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Background: The adverse effect of aflatoxin in broilers is well known. However, dietary supplementation of Saccharomyces cell wall and/or Nanocurcumin may decrease the negative effect of aflatoxin B1 because of the bio-adsorbing feature of the functional ingredients in Yeast Cell Wall and the detoxification effect of curcumin nanoparticles. The goal of this study was to see how Saccharomyces cell wall/Nanocurcumin alone or in combination with the aflatoxin-contaminated diet ameliorated the toxic effects of aflatoxin B1 on broiler development, blood and serum parameters, carcass traits, histology, immune histochemistry, liver gene expression, and aflatoxin residue in the liver and muscle tissue of broilers for 35 days. Moreover, the withdrawal time of aflatoxin was measured after feeding the aflatoxicated group an aflatoxin-free diet. Broiler chicks one day old were distributed into five groups according to Saccharomyces cell wall and/or nanocurcumin with aflatoxin supplementation. The G1 group was given a formulated diet without any supplements. The G2 group was supplemented with aflatoxin (0.25 mg/kg diet) in the formulated diet. The G3 group was supplemented with aflatoxin (0.25 mg/kg diet) and Saccharomyces cell wall (1 kg/ton diet) in the formulated diet. The G4 group was supplemented with aflatoxin (0.25 mg/kg diet) and nanocurcumin (400 mg/kg) in the formulated diet. The G5 group was supplemented with aflatoxin (0.25 mg/kg diet) and Saccharomyces cell wall (1 kg/ton diet) in combination with nanocurcumin (200 mg/kg) in the formulated diet. Results: According to the results of this study, aflatoxin supplementation had a detrimental impact on the growth performance, blood and serum parameters, carcass traits, and aflatoxin residue in the liver and muscle tissue of broilers. In addition, aflatoxin supplementation led to a liver injury that was indicated by serum biochemistry and pathological lesions in the liver tissue. Moreover, the shortening of villi length in aflatoxicated birds resulted in a decrease in both the crypt depth ratio and the villi length ratio. The expression of CYP1A1 and Nrf2 genes in the liver tissue increased and decreased, respectively, in the aflatoxicated group. In addition, the aflatoxin residue was significantly (P ≤ 0.05) decreased in the liver tissue of the aflatoxicated group after 2 weeks from the end of the experiment. Conclusion: Saccharomyces cell wall alone or with nanocurcumin attenuated these negative effects and anomalies and improved all of the above-mentioned metrics. [ABSTRACT FROM AUTHOR]

In fluctuating nutrient environments, isogenic microbial cells transition into "multicellular" communities composed of phenotypically heterogeneous cells, showing functional specialization. In fungi (such as budding yeast), phenotypic heterogeneity is often described in the context of cells switching between different morphotypes (e.g., yeast to hyphae/pseudohyphae or white/opaque transitions in Candida albicans). However, more fundamental forms of metabolic heterogeneity are seen in clonal Saccharomyces cerevisiae communities growing in nutrient-limited conditions. Cells within such communities exhibit contrasting, specialized metabolic states, and are arranged in distinct, spatially organized groups. In this study, we explain how such an organization can stem from self-organizing biochemical reactions that depend on special metabolites. These metabolites exhibit plasticity in function, wherein the same metabolites are metabolized and utilized for distinct purposes by different cells. This in turn allows cell groups to function as specialized, interdependent cross-feeding systems which support distinct metabolic processes. Exemplifying a system where cells exhibit either gluconeogenic or glycolytic states, we highlight how available metabolites can drive favored biochemical pathways to produce new, limiting resources. These new resources can themselves be consumed or utilized distinctly by cells in different metabolic states. This thereby enables cell groups to sustain contrasting, even apparently impossible metabolic states with stable transcriptional and metabolic signatures for a given environment, and divide labor in order to increase community fitness or survival. We speculate on possible evolutionary implications of such metabolic specialization and division of labor in isogenic microbial communities. [ABSTRACT FROM AUTHOR]

