The role of mitochondria in the maturation of cytosolic and nuclear iron-sulfur proteins
Prof. Dr. Roland Lill
Institut für Zytobiologie
Philipps-Universiät Marburg
- Fellow der Max Planck Gesellschaft
Robert-Koch-Str. 6
35037 Marburg - Germany
Fon: +49-6421-286
6449
+49-6421-286 6483
(Sekr.)
Fax: +49-6421-286
6414
E-mail: Lill@staff.uni-marburg.de
http://www.uni-marburg.de/fb20/cyto/
Project description
Iron-sulfur (Fe/S) proteins in (non-green) eukaryotes are localised in mitochondria, cytosol, and nucleus. Their biogenesis requires the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries and the cytosolic iron-sulfur protein assembly (CIA) apparatus (Fig. 1). Most ISC and CIA components are conserved from yeast to man and are indispensable for life. This is due to the existence of essential Fe/S proteins in the nucleus with a function in various steps of DNA and RNA metabolism. Malfunction of some biogenesis proteins or Fe/S proteins causes neurological, haematological or oncological diseases.
Mitochondrial Fe/S proteins require the iron-sulfur cluster (ISC) assembly machinery which was inherited from bacteria during evolution. Cytosolic and nuclear Fe/S protein assembly also depends on the function of this machinery, yet additionally requires the mitochondrial ISC export apparatus and the cytosolic iron-sulfur protein assembly (CIA) machinery.
Maturation of cytosolic-nuclear Fe/S proteins crucially depends on the function of the mitochondrial ISC assembly machinery which produces an unknown compound (termed X in Fig. 2). The mitochondrial ABC transporter Atm1 exports X to the cytosol where it is used for assembly of an Fe/S cluster on the cytosolic Fe/S scaffold proteins Cfd1 and Nbp35. To elucidate the molecular mechanisms of this complex process we have reconstituted Fe/S cluster formation on purified apo-Cfd1-Nbp35 using isolated mitochondria and radiolabeled 55Fe or 35S-cysteine. Synthesis of the Fe/S cluster depends on the mitochondrial ISC assembly and export machineries, and the addition of both iron and cysteine. Dissection of the reconstitution reaction in a mitochondria-dependent and -independent reaction showed that the former reaction required only cysteine but no iron. This suggests that X is a sulfur-containing (S) compound which, independently of mitochondria, is combined with cytosolic iron to de novo generate an Fe/S cluster on Cfd1-Nbp35.
The mitochondrial ABC transporter Atm1 of the ISC export machinery translocates a still unknown sulfur-containing compound (X) to the cytosol which is then used by components of the CIA machinery to assemble cytosolic and nuclear Fe/S proteins in two major steps involving the indicated CIA components.
In the new funding period we will investigate the molecular function of mitochondria as a sulfur-donating compartment for cytosolic-nuclear Fe/S protein maturation and other sulfur-requiring processes. Our work will concentrate on the following aspects.
1) Building on the Fe/S protein reconstitution assay we will identify compound X exported by mitochondrial Atm1. X will be radiolabeled with 35S-cysteine in isolated mitochondria, exported, and the mitochondrial supernatant will be analysed for X by HPLC and mass spectrometry. We then will analyze the molecular mechanism of the export reaction.
2) We will investigate the molecular role of the newly identified CIA proteins Tah18 and Dre2 which transfer electrons from NADPH to an early step of cytosolic-nuclear Fe/S protein biogenesis. Electron input may be crucial for using X in Fe/S cluster formation on Cfd1-Nbp35.
3) The dynamic interaction of these four (and potentially further new) CIA components will be analysed with the aim to mechanistically understand their functional cooperation during the assembly process.
4) The 3D structures of these CIA components and Atm1 will be solved to better understand the underlying molecular mechanisms of Fe/S cluster assembly on Cfd1-Nbp35.
Together, these studies should allow us to further improve our understanding of the crucial function of mitochondria in cytosolic-nuclear Fe/S protein maturation.
