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SnapShot: Eukaryotic Fe-S Protein Biogenesis
SnapShot: Eukaryotic Fe-S Protein Biogenesis Viktoria Désirée Paul and Roland Lill Institut für Zytobiologie und Zytopathologie, Philipps Universität Marburg, 35032 Marburg, Germany STEP 1
STEP 2
Electron supply
Fe-S cluster transfer
Fe-S cluster assembly
Fe-S cluster insertion into target proteins
GLRX3 Fe2+ MFRN1 MFRN2
2Fe-2S proteins Scaffold protein Core ISC assembly machinery
NADPH
FDXR
e-
FDX2
Ala
Cys
e-
HSCB GRPEL1 GRPEL2
ISCU
ACO2 BOLA3 ?
-SSH
FXN ISCU
NFS1/ISD11
GLRX5-GSH
HSPA9-ATP
-SH
BOLA3 ?
ISCA1 ISCA2 IBA57
NFU1
LIAS SDH ISC targeting factors
IND1
Complex I
ISC export machinery
ALR
MITOCHONDRIA
GSH ABCB7
CYTOSOL CIA machinery
CIA targeting complex
Scaffold protein complex
X- S
FAD
CFD1 FMN CIAPIN1
NBP35
NDOR1 e-
ABCE1
NBP35 CFD1
XPD RTEL1
IOP1
GLRX3 CIA1
ISC components HUMAN YEAST
ISC export components HUMAN YEAST
Iron trafficking proteins HUMAN YEAST
ISCU NFS1 ISD11 FXN FDX2 FDXR HSPA9 HSCB GRPEL1/2 GLRX5 ISCA1 ISCA2 IBA57 NFU1 BOLA3 IND1
ABCB7 ALR
MFRN1/2 GLRX3
Isu1/Isu2 Nfs1 Isd11 Yfh1 Yah1 Arh1 Ssq1 Jac1 Mge1 Grx5 Isa1 Isa2 Iba57 Nfu1 Aim1 Ind1
DPYD
MMS19
CIA2B
eNADPH
CIA1
Atm1 Erv1
POLD1
NUCLEUS
Mrs3/4 Grx3/4
CIA components HUMAN YEAST
Selected Fe-S proteins HUMAN YEAST
CFD1 NBP35 CIAPIN1 NDOR1 IOP1 CIA1 CIA2B CIA2A MMS19
ACO2 SDHB LIAS DPYD ABCE1 XPD RTEL1 POLD1 GPAT IRP1
Cfd1 Nbp35 Dre2 Tah18 Nar1 Cia1 Cia2 Absent Mms19
MMS19
Aco1 Sdh2 Lip5 absent Rli1 Rad3 absent Pol3 absent absent
CIA1
GPAT
CIA2B
Regulation of iron homeostasis IRP2 CIA2A CIA2A
384 Cell Metabolism 20, August 5, 2014 ©2014 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cmet.2014.07.010
IRP1
See online version for legends and references
SnapShot: Eukaryotic Fe-S Protein Biogenesis Viktoria Désirée Paul and Roland Lill Institut für Zytobiologie und Zytopathologie, Philipps Universität Marburg, 35032 Marburg, Germany Overview of the Biogenesis Pathways Iron-sulfur (Fe-S) clusters are important protein cofactors present in all forms of life. In eukaryotes, Fe-S proteins perform functions in electron transport, enzyme catalysis, and regulation, and they participate in essential cellular processes such as DNA replication, DNA repair, transcription, and translation. The synthesis of Fe-S clusters and their subsequent insertion into apoproteins is a conserved process involving some 30 proteins. Fe-S protein biogenesis is initiated by the mitochondrial ISC (iron-sulfur cluster) assembly machinery, which is involved in the biogenesis of virtually all cellular Fe-S proteins, including those located in the cytosol and nucleus. Maturation of the latter proteins additionally depends on a yet unknown, sulfur-containing compound (indicated here as “X-S”) that is provided by the mitochondrial ISC system and delivered by the ISC export machinery to the cytosol where the CIA (cytosolic iron-sulfur protein assembly) machinery completes biogenesis. The essential character of the biosynthetic process is explained by the function of Fe-S proteins involved in cytosolic protein translation (ABC protein ABCE1) or in numerous steps of nuclear DNA metabolism (e.g., DNA polymerase POLD1 or DNA helicase XPD). Functional insights into the molecular mechanism of (nonplant) Fe-S protein biogenesis