe-2s ] cluster ( ye et al . ,
2010 ) . a general scheme for biogenesis of fe - s clusters in mammalian cells .
( a ) nfs1 is a cysteine desulfurase that forms a dimer to which monomers of the primary scaffold protein iscu bind near the top and bottom of the complex . in eukaryotes
nfs1 also binds the cofactor pyridoxal phosphate ( not shown ) . structural and biochemical studies suggest that frataxin forms part of the initial fe - s cluster biogenesis complex , potentially occupying a pocket between nfs1 and iscu .
nfs1 donates inorganic sulfur , and cysteines from iscu provide the sulfur ligands that directly bind iron in the nascent fe - s cluster .
( b ) once the fe - s cluster is assembled , it must be transferred to recipient proteins .
work in bacteria and yeast model systems suggests that a dedicated chaperone  co - chaperone pair of proteins participates in cluster transfer from the primary scaffold , iscu , to recipient fe - s proteins . the co - chaperone is known to be hsc20 ( a dnaj protein ) , whereas the chaperone is an hsp70 homolog that has not yet been clearly identified in mammalian cells .
hsc20 binds iscu , and the hsc20-iscu complex probably then binds to its hsc70 partner through two different binding sites : hsc20 contacts the n - terminus of hsc70 and its binding partner , iscu , binds to the c - terminal substrate - binding domain region of hsc70 .
the j domain region of hsc20 contains three residues [ his ( h ) pro ( p ) and asp ( d ) ; hpd ] that activate the atpase activity of hsc70 . upon activation , a conformational change
is proposed to occur in the substrate - binding domain of hsc70 that affects bound iscu , resulting in extrusion of a peptide containing the residues lppvk from the iscu globular protein .
the lppvk peptide then binds to a groove in the substrate - binding domain of hsc70 , which consolidates or perhaps further enhances the conformational change in iscu , which might convert it to a conformation that facilitates donation of its cluster to recipient proteins . in this model
, hsc20 helps protect the vulnerable fe - s cluster bound to iscu as it dissociates from the multimeric assembly complex , and hsc20 then escorts iscu to form a trimeric complex with hsc70 .
the consumption of atp probably provides a powerful impetus to drive conformational changes of iscu and the substrate - binding domain of hsc70 ; these changes might facilitate release of the fe - s cluster from iscu . by capturing the energy released by atp hydrolysis and coupling it to conformational changes ,
the chaperone  co - chaperone pair help the fe - s cluster to reach its target proteins .
target proteins could include some direct targets , or proteins such as nfu1 , bola3 , nubpl or glrx5 that might function as intermediary scaffolds that then donate fe - s clusters to specific subsets of recipient proteins .
( c ) mutations in proteins acting at different points in the biogenesis pathway cause diseases with markedly different phenotypes ( described in table 1 ) .
after an fe - s cluster is formed by the core complex ( composed of nfs1 , isd11 , frataxin and iscu in mammalian cells ) ( tsai and barondeau , 2010 ; schmucker et al . , 2011 ; bridwell - rabb et al . , 2011
although many details of this transfer process remain undefined , much evidence indicates that a highly conserved chaperone  co - chaperone system participates in the next steps . a co - chaperone known as hsc20 in mammalian cells ( uhrigshardt et al . , 2010
) , hscb in bacteria ( vickery and cupp - vickery , 2007 ) and jac1 in s. cerevisiae ( craig and marszalek , 2002 ) binds to iscu . in bacteria
, hscb has been shown to form a complex with iscu and a co - chaperone partner , hsca ( ssq1 in s. cerevisiae ) , a member of the hsp70 heat shock protein family .
the mammalian hsp70 homolog with this function has not yet been identified , so references to mammalian hsc70 below and in fig .
homologs use the energy released by hydrolysis of atp to drive conformational changes and the refolding of target proteins .
recent studies indicate that bacterial hscb binds to iscu using highly conserved residues in a hydrophobic patch in the c - terminal portion of hscb ( fuzery et al . , 2011 ) .
