Binding of Yeast Frataxin to the Scaffold for Fe-S Cluster Biogenesis, Isu

Abstract

Friedreich ataxia is caused by reduced activity of frataxin, a conserved iron-binding protein of the mitochondrial matrix, thought to supply iron for formation of Fe-S clusters on the scaffold protein Isu. Frataxin binds Isu in an iron-dependent manner in vitro. However, the biological relevance of this interaction and whether in vivo the interaction between frataxin and Isu is mediated by adaptor proteins is a matter of debate. Here, we report that alterations of conserved, surface-exposed residues of yeast frataxin, which have deleterious effects on cell growth, impair Fe-S cluster biogenesis and interaction with Isu while altering neither iron binding nor oligomerization. Our results support the idea that the surface of the ␤-sheet, adjacent to the acidic, iron binding ridge, is important for interaction of Yfh1 with the Fe-S cluster scaffold and point to a critical role for frataxin in Fe-S cluster biogenesis.Friedreich ataxia (FRDA), 2 a progressive neurodegenerative disease, is caused by a decrease in the level, and in some cases activity, of frataxin, a highly conserved iron-binding protein of the mitochondrial matrix (1-3). The effects of deficiency of frataxin in human cells and Yfh1 in yeast are strikingly similar: decrease in the activity of Fe-S-containing enzymes and increase in intramitochondrial iron levels (4, 5). Not surprisingly considering their 65% amino acid identity, the tertiary structures of frataxin and Yfh1 are very similar, a ␤-sheet packed against two ␣-helices (6 -8). The larger helix and part of the adjacent ␤-strand form an acidic ridge that binds iron. While a Yfh1 monomer can bind 2 ferrous irons (8, 9), it also undergoes iron-dependent oligomerization to form trimers and ultimately oligomers having up to 48 subunits that bind Ͼ2000 iron atoms (10, 11). Alterations in the acidic residues of the ridge can affect oligomerization and thus robust iron binding, alleviating any ability to serve an iron storage function (12, 13).Evidence indicates that Yfh1/frataxin plays an important role in the formation of Fe-S clusters (5, 14) on the highly conserved scaffold Isu prior to transfer to recipient apoproteins (15). Its proposed role as an iron donor is supported by its high affinity, iron-dependent interaction with Isu and its ability to donate iron for cluster formation on Isu in vitro (16). In addition, evidence strongly indicates that Nfs1, an essential cysteine desulfurase, which is required for Fe-S cluster formation on Isu in vivo, functions as the sulfur donor (17, 18). Another essential protein, Isd11, is also required for in vivo Fe-S cluster formation (15,19). It forms a stable complex with Nfs1 and is required for Nfs1 to be stable in vivo.The manner in which the activities of these proteins, including their physical interactions, are coordinated to allow cluster formation on Isu in vivo is a matter of debate. Most relevant to this report, it has been recently argued that the relevant in vivo interaction between frataxin homologs and scaffolds is indirect, despite the fact th...

Frataxin Depletion in Yeast Triggers Up-regulation of Iron Transport Systems before Affecting Iron-Sulfur Enzyme Activities

“…The second one suggests that, rather than acting as the iron donor for ironsulfur biogenesis, frataxin senses mitochondrial iron content and regulates the rate of iron-sulfur biogenesis (53). This second hypothesis would explain the interactions observed between frataxin and members of the iron-sulfur biosynthetic machinery (11)(12)(13), as well as its iron binding properties (54). Of course, one of the early consequences of losing either an iron storage protein or an iron-sensing protein could be the deregulation of iron metabolism, as observed in the present study.…”

“…Many studies have provided evidence that frataxins act as iron donors in iron-sulfur maturation. Most of these studies are based on the finding of direct interactions between frataxins and ISC system proteins such as Isu1 (11)(12)(13). However, other findings contradict the hypothesis that frataxin plays a primary role in iron-sulfur biogenesis.…”

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein, Display Constitutive Activation of the Iron Starvation Response

“…S. cerevisiae YFH1, the budding yeast frataxin homolog, was earlier reported to directly participate in ISC biogenesis, because aconitase activity was reduced in cells lacking the protein, and this inactivation seemed to precede iron accumulation (9,47). In particular, yeast frataxin has been suggested to participate in ISC maturation as an iron donor, based on its reported interaction with Isu1 (19,48). However, it has also been demonstrated that restricting oxidative damage, either by decreasing ROS production (49) or by diminishing available iron (50), prevents aconitase inactivation.…”

Assembly of the Iron-binding Protein Frataxin in Saccharomyces cerevisiae Responds to Dynamic Changes in Mitochondrial Iron Influx and Stress Level

“…Unlike the yeast or E. coli orthologues, human frataxin assembles at physiological iron concentrations during expression in E. coli or in human heart but not in vitro (40,41), indicating that different mechanisms govern frataxin assembly in different species. In the monocellular eukaryote, Saccharomyces cerevisiae, frataxin most likely carries out iron delivery and detoxification concurrently, and indeed these functions are interdependent and difficult to separate (34,37,42). We therefore used S. cerevisiae as a model to begin to elucidate regulation of frataxin assembly in vivo.…”

“…However, little is still known about this process. Assembly of the yeast frataxin homologue (Yfh1) in vitro requires the presence of Fe 2ϩ and proceeds in an iron concentration-dependent manner, according to the progression ␣ 3 ␣ 3 3 ␣ 6 3 ␣ 12 3 ␣ 24 3 ␣ 48 (36,37). Although one group reported that Yfh1 oligomerization is dispensable in vivo (38), we later showed that mutations affecting Yfh1 assembly are asymptomatic in unstressed cells but become harmful during stress exposure and cause synthetic lethality in cells lacking copper-zinc superoxide dismutase (34).…”