Aft1 Nuclear Localization and Transcriptional Response to Iron Starvation Rely upon TORC2/Ypk1 Signaling and Sphingolipid Biosynthesis.

Iron scarcity provokes a cellular response consisting of the strong expression of high-affinity systems to optimize iron uptake and mobilization. Aft1 is a primary transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Aft1 responds to iron deprivation by translocating from the cytoplasm to the nucleus. Here, we demonstrate that the AGC kinase Ypk1, as well as its upstream regulator TOR Complex 2 (TORC2), are required for proper Aft1 nuclear localization following iron deprivation. We exclude a role for TOR Complex 1 (TORC1) and its downstream effector Sch9, suggesting this response is specific for the TORC2 arm of the TOR pathway. Remarkably, we demonstrate that Aft1 nuclear localization and a robust transcriptional response to iron starvation also require biosynthesis of sphingolipids, including complex sphingolipids such as inositol phosphorylceramide (IPC) and upstream precursors, e.g., long-chain bases (LCBs) and ceramides. Furthermore, we observe the deficiency of Aft1 nuclear localization and impaired transcriptional response in the absence of iron when TORC2-Ypk1 is impaired is partially suppressed by exogenous addition of the LCB dihydrosphingosine (DHS). This latter result is consistent with prior studies linking sphingolipid biosynthesis to TORC2-Ypk1 signaling. Taken together, these results reveal a novel role for sphingolipids, controlled by TORC2-Ypk1, for proper localization and activity of Aft1 in response to iron scarcity.

Bulk autophagy induction and life extension is achieved when iron is the only limited nutrient in Saccharomyces cerevisiae.

We have investigated the effects that iron limitation provokes in Saccharomyces cerevisiae exponential cultures. We have demonstrated that one primary response is the induction of bulk autophagy mediated by TORC1. Coherently, Atg13 became dephosphorylated whereas Atg1 appeared phosphorylated. The signal of iron deprivation requires Tor2/Ypk1 activity and the inactivation of Tor1 leading to Atg13 dephosphorylation, thus triggering the autophagy process. Iron replenishment in its turn, reduces autophagy flux through the AMPK Snf1 and the subsequent activity of the iron-responsive transcription factor, Aft1. This signalling converges in Atg13 phosphorylation mediated by Tor1. Iron limitation promotes accumulation of trehalose and the increase in stress resistance leading to a quiescent state in cells. All these effects contribute to the extension of the chronological life, in a manner totally dependent on autophagy activation.

Interactions of GMP with Human Glrx3 and with Saccharomyces cerevisiae Grx3 and Grx4 Converge in the Regulation of the Gcn2 Pathway.

The human monothiol glutaredoxin Glrx3 (PICOT) is ubiquitously distributed in cytoplasm and nuclei in mammalian cells. Its overexpression has been associated with the development of several types of tumors, whereas its deficiency might cause retardation in embryogenesis. Its exact biological role has not been well resolved, although a function as a chaperone distributing iron/sulfur clusters is currently accepted. Yeast humanization and the use of a mouse library have allowed us to find a new partner for PICOT: the human GMP synthase (hGMPs). Both proteins carry out collaborative functions regarding the downregulation of the Saccharomyces cerevisiae Gcn2 pathway under conditions of nutritional stress. Glrx3/hGMPs interact through conserved residues that bridge iron/sulfur clusters and glutathione. This mechanism is also conserved in budding yeast, whose proteins Grx3/Grx4, along with GUA1 (S. cerevisiae GMPs), also downregulate the integrated stress response (ISR) pathway. The heterologous expression of Glrx3/hGMPs efficiently complements Grx3/Grx4. Moreover, the heterologous expression of Glrx3 efficiently complements the novel participation in chronological life span that has been characterized for both Grx3 and Grx4. Our results underscore that the Glrx3/Grx3/Grx4 family presents an evolutionary and functional conservation in signaling events that is partly related to GMP function and contributes to cell life extension.IMPORTANCESaccharomyces cerevisiae is an optimal eukaryotic microbial model to study biological processes in higher organisms despite the divergence in evolution. The molecular function of yeast glutaredoxins Grx3 and Grx4 is enormously interesting, since both proteins are required to maintain correct iron homeostasis and an efficient response to oxidative stress. The human orthologous Glrx3 (PICOT) is involved in a number of human diseases, including cancer. Our research expanded its utility to human cells. Yeast has allowed the characterization of GMP synthase as a new interacting partner for Glrx3 and also for yeast Grx3 and Grx4, the complex monothiol glutaredoxins/GMPs that participate in the downregulation of the activity of the Gcn2 stress pathway. This mechanism is conserved in yeast and humans. Here, we also show that this family of glutaredoxins, Grx3/Grx4/Glrx3, also has a function related to life extension.

Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival.

Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.

The MAP kinase Slt2 is involved in vacuolar function and actin remodeling in Saccharomyces cerevisiae mutants affected by endogenous oxidative stress.

Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that grx3 grx4 and grx5 mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the grx3 grx4 mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of SLT2 in the grx3 grx4 mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the grx3 grx4 slt2 triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in sod1, grx5, and grx3 grx4 slt2 mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.

Mtl1 O-mannosylation mediated by both Pmt1 and Pmt2 is important for cell survival under oxidative conditions and TOR blockade.

Mtl1 is a cell surface sensor and member of the Pkc1-MAPK pathway that senses oxidative stress and nutrient starvation. Here we demonstrate that the Mtl1 cytoplasmic domain physically interacts with the GEF (GTPase Exchange Factor) protein Rom2 of the CWI (Cell wall Integrity) pathway. Mtl1 is N-glycosylated protein, highly O-mannosylated by Pmt1, Pmt4 and mostly by Pmt2. Mtl1 localises to the bud, septum, the tip of the shmoo and the cell periphery. The O-mannosylation deficiency that occurs in both the pmt1 and pmt2 mutants adversely affects the distribution of Mtl1 on the septum and also hinders Mtl1 localisation in the tip of the shmoo. Here we present results demonstrating that: (i) O-mannosylation and, more specifically that affecting Mtl1 protein is required for cell survival in response to both oxidative stress and TOR blockade; (ii) Slt2 activity is impaired upon rapamycin treatment in both pmt2 and mtl1 mutants; (iii) Mtl1 is transcriptionally upregulated in quiescent conditions, (iv) O-mannosylation mediated by Pmt1 and Pmt2 favours Mtl1 protein stability. We propose a relevant role for Mtl1 O-mannosylation mediated by both Pmt1 and Pmt2 in the response to oxidative stress and in rapamycin treatment.

Both human and soya bean ferritins highly improve the accumulation of bioavailable iron and contribute to extend the chronological life in budding yeast.

Ferritin proteins have an enormous capacity to store iron in cells. In search for the best conditions to accumulate and store bioavailable iron, we made use of a double mutant null for the monothiol glutaredoxins GRX3 and GRX4. The strain grx3grx4 accumulates high iron concentrations in the cytoplasm, making the metal easily available for ferritin chelation. Here, we perform a comparative study between human (L and H) and soya bean ferritins (H1 and H2) function in the eukaryotic system Saccharomyces cerevisiae. We demonstrate that the four human and soya bean ferritin chains are successfully expressed in our model system. Upon coexpression of either both human or soya bean ferritin chains, respiratory conditions along with iron supplementation led us to obtain the maximum yields of iron stored in yeast described to date. Human and soya bean ferritin chains are functional and present equivalent properties as promoters of cell survival in iron overload conditions. The best system revealed that the four human and soya bean ferritins possess a novel function as anti-ageing proteins in conditions of iron excess. In this respect, both ferritin chains with oxidoreductase capacity (human-H and soya bean-H2) bear the highest capacity to extend life suggesting the possibility of an evolutionary conservation.

