Split-Luciferase-Assay doctoralThesis ths PD Dr. Mühlenhoff Ulrich Mühlenhoff, Ulrich (PD Dr.) 2016-05-03 Analysis of post-translational modifications on Fe/S proteins and characterisation of protein-protein interactions between Fe/S assembly factors in mitochondria of S. cerevisiae mitochondria 2015-10-22 S. cerevisiae Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg Saccharomyces cerevisiae Protein-Fragment-Komplementationssystem In S. cerevisiae sind drei Proteinmaschinerien für die de novo Biogenese von Fe/S-Clustern und deren Inkorporation in Zielproteine verantwortlich: die mitochondriale ISC- (iron sulfur cluster) Assemblierungsmaschinerie, das ISC-Export-System sowie das CIA- (cytosolic iron-sulfur-protein assembly) System. Der Mechanismus der de novo Biogenese eines [2Fe-2S]-Clusters durch die Komponenten der Kern-ISC-Maschinerie ist bereits gut untersucht. Wenig ist hingegen über den Prozess der [2Fe-2S]- nach [4Fe-4S]-Cluster-Konversion bekannt, der durch die späten ISC-Komponenten katalysiert wird sowie über die Mechanismen, mit denen Fe/S-Zielproteine an die ISC-Maschinerie rekrutiert werden. Die vorliegende Dissertation beschäftigt sich mit diesen zwei Teilaspekten aus dem letzten Schritt der Fe/S-Cluster-Biogenese. Im ersten Teilprojekt dieser Arbeit wurde analysiert, in welcher Form Apo-Fe/S-Proteine an die ISC-Maschinerie herangeführt werden. Hierbei stand die Frage im Vordergrund, ob und wie die Zelle die freien Thiolgruppen von Fe/S-Cluster ligierenden Cysteinen in der Apo-Form vor oxidativen Modifikationen schützt. Mittels massenspektrometrischer Analyse wurde gezeigt, dass mitochondriale Fe/S-Proteine in Zellen mit Defekten in der ISC-Maschinerie mit mehreren Massenaddukten von +30 bis +32 Da modifiziert werden. Bei diesen Modifikationen handelt es sich wahrscheinlich um Schwefeladditionen an die Cysteinliganden der Apo-Fe/S-Proteine in Form von verbrückenden Polysulfiden. Diese Modifikationen treten ebenfalls in geringem Maße unter normalen Wachstumsbedingungen in Wildtypzellen auf und werden verstärkt unter Eisenmangelbedingungen beobachtet. Da die Cysteinaddukte reduktiv entfernt werden können, liegt der Schluss nahe, dass es sich bei der Modifikation der Cysteinliganden von Apo-Fe/S-Proteinen mit Per- oder Polysulfiden um einen physiologischen Schutzmechanismus vor oxidationsbedingter Degradation handelt. Im zweiten Teilprojekt wurde ein neues System zur systematischen Analyse von Protein-Protein-Interaktionen in Mitochondrien von S. cerevisiae entwickelt um Interaktionen der späten ISC-Komponenten Isa1, Isa2 und Iba57 zu charakterisieren. Die Daten dieses Renilla Split-Luciferase-Testsystems legen den Schluss nahe, dass Isa1, Isa2 und Iba57 einen trimeren Komplex ausbilden, dessen Assemblierung in vivo von einer funktionierenden ISC-Maschinerie abhängig ist. Isa1 und Isa2 interagieren weiterhin mit dem Monothiol Glutaredoxin Grx5, welches den in der frühen ISC-Maschinerie synthetisierten [2Fe-2S]-Cluster von dem Gerüstprotein Isu1 übernimmt und an [2Fe 2S]-Zielproteine weiterreicht. Die Interaktion mit Isa1 und Isa2 spricht dafür, dass Grx5 seinen [2Fe-2S]-Cluster ebenfalls an die späte ISC-Maschinerie zur Konversion in einen [4Fe-4S]-Cluster weiterreichen könnte. Zudem interagierte das mitochondriale Ferredoxin Yah1 mit den späten ISC-Komponenten. Dies könnte bedeuten, dass Yah1 Elektronen für einen reduktiven Reaktionsschritt während der Fe/S-Cluster-Konversion bereitstellt. Da das Renilla Split-Luciferase-Testsystem zu einer konzentrationsabhängigen Assemblierung der Teildomänen in Mitochondrien tendiert, sollten die hier beobachteten Interaktionen mit einer weiteren Methode verifiziert werden. 