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Roland Lill Philipps-Universität Marburg

Roland Lill Philipps-Universität Marburg. Biogenesis of iron-sulfur proteins in eukaryotes A finally identified essential biosynthetic pathway. What will we discuss in this lecture ?. Short introduction into Fe/S clusters and proteins

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Roland Lill Philipps-Universität Marburg

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  1. Roland Lill Philipps-Universität Marburg Biogenesis of iron-sulfur proteins in eukaryotesA finally identified essential biosynthetic pathway

  2. What will we discuss in this lecture ? • Short introduction into Fe/S clusters and proteins • Overview on current knowledge of Fe/S protein biogenesis in eukaryotes • Mitochondrial Fe/S protein biogenesis is a bacteria-derived process • Mechanisms of Fe/S proteins biogenesis inside and outside mitochondria • Why is this process essential ? • Fe/S protein biogenesis has relevance for certain diseases

  3. Spin states can be measured by EPR (electron paramagnetic resonance) spectroscopy Fe/S clusters: Structure and oxidative states The two simplest forms of Fe/S clusters S: always S2- Fe: Fe2+ or Fe3+, in proteins coordinated by Cys, His, CO, CN-, ...

  4. UV-Vis Spectrum of a [4Fe-4S] protein 1.5 1 Absorbance 420 nm Holo 0.5 Apo 0 290 390 490 590 Cys S2- Fe2/3+ Wavelength (nm) How Fe/S clusters are integrated into proteins Typical consensus Fe/S binding motifs: - CX4CX2CX~30C in [2Fe-2S] proteins - CX2CX2CX20-40C in [4Fe-4S] proteins Pro and Gly next to Cys act as helix breakers

  5. Succinate dehydrogenase = Complex II Hydrogenase (H2 H++ 2 e-) Bacterial hydrogenase Diverse functions of Fe/S proteins  Electron transfer Respiratory complex I Respiratory complex II  Respiratory complex III (Rieske Fe/S protein) Ferredoxine - Adrenodoxine

  6. Forth Fe: Not coordinated by Cys, but citrate or isocitrate, serves as Lewis acid Aconitase Cys Cys TCA cycle Cys Diverse functions of Fe/S proteins  Enzyme co-factor Aconitase Aconitase-like proteins involved in biosynthesis of Lys, Leu and in iron regulation

  7. 16 The Haber-Bosch process at 25°C and 1 atm 2 8 16 + 16 Pi N2ase+ N2ase- Diverse functions of Fe/S proteins  Enzyme co-factor Nitrogenase (azototrophic bacteria)

  8. Fe Fe IRP1 Diverse functions of Fe/S proteins  Sensor for regulatory processes FNR - Anaerobic/aerobic gene expression switch in bacteria IRP1 (Iron regulatory protein 1) - Iron uptake and storage in mammals Cytosolic aconitase RNA binding protein Transferrin receptor: mRNA stabilisation with IRP1 bound to IRE

  9. Eukaryotic cell Nucleus Mitochondrion Cytosol Subcellular localisation of Fe/S proteins Citric acid cycle Amino acid synthesis Adrenodoxin Lipoic acid & biotin synthesis Respiratory Chain DNA repair Amino acid synthesis Iron uptake regulation Ribosome assembly Unknown functions

  10. Apoprotein Holoprotein How to synthesise Fe/S proteins In vitro chemical reconstitution Minus O2, high S2-, high Fe2+ Highly toxic to the cell Sulfide precipitates metals Fe2+ undergoes Fenton reaction (radical formation)

  11. Eukaryotic cell Nucleus Mitochondrion Cytosol ISC export mach. Three different machineries co-operate to synthesise Fe/S proteins ISC assembly mach. CIA machinery Apoprotein Holoprotein

  12. ABC transporter Atm1 Fe S S Fe Isu1/2 Ala Cys Nfs1 Fe S S Fe pmf Fe S S Iron (Fe2+) Fe Biogenesis of cellular Fe/S proteins requires mitochondria 4 proteins CIA machinery Apo Holo Extra-mitochondrial Fe/S proteins ISC export machinery 3 proteins Cytosol Mitochondrium Scaffold Apo 1998 ISC assembly machinery Holo Conserved in Bacteria (ISC operon), D. Dean 1998 14 proteins Mitochondrial Fe/S proteins

  13. N2 NH3 NIF Bacterium ISC machinery ISC export Plant cell Eukaryotic cell Nucleus ISC assembly SUF machinery Mitochondrion Plastid CIA machinery Cytosol How Fe/S protein biogenesis machineries were inherited in evolution SUF machinery (+O2, low Fe) Endosymbiosis