Staff
Dr. Torsten Heidenreich, Post-doctoral
fellow
Dipl. Chem. Holger Webert, PhD student
Dipl. Biol. Victoria Paul, PhD student
Nadine Richter, technician
Publications since 2007
1) Stehling, O., Smith, P.M., Biederbick, A., Balk, J., Lill, R. & Mühlenhoff, U. (2007). Investigation of iron-sulfur protein maturation in eukaryotes. Methods Mol. Biol. 372, 325-342.
2) Molik, S., Lill, R., & Mühlenhoff, U. (2007). Methods for studying iron metabolism in yeast mitochondria. Meth. Cell Biol. 80, 261-280.
3) Mander, G.J. & Lill, R. (2008). Iron-sulfur protein biogenesis in eukaryotes: Machineries, mechanisms, and pathology. In: Iron metabolism and disease (Fuchs, H., ed.), Research Signpost, Kerala, India, ch. 14, 337-364.
4) Mühlenhoff, U., Gerl, M.J., Flauger, B., Pirner, H.M., Balser, S., Richhardt, N., Lill, R. and Stolz, J. (2007). The ISC proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae. Eukaryot. Cell 6, 495-504.
5) Nakai, Y., Nakai, M., Lill, R., Suzuki, T, Hayashi, H. (2007). Thio modification of yeast cytosolic tRNA is an iron-sulfur protein-dependent pathway. Mol. Cell. Biol. 27, 2841-2847.
6) Wang, J., Fillebeen, C., Chen, G., Biederbick, A., Lill, R., Pantopoulos, K. (2007). Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway. Mol. Cell. Biol. 27, 2423-2430.
7) Netz, D.J.A., Pierik, A.J., Stümpfig, M., Mühlenhoff, U., & Lill, R. (2007). The Cfd1/Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol. Nature Chem. Biol. 3, 278-286.
8) Srinivasan, V., Netz, D.J.A., Webert, H., Mascarenhas, J., Pierik, A.J., Michel, H., & Lill, R. (2007). Structure of the yeast WD40 domain protein Cia1, a component acting late in iron-sulfur protein biogenesis. Structure 15, 1246-1257.
9) Lill, R. (2007). Iron-sulfur clusters: Basic building blocks for life. Zellbiologie Aktuell 33-3, 17-20.
10) Gelling, C., Dawes, I.W., Richhardt, N., Lill, R.* &
Mühlenhoff, U. (2008). Mitochondrial Iba57p is required for Fe/S
cluster formation on aconitase and activation of radical SAM enzymes.
Mol. Cell. Biol. 28, 1851-1861.
* Corresponding author.
11) Goldberg, A.V.*, Molik, S.*, Tsaousis, A.D., Neumann, K., Kuhnke, G., Delbac, F., Vivares, C.P., Hirt, R.P., Lill, R.# & Embley, M.# (2008). Localization and functionality of microsporidian iron-sulphur cluster assembly proteins. Nature 452, 624-628.
12) Joint first authors; # joint corresponding authors.
13) Hausmann, A., Samans, B., Lill, R. & Mühlenhoff, U. (2008). Cellular and mitochondrial remodeling upon defects in iron-sulfur protein biogenesis. J. Biol. Chem. 283, 8318-8330.
14) Lill, R. & Mühlenhoff, U. (2008). Maturation of iron-sulfur proteins in eukaryotes: Mechanisms, connected processes and diseases. Annu. Rev. Biochem. 77, 669-700.
15) Bych, K., Kerscher, S., Netz, D.J.A., Pierik, A.J., Zwicker, K., Huynen, M.A., Lill, R., Brandt, U., & Balk, J. (2008). The iron-sulphur protein Ind1 is required for effective complex I assembly. EMBO J. 27, 1736-1746.
