Identification of client iron–sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana

Abstract Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

Download Full-text

A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3

International Journal of Molecular Sciences ◽

10.3390/ijms21218121 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8121

Author(s):

Nathalie Berger ◽

Florence Vignols ◽

Brigitte Touraine ◽

Maël Taupin-Broggini ◽

Valérie Rofidal ◽

...

Keyword(s):

De Novo ◽

Protein Localization ◽

Expression Patterns ◽

Specific Protein ◽

Protein Accumulation ◽

Loss Of Function ◽

Iron Sulfur ◽

Proteomic Approach ◽

Client Proteins ◽

S Proteins

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

Download Full-text

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.

Download Full-text

From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis

Biological Chemistry ◽

10.1515/hsz-2020-0117 ◽

2020 ◽

Vol 401 (6-7) ◽

pp. 855-876 ◽

Cited By ~ 13

Author(s):

Roland Lill

Keyword(s):

De Novo ◽

Current Knowledge ◽

Protein Stabilization ◽

S Protein ◽

Cellular Iron ◽

Iron Sulfur ◽

Protein Biogenesis ◽

Sulfur Protein ◽

Initial Discovery ◽

S Proteins

AbstractProtein cofactors often are the business ends of proteins, and are either synthesized inside cells or are taken up from the nutrition. A cofactor that strictly needs to be synthesized by cells is the iron-sulfur (Fe/S) cluster. This evolutionary ancient compound performs numerous biochemical functions including electron transfer, catalysis, sulfur mobilization, regulation and protein stabilization. Since the discovery of eukaryotic Fe/S protein biogenesis two decades ago, more than 30 biogenesis factors have been identified in mitochondria and cytosol. They support the synthesis, trafficking and target-specific insertion of Fe/S clusters. In this review, I first summarize what led to the initial discovery of Fe/S protein biogenesis in yeast. I then discuss the function and localization of Fe/S proteins in (non-green) eukaryotes. The major part of the review provides a detailed synopsis of the three major steps of mitochondrial Fe/S protein biogenesis, i.e. the de novo synthesis of a [2Fe-2S] cluster on a scaffold protein, the Hsp70 chaperone-mediated transfer of the cluster and integration into [2Fe-2S] recipient apoproteins, and the reductive fusion of [2Fe-2S] to [4Fe-4S] clusters and their subsequent assembly into target apoproteins. Finally, I summarize the current knowledge of the mechanisms underlying the maturation of cytosolic and nuclear Fe/S proteins.

Download Full-text

The Yeast Scaffold Proteins Isu1p and Isu2p Are Required inside Mitochondria for Maturation of Cytosolic Fe/S Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.24.11.4848-4857.2004 ◽

2004 ◽

Vol 24 (11) ◽

pp. 4848-4857 ◽

Cited By ~ 94

Author(s):

Jana Gerber ◽

Karina Neumann ◽

Corinna Prohl ◽

Ulrich Mühlenhoff ◽

Roland Lill

Keyword(s):

Iron Homeostasis ◽

De Novo ◽

Mitochondrial Targeting ◽

Protein Maturation ◽

Iron Sulfur Cluster ◽

Subcellular Compartment ◽

S Protein ◽

Iron Sulfur ◽

Protein Biogenesis ◽

S Proteins

ABSTRACT Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

Download Full-text

Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111540 ◽

2020 ◽

Vol 89 (1) ◽

pp. 471-499 ◽

Cited By ~ 28

Author(s):

Roland Lill ◽

Sven-A. Freibert

Keyword(s):

Molecular Mechanisms ◽

De Novo ◽

Lipid Synthesis ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Atp Production ◽

Cellular Iron ◽

Iron Sulfur ◽

Sulfur Protein ◽

S Proteins

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone–mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein–mediated maturation of mitochondrial Fe/S proteins and the importance for human disease.

