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Background: Many different stress signaling pathways converge in a common response: slowdown or arrest cell cycle in the G1 phase. The G1/S transition (called Start in budding yeast) is a key checkpoint controlled by positive and negative regulators. Among them, Whi7 and Whi5 are transcriptional repressors of the G1/S transcriptional program, yeast functional homologs of the Retinoblastoma family proteins in mammalian cells. Under standard conditions, Whi7 plays a lesser role than Whi5 in Start inhibition. However, under cell wall stress, Whi7 is induced and plays a more important role in G1/S control. In this work, we investigated the functional hallmarks of Whi7 and Whi5, which determine their strength as Start inhibitors under cell wall stress. Methods: The response of Saccharomyces cerevisiae to Calcofluor White was investigated to characterize the regulation and function of Whi7 and Whi5 under cell wall stress. To control their protein levels, we used dose-dependent β-estradiol-induced expression and auxin-induced degron protein fusions. We also performed Chromatin Immunoprecipitation assays to investigate Whi7 and Whi5 association with Start promoters and scored cell cycle arrest and re-entry using cell microscopy assays. Results: We found that cell wall stress promoted the specific upregulation of the Whi7 Start repressor. First, although cell wall stress increases Whi7 protein levels, this is not the only determinant behind the Whi7 function in promoting G1 arrest. Indeed, artificial induction of Whi5 at the same protein level resulted in a lower G1 block. Second, under cell wall stress, Whi7 was specifically recruited to SBF-target promoters, independent of the increase in its protein levels or cell cycle stage. Finally, we found that Whi7 protein instability further increased during cell wall stress and that Whi7 degradation triggered advanced cell cycle re-entry. Conclusions: Here, we show that cell wall stress signaling specifically enhances Whi7 function as a Start transcriptional repressor. Importantly, we identified new Whi7-specific regulatory mechanisms that do not operate in the Whi5 repressor. Our results indicate that cells may benefit from stress-specific repressors to ensure the stress-induced G1 arrest and that Whi7 rapid degradation may be particularly important to resume cell cycle upon adaptation. [ABSTRACT FROM AUTHOR]

Phosphoribosyl pyrophosphate synthetase (PRS EC 2.7.6.1) is a rate-limiting enzyme that irreversibly catalyzes the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate (ATP). This key metabolite is required for the synthesis of purine and pyrimidine nucleotides, the two aromatic amino acids histidine and tryptophan, the cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), all of which are essential for various life processes. Despite its ubiquity and essential nature across the plant and animal kingdoms, PRPP synthetase displays species-specific characteristics regarding the number of gene copies and architecture permitting interaction with other areas of cellular metabolism. The impact of mutated PRS genes in the model eukaryote Saccharomyces cerevisiae on cell signalling and metabolism may be relevant to the human neuropathies associated with PRPS mutations. Human PRPS1 and PRPS2 gene products are implicated in drug resistance associated with recurrent acute lymphoblastic leukaemia and progression of colorectal cancer and hepatocellular carcinoma. The investigation of PRPP metabolism in accepted model organisms, e.g., yeast and zebrafish, has the potential to reveal novel drug targets for treating at least some of the diseases, often characterized by overlapping symptoms, such as Arts syndrome and respiratory infections, and uncover the significance and relevance of human PRPS in disease diagnosis, management, and treatment. [ABSTRACT FROM AUTHOR]

Filamentous fungi maintain hyphal growth to continually internalize membrane proteins related to cell wall synthesis, transporting them to the hyphal tips. Endocytosis mediates protein internalization via target recognition by the adaptor protein 2 complex (AP-2 complex). The AP-2 complex specifically promotes the internalization of proteins important for hyphal growth, and loss of AP-2 complex function results in abnormal hyphal growth. In this study, deletion mutants of the genes encoding the subunits of the AP-2 complex (α, β2, μ2, or σ2) in the filamentous fungus Aspergillus nidulans resulted in the formation of conidiophores with abnormal morphology, fewer conidia, and activated the cell wall integrity pathway. We also investigated the localization of ChsB, which plays pivotal roles in hyphal growth in A. nidulans , in the Δμ2 strain. Quantitative analysis suggested that the AP-2 complex is involved in ChsB internalization at subapical collar regions. The absence of the AP-2 complex reduced ChsB localization at the hyphal tips. Our findings suggest that the AP-2 complex contributes to cell wall integrity by properly localizing ChsB to the hyphal tips. • The AP-2 complex recognizes a part of endocytic cargos. • Deletion of the AP-2 complex leads to diverse phenotypes in Aspergillus nidulans. • The cell wall integrity pathway is activated in the deletion mutants. • The AP-2 complex promotes the hyphal-tip-localization of ChsB. [ABSTRACT FROM AUTHOR]

