Subcellular Localization of Fad1p in Saccharomyces cerevisiae: A Choice at Post-Transcriptional Level?
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ABSTRACT:
SUBMITTER: Bruni F
PROVIDER: S-EPMC8470081 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Saccharomyces cerevisiae myosin type II-deficient (myo1?) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1? transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1? strain may provide a simplified paradigm for cell wall stress survival.To explore the post-transcriptional regulation of the myo1? stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1? strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2?-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1? strains. Deletion of GCN2 in myo1? abolished eIF2?p phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1? strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1? and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2?-P in myo1? strains.Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1? strains. Inhibition of translation initiation factor eIF2? (eIF2?-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2?-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1? strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2?-P in myo1? strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1? stress response and possibly other stresses in yeast.
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Project description:Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.
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Project description:The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM) loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA) media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L), changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.
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Project description:The gene SPI1, of Saccharomyces cerevisiae, encodes a cell wall protein that is induced in several stress conditions, particularly in the postdiauxic and stationary phases of growth. It has a paralogue, SED1, which shows some common features in expression regulation and in the null mutant phenotype. In this work we have identified homologues in other species of yeasts and filamentous fungi, and we have also elucidated some aspects of the origin of SPI1, by duplication and diversification of SED1. In terms of regulation, we have found that the expression in the post-diauxic phase is regulated by genes related to the PKA pathway and stress response (MSN2/4, YAK1, POP2, SOK2, PHD1, and PHO84) and by genes involved in the PKC pathway (WSC2, PKC1, and MPK1).
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Project description:Mating-type switching in the budding yeast Saccharomyces cerevisiae relies on the Sir protein complex to silence HML and HMR, the two loci containing copies of the alleles of the mating type locus, MAT. Sir-based transcriptional silencing has been considered locus-specific, but the recent discovery of rare and transient escapes from silencing at HMLα2 with a sensitive assay called to question if these events extend to the whole locus. Adapting the same assay, we measured that transient silencing failures at HML were more frequent for the α2 gene than α1, similarly to their expression level in unsilenced cells. By coupling a mating assay, at HML we found that one of the two genes at that locus can be transiently expressed while the other gene is maintained silent. Thus, transient silencing loss can be a property of the gene rather than the locus. Cells lacking the SIR1 gene experience epigenetic bistability at HML and HMR. Our previous result led us to ask if HML could allow for two independent epigenetic states within the locus in a sir1Δ mutant. A simple construct using a double fluorescent reporter at HMLα1 and HMLα2 ruled out this possibility. Each HML locus displayed a single epigenetic state. We revisited the question of the correlation between the states of two HML loci in diploid cells, and showed they were independent. Finally, we determined the relative strength of gene repression achieved by Sir-based silencing with that achieved by the a1-α2 repressor.
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Project description:Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.
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Project description:BACKGROUND:The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. RESULTS:We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. CONCLUSIONS:Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.
| S-EPMC3224389 | biostudies-literature
Project description:BACKGROUND: Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. METHODS: We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. RESULTS: We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. CONCLUSIONS: In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.
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