together , hsca ( hsc70 ) and hscb ( hsc20 ) might facilitate fe - s cluster transfer from iscu to target apoproteins or to other secondary scaffolds by facilitating dissociation from the initial scaffold protein complex ( vickery and cupp - vickery , 2007 ; bonomi et al . , 2011 )
it remains unclear how fe - s cluster target proteins are identified , because the process seems to be selective and might depend on specific interactions between apoprotein targets and binding surfaces of the chaperone  co - chaperone complex , particularly along the extensive potential interaction area provided by the unusually large and distinctive c - terminus of hsc20 ( kampinga and craig , 2010 ) . because fe - s clusters are notoriously vulnerable to oxidative stress , the biogenesis system might have evolved to protect and enshroud fe - s clusters during most of the biogenesis and transfer process .
interestingly , mammalian hsc20 contains a cysteine - rich n - terminus that binds zinc in the crystal structure ( bitto et al . , 2008 ) , which could potentially bind an fe - s cluster ( uhrigshardt et al .
it is likely that binding of human hsc20 protects the nascent cluster on iscu , and leads iscu into a new multisubunit complex that contains the hsp70 homolog along with hsc20 and iscu . using the energy derived from atpase activity of the hsp70 homolog , a conformational change in this complex
might facilitate protected transfer of the fe - s cluster from iscu directly to recipient apoproteins or to secondary scaffold proteins that then facilitate delivery of the fe - s cluster to a defined group of recipient proteins .
target apoproteins are likely to be tethered to the chaperone  co - chaperone complex when the fe - s cluster of iscu is released so that cluster release can be coupled to fe - s acquisition by apoproteins .
as discussed , the fe - s cluster biogenesis pathway is fundamental to a variety of cellular processes : fe - s proteins are required for the function of two proteins of the citric acid cycle , succinate dehydrogenase and aconitase ; for respiratory chain complexes i  iii ; and for numerous other proteins in the mitochondria , cytosol and nucleus of mammalian cells .
thus , it is not surprising that disruption of fe - s cluster biogenesis causes human disease .
however , defects in different parts of the pathway cause very different disease phenotypes ( fig . 1 , table 1 ) .
for example , markedly diminished expression of frataxin causes frda , which is characterized by cardiac failure and the death of specific neuronal cell types , including the dorsal root ganglia , which are responsible for sensory perception and maintenance of balance [ see table 1 and the review by martelli et al . in this issue of disease models & mechanisms ( martelli et al . , 2012 ) ] .
by contrast , iscu myopathy , a disease resulting from a splicing mutation of iscu , causes pathology almost exclusively in skeletal muscles , and cardiac involvement is rare , developing only when there is a specific inactivating mutation on one allele ( kollberg et al . , 2009 ) .
tissue - specific factors might exacerbate the loss of protein function in affected tissues ; indeed , it has been suggested that tissue - specific splicing factors drive abnormal splicing of iscu in the skeletal muscles of individuals with iscu myopathy ( nordin et al . , 2011 ) .
it seems likely that the cells in affected tissues in frda express less frataxin than unaffected tissues , and that the fe - s cluster biogenesis pathway is therefore more compromised at those sites .
however , this observation has not been closely examined , possibly because of challenges associated with obtaining tissues from patients or autopsies . because there has only been a single patient with sideroblastic anemia caused by glrx5 mutation ,
it is not possible to verify proposed causes of tissue specificity for this disease ( table 1 ) .
studies of three newly described diseases caused by mutations in nubpl , nfu1 or bola3 suggest that transfer of fe - s clusters to specific target proteins downstream of the iscu chaperone  co - chaperone complex depends on specific pathways that shepherd fe - s clusters to subsets of recipient proteins ( also discussed below ) .
for instance , mutations in the complex - i - dedicated chaperone protein nubpl have been reported to cause mitochondrial encephalomyopathy , a severe multisystem infantile disease attributable to loss of complex i function .
by contrast , mutations in nfu1 or bola3 interfere quite specifically with activity of an fe - s enzyme , lipoate synthase , which provides lipoic acid modifications to a small group of specific enzymes , including the e2 subunits of pyruvate dehydrogenase and -ketoglutarate dehydrogenase , and to enzymes involved in branched chain amino acid metabolism and glycine degradation ( cameron et al . , 2011 ; navarro - sastre et al . , 2011 ) .
