The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The 'plastid-type' NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase.

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Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms22041598 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1598

Author(s):

Amber L. Hendricks ◽

Christine Wachnowsky ◽

Brian Fries ◽

Insiya Fidai ◽

James A. Cowan

Keyword(s):

Cluster Assembly ◽

Paramagnetic Resonance ◽

Iron Sulfur Cluster ◽

Sulfur Transfer ◽

Disease States ◽

Isolation And Characterization ◽

Iron Sulfur ◽

Natural Iron ◽

Lipoyl Synthase

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

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[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015726 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18367-18378 ◽

Cited By ~ 1

Author(s):

Tamanna Azam ◽

Jonathan Przybyla-Toscano ◽

Florence Vignols ◽

Jérémy Couturier ◽

Nicolas Rouhier ◽

...

Keyword(s):

Bimolecular Fluorescence Complementation ◽

Back Reaction ◽

Cluster Assembly ◽

Carrier Proteins ◽

Respiratory Chain Complexes ◽

Visible Absorption ◽

Iron Sulfur ◽

Cluster Transfer ◽

Activity Measurements ◽

Uv Visible

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.

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The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011034 ◽

2020 ◽

Vol 295 (6) ◽

pp. 1727-1742 ◽

Cited By ~ 6

Author(s):

Mélanie Roland ◽

Jonathan Przybyla-Toscano ◽

Florence Vignols ◽

Nathalie Berger ◽

Tamanna Azam ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

De Novo ◽

Acid Synthesis ◽

Bimolecular Fluorescence Complementation ◽

Cluster Assembly ◽

Amino Acid Synthesis ◽

Cluster Transfer ◽

Client Proteins ◽

Branched Chain

Proteins incorporating iron–sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

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

GeroScience ◽

10.1007/s11357-021-00327-z ◽

2021 ◽

Author(s):

Yi Sheng ◽

Guang Yang ◽

Kaitlyn Casey ◽

Shayla Curry ◽

Mason Oliver ◽

...

Keyword(s):

Stress Response ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Mitochondrial Iron

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MRE11 Is Crucial for Malaria Parasite Transmission and Its Absence Affects Expression of Interconnected Networks of Key Genes Essential for Life

Cells ◽

10.3390/cells9122590 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2590

Author(s):

David S. Guttery ◽

Abhinay Ramaprasad ◽

David J. P. Ferguson ◽

Mohammad Zeeshan ◽

Rajan Pandey ◽

...

Keyword(s):

Genome Stability ◽

Malaria Parasite ◽

Mosquito Vector ◽

Parasite Transmission ◽

Biological Processes ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Parasite Plasmodium ◽

Damage Response

The meiotic recombination 11 protein (MRE11) plays a key role in DNA damage response and maintenance of genome stability. However, little is known about its function during development of the malaria parasite Plasmodium. Here, we present a functional, ultrastructural and transcriptomic analysis of Plasmodium parasites lacking MRE11 during its life cycle in both mammalian and mosquito vector hosts. Genetic disruption of Plasmodium berghei mre11 (PbMRE11) results in significant retardation of oocyst development in the mosquito midgut associated with cytoplasmic and nuclear degeneration, along with concomitant ablation of sporogony and subsequent parasite transmission. Further, absence of PbMRE11 results in significant transcriptional downregulation of genes involved in key interconnected biological processes that are fundamental to all eukaryotic life including ribonucleoprotein biogenesis, spliceosome function and iron–sulfur cluster assembly. Overall, our study provides a comprehensive functional analysis of MRE11′s role in Plasmodium development during the mosquito stages and offers a potential target for therapeutic intervention during malaria parasite transmission.