  14. 1. Regulatable yeast mutants (GAL promoter, ts cells) 2. In vivo radiolabelling +55Fe + Galactose ATM1 PromGAL1-10 [55Fe-S] + Glucose WT Gal-ATM1 Gal Glc Gal Glc Atm1 Cell lysate [55Fe-S] Porin [55Fe] Immunoprecipitation of reporter protein (+/- tag) ATM1 PromGAL1-10 Scintillation counting (Fe or Fe/S) Ho we measure 55Fe/S protein maturation in vivo

  15. Growth of Gal-ATM1 cells in glucose or galactose media 40 h Cells + 55Fe Cell lysate Fe/S protein IP Scintillation counting a-Aco1 a-Leu1 PIS PIS Glu Gal Atm1 Leu1 Immunostain The mitochondrial ABC transporter Atm1 is required for biosynthesis of cytosolic, but not mitochondrial Fe/S proteins Cytosolic Mitochondrial 125 100 75 Immunoprecipitated Fe/S protein (%) 50 25 0 GluGal GluGal C-source

  16. CIA machinery Apo Holo Extra-mitochondrial Fe/S proteins ISC export machinery ABC transporter Atm1 Fe S S Fe Cytosol Mitochondrium Scaffold Apo Isu1/2 ISC assembly machinery Holo 14 proteins Ala Cys Nfs1 Mitochondrial Fe/S proteins Fe S S Fe pmf Fe S S Iron (Fe2+) Fe Biogenesis of cellular Fe/S proteins requires mitochondria

  17. At which step of Fe/S cluster biosynthesis are ISC proteins needed? Isu1 Isu1 55Fe • Growth of “Gal-ISC” cells overexpressing ISU1 • In vivo labelling with 55Fe • Preparation of cell extracts • Immunoprecipitation ofIsu1  55Fe or 55Fe/S binding ? 55Fe 55Fe/S Fe/S protein ? ? ISC proteins

  18. 16 12 Immunoprecipitated 55Fe (nmol/ g cells) 8 4 0 Isu1  a-Isu1 Isu1  PIS WT a-Isu1 Isu1 binds iron in vivo

  19. Gal-NFS1 Gal-YAH1 Gal-ISA1 Gal-SSQ1 24 20 16 Immunoprecipitated 55Fe (nmol/ g cells) 12 8 4 0 Gal Gal Gal Gal Glc Glc Glc Glc Differential requirement of the ISC proteins for iron binding to Isu1

  20. Holo Extra-mitochondrial Fe/S proteins Cytosol ISC export machinery ABC transporter Atm1 Mitochondrion Fe S S Fe S S Fe Mitochondrial Fe/S proteins Fe Ala e-  NADH  Ferredoxin Nfs1/Isd11 Frataxin Isu1/2 CysSH Iron (Fe2+) A current model of Fe/S protein biogenesis in eukaryotes CIA machinery • Iron import across inner membrane requires • membrane potential (pmf) • reduced iron (Fe2+) • carrier proteins Mrs3/4 Isu1/2 ISC assembly machinery • Fe/S cluster synthesis on Isu1/2 requires • cysteine desulfurase (S donor) • ferredoxin plus reductase (electron donor from NADH) • iron donor (frataxin) ? Mrs3/4

  21. e- Ferredoxin [Fe-S] target proteins Cys Ala Isd11 Isd11 Isu1/2 scaffold Nfs1 Nfs1 HS- -SH HSS- -SSH PLP PLP PLP PLP S Fe Fe2+ Fe S Frataxin The mechanism of sulfur release from cysteine

  22. Holo Extra-mitochondrial Fe/S proteins Cytosol ISC export machinery ABC transporter Atm1 Mitochondrion Fe S S Fe S S Fe Mitochondrial Fe/S proteins Fe Ala e-  NADH  Ferredoxin Nfs1/Isd11 Frataxin Isu1/2 Cys Iron (Fe2+) A current model of Fe/S protein biogenesis in eukaryotes CIA machinery ? ? • Fe/S cluster transfer to target apoproteins requires • chaperones • Hsp70 (DnaK) + ATP • DnaJ-like co-chaperone • ADP/ATP exchange factor Isu1/2 Chaperones ATP LPPVK ISC assembly machinery Hsp70 interacts with conserved LPPVK motif of Isu1/2 ? Mrs3/4