the small number of individuals with nfu1 or bola3 mutations died within the first year of life , and their main symptoms of lactic acidosis and failure to thrive have thus far not been reported to be highly tissue specific ( see table 1 ) .
to understand the diseases that result from defects in the fe - s cluster biogenesis machinery , it is important to fully understand how this process occurs in mammalian cells .
insights into mammalian fe - s cluster biogenesis have been confounded by the fact that a dominating paradigm has been developed in the model system s. cerevisiae , in which it was proposed that fe - s cluster biogenesis occurred or was initiated solely in the mitochondria , but not cytosolic or nuclear compartments ( lill , 2009 ) .
specifically , it was proposed that the mitochondrial exporter atm1 exported either fully formed fe - s clusters or a special form of sulfur that was required for cytosolic fe - s cluster biogenesis ( lill , 2009 ) , and that loss of atm1 transporter activity led to the accumulation of iron within the mitochondrial matrix . in s.
sulfur compound from mitochondria has been proposed to contribute to cytosolic fe - s cluster biogenesis by proteins known as tah18 and dre2 ( netz et al . , 2010 ) .
it was proposed that a cluster is transferred from dre2 to other members of the cytosolic fe - s cluster assembly ( cia ) proteins ( sharma et al . , 2010 ) .
there are mammalian homologs of yeast cia proteins , including ndor1 and ciapin1 ( homologs of tah18 and dre2 , respectively ) , nubp1 and nubp2 [ homologs of yeast nbp35 and cfd1 , respectively ( stehling et al . , 2008 ) ] , narfl ( homolog of yeast nar1 ) and ciao1 ( homolog of yeast cia1 ) ( srinivasan et al . , 2007 ) .
however , the model purporting that cytosolic fe - s cluster biogenesis depends on export of a mitochondrial product was recently called into question because genetic manipulations that were initially used to inactivate atm1 also introduced wild - type leu2 into a leu2-null strain , which unknowingly resulted in repressed transcription of leu1 relative to the control strain .
leu1 encodes a cytosolic fe - s protein that was being used as a readout to quantify cytosolic fe - s cluster biogenesis . because leu1 activity was assessed as being low in atm1-null strains , this was misinterpreted as evidence that the mitochondrial exporter , atm1 , contributed an essential component to cytosolic fe - s cluster biogenesis .
recently , when the experiment was performed with appropriate controls , there was no evidence that loss of atm1 adversely affected leu1 activity ( bedekovics et al . , 2011 ) .
thus , the model that fe - s clusters are assembled solely in mitochondria of yeast cells lacks crucial experimental support , and some models based on work in s. cerevisiae must be reconsidered . in any case
, there is now substantial evidence that the paradigm that fe - s clusters are assembled or initiated solely in mitochondria is not true in mammalian cells , an issue that must be clarified to allow insights into disease pathophysiology .
it is well accepted that the basic fe - s cluster biosynthetic proteins are highly expressed in mammalian mitochondria , where they supply fe - s clusters to proteins of the citric acid cycle , the respiratory chain complexes and other pathways ( described above ) .
less appreciated and somewhat controversial , however , is the idea that some of these proteins are also present in the cytosolic and/or nuclear compartments of mammalian cells ( fig .
it has been reported that small amounts of the cysteine desulfurase nfs1 and its binding partner isd11 are expressed in the cytosolic and/or nuclear compartments of mammalian cells ( land and rouault , 1998 ; shi et al . , 2009 ) , where small amounts of the scaffold proteins iscu and nfu1 are also present ( tong et al . ,
2003 ; tong and rouault , 2006 ) . the observed extra - mitochondrial localization of these proteins is consistent with the fact that there are nuclear localization signals in mammalian nfs1 ( land and rouault , 1998 ) and isd11 ( shi et al . , 2009 ) , as well as in nfs1 of s. cerevisiae ( nakai et al . ,
2001 ) ; however , s. cerevisiae nfs1 is not believed to participate in extra - mitochondrial fe - s cluster biogenesis ( biederbick et al . , 2006 ) .