2015 https://archiv.ub.uni-marburg.de/diss/z2015/0574/cover.png opus:6412 posttranslationale Cysteinmodifikationen Klinische Zytobiologie und Zytopathologie urn:nbn:de:hebis:04-z2015-05748 In S. cerevisiae, three distinct protein machineries are responsible for the de novo biogenesis of Fe/S-clusters and their incorporation into target proteins: the mitochondrial ISC- (iron sulfur cluster) machinery, the ISC-export system and the CIA- (cytosolic iron-sulfur-protein assembly) system. The mechanism of the de novo biogenesis of a [2Fe-2S]-cluster by the core components of the ISC-machinery is well characterized on a functional level. On the contrary, little is known about the process of Fe/S-cluster conversion, catalyzed by the late acting ISC-components, or how Fe/S-target proteins are recruited to the ISC-machinery. This thesis focuses on the last step of Fe/S-cluster biogenesis; the incorporation of the Fe/S-cluster into target proteins. The goal of the first project was to analyze the status of the cysteine ligands of apo-Fe/S-proteins prior to the insertion of an Fe/S-cluster. Because the thiol-groups of these cysteines are exposed to the mitochondrial matrix, it seemed likely that the cell would manage to protect them against oxidative damage. Mass spectrometrical analysis of a mitochondrial Fe/S-protein revealed multiple increments in mass of +30 to +32 Da on Fe/S-proteins in cells with defects in the ISC-machinery. These modifications are most likely bridging polysulfides between the cysteine ligands of apo-Fe/S-proteins. Furthermore, these modifications were also found to a lesser degree in wildtype cells grown under standard conditions, and to a higher degree in cells which were cultured in iron poor medium. As the cysteine modifications could also be reductively cleaved, we suggest that the modification of the cysteine ligands of apo-Fe/S-proteins, with a per- or polysulfide, is actually a physiological protection mechanism against oxidation dependent protein degradation. In the second project, a protein-fragment complementation assay was established in mitochondria of S. cerevisiae in order to characterize interactions between the late acting ISC-components Isa1, Isa2 and Iba57. The data of this Renilla split-luciferase assay indicates a trimeric complex of these three proteins in vivo. The assembly of this complex seems to be dependent of a functional active ISC-machinery. Furthermore, Isa1 and Isa2 were found to interact with Grx5, the first ISC-component, which receives the de novo assembled [2Fe-2S]-cluster from Isu1 and transfers it to [2Fe-2S]-target proteins. The interaction with Isa1 and Isa2 suggests that Grx5 can also donate its Fe/S-cluster to the late acting ISC-components for conversion into a [4Fe-4S]-cluster. The Renilla Split-Luciferase assay revealed interactions between Yah1 and Isa1, Isa2 and Iba57. This data proposes that the process of Fe/S-cluster conversion depends on the supply with reducing equivalents. Because the Renilla Split-Luciferase assay displayed a concentration dependent assembly of the two Luciferase domains in mitochondria, the interactions described here have to be verified with an independent method. Analyse von posttranslationalen Modifikationen an Fe/S-Proteinen und Protein-Protein-Interaktionen zwischen Fe/S-Assemblierungsfaktoren in Mitochondrien von S. cerevisiae Rees DC (2002) Great metalloclusters in enzymology. Annual review of biochemistry 71: 221-246 Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24: 218-229 Murphy MP (2009) How mitochondria produce reactive oxygen species. The Biochemical journal 417: 1-13 Mansy SS, Xiong Y, Hemann C, Hille R, Sundaralingam M, Cowan JA (2002) Crystal structure and stability studies of C77S HiPIP: a serine ligated [4Fe-4S] cluster. Biochemistry 41: 1195-1201 Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic acids research 32: e115 Pierik AJ, Netz DJ, Lill R (2009) Analysis of iron-sulfur protein maturation in eukaryotes. Nature protocols 4: 753- 766 Wiedemann N, Urzica E, Guiard B, Muller