have been mainly gained from yeast, but the pathway seems highly conserved in human cells. The ISC and CIA components are not structurally related, yet they assist the biogenesis processes in mitochondria and cytosol along similar biosynthetic principles. The overall biogenesis processes in mitochondria or cytosol can be split into two main steps: First, a Fe-S cluster is assembled de novo on a scaffold protein, a reaction requiring a sulfur donor and electron supply. Scaffold proteins contain conserved cysteine residues and bind the Fe-S cluster in a labile and transient manner. Second, the loosely bound Fe-S cluster is released from the scaffold and may be transiently bound to dedicated targeting factors that facilitate the target-specific insertion into the polypeptide chain. Many of the ISC and CIA proteins contain Fe-S clusters (not shown here for simplicity). Biogenesis of Mitochondrial Fe-S Proteins Mitochondrial Fe-S protein biogenesis is initiated by the de novo synthesis of a [2Fe-2S] cluster on the scaffold protein ISCU. This reaction requires the cysteine desulfurase complex NFS1-ISD11, which converts cysteine to alanine and a NFS1-bound persulfide (-SSH), which serves as a sulfur donor. Frataxin (FXN) has been suggested as an allosteric regulator or a donor of iron that is imported into mitochondria by the carrier proteins mitoferrin (MFRN) 1 and 2. Fe-S cluster assembly on ISCU further depends on the supply of electrons from the transfer chain NADPH, ferredoxin reductase FDXR, and the [2Fe-2S] ferredoxin FDX2. Most likely the electrons are used for reduction of the cysteine sulfur (S0) to the sulfide (S2-) present in the Fe-S cluster. In the second step, the Fe-S cluster is released from ISCU and delivered to transfer proteins. The Fe-S cluster release from ISCU is facilitated by a chaperone system consisting of the Hsp70 protein HSPA9, its cochaperone HSCB, and the nucleotide exchange factor GRPEL1/GRPEL2. HSCB recruits holo-ISCU and directs it to the ATP-bound HSPA9. The Fe-S cluster released from ISCU is then transferred to the monothiol glutaredoxin GLRX5, which coordinates the [2Fe-2S] cluster by two additional glutathione (GSH) molecules. The components described so far comprise the core ISC assembly machinery, which is sufficient for both the maturation of mitochondrial [2Fe-2S] proteins and the production of the sulfur-containing compound X-S that is delivered to the cytosol. Maturation of mitochondrial [4Fe-4S] proteins such as aconitase (ACO2) additionally depends on ISCA1 and ISCA2 that functionally interact with IBA57. Some mitochondrial [4Fe-4S] proteins such as lipoate synthase (LIAS) and the respiratory chain complexes I and II (SDH) require additional dedicated targeting factors (NFU1 and IND1) for cluster delivery and insertion. The precise functional position of BOLA3 is still unclear. Biogenesis of Cytosolic and Nuclear Fe-S Proteins Biogenesis of cytosolic and nuclear Fe-S proteins requires mitochondrial NFS1-ISD11 and the other core ISC assembly proteins to generate the sulfur-containing compound X-S that is exported to the cytosol by the ABC transporter ABCB7. This reaction also involves GSH and the intermembrane space sulfhydryl oxidase ALR. In the first step, a [4Fe4S] cluster is assembled on the scaffold protein