Mitochondrial Iron-sulfur Cluster Assembly Machinery Research Articles

Protein-Interaction Affinity Gradient Drives [4Fe-4S] Cluster Insertion in Human Lipoyl Synthase.

Human lipoyl synthase (LIAS) is an enzyme containing two [4Fe–4S] clusters (named FeSRS and FeSaux) involved in the biosynthesis of the lipoyl cofactor. The mechanism by which a [4Fe–4S] cluster is inserted into LIAS has thus far remained elusive. Here we show that NFU1 and ISCA1 of the mitochondrial iron–sulfur cluster assembly machinery, via forming a heterodimeric complex, are the key factors for the insertion of a [4Fe–4S] cluster into the FeSRS site of LIAS. In this process, the crucial actor is the C-domain of NFU1, which, by exploiting a protein-interaction affinity gradient increasing from ISCA1 to LIAS, drives the cluster to its final destination.

A novel role of the mitochondrial iron-sulfur cluster assembly protein ISCU-1/ISCU in longevity and stress response.

As an ancient cellular co-factor ubiquitously present in all domains of life, nearly all iron-sulfur ([Fe-S]) clusters are assembled in the mitochondrion. Although multiple mitochondrion-derived signalings are known to be key players in longevity regulation, whether the mitochondrial [Fe-S] cluster assembly machinery modulates lifespan is previously unknown. Here, we find that ISCU-1, the C. elegans ortholog of the evolutionarily conserved iron-sulfur cluster (ISC) assembly machinery central protein ISCU, regulates longevity and stress response. Specifically, ISCU-1 accelerates aging in the intestine. Moreover, we identify the Nrf2 transcription factor SKN-1 and a nuclear hormone receptor NHR-49 as the downstream factors of ISCU-1. Lastly, a mitochondrial outer membrane protein phosphatase PGAM-5 appears to link ISCU-1 to SKN-1 and NHR-49 in lifespan regulation. Together, we have identified a novel function of mitochondrial ISC assembly machinery in longevity modulation and stress response.

A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis.

During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. Invivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, that is, a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.

Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery

In mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur cluster proteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 and IBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. We report here the use of an integrative approach, utilizing information from small-angle X-ray scattering (SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural model of the human mitochondrial [2Fe-2S]2+ ISCA2-IBA57 complex. In the applied experimental conditions, all the data converge to a structural organization of dimer of dimers for the [2Fe-2S]2+ ISCA2-IBA57 complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of the ISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified from the dimeric [2Fe-2S]2+ ISCA2-IBA57 structural model allowed us to define the molecular grounds of the pathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57 hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] protein biogenesis.

Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins.

Iron-sulfur (Fe/S) proteins are involved in numerous key biological functions such as respiration, metabolic processes, protein translation, DNA synthesis, and DNA repair. The simplest types of Fe/S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] forms that sometimes are present in multiple copies. De novo assembly of Fe/S cofactors and their insertion into apoproteins in living cells requires complex proteinaceous machineries that are frequently highly conserved. In eukaryotes such as yeast and mammals, the mitochondrial iron-sulfur cluster assembly machinery and the cytosolic iron-sulfur protein assembly system consist of more than 30 components that cooperate in the generation of some 50 cellular Fe/S proteins. Both the mechanistic dissection of the intracellular Fe/S protein assembly pathways and the identification and characterization of Fe/S proteins rely on tool boxes of in vitro and in vivo methods. These cell biological, biochemical, and biophysical techniques help to determine the extent, stability, and type of bound Fe/S cluster. They also serve to distinguish bona fide Fe/S proteins from other metal-binding proteins containing similar cofactor coordination motifs. Here, we present a collection of in vitro methods that have proven useful for basic biochemical and biophysical characterization of Fe/S proteins. First, we describe the chemical assembly of [2Fe-2S] or [4Fe-4S] clusters on purified apoproteins. Then, we summarize a reconstitution system reproducing the de novo synthesis of a [2Fe-2S] cluster in mitochondria. Finally, we explain the use of UV-vis, CD, electron paramagnetic resonance, and Mössbauer spectroscopy for the routine characterization of Fe/S proteins.

Formation of [4Fe-4S] clusters in the mitochondrial iron-sulfur cluster assembly machinery.

The generation of [4Fe-4S] clusters in mitochondria critically depends, in both yeast and human cells, on two A-type ISC proteins (in mammals named ISCA1 and ISCA2), which perform a nonredundant functional role forming in vivo a heterocomplex. The molecular function of ISCA1 and ISCA2 proteins, i.e., how these proteins help in generating [4Fe-4S] clusters, is still unknown. In this work we have structurally characterized the Fe/S cluster binding properties of human ISCA2 and investigated in vitro whether and how a [4Fe-4S] cluster is assembled when human ISCA1 and ISCA2 interact with the physiological [2Fe-2S](2+) cluster-donor human GRX5. We found that (i) ISCA2 binds either [2Fe-2S] or [4Fe-4S] cluster in a dimeric state, and (ii) two molecules of [2Fe-2S](2+) GRX5 donate their cluster to a heterodimeric ISCA1/ISCA2 complex. This complex acts as an "assembler" of [4Fe-4S] clusters; i.e., the two GRX5-donated [2Fe-2S](2+) clusters generate a [4Fe-4S](2+) cluster. The formation of the same [4Fe-4S](2+) cluster-bound heterodimeric species is also observed by having first one [2Fe-2S](2+) cluster transferred from GRX5 to each individual ISCA1 and ISCA2 proteins to form [2Fe-2S](2+) ISCA2 and [2Fe-2S](2+) ISCA1, and then mixing them together. These findings imply that such heterodimeric complex is the functional unit in mitochondria receiving [2Fe-2S] clusters from hGRX5 and assembling [4Fe-4S] clusters before their transfer to the final target apo proteins.

Iron-Induced Dissociation of the Aft1p Transcriptional Regulator from Target Gene Promoters Is an Initial Event in Iron-Dependent Gene Suppression

Aft1p is an iron-responsive transcriptional activator that plays a central role in the regulation of iron metabolism in Saccharomyces cerevisiae. Aft1p is regulated by accelerated nuclear export in the presence of iron, mediated by Msn5p. However, the transcriptional activity of Aft1p is suppressed under iron-replete conditions in the Δmsn5 strain, although Aft1p remains in the nucleus. Aft1p dissociates from its target promoters under iron-replete conditions due to an interaction between Aft1p and the monothiol glutaredoxin Grx3p or Grx4p (Grx3/4p). The binding of Grx3/4p to Aft1p is induced by iron repletion and requires binding of an iron-sulfur cluster to Grx3/4p. The mitochondrial transporter Atm1p, which has been implicated in the export of iron-sulfur clusters and related molecules, is required not only for iron binding to Grx3p but also for dissociation of Aft1p from its target promoters. These results suggest that iron binding to Grx3p (and presumably Grx4p) is a prerequisite for the suppression of Aft1p. Since Atm1p plays crucial roles in the delivery of iron-sulfur clusters from the mitochondria to the cytoplasm and nucleus, these results support the previous observations that the mitochondrial iron-sulfur cluster assembly machinery is involved in cellular iron sensing.