MDG1-mediated transcriptional reprogramming enhances cellulase production and alters thermal activity in recombinant Saccharomyces cerevisiae.

Base excision repair and homologous recombination are required for prevention of a chronic DNA damage response in Saccharomyces cerevisiae.

There is significant variation in the rate and spectrum of spontaneous mutations among taxa. How this variation is shaped by natural selection remains a subject of debate. The drift barrier hypothesis proposes that selection generally favors lower mutation rates due to the risk of deleterious mutations but acts less effectively against weak mutator alleles in smaller populations, allowing the mutation rate to increase due to genetic drift. Given this model, we propose that mutation rates may also be elevated in cell types that appear rarely in a population, where DNA replication and repair processes are subject to selection less often. We can begin to test this prediction in yeast species, some of which can be grown in either a haploid or diploid cell state. Existing data on the budding yeast Saccharomyces cerevisiae support this prediction, with a higher mutation rate observed in haploids, which is the rare cell type in natural populations. However, this pattern could also appear if haploidy is inherently mutagenic, regardless of the dominant cell type. To test these alternatives, we conducted a mutation accumulation experiment with haploid and diploid cells of the fission yeast Schizosaccharomyces pombe, in which diploidy is the rare cell type. In this species, we found a higher mutation rate in diploids, consistent with our prediction. In both species, the spectrum of mutations is also influenced by ploidy state. Our findings suggest that limits to selection on mutation may be evident as variation within species.

Mitogen-activated protein kinases (MAPKs) display remarkable regulatory plasticity across evolution, ranging from highly specialized pathways to broadly responsive global signaling hubs. In the budding yeastSaccharomyces cerevisiae, the high-osmolarity glycerol (HOG) network has served as a paradigm for largely stress-specific MAPK signaling, where the Hog1 MAPK coordinates osmoadaptation. This stands in sharp contrast to Hog1 orthologs in other fungi and humans, which respond not only to osmotic stress, but also diverse stresses including UV, heat shock, oxidative stress, and pathogen signals. Whether the relative osmospecificity ofS. cerevisiaeHog1 represents an ancestral feature or lineage-specific evolution remains unclear. The majority of foundational work onS. cerevisiaeHOG signaling has been performed in laboratory strains that are known to be genetic and phenotypic outliers, and we have been leveraging wildS. cerevisiaestrains to understand aspects of stress signaling that may have been lost in laboratory strains. Here, we examined the phenotypic effects ofhog1Δmutations in a commonly-used laboratoryS. cerevisiaestrain and diverse wild strains on stress cross protection and gene expression. Our findings demonstrate an expanded role in cross-stress protection for Hog1 in wild yeast strains compared to the laboratory strain. More strikingly, we identified a large number of Hog1-dependent genes for non-osmotic stresses in the wild strains that was completely absent in the lab strain. Notably, the Hog1 regulon in wild strains responding to non-osmotic stresses is largely distinct from the canonical osmotic stress response, which we show likely occurs through non-canonical cytoplasmic functions. These findings reveal surprising within-species plasticity for the highly conserved HOG network, suggesting that evolutionary transitions between specialist to generalist stress signaling may occur with relative ease.

Mitochondrial genetic diseases are complex disorders that impair cellular energy production, leading to diverse clinical manifestations across multiple organ systems. These diseases arise from mutations in either mitochondrial DNA or nuclear DNA. Among nuclear DNA-related cases, mutations in POLG and POLG2, which encode subunits of mitochondrial DNA polymerase γ, are particularly significant, causing conditions such as Alpers-Huttenlocher syndrome and progressive external ophthalmoplegia. Model organisms have been instrumental in elucidating POLG-related disease mechanisms and advancing therapeutic strategies. Saccharomyces cerevisiae (budding yeast) provided insights into fundamental mitochondrial functions, while Caenorhabditis elegans (roundworm) helped explore POLG's roles in multicellular organisms. Drosophila melanogaster (fruit fly) has been pivotal in studying neurological aspects, and Mus musculus (mouse) models contributed to understanding systemic effects in mammals. Recently, Danio rerio (zebrafish) has emerged as a promising vertebrate model for drug screening, due to its optical transparency and genetic tractability. Each model system offers unique advantages, collectively bridging the gap between basic research and clinical applications. This review will examine in vivo models used in POLG disorder research, highlighting their contributions to understanding disease mechanisms and therapeutic advancements.