16) Stehling, O., Netz, D.J.A., Niggemeyer, B., Rösser, R., Eisenstein, R.S., Puccio, H., Pierik, A.J., & Lill, R. (2008). Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis. Mol. Cell. Biol. 28, 5517-5528.
17) Boyd, J.M., Pierik, A.J., Netz, D.J.A., Lill, R., & Downs, D.M. (2008). Bacterial ApbC can bind and effectively transfer iron-sulfur clusters. Biochemistry 47, 8195-8202.
18) Miao, R., Martinho, M., Morales, J.G., Kim, H., Ellis, E.A., Lill, R., Hendrich, M.P., Münck, E., & Lindahl, P.A. (2008). EPR and Mössbauer spectroscopy of intact mitochondria isolated from Yah1p-depleted Saccharomyces cerevisiae. Biochemistry 47, 9888-9899.
19) Bych, K., Netz, D.J.A., Vigani, G., Bill, E., Lill, R., Pierik, A.J. & Balk, J. (2008). The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage. J. Biol. Chem. 283, 35797-35804.
20) Sheftel, A.D. & Lill, R. (2009). The power plant of the cell is also a smithy: The emerging role of mitochondria in cellular iron homeostasis. Ann. Med. 41, 82-99.
21) Pierik, A., Netz, D.J.A., & Lill, R. (2009). Analysis of iron-sulfur protein maturation in eukaryotes. Nature Protocols 4, 753-766.
22) Urzica, E., Pierik, A.J., Mühlenhoff, U., & Lill, R. (2009). Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1. Biochemistry 48, 4946-4958.
23) Stehling, O., Sheftel, A.D. & Lill, R. (2009). Controlled expression of iron-sulfur cluster assembly components for respiratory chain complexes in mammalian cells. Methods Enzymol. 456, 209-231.
24) Lill, R. (2009). Function and biogenesis of iron-sulphur proteins. Nature 460, 831-838. Podcast on http://www.nature.com/nature/podcast/index-metalloproteins-09-08-13.html
25) Naamati, A., Regev-Rudzki, N., Galperin, S., Lill, R., & Pines, O. (2009). Dual targeting of Nfs1 and discovery of its novel processing enzyme, Icp55. J. Biol. Chem. 284, 30200-30208.
26) Sheftel, A.D., Stehling, O., Pierik, A.J., Netz, D.J.A., Kerscher, S., Elsässer, H.P., Wittig, I., Balk, J., Brandt, U., & Lill R. (2009). Human Ind1, an iron-sulfur cluster assembly factor for respiratory complex I. Mol. Cell. Biol. 29, 6059-6073.
27) Lillig, C.H. & Lill, R. (2009). Lights on iron-sulfur clusters. Chem. Biol. 16, 1213-1214.
28) Schwenkert, S.*, Netz, D.J.A.*, Frazzon, J., Pierik, A.J., Bill, E., Gross, J., Lill, R., & Meurer, J. (2010). Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly. Biochem. J. 425, 207-214. * Joint first authors
29) Ihrig, J., Hausmann, A., Hain, A., Richter, N., Hamza, I., Lill, R., & Mühlenhoff, U. (2010). Iron regulation through the back door: Iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in Saccharomyces cerevisiae. Eukaryot. Cell 9, 460-471.
30) Sheftel, A., Stehling, O., & Lill R. (2010). Iron-sulfur proteins in health and disease. Trends Endocrinol. Metab. 21, 302-314.
31) Albrecht, A.G., Netz, D.J.A., Miethke, M., Pierik, A.J., Burghaus, O., Peuckert, F., Lill, R.*, & Marahiel, M.A.* (2010). SufU is an essential iron-sulfur cluster scaffold protein in Bacillus subtilis. J. Bacteriol. 192, 1643-1651. * Joint corresponding authors
32) Pierrel, F., Hamelin, O., Douky, T., Kieffer-Jaquinod, S., Mühlenhoff, U., Ozeir, M., Lill, R., and Fontecave, M. (2010). Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis. Chem. Biol. 17, 449-459. Commentary by Rötig, A. (2010) Chem. Biol. 17, 415-416.