the extra - mitochondrial isoforms of mammalian iscu and nfu1 are encoded by alternatively spliced transcripts that lack a recognizable mitochondrial targeting signal ( tong et al . , 2003 ; tong and rouault , 2006 ) , and there is functional evidence that the cytosolic iscu functions as a scaffold and source for extra - mitochondrial fe - s clusters in mammalian cells ( tong and rouault , 2006 ) . moreover , the co - chaperone hsc20 has been detected in cytosolic fractions ( uhrigshardt et al . ,
however , because proteins are synthesized in the cytosol , it is possible that some of the fe - s cluster biogenesis proteins found in this compartment represent proteins that were inefficiently imported into and processed by mitochondria .
notably , there is almost always much more of each protein in the mitochondrial matrix than in the extra - mitochondrial fractions .
proposed differences in the localization of fe - s cluster biogenesis proteins in 
 s. cerevisiae 
 versus mammalian cells .
( a ) yeast mitochondria contain the basic fe - s cluster biogenesis proteins , whereas cytosolic fe - s cluster biogenesis has been proposed to depend on the export of a sulfur compound from mitochondria through the transporter atm1 ( abcb7 in human cells ) .
further biogenesis in the cytoplasm depends on a distinct set of proteins , including tah18 , dre2 , nbp35 , cfd1 , nar1 and cia1 , which are collectively referred to as cia ( cytosolic iron - sulfur assembly ) proteins .
( b ) by contrast , in mammalian cells , it seems that most of the basic fe - s cluster biogenesis proteins are expressed in mitochondria as well as in the cytosolic and/or nuclear compartment ( nuclear localization of these proteins not represented in the figure ) .
in addition , there are human counterparts to the proteins implicated in cytosolic fe - s cluster biogenesis in yeast , including ndor1 ( homolog of tah18 ) , ciapin1 , ( homolog of dre2 ) , nubp1 and nubp2 ( homologs of nbp35 and cfd1 , respectively ) , and narfl and ciao1 ( homologs of nar1 and cia1 , respectively ) .
to explain mitochondrial iron overload ( which occurs as a consequence of disrupted fe - s cluster biogenesis ) , it is possible that mitochondria depend on fe - s cluster biogenesis to synthesize a molecule that functions as a regulatory signal . in the absence of this signal ( i.e. when fe - s cluster biogenesis is impaired )
, remodeling of nuclear transcription leads to mitochondrial iron overload , as the cell attempts to compensate for possible mitochondrial iron deficiency .
because mitochondrial iron overload probably contributes to disease pathogenesis in frda , iscu myopathy and possibly several other diseases , elucidation of the details of this pathway might pave the way to new therapeutic interventions . understanding the pathway by which fe - s clusters could be assembled outside
the mitochondrial compartment is becoming more important as the list of recognized cytosolic and nuclear fe - s proteins grows .
indeed , there might be many as - yet - undiscovered fe - s proteins in the mitochondrial , cytosolic and nuclear compartments of mammalian cells .
however , fe - s proteins lack a distinctive motif that permits easy recognition , and they tend to lose their sensitive prosthetic groups during purification .
importantly , a significant number of dna repair enzymes are fe - s proteins , including the protein responsible for excision - repair of uv damage that causes the disease xeroderma pigmentosum ( xpd ) when defective , and a related helicase that causes fanconi anemia when defective ( rudolf et al . , 2006 ) .
in addition , telomere maintenance depends on the nuclear fe - s protein rtel-1 ( uringa et al .
, 2011 ) , and eukaryotic dna polymerases depend on fe - s clusters for function ( netz et al . , 2011 )
. some of the cytosolic fe - s  assembly proteins previously identified in yeast might function in mammalian cells as secondary scaffold proteins that transfer clusters to specific classes of protein targets such as dna repair enzymes .
it is well known that fully formed fe - s clusters can readily move from one protein to another ; this mobility of transfer between proteins is one of the most distinctive features of fe - s clusters ( beinert et al . , 1997 ) .
thus , it is perhaps not surprising that mutations of nubp1 and nubp2 impair cell division ( christodoulou et al . , 2006 ) , because these proteins are implicated in cytosolic fe - s cluster biogenesis and some of their likely targets are involved in chromosomal duplication and in maintenance of nuclear integrity , processes that are necessary for proper cell division ( netz et al . , 2010 ) .
