Stress Response In Yeast Research Articles

The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

Differential stability of Gcn4p controls its cell-specific activity in differentiated yeast colonies.

Gcn4p belongs to conserved AP-1 transcription factors involved in many cellular processes, including cell proliferation, stress response, and nutrient availability in yeast and mammals. AP-1 activities are regulated at different levels, such as translational activation or protein degradation, which increases the variability of regulation under different conditions. Gcn4p activity in unstructured yeast liquid cultures increases upon amino acid deficiency and is rapidly eliminated upon amino acid excess. Gcn2p kinase is the major described regulator of Gcn4p that enables GCN4 mRNA translation via the uORFs mechanism. Here, we show that Gcn4p is specifically active in U cells in the upper regions and inactive in L cells in the lower regions of differentiated colonies. Using in situ microscopy in combination with analysis of mutants and strains with GFP at different positions in the translational regulatory region of Gcn4p, we show that cell-specific Gcn4p activity is independent of Gcn2p or other translational or transcriptional regulation. Genetically, biochemically, and microscopically, we identified cell-specific proteasomal degradation as a key mechanism that diversifies Gcn4p function between U and L cells. The identified regulation leading to active Gcn4p in U cells with amino acids and efficient degradation in starved L cells differs from known regulations of Gcn4p in yeast but shows similarities to the activity of AP-1 ATF4 in mammals during insulin signaling. These findings may open new avenues for understanding the parallel activities of Gcn4p/ATF4 and reveal a novel biological role for cell type-specific regulation of proteasome-dependent degradation.IMPORTANCEIn nature, microbes usually live in spatially structured communities and differentiate into precisely localized, functionally specialized cells. The coordinated interplay of cells and their response to environmental changes, such as starvation, followed by metabolic adaptation, is critical for the survival of the entire community. Transcription factor Gcn4p is responsible for yeast adaptation under amino acid starvation in liquid cultures, and its activity is regulated mainly at the level of translation involving Gcn2p kinase. Whether Gcn4p functions in structured communities was unknown. We show that translational regulation of Gcn4p plays no role in the development of colony subpopulations; the main regulation occurs at the level of stabilization of the Gcn4p molecule in the cells of one subpopulation and its proteasomal degradation in the other. This regulation ensures specific spatiotemporal activity of Gcn4p in the colony. Our work highlights differences in regulatory networks in unorganized populations and organized structures of yeast, which in many respects resemble multicellular organisms.

Improvement of cell growth in green algae Chlamydomonas reinhardtii through co-cultivation with yeast Saccharomyces cerevisiae.

CO2 fixation methods using green algae have attracted considerable attention because they can be applied for the fixation of dilute CO2 in the atmosphere. However, green algae generally exhibit low CO2 fixation efficiency under atmospheric conditions. Therefore, it is a challenge to improve the CO2 fixation efficiency of green algae under atmospheric conditions. Co-cultivation of certain microalgae with heterotrophic microorganisms can increase the growth potential of microalgae under atmospheric conditions. The objective of this study was to determine the culture conditions under which the growth potential of green algae Chlamydomonas reinhardtii is enhanced by co-culturing with the yeast Saccharomyces cerevisiae, and to identify the cause of the enhanced growth potential. When C. reinhardtii and S. cerevisiae were co-cultured with an initial green algae to yeast inoculum ratio of 1:3, the cell concentration of C. reinhardtii reached 133 × 105cells/mL on day 18 of culture, which was 1.5 times higher than that of the monoculture. Transcriptome analysis revealed that the expression levels of 363 green algae and 815 yeast genes were altered through co-cultivation. These included genes responsible for ammonium transport and CO2 enrichment mechanism in green algae and the genes responsible for glycolysis and stress responses in yeast. We successfully increased C. reinhardtiigrowth potential by co-culturing it with S. cerevisiae. The main reasons for this are likely to be an increase in inorganic nitrogen available to green algae via yeast metabolism and an increase in energy available for green algae growth instead of CO2 enrichment.

Sse1, Hsp110 chaperone of yeast, controls the cellular fate during endoplasmic reticulum stress.

Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as a nucleotide exchange factor (NEF), as a protein-disaggregase and as a chaperone linked to protein synthesis (CLIPS) are well documented. In the current study, we show that SSE1 genetically interacts with IRE1 and HAC1, the endoplasmic reticulum-unfolded protein response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, the absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signaling. Furthermore, cells lacking SSE1 show inefficient ER-stress-responsive reorganization of translating ribosomes from polysomes to monosomes that drive uninterrupted protein translation during tunicamycin stress. In consequence, the sse1Δ strain shows prominently faster reversal from ER-UPR activated state indicating quicker restoration of homeostasis, in comparison to the wild-type (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress-mediated cell division arrest, which is escaped in sse1Δ strain during chronic tunicamycin stress. Accordingly, sse1Δ strain shows significantly higher cell viability in comparison to WT yeast imparting the stark fitness following short-term as well as long-term tunicamycin stress. These data, all together, suggest that cytosolic chaperone Sse1 is an important modulator of ER stress response in yeast and it controls stress-induced cell division arrest and cell death during overwhelming ER stress induced by tunicamycin.

Differential Protein Expression in Set5p-Mediated Acetic Acid Stress Response and Novel Targets for Engineering Yeast Stress Tolerance.

Acetic acid is a prevalent inhibitor in lignocellulosic hydrolysate, which represses microbial growth and bioproduction. Histone modification and chromatin remodeling have been revealed to be critical for regulating eukaryotic metabolism. However, related studies in chronic acetic acid stress responses remain unclear. Our previous studies revealed that overexpression of the histone H4 methyltransferase Set5p enhanced acetic acid stress tolerance of the budding yeast Saccharomyces cerevisiae. In this study, we examined the role of Set5p in acetic acid stress by analyzing global protein expression. Significant activation of intracellular protein expression under the stress was discovered, and the functions of the differential proteins were mainly involved in chromatin modification, signal transduction, and carbohydrate metabolism. Notably, a substantial increase of Set5p expression was observed in response to acetic acid stress. Functional studies demonstrated that the restriction of the telomere capping protein Rtc3p, as well as Ies3p and Taf14p, which are related to chromatin regulation, was critical for yeast stress response. This study enriches the understanding of the epigenetic regulatory mechanisms underlying yeast stress response mediated by histone-modifying enzymes. The results also benefit the development of robust yeast strains for lignocellulosic bioconversion.

Replication stress response in fission yeast differentially depends on maintaining proper levels of Srs2 helicase and Rrp1, Rrp2 DNA translocases.

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome.

Transcriptomic changes in single yeast cells under various stress conditions

BackgroundThe stress response of Saccharomyces cerevisiae has been extensively studied in the past decade. However, with the advent of recent technology in single-cell transcriptome profiling, there is a new opportunity to expand and further understanding of the yeast stress response with greater resolution on a system level. To understand transcriptomic changes in baker’s yeast S. cerevisiae cells under stress conditions, we sequenced 117 yeast cells under three stress treatments (hypotonic condition, glucose starvation and amino acid starvation) using a full-length single-cell RNA-Seq method.ResultsWe found that though single cells from the same treatment showed varying degrees of uniformity, technical noise and batch effects can confound results significantly. However, upon careful selection of samples to reduce technical artifacts and account for batch-effects, we were able to capture distinct transcriptomic signatures for different stress conditions as well as putative regulatory relationships between transcription factors and target genes.ConclusionOur results show that a full-length single-cell based transcriptomic analysis of the yeast may help paint a clearer picture of how the model organism responds to stress than do bulk cell population-based methods.

BrCYP71A15 Negatively Regulates Hg Stress Tolerance by Modulating Cell Wall Biosynthesis in Yeast

Over the past two decades, heavy metal pollution has been a common problem worldwide, greatly threatening crop production. As one of the metal pollutants, Mercury (Hg) causes damage to plant cells and reduces cellular and biochemical activities. In this study, we identified a novel cytochrome P450 family gene, BrCYP71A15, which was involved in Hg stress response in yeast. In Chinese cabbage, the BrCYP71A15 gene was located on chromosome A01, which was highly expressed in roots. Additionally, the expression level of BrCYP71A15 was induced by different heavy metal stresses, and the BrCYP71A15 protein exhibited a strong interaction with other proteins. Overexpression of BrCYP71A15 in yeast cells showed no response to a number of heavy metal stresses (Cu, Al, Co, Cd) in yeast but showed high sensitivity to Hg stress; the cells grew slower than those carrying the empty vector (EV). Moreover, upon Hg stress, the growth of the BrCYP71A15-overexpressing cells increased over time, and Hg accumulation in yeast cells was enhanced by two-fold compared with the control. Additionally, BrCYP71A15 was translocated into the nucleus under Hg stress. The expression level of cell wall biosynthesis genes was significantly influenced by Hg stress in the BrCYP71A15-overexpressing cells. These findings suggested that BrCYP71A15 might participate in HG stress tolerance. Our results provide a fundamental basis for further genome editing research and a novel approach to decrease Hg accumulation in vegetable crops and reduce environmental risks to human health through the food chain.