complex CFD1-NBP35. Electrons required for this reaction are provided by NADPH, NDOR1, and CIAPIN1. Biogenesis involves the cytosolic multidomain glutaredoxin GLRX3, which plays a general role in intracellular iron supply. In the second step, Fe-S cluster transfer is accomplished by IOP1 and the three CIA-targeting complex components CIA1, CIA2B, and MMS19. Various subcomplexes may deliver the [4Fe-4S] clusters in a target-specific fashion to dedicated cytosolic Fe-S proteins such as DPYD, ABCE1, and GPAT or nuclear Fe-S proteins such as XPD, RTEL1, and POLD1. Human cells, but not yeast, encode a second CIA2 homolog, termed CIA2A, that performs a dedicated role in the maturation of cytosolic ACO2, the holoform of iron regulatory protein (IRP) 1. CIA2A also binds to IRP2, leading to its stabilization, and thus performs a dual role in cellular iron homeostasis in mammals. References Beilschmidt, L.K., and Puccio, H.M. (2014). Biochimie 100, 48–60. Kampinga, H.H., and Craig, E.A. (2010). Nat. Rev. Mol. Cell Biol. 11, 579–592. Lill, R. (2009). Nature 460, 831–838. Lill, R., and Mühlenhoff, U. (2008). Annu. Rev. Biochem. 77, 669–700. Lill, R., Hoffmann, B., Molik, S., Pierik, A.J., Rietzschel, N., Stehling, O., Uzarska, M.A., Webert, H., Wilbrecht, C., and Mühlenhoff, U. (2012). Biochim. Biophys. Acta 1823, 1491–1508. Netz, D.J., Mascarenhas, J., Stehling, O., Pierik, A.J., and Lill, R. (2014). Trends Cell Biol. 24, 303–312. Rouault, T.A. (2012). Dis. Model. Mech. 5, 155–164. Sharma, A.K., Pallesen, L.J., Spang, R.J., and Walden, W.E. (2010). J. Biol. Chem. 285, 26745–26751. Stehling, O., and Lill, R. (2013). Cold Spring Harb. Perspect. Biol. 5, a011312.
384.e1 Cell Metabolism 20, August 5, 2014 ©2014 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cmet.2014.07.010
STEP 2
Electron supply
Fe-S cluster transfer
Fe-S cluster assembly
Fe-S cluster insertion into target proteins
GLRX3 Fe2+ MFRN1 MFRN2
2Fe-2S proteins Scaffold protein Core ISC assembly machinery
NADPH
FDXR
e-
FDX2
Ala
Cys
e-
HSCB GRPEL1 GRPEL2
ISCU
ACO2 BOLA3 ?
-SSH
FXN ISCU
NFS1/ISD11
GLRX5-GSH
HSPA9-ATP
-SH
BOLA3 ?
ISCA1 ISCA2 IBA57
NFU1
LIAS SDH ISC targeting factors
IND1
Complex I
ISC export machinery
ALR
MITOCHONDRIA
GSH ABCB7
CYTOSOL CIA machinery
CIA targeting complex
Scaffold protein complex
X- S
FAD
CFD1 FMN CIAPIN1
NBP35
NDOR1 e-
ABCE1
NBP35 CFD1
XPD RTEL1
IOP1
GLRX3 CIA1
ISC components HUMAN YEAST
ISC export components HUMAN YEAST
Iron trafficking proteins HUMAN YEAST
ISCU NFS1 ISD11 FXN FDX2 FDXR HSPA9 HSCB GRPEL1/2 GLRX5 ISCA1 ISCA2 IBA57 NFU1 BOLA3 IND1
ABCB7 ALR
MFRN1/2 GLRX3
Isu1/Isu2 Nfs1 Isd11 Yfh1 Yah1 Arh1 Ssq1 Jac1 Mge1 Grx5 Isa1 Isa2 Iba57 Nfu1 Aim1 Ind1
DPYD
MMS19
CIA2B
eNADPH
CIA1
Atm1 Erv1
POLD1
NUCLEUS
Mrs3/4 Grx3/4
CIA components HUMAN YEAST
Selected Fe-S proteins HUMAN YEAST
CFD1 NBP35 CIAPIN1 NDOR1 IOP1 CIA1 CIA2B CIA2A MMS19
ACO2 SDHB LIAS DPYD ABCE1 XPD RTEL1 POLD1 GPAT IRP1
Cfd1 Nbp35 Dre2 Tah18 Nar1 Cia1 Cia2 Absent Mms19
MMS19
Aco1 Sdh2 Lip5 absent Rli1 Rad3 absent Pol3 absent absent
CIA1
GPAT
CIA2B
Regulation of iron homeostasis IRP2 CIA2A CIA2A
384 Cell Metabolism 20, August 5, 2014 ©2014 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cmet.2014.07.010
IRP1
See online version for legends and references