Pkc1 and actin polymerisation activities play a role in ribosomal gene repression associated with secretion impairment caused by oxidative stress.

In Saccharomyces cerevisiae, the cell integrity pathway plays a role in the oxidative stress response. In this study, we show that the Pkc1 protein mediates oxidative signalling by helping to downregulate ribosomal gene expression when cells are exposed to hydrogen peroxide. An active actin cytoskeleton is required for this function, because the cells blocked in actin polymerisation were unable to repress ribosomal gene transcription. Following the invertase secretion pattern, we hypothesize that oxidative stress induced by hydrogen peroxide could have affected the latter steps of secretion. This would explain why the Pkc1 function was required to repress ribosomal biogenesis.

The MAPK Slt2/Mpk1 plays a role in iron homeostasis through direct regulation of the transcription factor Aft1.

Iron is an essential element for life. Cells develop mechanisms to tightly regulate its homeostasis, in order to avoid abnormal accumulation and the consequent cell toxicity. In budding yeast, the high affinity iron regulon is under the control of the transcription factor Aft1. We present evidence demonstrating that the MAPK Slt2 of the cell wall integrity pathway (CWI), phosphorylates and negatively regulates Aft1 activity upon the iron depletion signal, both in fermentative or respiratory conditions. The lack of Slt2 provokes Aft1 dysfunction leading to a shorter chronological life span. The signal of iron scarcity is not transmitted to Slt2 through other signalling pathways such as TOR1, PKA, SNF1 or TOR2/YPK1. The observation that Slt2 physically binds Aft1 rather suggests a direct regulation.

The Cell Wall Integrity Receptor Mtl1 Contributes to Articulate Autophagic Responses When Glucose Availability Is Compromised.

Mtl1protein is a cell wall receptor belonging to the CWI pathway. Mtl1 function is related to glucose and oxidative stress signaling. In this report, we show data demonstrating that Mtl1 plays a critical role in the detection of a descent in glucose concentration, in order to activate bulk autophagy machinery as a response to nutrient deprivation and to maintain cell survival in starvation conditions. Autophagy is a tightly regulated mechanism involving several signaling pathways. The data here show that in Saccharomyces cerevisiae, Mtl1 signals glucose availability to either Ras2 or Sch9 proteins converging in Atg1 phosphorylation and autophagy induction. TORC1 complex function is not involved in autophagy induction during the diauxic shift when glucose is limited. In this context, the GCN2 gene is required to regulate autophagy activation upon amino acid starvation independent of the TORC1 complex. Mtl1 function is also involved in signaling the autophagic degradation of mitochondria during the stationary phase through both Ras2 and Sch9, in a manner dependent on either Atg33 and Atg11 proteins and independent of the Atg32 protein, the mitophagy receptor. All of the above suggest a pivotal signaling role for Mtl1 in maintaining correct cell homeostasis function in periods of glucose scarcity in budding yeast.

Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their Trx domains, which contributes to oxidative stress resistance.

Grx3 and Grx4 are two monothiol glutaredoxins of Saccharomyces cerevisiae that have previously been characterized as regulators of Aft1 localization and therefore of iron homeostasis. In this study, we present data showing that both Grx3 and Grx4 have new roles in actin cytoskeleton remodeling and in cellular defenses against oxidative stress caused by reactive oxygen species (ROS) accumulation. The Grx4 protein plays a unique role in the maintenance of actin cable integrity, which is independent of its role in the transcriptional regulation of Aft1. Grx3 plays an additive and redundant role, in combination with Grx4, in the organization of the actin cytoskeleton, both under normal conditions and in response to external oxidative stress. Each Grx3 and Grx4 protein contains a thioredoxin domain sequence (Trx), followed by a glutaredoxin domain (Grx). We performed functional analyses of each of the two domains and characterized different functions for them. Each of the two Grx domains plays a role in ROS detoxification and cell viability. However, the Trx domain of each Grx4 and Grx3 protein acts independently of its respective Grx domain in a novel function that involves the polarization of the actin cytoskeleton, which also determines cell resistance against oxidative conditions. Finally, we present experimental evidence demonstrating that Grx4 behaves as an antioxidant protein increasing cell survival under conditions of oxidative stress.

