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

Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical iron-requiring processes under changing environmental conditions.     

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.     

Inhibition of Fe-S cluster biosynthesis decreases mitochondrial iron export: evidence that Yfh1p affects Fe-S cluster synthesis

Decreased expression of Yfh1p in the budding yeast, Saccharomyces cerevisiae, and the orthologous human gene frataxin results in respiratory deficiency and mitochondrial iron accumulation. The absence of Yfh1p decreases mitochondrial iron export. We demonstrate that decreased expression of Nfs1p, the yeast cysteine desulfurase that plays a central role in Fe-S cluster synthesis, also results in mitochondrial iron accumulation due to decreased export of mitochondrial iron. In the absence of Yfh1p, activity of Fe-S-containing enzymes (aconitase, succinate dehydrogenase) is decreased, whereas the activity of a non-Fe-S-containing enzyme (malate dehydrogenase) is unaffected. Aconitase protein was abundant even though the activity of aconitase was decreased in both aerobic and anaerobic conditions. These results demonstrate a direct role of Yfh1p in the formation of Fe-S clusters and indicate that mitochondrial iron export requires Fe-S cluster biosynthesis.     

MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone

Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 A x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the approximately 650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a beta-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.     

AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis

In bacteria, yeast, and mammals, iron-sulfur (Fe-S) cluster-containing proteins are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. In humans, iron-storage diseases such as X-linked sideroblastic anemia and ataxia are caused by defects in Fe-S cluster availability. The biogenesis of Fe-S clusters involves several pathways, and in bacteria, the SufABCDSE operon has been shown to play a vital role in Fe-S biogenesis and repair during oxidative stress. Although Fe-S proteins play vital roles in plants, Fe-S cluster biogenesis and maintenance and physiological consequences of dysfunctional Fe-S cluster assembly remains obscure. Here we report that Arabidopsis plants deficient for the SufC homolog AtNAP7 show lethality at the globular stage of embryogenesis. AtNAP7 is expressed in developing embryos and in apical, root, and floral meristems and encodes an ATP-binding cassette/ATPase that can partially rescue growth defects in an Escherichia coli SufC mutant during oxidative stress. AtNAP7 is plastid-localized, and mutant embryos contain abnormal developing plastids with disorganized thylakoid structures. We found that AtNAP7 can interact with AtNAP6, a plastidic Arabidopsis SufD homolog, and because Arabidopsis plastids also harbor SufA, SufB, SufS, and SufE homologs, plastids probably contain a complete SUF system. Our results imply that AtNAP7 represents a conserved SufC protein involved in the biogenesis and/or repair of oxidatively damaged Fe-S clusters and suggest an important role for plastidic Fe-S cluster maintenance and repair during Arabidopsis embryogenesis.     

Differential diagnosis of lipoic acid synthesis defects

Lipoic acid (LA) is an essential cofactor required for the activity of five multienzymatic complexes that play a central role in the mitochondrial energy metabolism: four 2-oxoacid dehydrogenase complexes [pyruvate dehydrogenase (PDH), branched-chain ketoacid dehydrogenase (BCKDH), 2-ketoglutarate dehydrogenase (2-KGDH), and 2-oxoadipate dehydrogenase (2-OADH)] and the glycine cleavage system (GCS). LA is synthesized in a complex multistep process that requires appropriate function of the mitochondrial fatty acid synthesis (mtFASII) and the biogenesis of iron–sulphur (Fe-S) clusters. Defects in the biosynthesis of LA have been reported to be associated with multiple and severe defects of the mitochondrial energy metabolism. In recent years, disease-causing mutations in genes encoding for proteins involved in LA metabolism have been reported: NFU1, BOLA3, IBA57, LIAS, GLRX5, LIPT1, ISCA2, and LIPT2. These studies represented important progress in understanding the pathophysiology and molecular bases underlying these disorders. Here we review current knowledge regarding involvement of LA synthesis defects in human diseases with special emphasis on the diagnostic strategies for these disorders. The clinical and biochemical characteristics of patients with LA synthesis defects are discussed and a workup for the differential diagnosis proposed.     

Protein expression profiles in patients carrying NFU1 mutations. Contribution to the pathophysiology of the disease

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1 were recently identified in patients with fatal encephalopathy displaying a biochemical phenotype consistent with defects in lipoic acid-dependent enzymatic activities and respiratory chain complexes. This discovery highlighted the molecular function of NFU1 as an iron-sulfur(Fe-S) cluster protein necessary for lipoic acid biosynthesis and respiratory chain complexes activities. To understand the pathophysiological mechanisms underlying this disease we have characterized the protein expression profiles of patients carrying NFU1 mutations. Fibroblasts from patients with the p.Gly208Cys mutation showed complete absence of protein-bound lipoic acid and decreased SDHA and SDHB subunits of complex II. In contrast, subunits of other respiratory chain complexes were normal. Protein lipoylation was also decreased in muscle and liver but not in other tissues available (brain, kidney, lung) from NFU1 patients. Although levels of the respiratory chain subunits were unaltered in tissues, BN-PAGE showed an assembly defect for complex II in muscle, consistent with the low enzymatic activity of this complex. This study provides new insights into the molecular bases of NFU1 disease as well as into the regulation of NFU1 protein in human tissues. We demonstrate a ubiquitous expression of NFU1 protein and further suggest that defects in lipoic acid biosynthesis and complex II are the main molecular signature of this disease, particularly in patients carrying the p.Gly208Cys mutation.     

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.     

Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A.

The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype. HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1. Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells). The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation. These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.     

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.     

Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress.

A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD. In the yeast cells the fusion protein was imported into the mitochondrial matrix. However, the presequence was not cleaved. In a control set of experiments, the E. coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S. cerevisiae DL1Mn- cells. In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix. E. coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.     

J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.     

Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants.

cDNA clones coding for the nuclear-encoded mitochondrial Rieske iron-sulfur protein (RISP) have been isolated from maize and tobacco. Complementation analysis of hybrid proteins consisting of different domains of plant and yeast RISPs showed that the carboxyl two-thirds of the plant protein is functionally equivalent to that of the yeast protein. The amino terminus of the RISP, however, seems to be species specific because this region is not interchangeable between plant and yeast proteins. Complementation analysis of hybrid proteins also identified a structurally conserved domain probably essential for the function of bc1 complex RISPs. A specific domain from the plant RISP was found to cause temperature-sensitive respiratory growth in yeast. We have demonstrated that yeast can serve as a model system to study the structural and functional relationships of plant gene products that are enzymatic components of the mitochondrial respiratory chain.