Iron-sulfur clusters are involved in post-translational arginylation

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

Synthetic activation of yeast stress response improves secretion of recombinant proteins

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies. The availability of powerful genetic engineering and synthetic biology tools has facilitated improvement of this cell factory to overcome certain limitations. However, cell engineering to improve secretion often remains a trial-and-error approach and improvements are often specific to the protein produced. Where multiple genetic interventions are needed to remove bottlenecks in the process of recombinant protein secretion, this leads to a high number of combinatorial possibilities for creation of new production strains. Therefore, our aim was to exploit whole transcriptional programs (stress response pathways) in order to simplify the strain engineering of new production strains. Indeed, the artificial activation of the general stress response transcription factor Msn4, as well as synthetic versions thereof, could replace the secretion enhancing effect of several cytosolic chaperones. Greater than 4-fold improvements in recombinant protein secretion were achieved by overexpression of MSN4 or synMSN4, either alone or in combination with Hac1 or ER chaperones. With this concept we were able to successfully engineer strains reaching titers of more than 2.5 g/L scFv and 8 g/L VHH in bioreactor cultivations. This increased secretion capacity of different industrially relevant model proteins indicates that MSN4 overexpression most likely represents a general concept to improve recombinant protein production in yeast.

Evolutionary analysis of p38 stress-activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress.

p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specificity. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki, a close unicellular relative of animals, follows a phosphorylation-dephosphorylation pattern typical of p38 kinases. At the same time, Capsaspora's cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cell's reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. This study improves our understanding of p38 evolution and opens new perspectives for p38 research.

Model yeast as a versatile tool to examine the antioxidant and anti-ageing potential of flavonoids, extracted from medicinal plants.

The demand for the production of herbal extracts for cosmetics, food, and health supplements, known as plant-based medicine, is rising globally. Incorporating herbal extracts could help to create higher value products due to the functional properties of bioactive compounds. Because the phytochemical composition could vary depending on the processing methods, a simple bioassay of herbal bioactive compounds is an important screening method for the purposes of functional characterization and quality assurance. As a simplified eukaryotic model, yeast serves as a versatile tool to examine functional property of bioactive compounds and to gain better understanding of fundamental cellular processes, because they share similarities with the processes in humans. In fact, aging is a well-conserved phenomenon between yeast and humans, making yeast a powerful genetic tool to examine functional properties of key compounds obtained from plant extracts. This study aimed to apply a well-established model yeast, Saccharomyces cerevisiae, to examine the antioxidant and anti-aging potential of flavonoids, extracted from medicinal plants, and to gain insight into yeast cell adaptation to oxidative stress. Some natural quercetin analogs, including morin, kaempferol, aromadendrin, and steppogenin, protected yeast cells against oxidative stress induced by acetic acid, as shown by decreased cell sensitivity. There was also a reduction in intracellular reactive oxygen species following acetic acid treatment. Using the chronological aging assay, quercetin, morin, and steppogenin could extend the lifespan of wild-type S. cerevisiae by 15%–25%. Consistent with the fact that oxidative stress is a key factor to aging, acetic acid resistance was associated with increased gene expression of TOR1, which encodes a key growth signaling kinase, and MSN2 and MSN4, which encode stress-responsive transcription factors. The addition of the antioxidant morin could counteract this increased expression, suggesting a possible modulatory role in cell signaling and the stress response of yeast. Therefore, yeast represents a versatile model organism and rapid screening tool to discover potentially rejuvenescent molecules with anti-aging and anti-oxidant potential from natural resources and to advance knowledge in the molecular study of stress and aging.

