TY - JOUR A1 - Bühning, Martin A1 - Friemel, Martin A1 - Leimkühler, Silke T1 - Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil IscS and Human NFS1 JF - Biochemistry N2 - The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present. Y1 - 2017 U6 - https://doi.org/10.1021/acs.biochem.7b00627 SN - 0006-2960 VL - 56 SP - 4592 EP - 4605 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Friemel, Martin A1 - Marelja, Zvonimir A1 - Li, Kuanyu A1 - Leimkühler, Silke T1 - The N-Terminus of Iron-Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with L-Cysteine Desulfurase NFS1 JF - Biochemistry N2 - Assembly of iron sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by L-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondria) targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1. Y1 - 2017 U6 - https://doi.org/10.1021/acs.biochem.6b01239 SN - 0006-2960 VL - 56 SP - 1797 EP - 1808 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Lim, Sze Chern A1 - Friemel, Martin A1 - Marum, Justine E. A1 - Tucker, Elena J. A1 - Bruno, Damien L. A1 - Riley, Lisa G. A1 - Christodoulou, John A1 - Kirk, Edwin P. A1 - Boneh, Avihu A1 - DeGennaro, Christine M. A1 - Springer, Michael A1 - Mootha, Vamsi K. A1 - Rouault, Tracey A. A1 - Leimkühler, Silke A1 - Thorburn, David R. A1 - Compton, Alison G. T1 - Mutations in LYRM4, encoding ironsulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes JF - Human molecular genetics N2 - Ironsulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called ironsulfur or FeS proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203GT, p.R68L) was identified via massively parallel sequencing of 1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other FeS proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of FeS proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy. Y1 - 2013 U6 - https://doi.org/10.1093/hmg/ddt295 SN - 0964-6906 SN - 1460-2083 VL - 22 IS - 22 SP - 4460 EP - 4473 PB - Oxford Univ. Press CY - Oxford ER - TY - GEN A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The eukaryotic-specific Isd11 is a complex- orphan protein with ability to bind the prokaryotic IscS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 551 KW - sulfur cluster formation KW - Escherichia coli KW - cysteine desulfurase KW - interacting protein KW - bacterial frataxin KW - statistical-model KW - biogenesis KW - biosynthesis KW - NFS1 KW - deficiency Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-411906 SN - 1866-8372 IS - 551 ER - TY - JOUR A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS JF - PLoS one N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction. Y1 - 2016 U6 - https://doi.org/10.1371/journal.pone.0157895 SN - 1932-6203 VL - 11 SP - 383 EP - 395 PB - PLoS CY - San Fransisco ER -