Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Thio modification of yeast cytosolic tRNA is an iron-sulfur protein-dependent pathway.

ABSTRACT: Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.

SUBMITTER: Nakai Y

PROVIDER: S-EPMC1899921 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Iron-sulfur biology invades tRNA modification: the case of U34 sulfuration.

Project description:Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

| S-EPMC8053098 | biostudies-literature

Mitochondria export iron-sulfur and sulfur intermediates to the cytoplasm for iron-sulfur cluster assembly and tRNA thiolation in yeast.

Project description:Iron-sulfur clusters are essential cofactors of proteins. In eukaryotes, iron-sulfur cluster biogenesis requires a mitochondrial iron-sulfur cluster machinery (ISC) and a cytoplasmic iron-sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [35S]cysteine to detect cytoplasmic Fe-35S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe-S)int, needed for cytoplasmic iron-sulfur cluster assembly. The mitochondrial biosynthesis of (Fe-S)int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe-S)int via the Atm1 transporter in the inner membrane, and we detected (Fe-S)int in active form. When (Fe-S)int was added to cytoplasm, CIA utilized it for iron-sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron-sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe-S)int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron-sulfur cluster synthesis. Of note, the (Fe-S)int is distinct from the sulfur intermediate called Sint, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron-sulfur cluster assembly in yeast cells.

| S-EPMC6579456 | biostudies-literature

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

Project description:Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 ?M and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.

| S-EPMC4583046 | biostudies-literature

A role for IOP1 in mammalian cytosolic iron-sulfur protein biogenesis.

Project description:The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.

| S-EPMC2431034 | biostudies-literature

Mitochondria Export Sulfur Species Required for Cytosolic tRNA Thiolation.

Project description:In eukaryotes, mitochondria have been hypothesized to generate sulfur species required for tRNA thiolation in the cytosol, although no direct evidence thus far exists. Here we have detected these sulfur species, making use of our observation that isolated yeast cytosol alone is unable to thiolate tRNAs but can do so upon addition of mitochondria. Mitochondria were found to utilize the cysteine desulfurase Nfs1 to produce sulfur-containing species with masses ranging from 700 to 1,100 Da. Mitochondria exported these species via the Atm1 transporter in the inner membrane. Once exported to the cytosol, these sulfur species promoted cytosolic tRNA thiolation with no further requirement of mitochondria. Furthermore, we found that the Isu1/2 scaffolds but not the Ssq1 chaperone of the mitochondrial iron-sulfur cluster machinery were required for cytosolic tRNA thiolation, and thus the sulfur utilization pathway bifurcates at the Isu1/2 site for intra-organellar use in mitochondria or export to the cytosol.

| S-EPMC6014917 | biostudies-literature

The tRNA modification complex elongator regulates the Cdc42-dependent mitogen-activated protein kinase pathway that controls filamentous growth in yeast.

Project description:Signal transduction pathways control multiple aspects of cellular behavior, including global changes to the cell cycle, cell polarity, and gene expression, which can result in the formation of a new cell type. In the budding yeast Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth induces a dimorphic foraging response under nutrient-limiting conditions. How nutritional cues feed into MAPK activation remains an open question. Here we report a functional connection between the elongator tRNA modification complex (ELP genes) and activity of the filamentous growth pathway. Elongator was required for filamentous growth pathway signaling, and elp mutants were defective for invasive growth, cell polarization, and MAPK-dependent mat formation. Genetic suppression analysis showed that elongator functions at the level of Msb2p, the signaling mucin that operates at the head of the pathway, which led to the finding that elongator regulates the starvation-dependent expression of the MSB2 gene. The Elp complex was not required for activation of related pathways (pheromone response or high osmolarity glycerol response) that share components with the filamentous growth pathway. Because protein translation provides a rough metric of cellular nutritional status, elongator may convey nutritional information to the filamentous growth pathway at the level of MSB2 expression.

| S-EPMC2747823 | biostudies-other

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

Project description:The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

| S-EPMC9243173 | biostudies-literature

MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.

Project description:DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.

| S-EPMC4619598 | biostudies-literature

Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae.

Project description:An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol.

| S-EPMC4646409 | biostudies-literature

Phosphorylation of Elp1 by Hrr25 is required for elongator-dependent tRNA modification in yeast.

Project description:Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.

| S-EPMC4287497 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data