Recent work by Kentaro Ohkuni and colleagues exemplifies how a series of molecular mechanisms contribute to a cellular outcome--equal distribution of chromosomes. Failure to maintain structural and numerical integrity of chromosomes is one contributing factor in genetic diseases such as cancer. Specifically, the authors investigated molecular events surrounding centromeric histone H3 variant Cse4 deposition--a process important for chromosome segregation, using Saccharomyces cerevisiae as a model organism. This study illustrates an example of a post-translational modification--sumoylation--regulating a cellular process and the concept of genetic interactions (e.g., synthetic dosage lethality). Furthermore, the study highlights the importance of using diverse experimental approaches in answering a few key research questions. The authors used molecular biology techniques (e.g., qPCR), biochemical experiments (e.g., Ni-NTA/8His protein purification), as well as genetic approaches to understand the regulation of Cse4. At a bigpicture level, the study reveals how genetic changes can lead to subsequent molecular and cellular changes. [ABSTRACT FROM AUTHOR]

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4. Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1Δ, slx5Δ, and hir2Δ strains, GAL-cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation. [ABSTRACT FROM AUTHOR]

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability. [ABSTRACT FROM AUTHOR]

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome. [ABSTRACT FROM AUTHOR]

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation. [ABSTRACT FROM AUTHOR]

The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure. [ABSTRACT FROM AUTHOR]

Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1–4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an multicopy suppression of a budding defect 2 (Msb2)-dependent mitogen-activated protein (MAP) kinase (HOG2) and an ASM-1/Phd1/StuA/EFG1/SOK2 (APSES) transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans. [ABSTRACT FROM AUTHOR]

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5′ to 3′ bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells. [ABSTRACT FROM AUTHOR]

Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus. [ABSTRACT FROM AUTHOR]

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain’s ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition. [ABSTRACT FROM AUTHOR]

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p. [ABSTRACT FROM AUTHOR]

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus. [ABSTRACT FROM AUTHOR]

The canonical model of eukaryotic translation posits that efficient translation initiation increases protein expression and mRNA stability. Contrary to this model, we find that increasing initiation rate can decrease both protein expression and stability of certain mRNAs in the budding yeast Saccharomyces cerevisiae. These mRNAs encode a stretch of polybasic residues that cause ribosome stalling. Our computational modeling predicts that the observed decrease in gene expression at high initiation rates occurs when ribosome collisions at stalls stimulate abortive termination of the leading ribosome or cause endonucleolytic mRNA cleavage. Consistent with this prediction, the collision-associated quality-control factors Asc1 and Hel2 (orthologs of human RACK1 and ZNF598, respectively) decrease gene expression from stall-containing mRNAs only at high initiation rates. Remarkably, hundreds of S. cerevisiae mRNAs that contain ribosome stall sequences also exhibit lower translation efficiency. We propose that inefficient translation initiation allows these stall-containing endogenous mRNAs to escape collision-stimulated reduction in gene expression. [ABSTRACT FROM AUTHOR]

In all kingdoms of life, is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by prior to cell division to ensure each daughter cell receives the full complement of . DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life. [ABSTRACT FROM AUTHOR]

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs. [ABSTRACT FROM AUTHOR]

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We also provide evidence that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses RDT1 transcription until it is removed from the promoter in response to a dsDNA break at the MAT locus induced by HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating-type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating-type switching, thus providing a novel model for dissecting condensin function in vivo. [ABSTRACT FROM AUTHOR]

L-aspartate aminotransferase is a pyridoxal 5ʹ-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open–closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5ʹ-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5ʹ-phosphate-dependent enzymes. [ABSTRACT FROM AUTHOR]

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these “hungry spliceosome” conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of “intronization”, whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function. [ABSTRACT FROM AUTHOR]

