Yeast最新文献

Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.

To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest.

Smk1 is a MAPK homolog in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore wall assembly. During this program, haploid cells are surrounded by a layer of mannan and then a layer of glucan. These inner layers of the spore wall resemble the vegetative cell wall. Next, the outer layers consisting of chitin/chitosan and then dityrosine are assembled. The outer layers are spore-specific and provide protection against environmental stressors. Smk1 is required for the proper assembly of spore walls. However, the protective properties of the outer layers have limited our understanding of how Smk1 controls this morphogenetic program. Mutants lacking the chitin deacetylases, Cda1 and Cda2, form spores that lack the outer layers of the spore wall. In this study, cda1,2∆ cells were used to demonstrate that Smk1 promotes deposition of the glucan layer of the spore wall through the partially redundant glucan synthases Gsc2 and Fks3. Although Gsc2 is localized to sites of spore wall assembly in the wild type, it is mislocalized in the mother cell cytoplasm in the smk1∆ mutant. These findings suggest that Smk1 controls assembly of the spore wall by regulating the localization of Gsc2 during sporogenesis.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' → 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' → 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.

Meiotic crossovers play a vital role in proper chromosome segregation and evolution of most sexually reproducing organisms. Meiotic recombination can be visually observed in Saccharomyces cerevisiae tetrads using linked spore-autonomous fluorescent markers placed at defined intervals within the genome, which allows for analysis of meiotic segregation without the need for tetrad dissection. To automate the analysis, we developed a deep learning-based image recognition and classification pipeline for high-throughput tetrad detection and meiotic crossover classification. As a proof of concept, we analyzed a large image data set from wild-type and selected gene knock-out mutants to quantify crossover frequency, interference, chromosome missegregation, and gene conversion events. The deep learning-based method has the potential to accelerate the discovery of new genes involved in meiotic recombination in S. cerevisiae such as the underlying factors controlling crossover frequency and interference.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 ^T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from  d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

Fitness in micro-organisms can be proxied by growth parameters on different media and/or temperatures. This is achieved by measuring optical density at 600 nm using a spectrophotometer, which measures the effect of absorbance and side scattering due to turbidity of cells suspensions. However, when growth kinetics must be monitored in many 96-well plates at the same time, buying several 96-channel spectrophotometers is often beyond budgets. The MiniRead device presented here is a simple and inexpensive do-it-yourself 96-well temperature-controlled turbidimeter designed to measure the interception of white light via absorption or side scattering through liquid culture medium. Turbidity is automatically recorded in each well at regular time intervals for up to several days or weeks. Output tabulated text files are recorded into a micro-SD memory card to be easily transferred to a computer. We propose also an R package which allows (1) to compute the nonlinear calibration curves required to convert raw readings into cell concentration values, and (2) to analyze growth kinetics output files to automatically estimate proxies of growth parameters such as lag time, maximum growth rate, or cell concentration at the plateau.

Lipid binding domains and protein lipidations are essential features to recruit proteins to intracellular membranes, enabling them to function at specific sites within the cell. Membrane association can also be exploited to answer fundamental and applied research questions, from obtaining insights into the understanding of lipid metabolism to employing them for metabolic engineering to redirect fluxes. This review presents a broad catalog of membrane binding strategies focusing on the plasma membrane of Saccharomyces cerevisiae. Both lipid binding domains (pleckstrin homology, discoidin-type C2, kinase associated-1, basic-rich and bacterial phosphoinositide-binding domains) and co- and post-translational lipidations (prenylation, myristoylation and palmitoylation) are introduced as tools to target the plasma membrane. To provide a toolset of membrane targeting modules, respective candidates that facilitate plasma membrane targeting are showcased including their in vitro and in vivo properties. The relevance and versatility of plasma membrane targeting modules are further highlighted by presenting a selected set of use cases.