H, Lohaus C, Meyer HE, Ryan MT, Meisinger C, Muhlenhoff U, Lill R, Pfanner N (2006) Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins. The EMBO journal 25: 184-195 Sabharwal SS, Schumacker PT (2014) Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? Nature reviews. Cancer 14: 709-721 Rinalducci S, Murgiano L, Zolla L (2008) Redox proteomics: basic principles and future perspectives for the detection of protein oxidation in plants. Journal of experimental botany 59: 3781-3801 Modis K, Bos EM, Calzia E, van Goor H, Coletta C, Papapetropoulos A, Hellmich MR, Radermacher P, Bouillaud F, Szabo C (2014) Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part II. Pathophysiological and therapeutic aspects. British journal of pharmacology 171: 2123-2146 Pujol-Carrion N, Belli G, Herrero E, Nogues A, de la Torre-Ruiz MA (2006) Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae. Journal of cell science 119: 4554-4564 Py B, Gerez C, Angelini S, Planel R, Vinella D, Loiseau L, Talla E, Brochier -Armanet C, Garcia Serres R, Latour JM, Ollagnier-de Choudens S, Fontecave M, Barras F (2012) Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier. Molecular microbiology 86: 155-171 Solmaz SR, Hunte C (2008) Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer. The Journal of biological chemistry 283: 17542-17549 Srinivasan V, Netz DJ, Webert H, Mascarenhas J, Pierik AJ, 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 Willems P, Wanschers BF, Esseling J, Szklarczyk R, Kudla U, Duarte I, Forkink M, Nooteboom M, Swarts H, Gloerich J, Nijtmans L, Koopman W, Huynen MA (2013) BOLA1 is an aerobic protein that prevents mitochondrial morphology changes induced by glutathione depletion. Antioxidants + redox signaling 18: 129- 138 Parent A, Elduque X, Cornu D, Belot L, Le Caer JP, Grandas A, Toledano MB, D'Autreaux B (2015) Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols. Nature communications 6: 5686 Netz DJ, Pierik AJ, Stumpfig M, Bill E, Sharma AK, Pallesen LJ, Walden WE, Lill R (2012a) A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1 -Nbp35 complex as a scaffold in iron-sulfur protein maturation. The Journal of biological chemistry 287: 12365-12378 Ohashi K, Mizuno K (2014) A novel pair of split venus fragments to detect protein -protein interactions by in vitro and in vivo bimolecular fluorescence complementation assays. Methods in molecular biology 1174: 247- 262 Paul VD, Lill R (2015) Biogenesis of cytosolic and nuclear iron-sulfur proteins and their role in genome stability. Biochimica et biophysica acta 1853: 1528-1539 Mishanina TV, Libiad M, Banerjee R (2015) Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nature chemical biology 11: 457-464 Py B, Barras F (2010) Building Fe-S proteins: bacterial strategies. Nature reviews Microbiology 8: 436-446 Hausmann A, Samans B, Lill R, Muhl enhoff U (2008) Cellular and Mitochondrial Remodeling upon Defects in Iron-Sulfur Protein Biogenesis. The Journal of biological chemistry 283: 8318-8330 Muhlenhoff U, Gerber J, Richhardt N, Lill R (2003) Components involved in assembly and dislocation of iron - sulfur clusters on the scaffold protein Is u1p. The EMBO journal 22: 4815-4825 Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MB, Bachovchin DA, Mowen K, Baker D, Cravatt BF Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468: 790-795 Srinivasan V, Pierik AJ, Lill R (2014) Crystal structures of nucleotide-free and glutathione-bound mitochondrial ABC transporter Atm1. Science 343: 1137-1140 Moraes CT, Diaz F, Barrientos A (2004) Defects in the biosynthesis of mitochondri al heme c and heme a in yeast and mammals. Biochimica et biophysica acta 1659: 153-159 Magliery TJ, Wilson CG, Pan W, Mishler D, Ghosh I, Hamilton AD, Regan L (2005) Detecting protein -protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. Journal of the American Chemical