The Gene Ontology (GO) knowledgebase (https://geneontology.org) is a comprehensive resource describing the functions of genes. The GO knowledgebase is regularly updated and improved. We describe here the major updates that have been made in the past 3 years. The ontology and annotations have been expanded and revised, particularly in several areas of biology: cellular metabolism, multi-organism interactions (e.g. host-pathogen), extracellular matrix proteins, chromatin remodeling (e.g. the "histone code"), and noncoding RNA functions. We have released version 2 of a comprehensive set of integrated, reviewed annotations for human genes, which we call the "functionome." We have also dramatically increased the number of GO-CAM models, with over 1500 models of metabolic and signaling pathways, primarily in human, mouse, budding and fission yeast, and fruit fly. Finally, we discuss our current recommendations and future prospects of AI in the use and development of GO.

Yeast is a widely used model organism in biological and proteomics research. Conventional bottom-up proteomic analysis of yeast cells requires disruption of the rigid cell wall to extract proteins, which is often associated with lengthy procedures, significant technical variations, and noticeable sample loss. Here, we present an "in-cell proteomics" approach that eliminates cell lysis and digests proteins directly in the yeast cells after a rapid methanol fixation. The approach integrates all the sample processing into a single filter device, offering a simple yet highly effective and sensitive approach for yeast proteomics analysis. We applied this approach to characterize proteome dynamics in the budding yeast Saccharomyces cerevisiae during cell cycle progression and following DNA damage. With single-shot LC-MS, we were able to detect and quantify around 3500 yeast proteins from the in-cell digests. Our study introduces a novel in-cell approach for yeast proteomics analysis and presents a quantitative proteome map of yeast cell-cycle progression with high temporal resolution for cell division cycle (Cdc) proteins. It also provides a comprehensive, time-resolved view of proteome-wide dynamics and remodeling throughout the yeast cell cycle in response to methyl methanesulfonate (MMS)-induced DNA damage. SUMMARY: Yeast proteomics studies often require detergent-based and/or mechanical disruption procedures for cell lysis and protein digestion. We reported an "in-cell proteomics" approach that eliminates cell lysis and digests proteins directly in the yeast cells after a simple methanol fixation. The approach integrates all the sample processing into a single filter device, offering a rapid yet highly effective and sensitive approach for yeast proteomics analysis. Using this method, we were able to characterize proteome dynamics in the budding yeast Saccharomyces cerevisiae during cell cycle progression and following DNA damage.

Protein circuits organize cell biology, but synthetic dynamics are challenging to engineer due to stochastic genetic and biochemical variation. Genetically encoded oscillators (GEOs) built from bacterial MinDE-family ATPases and activators generate synthetic protein waves that act as novel frequency-domain imaging barcodes in eukaryotic cells, providing a platform for understanding, engineering, and applying synthetic protein dynamics. Using budding yeast, we disentangle how expression levels and expression noise govern the GEO waveform and encodability. While the GEO amplitude is sensitive to extrinsic noise, the GEO frequency is stably encoded by the activator:ATPase ratio. By integrating GEO components into the yeast modular cloning toolkit, we developed different noise-guided expression strategies that act like filters on the GEO waveform. We paired these filters with hundreds of biochemically distinct GEO variants to engineer clonal populations that oscillate at distinct frequencies and to design waveform libraries with customizable spectral features and tunable waveform variation. Our work establishes a robust platform for precision genetic encoding of synthetic GEO oscillations and highlights the utility of noise-guided strategies for dynamic protein circuit design.