Two proteins from Saccharomyces cerevisiae: Pfy1 and Pkc1, play a dual role in activating actin polymerization and in increasing cell viability in the adaptive response to oxidative stress.

In this work, we show that the proteins Pkc1 and Pfy1 play a role in the repolarization of the actin cytoskeleton and in cell survival in response to oxidative stress. We have also developed an assay to determine the actin polymerization capacity of total protein extracts using fluorescence recovery after photobleaching techniques and actin purified from rabbit muscle. This assay allowed us to demonstrate that Pfy1 promotes actin polymerization under conditions of oxidative stress, while Pkc1 induces actin polymerization and cell survival under all the conditions tested. Our assay also points to a relationship between Pkc1 and Pfy1 in the actin cytoskeleton polymerization that is required to adapt to oxidative stress.

A genome-wide transcriptional study reveals that iron deficiency inhibits the yeast TORC1 pathway.

Iron is an essential micronutrient that participates as a cofactor in a broad range of metabolic processes including mitochondrial respiration, DNA replication, protein translation and lipid biosynthesis. Adaptation to iron deficiency requires the global reorganization of cellular metabolism directed to optimize iron utilization. The budding yeast Saccharomyces cerevisiae has been widely used to characterize the responses of eukaryotic microorganisms to iron depletion. In this report, we used a genomic approach to investigate the contribution of transcription rates to the modulation of mRNA levels during adaptation of yeast cells to iron starvation. We reveal that a decrease in the activity of all RNA polymerases contributes to the down-regulation of many mRNAs, tRNAs and rRNAs. Opposite to the general expression pattern, many genes including components of the iron deficiency response, the mitochondrial retrograde pathway and the general stress response display a remarkable increase in both transcription rates and mRNA levels upon iron limitation, whereas genes encoding ribosomal proteins or implicated in ribosome biogenesis exhibit a pronounced fall. This expression profile is consistent with an activation of the environmental stress response. The phosphorylation stage of multiple regulatory factors strongly suggests that the conserved nutrient signaling pathway TORC1 is inhibited during the progress of iron deficiency. These results suggest an intricate crosstalk between iron metabolism and the TORC1 pathway that should be considered in many disorders.

Coping with oxidative stress. The yeast model.

Saccharomyces cerevisiae is an optimal model to study stress responses for various reasons: i) budding yeast genome presents a high degree of homology with the human genome; ii) there are many proteins that show an elevated functional homology with specific human proteins; iii) it is a system whose genetic manipulation is reasonably easy and cheaper than other models; iv) the possibility of working with an haploid state facilitates the study of multiple processes; v) databases are the most complete of all the eukaryotic models. Due to the latest information derived from proteomic and genomic analyses, the genetic, biochemical and molecular information available relative to this biological system is extraordinarily big and complete. In this review, we present an overview of the mechanisms unravelling sensing and transducing oxidative stress. TOR, RAS/PKA, CWI, SNF1, and HOG are the main pathways involved both in the oxidative response and in the correct entry in stationary phase. In general, TOR and RAS/PKA dowregulation and SNF1 and CWI upregulation favour both a correct defence against oxidative damage and the entry in the quiescent state. All of these pathways have counterparts in humans. The actin cytoskeleton plays a dual function as sensor and target of oxidation, in tight connection with the former signalling cascades. In budding yeast, progression through stationary phase and quiescence constitute an accepted current model to study some of the mechanisms that determine life span. Aging is a process associated to oxidative stress and it is in tight relationship with bulk autophagy and mitophagy, both are mechanisms belonging to the oxidative defence and promoters of life extension when correctly regulated by, among other elements, the signalling cascades.