Society 127: 146-157 Makmura L, Hamann M, Areopagita A, Furuta S, Munoz A, Momand J (2001) Development of a sensitive assay to detect reversibly oxidized protein cystei ne sulfhydryl groups. Antioxidants + redox signaling 3: 1105-1118 Sundquist T. (2011) Dual -Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays? Vilella F, Alves R, Rodriguez-Manzaneque MT, Belli G, Swaminathan S, Sunnerhagen P, Herrero E (2004) Evolution and cellular function of monothiol glutaredoxins: involvement in iron -sulphur cluster assembly. Comparative and functional genomics 5: 328-341 Robinson KM, Lemire BD (1995) Flavinylation of succinate: ubiquinone oxidored uctase from Saccharomyces cerevisiae. Methods Enzymol 260: 34-51 V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti -Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415: 141-147 Webert H, Freibert SA, Gallo A, Heidenreich T, Linne U, Amlacher S, Hurt E, Muhlenhoff U, Banci L, Lill R (2014) Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin. Nature communications 5: 5013 Sherman F (2002) Getting started with yeast. Methods in enzymology 350: 3-41 Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O'Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425: 737-741 Qi W, Li J, Chain CY, Pasquevich GA, Pasquevich AF, Cowan JA (2012) Glutathione complexed Fe-S centers. Journal of the American Chemical Society 134: 10745-10748 NEB. Guidelines for PCR Optimization with Phusion High-Fidelity DNA Polymerase. New England BioLabs, Ipswich, MA, USA. Stehling O, Mascarenhas J, Vashisht AA, Sheftel AD, Niggemeyer B, Rosser R, Pierik AJ, Wohlschlegel JA, Lill R (2013) Human CIA2A-FAM96A and CIA2B-FAM96B integrate iron homeostasis and maturation of different subsets of cytosolic-nuclear iron-sulfur proteins. Cell metabolism 18: 187-198 Tsai CL, Barondeau DP (2010) Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex. Biochemistry 49: 9132-9139 Thorne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and o ther bioluminescent reporters used in chemical biology. Chemistry + biology 17: 646-657 Pallesen LJ, Solodovnikova N, Sharma AK, Walden WE (2013) Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold. The Journal of biological chemistry 288: 23358-23367 Orij R, Postmus J, Ter Beek A, Brul S, Smits GJ (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155: 268-278 Pierrel F, Hamelin O, Douki T, Kieffer-Jaquinod S, Muhlenhoff U, Ozeir M, Lill R, Fontecave M (2010) Involvement of mitochondrial ferredoxin and para -aminobenzoic acid in yeast coenzyme Q biosynthesis. Chemistry + biology 17: 449-459 Xu XM, Moller SG (2011) Iron-sulfur clusters: biogenesis, molecular mechanisms, and their functional significance. Antioxidants + redox signaling 15: 271-307 Roche B, Aussel L, Ezraty B, Mandin P, Py B, Barras F (2013) Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity. Biochimica et biophysica acta 1827: 455-469 Sipos K, Lange H, Fekete Z, Ullmann P, Lill R, Kispal G (2002) Maturation of cytosolic iron -sulfur proteins requires glutathione. The Journal of biological chemistry 277: 26944-26949 Molik S, Lill R, Muhlenhoff U (2007) Methods for studying iron metabolism in yeast mitochondria. Methods in cell biology 80: 261-280 Pelzer W, Muhlenhoff U, Diekert K, Siegmund K, Kispal G, Lill R (2000) Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins. FEBS letters 476: 134-139 Murphy MP (2012) Mitochondrial thiols in antioxidant protection and redox signaling: distinct roles for glutathionylation and other thiol modifications. Antioxidants + redox signaling 16: 476-495 Paulmurugan R, Gambhir SS (2003) Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Analytical chemistry 75: 1584-1589 Tonduti D, Dorboz I, Imbard A, Slama A, Boutron A, Pichard S, Elmaleh M, Vallee L, Benoist J, Ogier H, Boespflug-Tanguy O (2015) New spastic paraplegia phenotype assoc iated to