Post-doc, Tianjin University
Exploring ∆Grx3/4(BY) suppressors to decipher iron-activation for iron-requiring processes
Iron is an essential nutrient for virtually all organisms because it functions as a cofactor in central cellular processes such as respiration, DNA synthesis and repair, ribosome biogenesis, and metabolism. However, as it is toxic at higher concentrations, cells have developed sophisticated systems for ensuring a tightly regulated iron homeostasis. Saccharomyces cereviceae, a well studied model organism in metal metabolism, maintains iron homeostasis via conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 which utilize their bound Fe-S cofactor for intracellular iron trafficking. In the recent past investigations have revealed that depletion of Grx3/4 leads to functional impairment of virtually all iron-dependent processes, including heme biosynthesis, mitochondrial and cytosolic Fe-S protein biogenesis, and the formation of di-iron centers in mitochondria and the cytosol, eventually leading to the loss of cell viability. However, double mutant ∆Grx3/4 (BY) background yeast strains are viable with a slower growth rate when grown on YPD medium and form tiny colonies compared to wild type BY4741/BY4742 strains. Interestingly, upon re culture of ∆Grx3/4(BY) strains for subsequent generations, few colonies escape from glutaredoxin deficiency accumulating suppressor mutation(s) to make use of essential iron for cell physiology. Thus, we are aiming at deciphering the nature of suppressor mutation(s) responsible for restoring the wild type phenotype in double mutant ∆Grx3/4(BY) yeast strains to correlate the mechanisms of iron activation for iron-requiring processes as these proteins are evolutionarily conserved from bacteria to humans.
Abstract: The β(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The β(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Tyr(•) formation, cells lacking β' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β' is required for iron loading and Tyr(•) formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in β. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β' and is inhibited by adding an iron chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ββ' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.
Pub.: 21 Sep '11, Pinned: 31 Aug '17
Abstract: Iron is an essential nutrient for cells. It is unknown how iron, after its import into the cytosol, is specifically delivered to iron-dependent processes in various cellular compartments. Here, we identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing. Depletion of Grx3/4 specifically impaired all iron-requiring reactions in the cytosol, mitochondria, and nucleus, including the synthesis of Fe/S clusters, heme, and di-iron centers. These defects were caused by impairment of iron insertion into proteins and iron transfer to mitochondria, indicating that intracellular iron is not bioavailable, despite highly elevated cytosolic levels. The crucial task of Grx3/4 is mediated by a bridging, glutathione-containing Fe/S center that functions both as an iron sensor and in intracellular iron delivery. Collectively, our study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms.
Pub.: 05 Oct '10, Pinned: 31 Aug '17
Abstract: Iron is a redox active element that functions as an essential cofactor in multiple metabolic pathways, including respiration, DNA synthesis and translation. While indispensable for eukaryotic life, excess iron can lead to oxidative damage of macromolecules. Therefore, living organisms have developed sophisticated strategies to optimally regulate iron acquisition, storage and utilization in response to fluctuations in environmental iron bioavailability. In the yeast Saccharomyces cerevisiae, transcription factors Aft1/Aft2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to producing and assembling iron cofactors, mitochondrial iron-sulfur (Fe/S) cluster biogenesis has emerged as a central player in iron sensing. A mitochondrial signal derived from Fe/S synthesis is exported and converted into an Fe/S cluster that interacts directly with Aft1/Aft2 and Yap5 proteins to regulate their transcriptional function. Various conserved proteins, such as ABC mitochondrial transporter Atm1 and, for Aft1/Aft2, monothiol glutaredoxins Grx3 and Grx4 are implicated in this iron-signaling pathway. The analysis of a wide range of S. cerevisiae strains of different geographical origins and sources has shown that yeast strains adapted to high iron display growth defects under iron-deficient conditions, and highlighted connections that exist in the response to both opposite conditions. Changes in iron accumulation and gene expression profiles suggest differences in the regulation of iron homeostasis genes.
Pub.: 21 Mar '17, Pinned: 31 Aug '17
Abstract: Glutathione is the most abundant cellular low molecular weight thiol in the majority of organisms in all kingdoms of life. Therefore, functions of glutathione and disturbed regulation of its concentration are associated with numerous physiological and pathological situations. Recent advances: The function of glutathione as redox buffer or antioxidant is increasingly being questioned. New functions, especially functions connected to the cellular iron homeostasis were elucidated. Via the formation of iron complexes, glutathione is an important player in all aspects of iron metabolism: sensing and regulation of iron levels, iron trafficking, and biosynthesis of iron cofactors. The variety of glutathione coordinated iron complexes and their functions with a special focus on FeS-glutaredoxins are summarized in this review. Interestingly, glutathione analogues that function as major low molecular weight thiols in organisms lacking glutathione resemble the functions in iron homeostasis.Since these iron-related functions are most likely also connected to thiol redox chemistry, it is difficult to distinguish between mechanisms related to either redox or iron metabolisms.The ability of glutathione to coordinate iron in different complexes with or without proteins needs further investigation. The discovery of new Fe-glutathione complexes and their physiological functions will significantly advance our understanding of cellular iron homeostasis.
Pub.: 26 May '17, Pinned: 31 Aug '17