Two stress-activated MAP kinase (SAPK) pathways in Saccharomyces cerevisiae respond to osmotic imbalances. The High Osmolarity Glycerol (HOG) pathway is activated in response to hyper-osmotic stress, whereas the Cell Wall Integrity (CWI) pathway is activated in response to hypo-osmotic stress. However, there is also evidence of complex interplay and crosstalk between the two pathways. For example, treatment with zymolyase, a mixture of cell wall degrading enzymes, is known to activate the SAPK Hog1 of the HOG pathway and the SAPK Mpk1 of the CWI pathway sequentially, with Mpk1 activation dependent upon Hog1. Additionally, the PTP2 - and PTP3 -encoded tyrosine-specific protein phosphatases play a key role in down-regulation of Hog1, but may also down-regulate Mpk1. In this study, we show that hyperactivation of Mpk1 in a ptp2 ptp3 null mutant is an indirect consequence of Hog1 hyperactivation, which induces accumulation of intracellular glycerol and an attendant hypo-osmotic stress. Mpk1 hyperactivity in the absence of PTP2 and PTP3 was suppressed by a hog1 null mutation, or by restoration of osmotic balance with a constitutive form of the glycerol channel Fps1. We found similarly that activation of Mpk1 in response to zymolyase treatment is partly a consequence of Hog1-driven glycerol accumulation. Thus, we have identified two conditions in which glycerol serves as a mediator of crosstalk between the HOG and CWI pathways. [ABSTRACT FROM AUTHOR]

Slt2, the MAPK of the cell wall integrity (CWI) pathway, connects different signaling pathways and performs different functions in the protective response of S. cerevisiae to stress. Previous work has evidenced the relation of the CWI pathway and the unfolded protein response (UPR), a transcriptional program activated upon endoplasmic reticulum (ER) stress. However, the mechanisms of crosstalk between these pathways and the targets regulated by Slt2 under ER stress remain unclear. Here, we demonstrated that ectopic expression of GFA1, the gene encoding the first enzyme in the synthesis of UDP-GlcNAc by the hexosamine biosynthetic pathway (HBP) or supplementation of the growth medium with glucosamine (GlcN), increases the tolerance of slt2 mutant cells to different ER-stress inducers. Remarkably, GlcN also alleviates the sensitivity phenotype of cells lacking IRE1 or HAC1, the main actors in controlling the UPR. The exogenous addition of GlcN reduced the abundance of glycosylated proteins and triggered autophagy. We also found that TORC1, the central stress and growth controller, is inhibited by tunicamycin exposure in cells of the wild-type strain but not in those lacking Slt2. Consistent with this, the tunicamycin-induced activation of autophagy and the increased synthesis of ATP in response to ER stress were absent by knock-out of SLT2. Altogether, our data placed Slt2 as an essential actor of the ER stress response by regulating the HBP activity and the TORC1-dependent signaling.

The Saccharomyces cerevisiae cell wall integrity (CWI) pathway took this name when its role in the cell response to cell wall aggressions was clearly established. The receptors involved in sensing the damage, the relevant components operating in signaling to the MAPK Slt2, the transcription factors activated by this MAPK, as well as some key regulatory mechanisms have been identified and characterized along almost 30 years. However, other stimuli that do not alter specifically the yeast cell wall, including protein unfolding, low or high pH, or plasma membrane, oxidative and genotoxic stresses, have been also found to trigger the activation of this pathway. In this review, we compile almost forty non-cell wall-specific compounds or conditions, such as tunicamycin, hypo-osmotic shock, diamide, hydroxyurea, arsenate, and rapamycin, which induce these stresses. Relevant aspects of the CWI-mediated signaling in the response to these non-conventional pathway activators are discussed. The data presented here highlight the central and key position of the CWI pathway in the safeguard of yeast cells to a wide variety of external aggressions. [ABSTRACT FROM AUTHOR]

Ampicillin has been widely used to treat bacterial infections. When we used ampicillin to eliminate bacterial contamination in yeast cultures, we observed induction of phosphorylation of MAP kinase 1 (Mpk1), a previously unknown function of ampicillin. We therefore investigated whether ampicillin activates the signal transduction pathway. Phosphorylation of Saccharomyces cerevisiae Mpk1 was induced by ampicillin in a dose- and time-dependent manner through the PKC1-CWI pathway. Mpk1 phosphorylation was maximal after treatment with 3 mM ampicillin for 90 min. Despite activation of Mpk1 phosphorylation, ampicillin did not influence yeast cell growth. Ampicillin reduced miconazole antifungal activity; miconazole had a minimum inhibitory concentration of 3.125 μg/mL against Candida albicans, which increased to 25 μg/mL after 48 h of treatment with 3 mM ampicillin. Finally, ampicillin activated phosphorylation of ERK1/2 (a mammalian homolog of Mpk1), with maximum effect at 3 mM ampicillin, in human HepG2 cells, but did not influence cell viability. The results of this study clearly indicate that ampicillin activated Mpk1 phosphorylation in yeast and ERK1/2 phosphorylation in HepG2 cells. In addition to its clinical application to eliminate bacteria, ampicillin could also be used to activate Mpk1 or ERK1/2 in the laboratory. [ABSTRACT FROM AUTHOR]

