scholarly journals SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells

2021 ◽  
Author(s):  
Ying Wang ◽  
Frederick S Yen ◽  
Xiphias Ge Zhu ◽  
Rebecca C Timson ◽  
Ross Weber ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Redox Homeostasis ◽  
Protein Abundance ◽  
Iron Sulfur Cluster ◽  
Oxidative Reactions ◽  
Iron Sulfur ◽  
Molecular Machinery ◽  
Mitochondrial Glutathione ◽  
Proliferation In Vitro

Glutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions. GSH is exclusively synthesized in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remain elusive. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, to regulate GSH transport into mitochondria. SLC25A39 loss reduces mitochondrial GSH import and abundance without impacting whole cell GSH levels. Cells lacking both SLC25A39 and its paralog SLC25A40 exhibit defects in the activity and stability of iron-sulfur cluster containing proteins. Moreover, mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Remarkably, the heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enabled mitochondrial GSH production and ameliorated the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH import machinery.

scholarly journals Iron–sulfur cluster biosynthesis

2008 ◽  
Vol 36 (6) ◽  
pp. 1112-1119 ◽  
Author(s):  
Sibali Bandyopadhyay ◽  
Kala Chandramouli ◽  
Michael K. Johnson
Keyword(s):  
Scaffold Proteins ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Eukaryotic Organisms ◽  
Almost All ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

scholarly journals Biofilm-Constructing Variants of Paraburkholderia phytofirmans PsJN Outcompete the Wild-Type Form in Free-Living and Static Conditions but Not In Planta

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Marine Rondeau ◽  
Qassim Esmaeel ◽  
Jérôme Crouzet ◽  
Pauline Blin ◽  
Isabelle Gosselin ◽  
...  
Keyword(s):  
Wild Type ◽  
In Planta ◽  
Free Living ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Iron Sulfur ◽  
Type Form ◽  
Static Conditions ◽  
Wild Type Form

ABSTRACT Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In a spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several microbe-associated molecular patterns (MAMPs), including exopolysaccharides (EPS), flagellin, and GroEL. Interestingly, the variants induce low PR1 and PDF1.2 expression compared to that of the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an iron-sulfur cluster: hscA (encoding a cochaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in a heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and coexpress a variant that outcompete the wild-type form in free-living and static conditions but not in planta. IMPORTANCE Paraburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from the wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta, and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneous mutations in an iron-sulfur cluster. Overall, the data provided here allow a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in a heterogeneous environment.

scholarly journals Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

2013 ◽  
Vol 288 (20) ◽  
pp. 14200-14211 ◽  
Author(s):  
Sylvain Boutigny ◽  
Avneesh Saini ◽  
Edward E. K. Baidoo ◽  
Natasha Yeung ◽  
Jay D. Keasling ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Carrier Protein ◽  
Cluster Assembly ◽  
Carrier Proteins ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Initial Cluster ◽  
Initial Assembly

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-l-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

scholarly journals Mitochondrial Arabidopsis thaliana TRXo Isoforms Bind an Iron–Sulfur Cluster and Reduce NFU Proteins In Vitro

Antioxidants ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 142 ◽  
Author(s):  
Flavien Zannini ◽  
Thomas Roret ◽  
Jonathan Przybyla-Toscano ◽  
Tiphaine Dhalleine ◽  
Nicolas Rouhier ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Function Analysis ◽  
Active Form ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Regulation Mechanism ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur

In plants, the mitochondrial thioredoxin (TRX) system generally comprises only one or two isoforms belonging to the TRX h or o classes, being less well developed compared to the numerous isoforms found in chloroplasts. Unlike most other plant species, Arabidopsis thaliana possesses two TRXo isoforms whose physiological functions remain unclear. Here, we performed a structure–function analysis to unravel the respective properties of the duplicated TRXo1 and TRXo2 isoforms. Surprisingly, when expressed in Escherichia coli, both recombinant proteins existed in an apo-monomeric form and in a homodimeric iron–sulfur (Fe-S) cluster-bridged form. In TRXo2, the [4Fe-4S] cluster is likely ligated in by the usual catalytic cysteines present in the conserved Trp-Cys-Gly-Pro-Cys signature. Solving the three-dimensional structure of both TRXo apo-forms pointed to marked differences in the surface charge distribution, notably in some area usually participating to protein–protein interactions with partners. However, we could not detect a difference in their capacity to reduce nitrogen-fixation-subunit-U (NFU)-like proteins, NFU4 or NFU5, two proteins participating in the maturation of certain mitochondrial Fe-S proteins and previously isolated as putative TRXo1 partners. Altogether, these results suggest that a novel regulation mechanism may prevail for mitochondrial TRXs o, possibly existing as a redox-inactive Fe-S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation in specific physiological conditions.

scholarly journals Two Distinct L-Lactate Dehydrogenases Play a Role in the Survival of Neisseria gonorrhoeae in Cervical Epithelial Cells

2019 ◽  
Vol 221 (3) ◽  
pp. 449-453 ◽  
Author(s):  
Nathan H Chen ◽  
Cheryl-Lynn Y Ong ◽  
Jonathan O’Sullivan ◽  
Ines Ibranovic ◽  
Krystelle Davey ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Functional Role ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Cervical Epithelial Cells ◽  
Lactate Dehydrogenases

Abstract L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

scholarly journals Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Sven-A. Freibert ◽  
Alina V. Goldberg ◽  
Christian Hacker ◽  
Sabine Molik ◽  
Paul Dean ◽  
...  
Keyword(s):  
Evolutionary Conservation ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
In Vitro Reconstitution ◽  
Cluster Biosynthesis

Biogenesis and Transfer of Iron-Sulfur Clusters from Acidithiobacillus ferrooxidans

2013 ◽  
Vol 825 ◽  
pp. 198-201 ◽  
Author(s):  
Jian She Liu ◽  
Lin Qian ◽  
Chun Li Zheng
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Binding Protein ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins

Iron-sulfur (Fe-S) proteins are ubiquitous and participate in multiple essential functions of life. However, little is currently known about the mechanisms of iron-sulfur biosynthesis and transfer in acidophilic microorganisms. In this study, the IscS, IscU and IscA proteins from Acidithiobacillus ferrooxidans were successfully expressed in Escherichia coli and purified by affinity chromatography. The IscS was a cysteine desulfurase which catalyzes desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly. Purified IscU did not have an iron-sulfur cluster but could act as a scaffold protein to assemble the [2Fe-2S] cluster in vitro. The IscA was a [4Fe-4S] cluster binding protein, but it also acted as an iron binding protein. Further studies indicated that the iron sulfur clusters could be transferred from pre-assembled scaffold proteins to apo-form iron sulfur proteins, the reconstituted iron sulfur proteins could restore their physiological activities.

scholarly journals IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

2013 ◽  
Vol 288 (23) ◽  
pp. 16680-16689 ◽  
Author(s):  
Mineaki Seki ◽  
Yukiko Takeda ◽  
Kazuhiro Iwai ◽  
Kiyoji Tanaka
Keyword(s):  
Cluster Assembly ◽  
Core Complex ◽  
Core Component ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
The Core ◽  
Iron Sulfur ◽  
External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

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