Investigating Gene Regulation by the Tup1-Cyc8 (Ssn6) Complex in the yeast Saccharomyces cerevisiae

The Tup1-Cyc8 (Ssn6) co-repressor complex is a powerful epigenetic regulator of genes in the yeast Saccharomyces cerevisiae. The highly conserved complex brings about a repressive chromatin structure at regulatory regions of its target genes or prevents the recruitment of the factors needed for activation of transcription. The FLO family of genes are repressed by the Tup1-Cyc8 complex, these genes encode the proteins required for flocculation, a stress response in yeast where the cells aggregate, or form flocs, to protect cells within the floc. Interestingly each mutant strain (tup1, cyc8 and tup1 cyc8) has a distinct flocculant phenotype. The tup1 strain displays large, dense flocs compared to smaller, more dispersed flocs associated with the cyc8 strain, whereas the tup1 cyc8 strain displays an intermediate flocculant phenotype. RT-qPCR showed that FLO1, considered to be the dominant member of this family of genes, is highly de-repressed in the tup1 and tup1 cyc8 deletion strains. This suggests that Tup1 makes the dominant contribution to repression of this gene. However, this pattern is not seen at all target genes. The results of RNA-Sequencing show a core set of 429 genes significantly upregulated in all three mutant strains. These genes, on average, show the highest de-repression in the cyc8 and tup1 cyc8 mutant strains. Indicating that Cyc8 makes the dominant contribution to repression at the majority of target genes. Together these results indicate that each of the subunits of the Tup1-Cyc8 complex may be dominant in bringing about repression at different sets of genes.

Vitamin D/vitamin D receptor, autophagy, and infection.

Vitamin D plays an important role in mineral and bone homeostasis, immune responses, cardiovascular function and keratinocyte proliferation and differentiation. Vitamin D performs most of its functions by binding to vitamin D receptors (VDR), which interact with other intracellular signaling pathways to regulate bone metabolism, inflammation, immunity, cell cycle progression and apoptosis. Autophagy is a basic stress response in yeast, plants and mammals, and plays a critical role in maintaining optimal functional states at the level of cells and organs. Vitamin D/VDR plays an anti-infection role via inducing and regulating autophagy.

Involvement of Sec71 and Ubp2 in tunicamycin-induced ER stress response in the fission yeast.

Accumulation of unfolded or misfolded proteins in the cellular environment result in ER stress and activates the unfolded protein response (UPR). The UPR alleviates ER stress and restores homeostasis, but it triggers cell death under prolonged stress. Here, we aimed to investigate the involvement of Sec71, an Arf-GEF involved in vesicular transport, in the tunicamycin-induced ER stress response. Since deubiquitinases and ER stress are known to be closely linked, we investigated this response by evaluating the potential role of Ubp2, a deubiquitinase, in the ER stress response in fission yeast. Tunicamycin-induced ER stress responses were assessed by analyzing cell viability, apoptosis, intracellular oxidation levels, and proteasomal activities in sec71 and ubp2-deficient cells. The cell viability of Δsec71 and Δubp2 decreased after exposure to 0.5µg/mL tunicamycin. Deleting either ubp2 or sec71 genes significantly decreased proteasomal activity and sensitized cells to ER stress, resulting in increased apoptosis compared with wild-type cells after tunicamycin treatment. DCFDA (2,7-dichlorodihydrofluorescein diacetate) reduction increased in correlation with apoptosis observed in the mutant cells, indicating higher levels of reactive oxygen species. The results highlight the involvement of S. pombe Ubp2 in the known role of the ubiquitin-proteasome system in the ER stress response. We hypothesise that Sec71 is associated with ER homeostasis, and our findings on Sec71 provide new insight into the regulation of cell death mechanisms arising from the ER stress.

Intrinsically disordered signaling proteins: Essential hub players in the control of stress responses in Saccharomyces cerevisiae.