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Multiple Sense and Antisense Promoters Contribute to the Regulated Expression of the isc-suf Operon for Iron-Sulfur Cluster Assembly in Rhodobacter

Microorganisms ◽

10.3390/microorganisms7120671 ◽

2019 ◽

Vol 7 (12) ◽

pp. 671 ◽

Cited By ~ 1

Author(s):

Xin Nie ◽

Bernhard Remes ◽

Gabriele Klug

Keyword(s):

Rhodobacter Sphaeroides ◽

Photosynthetic Electron Transport ◽

Regulatory Proteins ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Iron Sulfur Clusters ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Photosynthetic Complexes ◽

The One

A multitude of biological functions relies on iron-sulfur clusters. The formation of photosynthetic complexes goes along with an additional demand for iron-sulfur clusters for bacteriochlorophyll synthesis and photosynthetic electron transport. However, photooxidative stress leads to the destruction of iron-sulfur clusters, and the released iron promotes the formation of further reactive oxygen species. A balanced regulation of iron-sulfur cluster synthesis is required to guarantee the supply of this cofactor, on the one hand, but also to limit stress, on the other hand. The phototrophic alpha-proteobacterium Rhodobacter sphaeroides harbors a large operon for iron-sulfur cluster assembly comprising the iscRS and suf genes. IscR (iron-sulfur cluster regulator) is an iron-dependent regulator of isc-suf genes and other genes with a role in iron metabolism. We applied reporter gene fusions to identify promoters of the isc-suf operon and studied their activity alone or in combination under different conditions. Gel-retardation assays showed the binding of regulatory proteins to individual promoters. Our results demonstrated that several promoters in a sense and antisense direction influenced isc-suf expression and the binding of the IscR, Irr, and OxyR regulatory proteins to individual promoters. These findings demonstrated a complex regulatory network of several promoters and regulatory proteins that helped to adjust iron-sulfur cluster assembly to changing conditions in Rhodobacter sphaeroides.

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Regulation of the HscA ATPase Reaction Cycle by the Co-chaperone HscB and the Iron-Sulfur Cluster Assembly Protein IscU

Journal of Biological Chemistry ◽

10.1074/jbc.m410117200 ◽

2004 ◽

Vol 279 (52) ◽

pp. 53924-53931 ◽

Cited By ~ 59

Author(s):

Jonathan J. Silberg ◽

Tim L. Tapley ◽

Kevin G. Hoff ◽

Larry E. Vickery

Keyword(s):

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Reaction Cycle ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Atpase Reaction

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Crucial Players for Inter-Organelle Communication: PI5P4Ks and Their Lipid Product PI-4,5-P2 Come to the Surface

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.791758 ◽

2022 ◽

Vol 9 ◽

Author(s):

Archna Ravi ◽

Lavinia Palamiuc ◽

Brooke M. Emerling

Keyword(s):

Stress Response ◽

Human Diseases ◽

Type Ii ◽

Metabolic Homeostasis ◽

Cellular Homeostasis ◽

Evolutionarily Conserved ◽

Lipid Product ◽

Canonical Type ◽

Organelle Communication ◽

A Minor

While organelles are individual compartments with specialized functions, it is becoming clear that organellar communication is essential for maintaining cellular homeostasis. This cooperation is carried out by various interactions taking place on the membranes of organelles. The membranes themselves contain a multitude of proteins and lipids that mediate these connections and one such class of molecules facilitating these relations are the phospholipids. There are several phospholipids, but the focus of this perspective is on a minor group called the phosphoinositides and specifically, phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). This phosphoinositide, on intracellular membranes, is largely generated by the non-canonical Type II PIPKs, namely, Phosphotidylinositol-5-phosphate-4-kinases (PI5P4Ks). These evolutionarily conserved enzymes are emerging as key stress response players in cells. Further, PI5P4Ks have been shown to modulate pathways by regulating organelle crosstalk, revealing roles in preserving metabolic homeostasis. Here we will attempt to summarize the functions of the PI5P4Ks and their product PI-4,5-P2 in facilitating inter-organelle communication and how they impact cellular health as well as their relevance to human diseases.

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