in summary , although the scenario in yeast remains in dispute , the accumulating evidence that fe - s cluster biogenesis can occur de novo in the mitochondrial matrix as well as in extra - mitochondrial compartments of mammalian cells is strong and consistent .
it is crucial to correctly analyze the basics of the mammalian fe - s cluster biogenesis pathway , because it sets the stage for understanding one of the most common complications of impaired fe - s cluster biogenesis : the propensity to develop marked mitochondrial iron overload and subsequent iron - related mitochondrial damage .
evidence suggests that interruption of mitochondrial fe - s cluster biogenesis leads to mitochondrial iron overload and cytosolic iron depletion in mammalian cells , which is associated with disease pathology .
for example , mitochondrial iron overload is a distinctive feature of the heart and some neurons of frda patients ( koeppen , 2011 ) , of the skeletal muscle cells of individuals with iscu myopathy ( mochel et al . , 2008 ) , and of the immature red blood cells of a patient with sideroblastic anemia attributable to deficiency of glrx5 ( ye et al . , 2010 ) .
in yeast , mutations of frataxin ( babcock et al . , 1997 ) , of the chaperone ssq1 ( hsp70 homolog ) ( knight et al . , 1998 ) or the j - domain co - chaperone jac1 ( hsc20 homolog ) ( kim et al . , 2001 )
compromise the biogenesis of fe - s clusters , and also cause mitochondrial iron overload . in mammalian cells ,
mitochondrial iron overload has been observed to occur in concert with cytosolic iron depletion , as judged by activities of cytosolic iron regulatory proteins ( li et al . , 2008 ; shi et al . , 2009 ) .
in both yeast and human cells , this mitochondrial iron overload can be readily reversed by restoration of the missing fe - s cluster biogenesis protein ( babcock et al . , 1997 ; ye et al . , 2010
one early hypothesis referred to above was that iron was able to leave mitochondria only when it had been incorporated into an fe - s cluster ( kispal et al . , 1999 ) .
subsequently , this hypothesis was modified to suggest that abcb7 ( the mammalian homolog of yeast atm1 ) exports a special type of sulfur , but not iron , from mitochondria ( lill , 2009 ; netz et al . , 2010 ) .
however , the revised theory does not offer an explanation for why iron accumulates in mitochondria : the idea that iron accumulates in mitochondria passively because it can not be incorporated into fe - s clusters does not take into account the possibility that mitochondrial iron metabolism is a highly regulated process that might be imbalanced in cells with defective fe - s cluster biogenesis . in support of this idea , transcriptional expression of the mitochondrial iron importer mitoferrin is increased in frataxin - deficient mouse hearts ( huang et al . ,
remodeling transcription of this nuclear gene might represent a response to signals of iron deficiency from mitochondria .
how might mitochondria generate a signal that could instruct the nucleus to upregulate the expression of mitoferrin ?
one possibility is that mitochondria export a product that is made only when the fe - s cluster biogenesis pathway is intact , and absence of this signal triggers a coordinated nuclear transcriptional response .
mitochondrial iron overload also occurs in individuals with x - linked sideroblastic anemia and ataxia ( allikmets et al . , 1999 )
caused by mutations in the abcb7 mitochondrial iron transporter , as well as in individuals with sideroblastic anemia caused by mutations in the red - blood - cell - specific amino - levulinate synthase alas2 , a mitochondrial protein that is involved in heme biosynthesis ( table 2 ) .
it is possible that both alas2 and abcb7 participate in a signaling pathway that regulates mitochondrial iron homeostasis , and , furthermore , that abcb7 transports a crucial regulatory molecule related to the product of alas2 . because the abcb7 substrate ( or that of its yeast homolog atm1 ) ( kuhnke et al . , 2006 ) has not yet been identified , it might be time to think more broadly about the regulation of mitochondrial iron homeostasis and to integrate the phenotypes observed upon mutation of alas2 and abcb7 into a single overarching model for mitochondrial iron regulation .
for instance , it will be important to determine whether mitochondrial iron overload occurs in cells from individuals with multiple mitochondrial dysfunctions syndrome caused by mutations in nfu1 or bola3 ( cameron et al . , 2011 ) ( table 1 ) .