Highlights: [•] The main cell-wall digesting enzymes of zymolyase were individually inhibited. [•] β-1,3-Glucanase and protease activities are required for CWI MAPK pathway activation. [•] Hkr1 mucin is essential for signaling cell-wall damage caused by zymolyase in yeast. [Copyright &y& Elsevier]

Living cells exposed to stressful environmental situations can elicit cellular responses that guarantee maximal cell survival. Most of these responses are mediated by mitogen-activated protein kinase (MAPK) cascades, which are highly conserved from yeast to humans. Cell wall damage conditions in the yeast Saccharomyces cerevisiae elicit rescue mechanisms mainly associated with reprogramming specific transcriptional responses via the cell wall integrity (CWI) pathway. Regulation of gene expression by this pathway is coordinated by the MAPK Slt2/Mpk1, mainly via Rlm1 and, to a lesser extent, through SBF (Swi4/Swi6) transcription factors. In this review, we summarize the molecular mechanisms controlling gene expression upon cell wall stress and the role of chromatin structure in these processes. Some of these mechanisms are also discussed in the context of other stresses governed by different yeast MAPK pathways. Slt2 regulates both transcriptional initiation and elongation by interacting with chromatin at the promoter and coding regions of CWI-responsive genes but using different mechanisms for Rlm1- and SBF-dependent genes. Since MAPK pathways are very well conserved in eukaryotic cells and are essential for controlling cellular physiology, improving our knowledge regarding how they regulate gene expression could impact the future identification of novel targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Slt2, the MAPK of the cell wall integrity (CWI) pathway, connects different signaling pathways and performs different functions in the protective response of S. cerevisiae to stress. Previous work has evidenced the relation of the CWI pathway and the unfolded protein response (UPR), a transcriptional program activated upon endoplasmic reticulum (ER) stress. However, the mechanisms of crosstalk between these pathways and the targets regulated by Slt2 under ER stress remain unclear. Here, we demonstrated that ectopic expression of GFA1, the gene encoding the first enzyme in the synthesis of UDP-GlcNAc by the hexosamine biosynthetic pathway (HBP) or supplementation of the growth medium with glucosamine (GlcN), increases the tolerance of slt2 mutant cells to different ER-stress inducers. Remarkably, GlcN also alleviates the sensitivity phenotype of cells lacking IRE1 or HAC1, the main actors in controlling the UPR. The exogenous addition of GlcN reduced the abundance of glycosylated proteins and triggered autophagy. We also found that TORC1, the central stress and growth controller, is inhibited by tunicamycin exposure in cells of the wild-type strain but not in those lacking Slt2. Consistent with this, the tunicamycin-induced activation of autophagy and the increased synthesis of ATP in response to ER stress were absent by knock-out of SLT2. Altogether, our data placed Slt2 as an essential actor of the ER stress response by regulating the HBP activity and the TORC1-dependent signaling. [ABSTRACT FROM AUTHOR]

Growing numbers of nanotoxicity research demonstrating that mechanical damage and oxidative stress are potential modes of nanoparticles (NPs) induced toxicity. However, the underlying mechanisms by which NPs interact with the eukaryotic cell and affect their physiological and metabolic functions are not fully known. We investigated the toxic effects of zinc oxide nanoparticles (ZnO-NPs) on budding yeast, Saccharomyces cerevisiae and elucidated the underlying mechanism. We observed cell wall damage and accumulation of reactive oxygen species (ROS) leading to cell death upon ZnO-NPs exposure. We detected a significant change in the cellular distribution of lipid biosynthetic enzymes (Fas1 and Fas2). Furthermore, exposure of ZnO-NPs altered the architecture of endoplasmic reticulum (ER) and mitochondria as well as ER-mitochondria encounter structure (ERMES) complex causing cellular toxicity due to lipid disequilibrium and proteostasis. We also observed significant changes in heat shock and unfolded protein responses, monitored by Hsp104-GFP localization and cytosolic Hac1 splicing respectively. Moreover, we observed activation of MAP kinases of CWI (Mpk1) and HOG (Hog1) pathways upon exposure to ZnO-NPs. Transcript level analyses showed induction of chitin synthesis and redox homeostasis genes. Finally, we observed induction in lipid droplets (LDs) formation, distorted vacuolar morphology and induction of autophagy as monitored by localization of Atg8p. However, we did not observe any significant change in epigenetic marks, examined by western blotting. Altogether, we provide evidence that exposure of ZnO-NPs results in cell death by affecting cell wall integrity and ER homeostasis as well as accumulation of ROS and saturated free fatty acids., (Copyright © 2018 Elsevier Ltd. All rights reserved.)