Cells have developed diverse mechanisms to monitor changes in their surroundings. This allows them to establish effective responses to cope with adverse environments. Some of these mechanisms have been well characterized in the budding yeast Saccharomyces cerevisiae, an excellent experimental model to explore and elucidate some of the strategies selected in eukaryotic organisms to adjust their growth and development in stressful conditions. The relevance of structural disorder in proteins and the impact on their functions has been uncovered for proteins participating in different processes. This is the case of some transcription factors (TFs) and other signaling hub proteins, where intrinsically disordered regions (IDRs) play a critical role in their function. In this work, we present a comprehensive bioinformatic analysis to evaluate the significance of structural disorder in those TFs (170) recognized in S. cerevisiae. Our findings show that 85.2% of these TFs contain at least one IDR, whereas ~30% exhibit a higher disorder level and thus were considered as intrinsically disordered proteins (IDPs). We also found that TFs contain a higher number of IDRs compared to the rest of the yeast proteins, and that intrinsically disordered TFs (IDTFs) have a higher number of protein-protein interactions than those with low structural disorder. The analysis of different stress response pathways showed a high content of structural disorder not only in TFs but also in other signaling proteins. The propensity of yeast proteome to undergo a liquid-liquid phase separation (LLPS) was also analyzed, showing that a significant proportion of IDTFs may undergo this phenomenon. Our analysis is a starting point for future research on the importance of structural disorder in yeast stress responses.

Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth.

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Roles for L o microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast.

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (Lo) microdomains, sterol-rich raftlike regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during Lo formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here we report that ER stress induced by lipid imbalance and other stressors induces Lo microdomain formation. This process is ESCRT independent and dependent on Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or Lo microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce Lo microdomains, µLP in response to these stressors is ESCRT dependent and Lo microdomain independent. Our findings reveal that Lo microdomain formation is a yeast stress response, and stress-induced Lo microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and Lo microdomains play functionally distinct roles in LD uptake during stress-induced µLP.

A Bacterial Quorum Sensing Molecule Elicits a General Stress Response in Saccharomyces cerevisiae.

Bacteria assess their population density through a chemical communication mechanism termed quorum sensing, in order to coordinate group behavior. Most research on quorum sensing has focused primarily on its role as an intraspecies chemical signaling mechanism that enables the regulation of certain phenotypes through targeted gene expression. However, in recent years several seminal studies have revealed important phenomena in which quorum sensing molecules appear to serve additional roles as interspecies signals that may regulate microbial ecology. In this study, we asked whether the budding yeast Saccharomyces cerevisiae can sense chemical signals from prokaryotes. When exposed to a variety of quorum sensing molecules from different bacterial species and from Candida albicans we found that N-(3-oxododecanoyl)-L-homoserine lactone (C12) from the opportunistic human pathogen Pseudomonas aeruginosa induces a remarkable stress response in yeast. Microarray experiments confirmed and aided in interpreting these findings, showing a unique and specific expression pattern that differed significantly from the response to previously described stress factors. We further characterized this response and report preliminary findings on the molecular basis for the recognition of C12 by the yeast.

Cowpea NAC Transcription Factors Positively Regulate Cellular Stress Response and Balance Energy Metabolism in Yeast via Reprogramming of Biosynthetic Pathways.

Yeast is a dominant host for recombinant production of heterologous proteins, high-value biochemical compounds, and microbial fermentation. During bioprocess operations, pH fluctuations, organic solvents, drying, starvation, osmotic pressure, and often a combination of these stresses cause growth inhibition or death, markedly limiting its industrial use. Thus, stress-tolerant yeast strains with balanced energy-bioenergetics are highly desirous for sustainable improvement of quality biotechnological production. We isolated two NAC transcription factors (TFs), VuNAC1 and VuNAC2, from a wild cowpea genotype, improving both stress tolerance and growth when expressed in yeast. The GFP-fused proteins were localized to the nucleus. Y2H and reporter assay demonstrated the dimerization and transactivation abilities of the VuNAC proteins having structural folds similar to rice SNAC1. The gel-shift assay indicated that the TFs recognize an "ATGCGTG" motif for DNA-binding shared by several native TFs in yeast. The heterologous expression of VuNAC1/2 in yeast improved growth, biomass, lifespan, fermentation efficiency, and altered cellular composition of biomolecules. The transgenic strains conferred tolerance to multiple stresses such as high salinity, osmotic stress, freezing, and aluminum toxicity. Analysis of the metabolome revealed reprogramming of major pathways synthesizing nucleotides, vitamin B complex, amino acids, antioxidants, flavonoids, and other energy currencies and cofactors. Consequently, the transcriptional tuning of stress signaling and biomolecule metabolism improved the survival of the transgenic strains during starvation and stress recovery. VuNAC1/2-based synthetic gene expression control may contribute to designing robust industrial yeast strains with value-added productivity.