Signal flow between CWI/TOR and CWI/RAS in budding yeast under conditions of oxidative stress and glucose starvation.

The CWI pathway cross-talks with TOR and RAS in both the oxidative and glucose starvation responses. Mtl1 is the cell-wall protein in charge of sensing and regulating this response. Rom2 and Rho1, which are the upper elements in the pathway, mediate this signal. Several outputs are involved and required for this response, one of which, ribosomal gene expression, seems to be regulated by Sfp1, amongst other possible transcription factors. Moreover, cross-talk also occurs in a reverse flow from TOR and RAS to the CWI pathway. Thus Tor1 and Ras2 inhibition also activates Slt2 in the absence of the Mtl1 protein and assures the proper adaptive response to oxidation and glucose deprivation.

How budding yeast sense and transduce the oxidative stress signal and the impact in cell growth and morphogenesis.

The eukaryotic microorganism Saccharomyces cerevisiae is a current model system in which to study the signal transduction pathways involved in the oxidative stress response. In this review we present the current evidence demonstrating that in S. cerevisiae several MAPK and signalling routes participate in this response (PKC1-MAPK, TOR, RAS-PKA-cAMP). The signalling processes converge in the activation of a number of transcription factors (Yap1, Skn7, Rlm1, Msn2/Msn4, Sfp1, among others) required for the expression of certain genes involved in the oxidative stress response. Another important output of these signalling pathways is the actin cytoskeleton, a known target for oxidation and whose organisation needs to be tightly controlled since it is essential for the integrity of the cell. We know about the existence of different levels of cross-talk between these signalling pathways, which gives strength to the enormous importance of keeping a correct redox homeostasis in cells. S cerevisiae maintains a safeguard mechanism assuring that cells always respond properly to oxidation, by means of mechanisms described in the current review.

Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae.

Grx3 and Grx4, two monothiol glutaredoxins of Saccharomyces cerevisiae, regulate Aft1 nuclear localisation. We provide evidence of a negative regulation of Aft1 activity by Grx3 and Grx4. The Grx domain of both proteins played an important role in Aft1 translocation to the cytoplasm. This function was not, however, dependent on the availability of iron. Here we demonstrate that Grx3, Grx4 and Aft1 interact each other both in vivo and in vitro, which suggests the existence of a functional protein complex. Interestingly, each interaction occurred independently on the third member of the complex. The absence of both Grx3 and Grx4 induced a clear enrichment of G1 cells in asynchronous cultures, a slow growth phenotype, the accumulation of intracellular iron and a constitutive activation of the genes regulated by Aft1. The grx3grx4 double mutant was highly sensitive to the oxidising agents hydrogen peroxide and t-butylhydroperoxide but not to diamide. The phenotypes of the double mutant grx3grx4 characterised in this study were mainly mediated by the Aft1 function, suggesting that grx3grx4 could be a suitable cellular model for studying endogenous oxidative stress induced by deregulation of the iron homeostasis. However, our results also suggest that Grx3 and Grx4 might play additional roles in the oxidative stress response through proteins other than Aft1.

Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress.

In this study we analyze the participation of the PKC1-MAPK cell integrity pathway in cellular responses to oxidative stress in Saccharomyces cerevisiae. Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidizing agents, diamide and hydrogen peroxide. Mtl1 is characterized for the first time as a cell-wall sensor of oxidative stress. We also show that the actin cytoskeleton is a cellular target for oxidative stress. Both diamide and hydrogen peroxide provoke a marked depolarization of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restore the correct actin organization. Diamide induces the formation of disulfide bonds in newly secreted cell-wall proteins. This mainly provokes structural changes in the cell outer layer, which activate the PKC1-MAPK pathway and hence the protein kinase Slt2. Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the machinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation.

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast.

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).