mutation of NFU1. Orphanet journal of rare diseases 10: 13 Manicki M, Majewska J, Ciesielski S, Schilke B, Blenska A, Kominek J, Marszalek J, Craig EA, Dutkiewicz R (2014) Overlapping binding sites of the frataxin homologue assembly factor and the heat shock protein 70 transfer factor on the Isu iron-sulfur cluster scaffold protein. The Journal of biological chemistry 289: 30268-30278 Greiner R, Palinkas Z, Basell K, Becher D, Antelmann H, Nagy P, Dick TP (2013) Polysulfides link H2S to protein thiol oxidation. Antioxidants + redox signaling 19: 1749-1765 Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods in molecular biology 112: 531-552 Matthews JC, Hori K, Cormier MJ (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry 16: 85-91 Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, Suematsu M, Motohashi H, Fujii S, Matsunaga T, Yamamoto M, Ono K, Devari e-Baez NO, Xian M, Fukuto JM, Akaike T (2014) Reactive cysteine persulfides and S- polythiolation regulate oxidative stress and redox signaling. Proceedings of the National Academy of Sciences of the United States of America 111: 7606-7611 Szabo C, Ransy C, Modis K, Andriamihaja M, Murghes B, Coletta C, Olah G, Yanagi K, Bouillaud F (2014) Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. British journal of pharmacology 171: 2099-2122 Promega. (2011) Renilla-Glo™ Luciferase Assay System -INSTRUCTIONS FOR USE OF PRODUCTS E2710, E2720, AND E2750. Promega Corporation, Fitchburg, WI, USA. Salinovich O, Montelaro RC (1986) Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analytical biochemistry 156: 341-347 West RW, Jr., Yocum RR, Ptashne M (1984) Saccharomyces cerevisiae GAL1 -GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Molecular and cellular biology 4: 2467-2478 Paul VD, Lill R (2014) SnapShot: eukaryotic Fe-S protein biogenesis. Cell metabolism 20: 384-384 e381 Syrovy I, Hodny Z (1991) Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. Journal of chromatography 569: 175-196 Sau AK, Chen CA, Cowan JA, Mazumdar S, Mitra S (2001) Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo-and apo-forms. Biophysical journal 81: 2320-2330 Roret T, Tsan P, Couturier J, Zhang B, Johnson MK, Rouhier N, Didierjean C (2014) Structural and spectroscopic insights into BolA-glutaredoxin complexes. The Journal of biological chemistry 289: 24588-24598 Walden WE, Selezneva AI, Dupuy J, Volbeda A, Fontecilla -Camps JC, Theil EC, Volz K (2006) Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA. Science 314: 1903-1908 Webert H (2011) Strukturelle und funktionelle Charakterisierung von Komponenten der eukaryotischen Eisen- Schwefel-Cluster-Biogenese-Maschinerie Fachbereich Chemie, Philipps -Universität Marburg, Marburg Smith AD, Agar JN, Johnson KA, Frazzon J, Amster IJ, Dean DR, Johnson MK (2001) Sulfur transfer from IscS to IscU: the first step in iron-sulfur cluster biosynthesis. Journal of the American Chemical Society 123: 11103- 11104 Strain J, Lorenz CR, Bode J, Garland S, Smolen GA, Ta DT, Vickery LE, Culotta VC (1998) Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. Identification of proteins predicted to mediate iron-sulfur cluster assembly. The Journal of biological chemistry 273: 31138-31144 Hudson EN, Weber G (1973) Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry 12: 4154-4161 Netz DJ, Stumpfig M, Dore C, Muhlenhoff U, Pierik AJ, Lill R (2010) Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis. Nature chemical biology 6: 758-765 Netz DJ, Pierik AJ, Stumpfig M, Muhlenhoff U, Lill R (2007) The Cfd1 -Nbp35 complex acts as a scaffold for iron- sulfur protein assembly in the yeast cytosol. Nature chemical biology 3: 278-286 Hausmann A, Aguilar Netz DJ, Balk J, Pierik AJ, Muhlenhoff U, Lill R (2005) Th e eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery. Proceedings of the National Academy of Sciences of the United States