SnapShot: Eukaryotic Fe-S Protein Biogenesis Viktoria Désirée Paul and Roland Lill Institut für Zytobiologie und Zytopathologie, Philipps Universität Marburg, 35032 Marburg, Germany Overview of the Biogenesis Pathways Iron-sulfur (Fe-S) clusters are important protein cofactors present in all forms of life. In eukaryotes, Fe-S proteins perform functions in electron transport, enzyme catalysis, and regulation, and they participate in essential cellular processes such as DNA replication, DNA repair, transcription, and translation. The synthesis of Fe-S clusters and their subsequent insertion into apoproteins is a conserved process involving some 30 proteins. Fe-S protein biogenesis is initiated by the mitochondrial ISC (iron-sulfur cluster) assembly machinery, which is involved in the biogenesis of virtually all cellular Fe-S proteins, including those located in the cytosol and nucleus. Maturation of the latter proteins additionally depends on a yet unknown, sulfur-containing compound (indicated here as “X-S”) that is provided by the mitochondrial ISC system and delivered by the ISC export machinery to the cytosol where the CIA (cytosolic iron-sulfur protein assembly) machinery completes biogenesis. The essential character of the biosynthetic process is explained by the function of Fe-S proteins involved in cytosolic protein translation (ABC protein ABCE1) or in numerous steps of nuclear DNA metabolism (e.g., DNA polymerase POLD1 or DNA helicase XPD). Functional insights into the molecular mechanism of (nonplant) Fe-S protein biogenesis have been mainly gained from yeast, but the pathway seems highly conserved in human cells. The ISC and CIA components are not structurally related, yet they assist the biogenesis processes in mitochondria and cytosol along similar biosynthetic principles. The overall biogenesis processes in mitochondria or cytosol can be split into two main steps: First, a Fe-S cluster is assembled de novo on a scaffold protein, a reaction requiring a sulfur donor and electron supply. Scaffold proteins contain conserved cysteine residues and bind the Fe-S cluster in a labile and transient manner. Second, the loosely bound Fe-S cluster is released from the scaffold and may be transiently bound to dedicated targeting factors that facilitate the target-specific insertion into the polypeptide chain. Many of the ISC and CIA proteins contain Fe-S clusters (not shown here for simplicity). Biogenesis of Mitochondrial Fe-S Proteins Mitochondrial Fe-S protein biogenesis is initiated by the de novo synthesis of a [2Fe-2S] cluster on the scaffold protein ISCU. This reaction requires the cysteine desulfurase complex NFS1-ISD11, which converts cysteine to alanine and a NFS1-bound persulfide (-SSH), which serves as a sulfur donor. Frataxin (FXN) has been suggested as an allosteric regulator or a donor of iron that is imported into mitochondria by the carrier proteins mitoferrin (MFRN) 1 and 2. Fe-S cluster assembly on ISCU further depends on the supply of electrons from the transfer chain NADPH, ferredoxin reductase FDXR, and the [2Fe-2S] ferredoxin FDX2. Most likely the electrons are used for reduction of the cysteine sulfur (S0) to the sulfide (S2-) present in the Fe-S cluster. In the second step, the Fe-S cluster is released from ISCU and delivered to transfer proteins. The Fe-S cluster release