DNA end resection to generate 3' single-stranded DNA (ssDNA) overhangs is the first step in homology-directed mechanisms of double-strand break (DSB) repair. While end resection has been extensively studied in the repair of endonuclease-induced DSBs, little is known about how resection proceeds at DSBs generated during DNA replication. We previously established a system to generate replication-dependent double-ended DSBs at the sites of nicks induced by the Cas9D10A nickase in the budding yeast genome. Here, we suggest that these DSB ends form in an asymmetric manner, with one being blunt or near blunt and the other bearing a 3' ssDNA overhang of up to the size of an Okazaki fragment. We find that Mre11 preferentially binds blunt ends and is required to evict Ku from these DSB ends and to promote end resection. In contrast, the ends predicted to have 3' overhangs have minimal Ku binding, and resection at these break ends can proceed in a mostly Mre11-independent manner through either the Exo1 or Dna2-Sgs1 long-range resection mechanisms. These findings indicate that resection proceeds differently at replication-dependent DSBs than at canonical DSBs and reveal that Ku selectively binds blunt ends, potentially explaining why replication-dependent DSBs are poorly repaired by nonhomologous end joining.

The budding yeast Saccharomyces cerevisiae is a well-established model for studying the genetic basis of complex traits, and it is a powerful system for investigating mechanisms of aging. Here, we examine the genomic and transcriptomic factors contributing to increased replicative age in recombinant yeast populations harboring standing genetic variation. Using Fluorescence-Activated Cell Sorting (FACS), we isolated young and aged cohort pairs across twelve biological replicates and sequenced their progeny to assess patterns of differentiation at the nucleotide and transcription levels. Most differentiated alleles were located in coding regions, including significant variants within 132 unique genes. Transcriptomic analysis revealed 60 differentially expressed genes in aged populations, including 18 genes with increased expression in aged cohorts, and 42 genes with decreased expression. Although only two genes (RFA3 and WSC4) were implicated in both genomic and transcriptomic analyses, functional overlap associated with protein homeostasis, DNA repair, and cell cycle regulation was evident across datasets. Notably, we found no strong evidence that differentially expressed genes were more likely to occur in close proximity to significant gene variants. This suggests that late-life survival is not predominantly governed by local cis-regulatory interactions (e.g. variants within or near coding regions). These findings underscore the power of integrating genomic and transcriptomic data to elucidate the genetics of complex traits such as aging, demonstrating how multi-omics approaches can reveal functional relationships that may be overlooked by single-layer analyses.

Cells must respond rapidly to heat stress by activating multiple signaling pathways that preserve proteostasis. In budding yeast, this includes induction of Hsf1 and Msn2/4-mediated transcription, cell integrity signaling, stress-triggered phase separation of proteins, and inhibition of translation. How these pathways are so rapidly activated and coordinated remains unclear. We show that the mechanosensor Mid2 senses heat-induced membrane stretch and leads to rapid phosphorylation of the cytosolic Hsp70 Ssa1 at a well-conserved threonine (T492). Phosphorylation of T492 leads to epichaperome rearrangement promoting fine-tuning of multiple cellular processes including translational pausing, HSF activity, MAPK signaling and stress granule resolution. Taken together, these results provide a comprehensive, unified theory of the global yeast heat shock response mediated by the Hsp70 chaperone code.

The Golgi apparatus is the central hub of secretory and endosomal pathways in a eukaryotic cell. Despite having a conserved basic organization, the Golgi varies greatly in structure and operation mode between different cell types, ranging from dispersed cisternae in the budding yeast to the ribbon of cisternae stacks in most mammalian cells. Cell shape and secretory demands dictate structural and functional properties of the Golgi. Neurons are a particularly interesting type of secretory cells that have a highly polarized architecture and a large and diverse secretome. The neuronal Golgi complex evolved into an elaborate set of compartmentalized organelles that process and sort diverse neuronal cargos, including synaptic proteins, neuropeptides, and neurotrophic factors. In this review, we describe the structural adaptations of the Golgi to neuronal architecture and discuss the principles of neuronal cargo sorting. We also highlight structural rearrangements of the neuronal Golgi in neurodegenerative diseases and discuss the role of mutations in Golgi-related proteins in neurodevelopment.

