Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/NFS1/ISD11 Iron-Sulfur Assembly Complex

et al. (2011) Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/NFS1/ISD11 Iron-Sulfur Assembly Complex. PLoS ONE 6(1): e16199. doi:10.1371/journal.pone.0016199 Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/ NFS1/ISD11 Iron-Sulfur Assembly Complex Ste phane Schmucker 0 Alain Martelli 0 Florent Colin 0 Adeline Page 0 Marie 0 Wattenhofer-Donze 0 Laurence Reutenauer 0 He le` ne Puccio 0 Francesc Palau, Instituto de Ciencia de Materiales de Madrid - Instituto de Biomedicina de Valencia, Spain 0 1 Department of Translational Medicine and Neurogenetics, Institut de Ge ne tique et de Biologie Mole culaire et Cellulaire (IGBMC) , Illkirch, France, 2 Inserm U596, Illkirch, France, 3 CNRS UMR7104, Illkirch , France , 4 Universite de Strasbourg, Strasbourg, France, 5 Chaire de Ge ne tique Humaine, Colle`ge de France , Illkirch , France Background: Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis. Methodology/Principal Findings: We present data providing new insights into the interactions of mammalian frataxin with the Fe-S assembly complex by combining in vitro and in vivo approaches. Through immunoprecipitation experiments, we show that the main endogenous interactors of a recombinant mature human frataxin are ISCU, NFS1 and ISD11, the components of the core Fe-S assembly complex. Furthemore, using a heterologous expression system, we demonstrate that mammalian frataxin interacts with the preformed core complex, rather than with the individual components. The quaternary complex can be isolated in a stable form and has a molecular mass of <190 kDa. Finally, we demonstrate that the mature human FXN81-210 form of frataxin is the essential functional form in vivo. Conclusions/Significance: Our results suggest that the interaction of frataxin with the core ISCU/NFS1/ISD11 complex most likely defines the essential function of frataxin. Our results provide new elements important for further understanding the early steps of de novo Fe-S cluster biosynthesis. - Funding: This work was supported by the French National Agency for Research (ANR-05-MRAR-013-01) and the EC under the European Research Council (ERC) grant 206634/ISCATAXIA and under the FP7 grant 242193/EFACTS to H.P. S.S. was supported by the French Ministry for Research (MRT award), A.M. by the American Friedreich Ataxia Research Alliance, and M.W.D. by the Association Francaise pour lAtaxie de Friedreich. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Human frataxin is the protein deficient in Friedreich ataxia (FRDA), a devastating autosomal recessive neurodegenerative disease associated with hypertophic cardiomyopthy, affecting 1/ 40,000 in the caucasian population [1,2]. Frataxin is a highly conserved mitochondrial protein from bacteria to humans [2,3]. Genetic and biochemical studies support a role of frataxin as a multifunctional protein in different iron-dependent mitochondrial pathways [2,3], through its ability to bind iron in vitro [4,5] and to deliver iron to different acceptors [6,7]. Moreover, binding of iron can trigger frataxin oligomerization in vitro, a process that was proposed to scavenge toxic iron in a bioavailable form and to be essential for frataxin function [8]. Both monomeric and oligomeric forms of frataxin were shown to interact with various potential iron acceptors. Frataxin was shown to interact in vitro with ferrochelatase and to provide the iron that is needed in the last step of heme biosynthesis [7,9]. Frataxin was also proposed to interact with mitochondrial aconitase, a Fe-S-containing protein, providing protection against the disassembly of the Fe-S cluster by facilitating iron transfer to aconitase [10]. Similarly, and more extensively, both monomeric and oligomeric forms of frataxin were proposed to be the iron donor protein for de novo Fe-S cluster biosynthesis [6,1116]. Fe-S clusters are critical prosthetic groups present in proteins involved in essential cellular processes ranging from nuclear genome stability, protein translation to mitochondrial metabolism [17]. Within the past decade, the biogenesis of Fe-S proteins has been extensively studied in bacteria and yeast demonstrating that it is a complex process involving multiple highly conserved components [17,18]. De novo Fe-S cluster assembly, a mitochondrial process in eukaryotes, relies on the assembly of a Fe-S cluster on a scaffold protein (IscU (bacteria), Isu1 (yeast), ISCU (mammalian)) from inorganic iron and sulfur, followed by the transfer of the scaffoldbound Fe-S cluster to the target apoproteins. Both the synthesis and the final transfer to apoproteins require the help of additional proteins. The sulfur is provided through a persulfide intermediate by a pyridoxal phosphate-dependent cysteine desulfurase, IscS in bacteria and the Nfs1/Isd11 complex in eukaryotes, that interacts with the scaffold protein to form a complex in which Fe-S cluster biosynthesis was proposed to occur in vivo [19]. The exact function of the eukaryotic protein Isd11 is unknown, but it has been proposed to stabilize Nfs1 through a direct interaction [20,21]. The bacterial, yeast and human frataxins (CyaY, Yfh1 and FXN, respectively) were shown to interact with multiple core components of the biosynthesis machinery [1116]. Furthermore, in vitro, iron loaded human frataxin was shown to deliver iron to ISCU [6]. However, how frataxin interacts with the Fe-S cluster biosynthesis components remains unclear as direct one-to-one interactions with each component were reported (IscS [12,22], IscU/Isu1 [6,11,16] or ISD11/Isd11 [14,15]). Finally, the iron-donor function of frataxin has recently been challenged as in vitro kinetic studies of Fe-S cluster biosynthesis using the bacterial components revealed that CyaY behaves as an iron-dependent inhibitor of Fe-S cluster assembly through a specific interaction with IscS [22]. Additional experiments are thus required to clarify both the function(s) and the molecular network(s) of the frataxin protein. To obtain a comprehensive insight into the multiple functions of frataxin, we decided to identify and characterize the interactions of human frataxin. In the present work, we show that endogenous mitochondrial ISCU, NFS1, and ISD11 are the main interactors of frataxin. Using complementary in vitro and in vivo experiments, we infer that the essential function of mammalian frataxin consists in interacting with the preformed ISCU/NFS1/ISD11 complex, forming a <190 kDa quate (...truncated)