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

Abstract

BackgroundFrataxin, 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 FindingsWe 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/SignificanceOur 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.

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

“…Only recently it was demonstrated that FXN associates with the early components of the mitochondrial Fe-S cluster biogenesis pathway, NFS1 and ISCU 2,23 . It was then reported that in the presence of ISCU, mammalian FXN stimulates sulfide production by NFS1 and this was correlated with stimulation of Fe-S cluster assembly 11,14 .…”

“…The ISC machinery comprises ISCU, a scaffold protein on which Fe-S clusters are assembled, NFS1, a pyridoxal phosphatedependent cysteine desulfurase, ISD11 and FXN [20][21][22][23] . The eukaryotic ISC machinery has evolved from bacteria and thus some of the primary steps of Fe-S cluster assembly have been conserved 16,24,25 .…”

“…1a) by the NFS1-ISD11 complex (NI). The NFS1-ISD11 complex (NI), ISCU (U) and FXN (F) have been shown to form homodimeric ternary and quaternary complexes with NI:U and NI:U:F stoichiometries of 1:1 and 1:1:1 for each subunit, respectively 11,14,23 , which will be denoted NIU and NIUF thereafter. We confirmed here by size exclusion chromatography that when incubated together, these proteins form the NIU and NIUF complex and that FXN does no interact with the NI complex in the absence of ISCU ( Supplementary Fig.…”

Missense Mutations Linked to Friedreich Ataxia Have Different but Synergistic Effects on Mitochondrial Frataxin Isoforms

“…17 and 18). Whereas oligomeric FXN forms stable complexes with NFS1-ISD11 in the absence or presence of ISCU (15), monomeric FXN 81-210 only binds to a preformed NFS1-ISD11-ISCU complex (15,16,19). Both isoforms are normally present in cultured cells and tissues and are thought to ensure incremental rates of iron-sulfur cluster synthesis depending on iron availability and metabolic requirements (8).…”

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold

“…Yfh1, the yeast frataxin homolog, has been proposed to serve as an iron donor and/or a regulator of Nfs1 function (9-11). Isu, Nfs1, and Yfh1 can bind with one another to form a so-called "Fe-S cluster assembly" complex (11)(12)(13). Transfer of the assembled clusters to recipient proteins requires a specialized Hsp70 chaperone system composed of the Hsp70 Ssq1 and the J-protein cochaperone Jac1, as well as the nucleotide release factor Mge1 (14,15).…”