Download Full-text

Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bj20111993 ◽

2012 ◽

Vol 444 (2) ◽

pp. 227-237 ◽

Cited By ~ 10

Author(s):

Kaushik Saha ◽

Michael E. Webb ◽

Stephen E. J. Rigby ◽

Helen K. Leech ◽

Martin J. Warren ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Quaternary Structure ◽

Specific Activity ◽

Close Association ◽

Physiological Role ◽

Cysteine Residue ◽

In Planta ◽

Iron Sulfur ◽

Tetrapyrrole Metabolism

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe–S] cluster. We determined the cluster to be a [4Fe–4S] type, which quickly oxidizes to a [2Fe–2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys135, is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe–4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe–S cluster or Cys135 retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe–S cluster in planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

Download Full-text

Differences in the Manifestation of Cell Pluripotence In Vivo and In Vitro in the Mutant Arabidopsis thaliana with the Phenotype of Cell Memory Disorder

Russian Journal of Plant Physiology ◽

10.1134/s1021443721010106 ◽

2021 ◽

Vol 68 (1) ◽

pp. 46-55

Author(s):

E. V. Kupriyanova ◽

E. R. Denisova ◽

M. A. Baier ◽

T. A. Ezhova

Keyword(s):

Arabidopsis Thaliana ◽

Shoot Regeneration ◽

Cultured Cells ◽

De Novo ◽

Epigenetic Memory ◽

In Planta ◽

Pluripotency Genes

Abstract Plant cells cultivated in vitro are a convenient model for studying the genetic and physiological mechanisms necessary for the cells to acquire a state of pluripotency. Earlier studies on a model plant Arabidopsis thaliana (L.) Heynh. have identified the key role of genes that determine the pluripotency of cells in the shoot apical meristem in de novo shoot regeneration in tissue culture. In accordance with this, cells of mutant plants with a higher level of expression of pluripotency genes were characterized by an increased potential for de novo shoot regeneration. The tae mutant was the exception to this rule. The mutant resumed the expression of pluripotency genes and cell proliferation at the late stages of leaf development, which indicates a violation of the mechanisms for maintaining epigenetic cellular memory. At the same time, leaf cells cultured in vitro showed a lower proliferative activity compared to the wild type and were not capable of de novo regeneration of shoots. A decrease in the regenerative potential of cultured cells of the tae mutant indicates an important role of epigenetic memory in the response of cells to exogenous hormones. Impaired epigenetic memory of leaf cells of the tae mutant and differences in their proliferative and regenerative capacities in planta and in vitro make this mutant a unique model for studying the role of epigenetic modifications in the regulation of cell pluripotency.

Download Full-text

Screening and Identification of Candidate GUN1-Interacting Proteins in Arabidopsis thaliana

International Journal of Molecular Sciences ◽

10.3390/ijms222111364 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11364

Author(s):

Linjuan Wang ◽

Xingqi Huang ◽

Kui Li ◽

Shuyuan Song ◽

Yunhe Jing ◽

...

Keyword(s):

Molecular Mechanisms ◽

Bimolecular Fluorescence Complementation ◽

Positive Interactions ◽

Interacting Proteins ◽

In Planta ◽

Photooxidative Damage ◽

Screening And Identification ◽

Bimolecular Fluorescence Complementation Assay ◽

Regulatory Functions ◽

Partner Proteins

Chloroplasts are semi-autonomous organelles governed by the precise coordination between the genomes of their own and the nucleus for functioning correctly in response to developmental and environmental cues. Under stressed conditions, various plastid-to-nucleus retrograde signals are generated to regulate the expression of a large number of nuclear genes for acclimation. Among these retrograde signaling pathways, the chloroplast protein GENOMES UNCOUPLED 1 (GUN1) is the first component identified. However, in addition to integrating aberrant physiological signals when chloroplasts are challenged by stresses such as photooxidative damage or the inhibition of plastid gene expression, GUN1 was also found to regulate other developmental processes such as flowering. Several partner proteins have been found to interact with GUN1 and facilitate its different regulatory functions. In this study, we report 15 possible interacting proteins identified through yeast two-hybrid (Y2H) screening, among which 11 showed positive interactions by pair-wise Y2H assay. Through the bimolecular fluorescence complementation assay in Arabidopsis protoplasts, two candidate proteins with chloroplast localization, DJC31 and HCF145, were confirmed to interact with GUN1 in planta. Genes for these GUN1-interacting proteins showed different fluctuations in the WT and gun1 mutant under norflurazon and lincomycin treatments. Our results provide novel clues for a better understanding of molecular mechanisms underlying GUN1-mediated regulations.

Download Full-text