from ISCU is facilitated by a chaperone system consisting of the Hsp70 protein HSPA9, its cochaperone HSCB, and the nucleotide exchange factor GRPEL1/GRPEL2. HSCB recruits holo-ISCU and directs it to the ATP-bound HSPA9. The Fe-S cluster released from ISCU is then transferred to the monothiol glutaredoxin GLRX5, which coordinates the [2Fe-2S] cluster by two additional glutathione (GSH) molecules. The components described so far comprise the core ISC assembly machinery, which is sufficient for both the maturation of mitochondrial [2Fe-2S] proteins and the production of the sulfur-containing compound X-S that is delivered to the cytosol. Maturation of mitochondrial [4Fe-4S] proteins such as aconitase (ACO2) additionally depends on ISCA1 and ISCA2 that functionally interact with IBA57. Some mitochondrial [4Fe-4S] proteins such as lipoate synthase (LIAS) and the respiratory chain complexes I and II (SDH) require additional dedicated targeting factors (NFU1 and IND1) for cluster delivery and insertion. The precise functional position of BOLA3 is still unclear. Biogenesis of Cytosolic and Nuclear Fe-S Proteins Biogenesis of cytosolic and nuclear Fe-S proteins requires mitochondrial NFS1-ISD11 and the other core ISC assembly proteins to generate the sulfur-containing compound X-S that is exported to the cytosol by the ABC transporter ABCB7. This reaction also involves GSH and the intermembrane space sulfhydryl oxidase ALR. In the first step, a [4Fe4S] cluster is assembled on the scaffold protein complex CFD1-NBP35. Electrons required for this reaction are provided by NADPH, NDOR1, and CIAPIN1. Biogenesis involves the cytosolic multidomain glutaredoxin GLRX3, which plays a general role in intracellular iron supply. In the second step, Fe-S cluster transfer is accomplished by IOP1 and the three CIA-targeting complex components CIA1, CIA2B, and MMS19. Various subcomplexes may deliver the [4Fe-4S] clusters in a target-specific fashion to dedicated cytosolic Fe-S proteins such as DPYD, ABCE1, and GPAT or nuclear Fe-S proteins such as XPD, RTEL1, and POLD1. Human cells, but not yeast, encode a second CIA2 homolog, termed CIA2A, that performs a dedicated role in the maturation of cytosolic ACO2, the holoform of iron regulatory protein (IRP) 1. CIA2A also binds to IRP2, leading to its stabilization, and thus performs a dual role in cellular iron homeostasis in mammals. References Beilschmidt, L.K., and Puccio, H.M. (2014). Biochimie 100, 48–60. Kampinga, H.H., and Craig, E.A. (2010). Nat. Rev. Mol. Cell Biol. 11, 579–592. Lill, R. (2009). Nature 460, 831–838. Lill, R., and Mühlenhoff, U. (2008). Annu. Rev. Biochem. 77, 669–700. Lill, R., Hoffmann, B., Molik, S., Pierik, A.J., Rietzschel, N., Stehling, O., Uzarska, M.A., Webert, H., Wilbrecht, C., and Mühlenhoff, U. (2012). Biochim. Biophys. Acta 1823, 1491–1508. Netz, D.J., Mascarenhas, J., Stehling, O., Pierik, A.J., and Lill, R. (2014). Trends Cell Biol. 24, 303–312. Rouault, T.A. (2012). Dis. Model. Mech. 5, 155–164. Sharma, A.K., Pallesen, L.J., Spang, R.J., and Walden, W.E. (2010). J. Biol. Chem. 285, 26745–26751. Stehling, O., and Lill, R. (2013). Cold Spring Harb. Perspect. Biol. 5, a011312.
384.e1 Cell Metabolism 20, August 5, 2014 ©2014 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cmet.2014.07.010