of America 102: 3266-3271 Sheftel AD, Wilbrecht C, Stehling O, Niggemeyer B, Elsasser HP, Muhlenhoff U, Lill R (2012) The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation. Molecular biology of the cell 23: 1157-1166 Pugh RA, Honda M, Leesley H, Thomas A, Li n Y, Nilges MJ, Cann IK, Spies M (2008) The iron-containing domain is essential in Rad3 helicases for coupling of ATP hydrolysis to DNA translocation and for targeting the helicase to the single-stranded DNA-double-stranded DNA junction. The Journal of biological chemistry 283: 1732-1743 Muhlenhoff U, Gerl MJ, Flauger B, Pirner HM, Balser S, Richhardt N, Lill R, Stolz J (2007) The ISC [corrected] 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. Eukaryotic cell 6: 495-504 Pondarre C, Antiochos BB, Campagna DR, Clarke SL, Greer EL, Deck KM, McDonald A, Han AP, Medlock A, Kutok JL, Anderson SA, Eisenstein RS, Fleming MD (2006) The mitochondrial ATP -binding cassette transporter Abcb7 is essential in mice and participates in cytosolic iron-sulfur cluster biogenesis. Human molecular genetics 15: 953-964 Mueller EG (2006) Trafficking in persulfides: delivering sulfur in biosynthetic pathways. Nature chemical biology 2: 185-194 Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in enzymology 350: 87-96 Hjorth E, Hadfi K, Zauner S, Maier UG (2005) Unique genetic compartmentalization of the SUF system in cryptophytes and characterization of a SufD mutant in Arabidopsis thaliana. FEBS Letters 579: 1129-1135 Michnick SW, Ear PH, Manderson EN, Remy I, Stefan E (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nature reviews. Drug discovery 6: 569-582 Haack TB, Rolinski B, Haberberger B, Zimmermann F, Schum J, Strecker V, Graf E, Athing U, Hoppen T, Wittig I, Sperl W, Freisinger P, Mayr JA, Strom TM, Meitinger T, Prokisch H (2013) Homozygous missense mutation in BOLA3 causes multiple mitochondrial dysfunctions syndrome in two siblings. Journal of inherited metabolic disease 36: 55-62 Prischi F, Konarev PV, Iannuzzi C, Pastore C, Adinolfi S, Martin SR, Svergun DI, Pastore A (2010) Structural bases for the interaction of frataxin with the central components of iron-sulphur cluster assembly. Nature communications 1: 95 Hida N, Awais M, Takeuchi M, Ueno N, Tashiro M, Takagi C, Singh T, Hayashi M, Ohmiya Y, Ozawa T (2009 ) High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS One 4: e5868 Qi W, Li J, Cowan JA (2014) A structural model for glutathione-complexed iron-sulfur cluster as a substrate for ABCB7-type transporters. Chemical communications 50: 3795-3798 Mortimer RK, Johnston JR (1986) Genealogy of principal strains of the yeast genetic stock center. Genetics 113: 35-43 Sambrook J, Russel DW (2001) Molecular Cloning -A laboratory manual, 3rd edition, 3rd edn. ColdSpring Harbour, USA: CSH Laboratory Press. Nogi T, Fathir I, Kobayashi M, Nozawa T, Miki K (2000) Crystal structures of photosynthetic reaction center and high-potential iron-sulfur protein from Thermochromatium tepidum: thermostability and electron transfer. Proceedings of the National Academy of Sciences of the United States of America 97: 13561-13566 Schilke B, Voisine C, Beinert H, Craig E (1999) Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America 96: 10206-10211 Gelling C, Dawes IW, Richhardt N, Li ll R, Muhlenhoff U (2008) Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes. Molecular and cellular biology 28: 1851-1861 Woo J, Howell MH, von Arnim AG (2008) Structure-function studies on the active site of the coelenterazine- dependent luciferase from Renilla. Protein science : a publication of the Protein Society 17: 725-735 Wallander ML, Leibold EA, Eisenstein RS (2006) Molecular control of vertebrate iron homeostasis by iron regulatory proteins. Biochimica et biophysica acta 1763: 668-689 Sheftel AD, Stehling O, Pierik AJ, Netz DJ, Kerscher S, Elsasser HP, Wittig