33) Sheftel, A.D., Stehling, O., Pierik, A.J., Elsässer, H.P., Mühlenhoff, U., Webert, H., Hobler, A., Hannemann, F., Bernhardt, R., & Lill, R. (2010). Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct functions in steroidogenesis, heme and Fe/S cluster biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 107, 11775-11780.
34) Mühlenhoff, U., Molik, S., Godoy, J.R., Uzarska, M.A., Richter, N., Seubert, A., Zhang, Y., Stubbe, J., Pierrel, F., Herrero, E., Lillig, C.H. & Lill, R. (2010). Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster. Cell Metab. 12 , 373-385.
35) Netz, D.J.A., Stümpfig, M., Doré, C., Mühlenhoff,
U., Pierik, A.J. & Lill, R. (2010). Tah18 transfers electrons to
Dre2 in cytosolic iron-sulfur protein biogenesis. Nature Chem.
Biol. 6, 758-765.
Commentary by DosSantos and Dean (2010) Nature Chem. Biol.
6, 700-701.
36) Hoffmann, B., Uzarska, M.A., Berndt, C., Godoy, J.R., Haunhorst, P., Lillig, C.H., Lill, R., & Mühlenhoff, U. (2011). The multidomain thioredoxin-monothiol glutaredoxins represent a distinct functional group. Antioxid. Redox Signal. 15, 19-30.
37) Castells-Roca, L., Mühlenhoff, U., Lill, R., Herrero, E., & Belli, G. (2011). The oxidative stress response in yeast cells involves changes in the stability of Aft1 regulon mRNAs. Mol. Microbiol. 81, 232-248.
38) Ozeir, M., Mühlenhoff, U., Webert, H., Lill, R., Fontecave, M., & Pierrel, F. (2011). Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency. Chem. Biol. 18, 1134-42. Commentary by Clarke, C.F. (2011) Chem. Biol. 18, 1069-1070.
39) Mühlenhoff, U., Richter, N., Pines, O., Pierik, A.J., & Lill, R. (2011). Specialized function of yeast Isa1 and Isa2 in the maturation of mitochondrial [4Fe-4S] proteins. J. Biol. Chem. 286, 41205-41216.
40) Navarro-Sastre, A.*, Tort, F.*, Stehling, O*., Uzarska, M.A.*, Arranz, J.A., del Toro, M., Labayru, M.T., Landa, J., Font, A., Garcia-Villoria, J., Merinero, B., Ugarte, M., Gutierrez-Solana, L.G., Campistol, J., Garcia-Cazorla, A., Vaquerizo, J., Riudor, E., Briones, P., Elpeleg, O., Ribes, A., & Lill, R.# (2011). A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins. Am. J. Hum. Genet. 89, 656-667. * Joint first authors; # joint corresponding authors.
41) Netz, D.J., Stith, C.M., Stümpfig, M., Köpf, G., Vogel, D., Genau, H.M., Stodola, J.L., Lill, R. #, Burgers, P.M. #, Pierik, A.J. # (2012). Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes. Nat. Chem. Biol. 8, 125-132. # Joint corresponding authors. Commentary by Bailey, S. (2012) Nat. Chem. Biol. 8, 24-25.
42 ) Sheftel, A.D.*, Wilbrecht, C.*, Stehling, O., Niggemeyer, B., Elsässer, H.P., Mühlenhoff, U., & Lill, R. (2012). The human mitochondrial ISCA1, ISCA2, AND IBA57 proteins are required for [4Fe-4S] protein maturation. Mol. Biol. Cell 23, in press. * Joint first authors.
43) Muckenthaler, M.U. & Lill, R. (2012). Cellular Iron Physiology. In “Iron Physiology and Pathophysiology in Humans”. (Eds. Anderson, G. & McLaren, G.). Humana Press, Totowa, NJ, USA. In press.
