Functional and regulatory profiling of energy metabolism in fission yeast

Background: The control of energy metabolism is fundamental for cell growth and function and anomalies in it are implicated in complex diseases and ageing. Metabolism in yeast cells can be manipulated by supplying different carbon sources: yeast grown on glucose rapidly proliferates by fermentation, analogous to tumour cells growing by aerobic glycolysis, whereas on non-fermentable carbon sources metabolism shifts towards respiration. Results: We screened deletion libraries of fission yeast to identify over 200 genes required for respiratory growth. Growth media and auxotrophic mutants strongly influenced respiratory metabolism. Most genes uncovered in the mutant screens have not been implicated in respiration in budding yeast. We applied gene-expression profiling approaches to compare steady-state fermentative and respiratory growth and to analyse the dynamic adaptation to respiratory growth. The transcript levels of most genes functioning in energy metabolism pathways are coherently tuned, reflecting anticipated differences in metabolic flows between fermenting and respiring cells. We show that acetyl-CoA synthase, rather than citrate lyase, is essential for acetyl-CoA synthesis in fission yeast. We also investigated the transcriptional response to mitochondrial damage by genetic or chemical perturbations, defining a retrograde response that involves the concerted regulation of distinct groups of nuclear genes that may avert harm from mitochondrial malfunction. Conclusions: This study provides a rich framework of the genetic and regulatory basis of energy metabolism in fission yeast and beyond, and it pinpoints weaknesses of commonly used auxotroph mutants for investigating metabolism. As a model for cellular energy regulation, fission yeast provides an attractive and complementary system to budding yeast

PBF complex regulates directly Ace2p-target genes in Schizosaccharomyces pombe

Resumen del póster presentado a la EMBO Conference on Fission Yeast: Pombe 2013; 7th International Fission Yeast Meeting celebrada en Londres (UK) del 24 al 29 de junio de 2013.Yeast division cycle is completed with the activation of a cell separation program that involves dissolution of the septum assemblied during cytokinesis between the two daughter cells, allowing them to become independent entities. Gene expression of hydrolytic enzymes (eng1+ and agn1+) responsible for septum dissolution is periodically activated at the end of the cell cycle by the specific transcription factor Ace2p (Alonso-Núñez et al., 2005). ace2+ expression is, in turn, periodically activated during mitosis by the transcriptional complex PBF (>PCB Binding Factor>). This complex consists of at least two forkhead-like proteins Sep1p, Fkh2p and a MADS box-like protein Mbx1p, although more recently it has been shown that other proteins participating in different events at the end of the cell cycle, as the kinase Plo1p, the phosphatase Clp1p and the anillin-like protein Mid1p, are also components of PBF (McInerny, JC 2011). In our laboratory, we have found that Ace2p-dependent genes show in their upstream promoter region sites for Ace2p binding but also PCB sites (>Pombe Cell-Cycle Box>) for PBF complex binding. Chromatin immunoprecipitation analyses (ChIP-qPCR) showed that at least Fkh2p and Sep1 bind in vivo to the eng1+ promoter. ChIP analyses through the cell cycle also revealed that Ace2p binding was coincident with maximum level of eng1+ mRNA, while Fkh2p was found to bind later, when mRNA level of this gene is low. Additionally, RT-qPCR assays in ace2Δ, fkh2Δ, sep1Δ, and mbx1Δ mutants show that expression of the genes under the control of Ace2p is affected in a different way by PBF components, indicating that transcriptional regulation is more complex than initially expected.Peer Reviewe

A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast

Iron is an essential cofactor, but it is also toxic at high levels. In Schizosaccharomyces pombe, the sensor glutaredoxin Grx4 guides the activity of the repressors Php4 and Fep1 to mediate a complex transcriptional response to iron deprivation: activation of Php4 and inactivation of Fep1 leads to inhibition of iron usage/storage, and to promotion of iron import, respectively. However, the molecular events ruling the activity of this double-branched pathway remained elusive. We show here that Grx4 incorporates a glutathione-containing iron-sulfur cluster, alone or forming a heterodimer with the BolA-like protein Fra2. Our genetic study demonstrates that Grx4-Fra2, but not Fep1 nor Php4, participates not only in iron starvation signaling but also in iron-related aerobic metabolism. Iron-containing Grx4 binds and inactivates the Php4 repressor; upon iron deprivation, the cluster in Grx4 is probably disassembled, the proteins dissociate, and Php4 accumulates at the nucleus and represses iron consumption genes. Fep1 is also an iron-containing protein, and the tightly bound iron is required for transcriptional repression. Our data suggest that the cluster-containing Grx4-Fra2 heterodimer constitutively binds to Fep1, and upon iron deprivation the disassembly of the iron cluster between Grx4 and Fra2 promotes reverse metal transfer from Fep1 to Grx4-Fra2, and de-repression of iron-import genes. Our genetic and biochemical study demonstrates that the glutaredoxin Grx4 independently governs the Php4 and Fep1 repressors through metal transfer. Whereas iron loss from Grx4 seems to be sufficient to release Php4 and allow its nuclear accumulation, total or partial disassembly of the Grx4-Fra2 cluster actively participates in iron-containing Fep1 activation by sequestering its iron and decreasing its interaction with promoters.This work was supported by the Spanish Ministry of Science and Innovation (BFU2012-32045), PLAN E and FEDER, and by SGR2009-196 from Generalitat de Catalunya (Spain) to EH. EH and JA are recipients of ICREA Academia Awards (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase

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.This work was supported by the Ministerio de Economía y Competitividad (Spain), PLAN E and FEDER (BFU2015-68350-P to EH, BFU2015-66347-P to JA, BFU2014-58429-P to MCB), and by 2014-SGR-154 from Generalitat de Catalunya (Spain) to EH and JA. AD is recipient of a pre-doctoral fellowship from Generalitat de Catalunya (Spain). EH is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Glucanases and Chitinases

In many yeast and fungi, β-(1,3)-glucan and chitin are essential components of the cell wall, an important structure that surrounds cells and which is responsible for their mechanical protection and necessary for maintaining the cellular shape. In addition, the cell wall is a dynamic structure that needs to be remodelled along with the different phases of the fungal life cycle or in response to extracellular stimuli. Since β-(1,3)-glucan and chitin perform a central structural role in the assembly of the cell wall, it has been postulated that β-(1,3)-glucanases and chitinases should perform an important function in cell wall softening and remodelling. This review focusses on fungal glucanases and chitinases and their role during fungal morphogenesis.This work was supported by grants from the Spanish Government to CR (BFU2017-84508-P) and CRV (BIO2015-70195-C2-1-R) and from Junta de Castilla y León to CR (SA116G19). The IBFG is supported by Programa “Escalera de Excelencia” from Junta de Castilla y León (CLU-2017-03) and University of Salamanca. All Spanish funding is co-sponsored by the European Union FEDER programme.Peer reviewe