I, Balk J, Brandt U, Lill R (2009) Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I. Molecular and cellular biology 29: 6059- 6073 Requejo R, Hurd TR, Costa NJ, Murphy MP (2010) Cysteine residues exposed on protein surfaces are the dominant intramitochondrial thiol and may protect against oxidative damage. The FEBS journal 277: 1465-1480 Huang H, Choi SY, Frohman MA (2010) A quantitative assay for mitochondrial fusion using Renilla luciferase complementation. Mitochondrion 10: 559-566 Sharma AK, Pallesen LJ, Spang RJ, Walden WE (2010) Cytosolic iron-sulfur cluster assembly (CIA) system: factors, mechanism, and relevance to cellular iron regulation. The Journal of biological chemistry 285: 26745- 26751 Mumberg D, Muller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic acids research 22: 5767-5768 Navarro-Sastre A, Tort F, Stehling O, Uzarska MA, Arranz JA, Del Toro M, Labayru MT, Landa J, Font A, Garcia - Villoria J, Merinero B, Ugarte M, Gutierrez-Solana LG, 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. American journal of human genetics 89: 656-667 Netz DJ, Stith CM, Stumpfig M, Kopf G, Vogel D, Genau HM, Stodola JL, Lill R, Burgers PM, Pierik AJ (2012b) Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes. Nature chemical biology 8: 125-132 Roux KJ, Kim DI, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of cell biology 196: 801-810 Muhlenhoff U, Richter N, Pines O, Pierik AJ, Lill R (2011) Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins. The Journal of biological chemistry 286: 41205-41216 Stehling O, Vashisht AA, Mascarenhas J, Jonsson ZO, Sharma T, Netz DJ, Pierik AJ, Wohlschlegel JA, Lill R (2012) MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity. Science 337: 195- 199 Hebert A, Forquin-Gomez MP, Roux A, Aubert J, Junot C, Heilier JF, Landaud S, Bonnarme P, Beckerich JM (2013) New insights into sulfur metabolism in yeasts as revealed by studies of Yarrowia lipolytica. Applied and environmental microbiology 79: 1200-1211 Uzarska MA, Dutkiewicz R, Freibert SA, Lill R, Muhlenhoff U (2013) The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation. Molecular biology of the cell 24: 1830- 1841 Haunhorst P, Hanschmann EM, Brautigam L, Stehling O, Hoffmann B, Muhlenhoff U, Lil l R, Berndt C, Lillig CH (2013) Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation. Molecular biology of the cell 24: 1895-1903 Lorenz WW, McCann RO, Longiaru M, Cormier MJ (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proceedings of the National Academy of Sciences of the United States of America 88: 4438-4442 Rodriguez-Manzaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E (1999) Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Molecular and cellular biology 19: 8180-8190 Quinlan CL, Perevoshchikova IV, Hey-Mogensen M, Orr AL, Brand MD (2013) Sites of reactive oxygen species generation by mitochondria oxidizing different substrates. Redox biology 1: 304-312 Pilon M, Abdel-Ghany SE, Van Hoewyk D, Ye H, Pilon-Smits EA (2006) Biogenesis of iron-sulfur cluster proteins in plastids. Genetic engineering 27: 101-117 Muhlenhoff U, Balk J, Richhardt N, Kaiser JT, Sipos K, Kispal G, Lill R (2004) Functional characterization of the eukaryotic cysteine desulfurase Nfs1p from Saccharomyces cerevisiae. The Journal of biological chemistry 279: 36906-36915 Schaedler TA, Thornton JD, Kruse I, Schwarzlander M, Meyer AJ, van Veen HW, Balk J (2014) A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly. The Journal of biological chemistry 289: 23264-23274 https://doi.org/10.17192/z2015.0574 Philipps-Universität Marburg protein fragment complementation assay German Fe/S-Proteine post-translational cysteine modifications Christ, Stefan Christ Stefan 128 application/pdf monograph Medizin Natural sciences + mathematics Naturwissenschaften Mitochondrium Fe/S-proteins split luciferase assay