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.

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.

Mtl1 is required to activate general stress response through Tor1 and Ras2 inhibition under conditions of glucose starvation and oxidative stress.

Mtl1 is a member of the cell wall integrity (CWI) pathway of Saccharomyces cerevisiae, which functions as a cell wall sensor for oxidative stress. Genome-wide transcriptional analysis revealed a cluster of genes that were down-regulated in the absence of Mtl1. Many of these genes were potentially regulated by the general stress response factor Msn2/Msn4. In response to rapamycin, caffeine, glucose starvation and oxidative stress provoked by H(2)O(2), mtl1 presents a significant loss of viability as well as a deficiency in the transcriptional response mediated by Msn2/Msn4. The Mtl1 function was required (i) to induce ribosomal gene repression, (ii) to induce the general stress response driven by the transcription factor Msn2/Msn4, and (iii) to activate the CWI pathway in response to both glucose starvation and oxidative stress. We also detected higher cAMP levels in the mtl1 mutant than in wild type cells indicative of up-regulated RAS2-PKA activity. Disruption of TOR1, disruption of RAS2, or hyperactivation of Rho1 restored both the viability and the transcriptional function (both ribosomal and Msn2/Msn4-dependent gene expression) in the mtl1 mutant to almost wild type levels when cells were starved of glucose or stressed with H(2)O(2). Taking our results together, we propose an essential role for Mtl1 in signaling oxidative stress and quiescence to the CWI pathway and to the general stress response through Rho1 and the inhibition of either the TOR1 or RAS2 functions. These mechanisms would be required to allow cells to adapt to both oxidative and nutritional stresses.

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.

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.

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.

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins.

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.