The centromeric protein-A (CENP-A) is an evolutionarily conserved histone H3 variant that marks the identity of the centromeres. Several mechanisms regulate the centromeric deposition of CENP-A as its mislocalization causes erroneous chromosome segregation, leading to aneuploidy-based diseases, including cancers. The most crucial deposition factor is a CENP-A specific chaperone, HJURP (Scm3 in budding yeast), which specifically binds to CENP-A. However, the discovery of HJURP as a DDR (DNA damage repair) protein and evidence of its binding to Holliday junctions in vitro indicate a CENP-A-deposition-independent role of these chaperones. In this study, using budding yeast, we demonstrate that Scm3 is crucial for the DDR pathway as Scm3-depleted cells are sensitive to DNA damage. We further observe that Scm3 depletion genetically interacts with the rad52 DDR mutant and is compromised in activating DDR-mediated arrest. We demonstrate that Scm3 associates with DNA damage sites and undergoes posttranslational modifications upon DNA damage. Overall, from this report and earlier studies on HJURP, we conclude that DDR functions of CENP-A chaperones are conserved across eukaryotes. The revelation that these chaperones promote genome stability in more than one pathway has clinical significance.

The human fungal pathogen Candida albicans undergoes a morphological transition from a budding yeast to a filamentous form, which is associated with pathogenesis. Various cues mediate this transition including intracellular reorganization. The cytoplasm is densely packed with proteins including large macromolecular complexes, such as ribosomes, and hence, molecular crowding can impact a range of cellular processes. However, the relationship between cytoplasmic molecular crowding and morphological growth states is unclear. Using a fluorescent microrheological probe and single particle tracking, we observed a striking decrease in molecular crowding during filamentous growth in C. albicans. On the basis of simulations, proteomics and structural data from in situ cryogenic electron microscopy, we show that the reduction in crowding is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume, leading to a dilution of ribosomes. Filamentation was enhanced in a mutant defective in ribosome biogenesis, while translation was not affected, suggesting that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis. Overall, we show that filamentous growth is associated with reduced cytoplasmic crowding via changes in ribosome concentration, suggesting that combination therapies in which ribosome biogenesis is also targeted may be advantageous.

The budding yeast Saccharomyces cerevisiae has ‘point’ centromeres, which are much smaller and simpler than centromeres of most other eukaryotes and have a defined DNA sequence. Other yeast taxa have different and highly diverse centromere structures, but a clear picture of how yeast centromeres have evolved is lacking. Here, we investigated nine yeast species in two taxonomic orders that are close outgroups to S. cerevisiae. We find that they have a wide diversity of centromere structures, indicating that multiple transitions of structure have occurred within the last 200 Myr. Some species have centromeres with defined sequence motifs (17 – 200 bp), others consist of Inverted Repeats (IRs), and others have Ty5-like retroelement clusters. Strikingly, the chromosomal locations of centromeres have largely been conserved across taxonomic orders, even as their structures have changed, which suggests that structure replacement occurs in situ. In some Barnettozyma species we find that a single genome can contain chromosomes with different centromere structures – some with IRs and some without – which suggests that a structural transition is underway in this genus. We identified only one example of a centromere moving by a long distance: a new centromere formed recently at the MAT locus of Barnettozyma californica, 250 kb from the previous centromere on that chromosome.

Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well-conserved heterochromatin epigenomes and their reader molecules. However, the details of the importance of them in heterochromatin formation still remain unclear. One reason for this is system redundancy, as multiple HP1 family proteins exist, as well as HP1 itself serving as hubs for various effector factors. To overcome these issues, we took a synthetic biology approach and introduced H3K9 methylation catalyzed by mouse H3K9 methyltransferases and HP1s into budding yeast, Saccharomyces cerevisiae (S. cerevisiae) which doesn't have this system, and examined its impact on transcription and chromatin compaction. We observed that the mammalian H3K9 methyltransferase can induce genome-wide H3K9 di- and tri-methylation in the S. cerevisiae, mainly in the gene body region, and HP1 accumulates over the H3K9 methylated regions. The forced expression of H3K9 methyltransferase and HP1 had little impact on transcription. Furthermore, Hi-C-seq analysis revealed no significant effects on the chromatin 3D structure. These results suggest that although H3K9 methylation and the recruitment of HP1 play essential roles in the epigenetic regulation of heterochromatin, they alone are not sufficient to alter the higher-order chromatin structure, at least in the gene body regions in S. cerevisiae.

Spermine and thermospermine synthases emerged multiple times during eukaryote evolution.

Age-dependent topoisomerase I depletion alters recruitment of rDNA silencing complexes.

The eukaryotic DNA damage and replication stress checkpoint is an essential component of the DNA damage response and crucial for genome maintenance. In budding yeast, the apical kinase Mec1 (ATR ortholog), along with binding partner Ddc2 (ATRIP ortholog), senses persistent RPA-bound ssDNA in the cell. Mec1 is activated by interaction with a Mec1-activating protein. One such activator, Dpb11 (TopBP1 ortholog), is recruited to a 5’ ss-dsDNA junction via the 9-1-1 checkpoint clamp. Due to their differential DNA binding preferences, it remains to be determined how Mec1 encounters its activators on damaged DNA. Using real-time single-molecule imaging of checkpoint proteins binding to dsDNA containing a long ssDNA gap, we show that, even in the absence of 9-1-1, Dpb11 binds to ssDNA and localizes to ss-dsDNA junctions in an RPA-dependent manner. Importantly, we directly visualize that Dpb11 recruits Mec1-Ddc2 to ss-dsDNA junctions. Additionally, single-molecule force spectroscopy was used to demonstrate that Dpb11 can interact with multiple DNA sites simultaneously to form bridges both alone and in the presence of RPA, stabilizing ssDNA loops and reducing the end-to-end distance of gapped DNA. Taken together, these data support a model in which Dpb11 facilitates Mec1 colocalization with its activators both directly by recruiting Mec1 to gap junctions and indirectly by decreasing the effective gap length.

Accelerating climate change and extreme temperatures urge us to better understand the potential of populations to tolerate and adapt to thermal challenges. Interspecific hybridization can facilitate adaptation to novel or extreme environments. However, predicting the long-term fitness effects of hybridization remains a major challenge in evolutionary and conservation biology. Experimental evolution with microbes provides a powerful tool for tracking adaption, across generations and in real time. We investigated the thermal adaptation dynamics of four species of budding yeast (Saccharomyces) and their interspecific F2 hybrids, for 140 generations under cold (5°C) and warm (31°C) conditions. We found significant variation in the evolutionary potential of species and hybrids, strongly determined by their natural temperature tolerance. The largest fitness improvements occurred in hybrids, with some populations nearly quadrupling in fitness in the cold environment, exceeding both parents in thermal adaptive potential. While adaption rates in some hybrid populations were high, their absolute fitness by the end of evolution was comparable to that of their parents. Reciprocal transplanting of evolved populations from the endpoint of evolution into opposite temperatures revealed that hybrids had greater resilience when challenged with sudden temperature shifts. Our results highlight that hybridization alters the fitness outcomes of long-term adaptation to extreme environments and may render populations more resilient to sudden environmental change, presenting both opportunities and challenges for conservation and sustainable agriculture.