nfu1 probably acts as a scaffold downstream of iscu during fe - s cluster biogenesis ( navarro - sastre et al . , 2011 ) , whereas bola3 is related to proteins that bind glutaredoxins , which play an unknown role in fe - s cluster biogenesis .
mutations in either nfu1 or bola3 disrupt the function of lipoic acid synthetase ( lias ) , which depends on a [ 4fe-4s ] cluster for activity .
lias adds a lipoate moiety to key subunits of pyruvate dehydrogenase and several other enzymes ( table 1 ) .
the fact that a specific subset of fe - s proteins is affected in these newly described diseases suggests that target specificity is achieved through use of specific scaffold proteins that function as intermediates between iscu and target proteins . because loss of glrx5 causes mitochondrial iron overload , and glrx5 might function as a bola3 partner ( huynen et al . , 2005 )
, it seems likely that the mitochondrial iron overload phenotype will also be observed in this newly described disease .
proposed roles of proteins encoded by genes associated with human sideroblastic anemias ( characterized by mitochondrial iron overload ) mitochondrial iron overload develops in several other diseases that do not involve defects in fe - s cluster biogenesis .
these diseases include those caused by mutations in an enzyme known as pus1 , which is expressed in both mitochondria and the nucleus and is required for pseudouridylate processing on transfer rnas ( trnas ; see table 2 ) , and by mutations in yars2 , a gene that encodes a mitochondrial tyrosine trna synthetase .
these results imply that a molecule produced by the mitochondrial protein synthesis apparatus also contributes to regulation of mitochondrial iron homeostasis .
the list of diseases attributable to defects in fe - s cluster biogenesis is growing . in the human genome , many of the genes encoding fe - s cluster biogenesis proteins , including hsc20 and iscu , are represented only once .
the lack of duplication of these essential genes might enhance the likelihood that deleterious phenotypes will arise from mutations that affect fe - s cluster biogenesis proteins .
notably , mutations in nfu1 , bola3 and nubpl cause severe disease and mortality in early infancy , and there is no evidence of significant tissue specificity .
identification of new disease genes in humans should accelerate progress in understanding fe - s cluster biogenesis , and should shed light on how specific target proteins are identified .
better characterization of several newly recognized diseases offers the prospect of making progress on details of the second part of the fe - s biogenesis pathway , particularly downstream of the chaperone  co - chaperone proteins , when fe - s clusters are ligated to specific recipient apoproteins .
further work will help to clarify how mitochondrial iron homeostasis is regulated , and learning what drives mitochondrial iron overload should yield better therapeutic approaches for diseases in which this feature contributes to pathology .
 iron - sulfur ( fe - s ) clusters are ubiquitous cofactors composed of iron and inorganic sulfur . 
 they are required for the function of proteins involved in a wide range of activities , including electron transport in respiratory chain complexes , regulatory sensing , photosynthesis and dna repair . 
 the proteins involved in the biogenesis of fe - s clusters are evolutionarily conserved from bacteria to humans , and many insights into the process of fe - s cluster biogenesis have come from studies of model organisms , including bacteria , fungi and plants . 
 it is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of fe - s cluster biogenesis . 
 although these diseases which include friedreich s ataxia ( frda ) , iscu myopathy , a rare form of sideroblastic anemia , an encephalomyopathy caused by dysfunction of respiratory chain complex i and multiple mitochondrial dysfunctions syndrome  affect different tissues , a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in fe - s cluster biogenesis . 
 this commentary outlines the basic steps of fe - s cluster biogenesis as they have been defined in model organisms . 
 in addition , it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of fe - s cluster biogenesis proteins in some eukaryotes , including mammals . 
 finally , it outlines several important unresolved questions in the field that , once addressed , should offer important clues into how mitochondrial iron homeostasis is regulated , and how dysfunction in fe - s cluster biogenesis can contribute to disease . 
Introduction
Basic biochemistry and function of Fe-S clusters
Fe-S cluster biogenesis: the basic pathway
Defects in Fe-S cluster biogenesis as causes of human disease
Subcellular distribution of Fe-S cluster biogenesis proteins in eukaryotic cells
A unifying hypothesis to integrate defective Fe-S cluster biogenesis with mitochondrial iron overload
Perspective