Abstract
The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.
Highlights
Cysteine residues are not very abundant in proteins, but they are over-represented in functional regions of proteins, such as surfaces and catalytic centers [1]
ribonucleotide reductase (RNR) is tightly regulated in a cell cycle-dependent manner at different levels
We show that cytosolic thioredoxin Trx1 is the primary electron donor for RNR in fission yeast, and that trx1 transcript and protein levels are up-regulated at G1-toS phase transition
Summary
Cysteine residues are not very abundant in proteins, but they are over-represented in functional regions of proteins, such as surfaces and catalytic centers [1]. The thiol group of cysteines is subject of post-translational modifications altering its redox state; several of these oxidation states are reversible, such as sulfenic acid and disulfides. Reversible thiol to disulfide switches happen as a consequence of cellular responses to oxidative stress, and several proteins with reactive cysteine residues undergo oxidations as part of their catalytic cycles (for a review, see [2]). Trxs and Grxs catalyze thiol-disulfide exchange reactions, and share a motif known as the Trx fold [3]. Both types of reductants use as the ultimate electron donor NADPH [4]. In the case of Trxs, Trx reductase is the intermediate between NADPH and Trx, while GSH reduces oxidized Grxs, GSH reductase being the link between NADPH and oxidized GSH
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