  23. Ein Gedi Qibbuz, Dead Sea, Israel

  24. Holo Extra-mitochondrial Fe/S proteins Cytosol ISC export machinery ABC transporter Atm1 Mitochondrion Fe S S Fe S S Fe Mitochondrial Fe/S proteins Fe Ala e-  NADH  Ferredoxin Nfs1/Isd11 Frataxin Isu1/2 Cys Iron (Fe2+) A current model of Fe/S protein biogenesis in eukaryotes CIA machinery ? ? • Atm1 might transport • a chelated Fe/S cluster • a sulfur-carrier (-SSH?) • a compound generated by a mitochondrial Fe/S protein Isu1/2 Chaperones ATP ISC assembly machinery Purified Atm1 in proteoliposomes:  ATPase of Atm1 is stimulated by peptidic thiol groups ? Mrs3/4

  25. CIA machinery Holo Extra-mitochondrial Fe/S proteins Cytosol ISC export machinery ABC transporter Atm1 Mitochondrion Fe S S Fe Isu1/2 S S Chaperones ATP Fe Mitochondrial Fe/S proteins Fe ISC assembly machinery Ala e-  NADH  Ferredoxin Nfs1/Isd11 Frataxin Isu1/2 Cys ? Mrs3/4 Iron (Fe2+) A current model of Fe/S protein biogenesis in eukaryotes GSH Erv1 (2 -SH + O2 S-S +H2O2) • Export also requires • Sulfhydryloxidase Erv1 • glutathione (GSH)

  26. FeS FeS FeS FeS FeS + Nbp35 Nbp35 Nbp35 Nbp35 Nbp35 Cia1 FeS FeS Cfd1 Cfd1 Cfd1 Cfd1 Cfd1 FeS FeS Nar1 Nar1 + A (brandnew) model of how Fe/S proteins are made in the cytosol by the CIA machinery 1) Two soluble P-loop ATPase form a complex 2) Two Fe/S clusters are assembled on the complex 3) Nar1, a potential electron donor binds to the complex 4) Interactions are mediated by a WD40 protein (platform)

  27. FeS FeS Nbp35 Nbp35 + Cia1 FeS FeS Cfd1 Cfd1 Apo FeS FeS Nar1 Nar1 Holo A (brandnew) model of how Fe/S proteins are made in the cytosol by the CIA machinery 5) Fe/S clusters are transferred to the apoproteins Cytosolic/nuclear Fe/S proteins • Sulfur and iron donors are unknown • More components will be identified • Energetics of the reaction unclear • ...

  28. CIA machinery Cia1 Cfd1/Nbp35 Nar1 Holo Rli1 essential Extra-mitochondrial Fe/S proteins Cytosol No essential GSH Erv1 ISC export machinery ABC transporter Atm1 Mitochondrion Fe S S Fe Isu1/2 S S /ATP Ssq1 Jac1/Mge1 Fe Mitochondrial Fe/S proteins Fe Grx5 ISC assembly machinery Ala /NADH  Yah1  Arh1 Nfs1/Isd11 Yfh1p Isa1/2 Nfu1 Isu1/2 Cys ? Mrs3/4 Iron (Fe2+) Nfs1/Isd11 Why is Fe/S protein biogenesis essential ? Function ? Encoded by essential genes

  29. Apo Holo Extra-mitochondrial Fe/S proteins Cytosol ISC export machinery ABC transporter Atm1 Fe S S Fe Mitochondrion Apo Isu1/2 ISC assembly machinery Holo Ala Cys Nfs1 Mitochondrial Fe/S proteins Fe S S Fe pmf Fe S S Iron Fe An essential role of mitochondria in the biogenesis of ribosomes CIA machinery Rli1 Fe/S ABC protein ribosome biogenesis Proteins

  30. Holo Defects are associated with neurodegenerative/hematological disorders Extra-mitochondrial Fe/S proteins Cytosol X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A) ISC export machinery ABC transporter Atm1 Fe S S Fe Mitochondrion Apo Isu1/2 Frataxin Friedreich Ataxia ISC assembly machinery Holo Ala Cys Nfs1 Mitochondrial Fe/S proteins Fe S S Fe pmf Fe S S Iron Fe Fe/S protein biogenesis is conserved in eukaroytesand of importance for human disease CIA machinery

  31. Classical functions Novel functions Oxidative phosphorylation (ATP) TCA cycle b-Oxidation of fatty acids Heme biosynthesis Urea cycle Amino acid biogenesis Phospholipid biosynthesis Co-factor biosynthesis DNA replication Transcription Protein biosynthesis Apoptosis Involvement in tumorigenesis ROS production Iron-sulfur protein biogenesis The only known essential process, at least in yeast Mitochondria: Why do we need these organelles? You now understand WHY!

  32. Thank you !! The Marburg Pipette in the 1960s

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