Phagocytosis is a receptor-mediated process critical to innate immune clearance of pathogens. It proceeds in a regulated sequence of stages: (a) migration of phagocytes towards pathogens, (b) recognition of PAMPs and binding through PRRs, (c) engulfment and internalisation into phagosomes, (d) phagosome maturation, and (e) killing of pathogen or host cells. However, little is known about the role that individual receptors play in these discrete stages in the recognition of fungal cells. In a previous study, we found that dectin-2 deficiency impacted some but not all stages of macrophage-mediated phagocytosis of Candida glabrata. Because the C-type lectin receptor dectin-2 critically requires coupling to the FcRγ chain for signalling, we hypothesised that this coupling may be important for regulating phagocytosis of fungal cargo. We therefore examined how deficiency in FcRγ itself or two receptors to which it couples (dectin-2 and mincle) impacts phagocytosis of six fungal organisms representing three different fungal taxa. Our data show that deficiency in these proteins impairs murine bone marrow-derived macrophage migration, engulfment, and phagosome maturation, but not macrophage survival. Therefore, FcRγ engagement with selective C-type lectin receptors (CLRs) critically affects the spatio-temporal dynamics of fungal phagocytosis. [ABSTRACT FROM AUTHOR]

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. [ABSTRACT FROM AUTHOR]

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes’ effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes’ adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection. [ABSTRACT FROM AUTHOR]

In the alcoholic fermentation process, Saccharomyces cerevisiae strains present differences in their nitrogen consumption profiles, these phenotypic outcomes have complex genetic and molecular architectures. In this sense, variations in nitrogen signaling pathways regulated by TORC1 represent one of the main sources of phenotypic diversity in nitrogen consumption. This emphasizes the possible roles that allelic variants from the TORC1 pathway have in the nitrogen consumption differences observed in yeast during the alcoholic fermentation. Here, we studied the allelic diversity in the TORC1 pathway across four yeast strains and determined how these polymorphisms directly impact nitrogen consumption during alcoholic fermentation. Using a reciprocal hemizygosity approach combined with phenotyping under fermentative conditions, we found that allelic variants of GTR1, TOR2, SIT4, SAP185, EAP1, NPR1 and SCH9 underlie differences in the ammonium and amino acids consumption phenotypes. Among these, GTR1 alleles from the Wine/European and West African genetic backgrounds showed the greatest effects on ammonium and amino acid consumption, respectively. Furthermore, we identified allelic variants of SAP185, TOR2, SCH9 and NPR1 from an oak isolate that increased the amino acid consumption preference over ammonium; representing putative candidates coming from a non-domesticated strain that could be used for genetic improvement programs. In conclusion, our results demonstrated that a large number of allelic variants within the TORC1 pathway significantly impacts on regulatory mechanisms of nitrogen assimilation during alcoholic fermentation. [ABSTRACT FROM AUTHOR]

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein–protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules. [ABSTRACT FROM AUTHOR]

The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted proteins. However, studies have shown that mSMP recruitment to the ER in eukaryotes can occur in a manner that is independent of the ribosome, translational control, and the signal recognition particle, although the mechanism remains largely unknown. Here, we identify a cis-acting RNA sequence motif that enhances mSMP localization to the ER and appears to increase mRNA stability, and both the synthesis and secretion of secretome proteins. Termed SECReTE, for ecretion-nhancing is gulatory argeting lement, this motif is enriched in mRNAs encoding secretome proteins translated on the ER in eukaryotes and on the inner membrane of prokaryotes. SECReTE consists of ≥10 nucleotide triplet repeats enriched with pyrimidine (C/U) every third base (i.e. NNY, where N = any nucleotide, Y = pyrimidine) and can be present in the untranslated as well as the coding regions of the mRNA. Synonymous mutations that elevate the SECReTE count in a given mRNA (e.g. SUC2, HSP150, and CCW12) lead to an increase in protein secretion in yeast, while a reduction in count led to less secretion and physiological defects. Moreover, the addition of SECReTE to the 3’UTR of an mRNA for an exogenously expressed protein (e.g. GFP) led to its increased secretion from yeast cells. Thus, SECReTE constitutes a novel RNA motif that facilitates ER-localized mRNA translation and protein secretion. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21–163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2–9 or 10–14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2–9 or 2–14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15–20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another. [ABSTRACT FROM AUTHOR]