The NuA4/TIP60 acetyltransferase complex is required for gene regulation, DNA repair and cell cycle progression. The limited structural information impeded understanding of NuA4/TIP60 assembly and regulatory mechanism. Here, we report the 4.7 Å cryo-electron microscopy (cryo-EM) structure of a NuA4/TIP60 TEEAA assembly (Tra1, Eaf1, Eaf5, actin and Arp4) and the 7.6 Å cryo-EM structure of a TEEAA-piccolo assembly (Esa1, Epl1, Yng2 and Eaf6). The Tra1 and Eaf1 constitute the assembly scaffold. The Eaf1 SANT domain tightly binds to the LBE and FATC domains of Tra1 by ionic interactions. The actin/Arp4 peripherally associates with Eaf1 HSA domain. The Eaf5/7/3 (TINTIN) and piccolo modules largely pack against the FAT and HEAT repeats of Tra1 and their association depends on Eaf1 N-terminal and HSA regions, respectively. These structures elucidate the detailed architecture and molecular interactions between NuA4 subunits and offer exciting insights into the scaffolding and regulatory mechanisms of Tra1 pseudokinase.

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8-an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The S. cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the positioning of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleosomal DNA and the H2A-H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.

Utilising one-carbon substrates such as carbon dioxide, methane, and methanol is vital to address the current climate crisis. Methylotrophic metabolism enables growth and energy generation from methanol, providing an alternative to sugar fermentation. Saccharomyces cerevisiae is an important industrial microorganism for which growth on one-carbon substrates would be relevant. However, its ability to metabolize methanol has been poorly characterised. Here, using adaptive laboratory evolution and 13C-tracer analysis, we discover that S. cerevisiae has a native capacity for methylotrophy. A systems biology approach reveals that global rearrangements in central carbon metabolism fluxes, gene expression changes, and a truncation of the uncharacterized transcriptional regulator Ygr067cp supports improved methylotrophy in laboratory evolved S. cerevisiae. This research paves the way for further biotechnological development and fundamental understanding of methylotrophy in the preeminent eukaryotic model organism and industrial workhorse, S. cerevisiae.

Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.

Saccharomyces cerevisiae is widely employed as a cell factory for the production of biofuels. However, product toxicity has hindered improvements in biofuel production. Here, we engineer the actin cytoskeleton in S. cerevisiae to increase both the cell growth and production of n-butanol and medium-chain fatty acids. Actin cable tortuosity is regulated using an n-butanol responsive promoter-based autonomous bidirectional signal conditioner in S. cerevisiae. The budding index is increased by 14.0%, resulting in the highest n-butanol titer of 1674.3 mg L-1. Moreover, actin patch density is fine-tuned using a medium-chain fatty acid responsive promoter-based autonomous bidirectional signal conditioner. The intracellular pH is stabilized at 6.4, yielding the highest medium-chain fatty acids titer of 692.3 mg L-1 in yeast extract peptone dextrose medium. Engineering the actin cytoskeleton in S. cerevisiae can efficiently alleviate biofuels toxicity and enhance biofuels production.

The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors.

The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

U1 snRNP plays a critical role in 5'-splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. Here, we report the cryoEM structure of yeast U1 snRNP at 3.6 Å resolution with atomic models for ten core proteins, nearly all essential domains of its RNA, and five stably associated auxiliary proteins. The foot-shaped yeast U1 snRNP contains a core in the "ball-and-toes" region architecturally similar to the human U1 snRNP. All auxiliary proteins are in the "arch-and-heel" region and connected to the core through the Prp42/Prp39 paralogs. Our demonstration that homodimeric human PrpF39 directly interacts with U1C-CTD, mirroring yeast Prp42/Prp39, supports yeast U1 snRNP as a model for understanding how transiently associated auxiliary proteins recruit human U1 snRNP in alternative splicing.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Genomic methods have been valuable for identifying RNA-binding proteins (RBPs) and the genes, pathways, and processes they regulate. Nevertheless, standard motif descriptions cannot be used to predict all RNA targets or test quantitative models for cellular interactions and regulation. We present a complete thermodynamic model for RNA binding to the S. cerevisiae Pumilio protein PUF4 derived from direct binding data for 6180 RNAs measured using the RNA on a massively parallel array (RNA-MaP) platform. The PUF4 model is highly similar to that of the related RBPs, human PUM2 and PUM1, with one marked exception: a single favorable site of base flipping for PUF4, such that PUF4 preferentially binds to a non-contiguous series of residues. These results are foundational for developing and testing cellular models of RNA-RBP interactions and function, for engineering RBPs, for understanding the biophysical nature of RBP binding and the evolutionary landscape of RNAs and RBPs.

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.

Srs2 is an Sf1a helicase that helps maintain genome stability in Saccharomyces cerevisiae through its ability to regulate homologous recombination. Srs2 downregulates HR by stripping Rad51 from single-stranded DNA, and Srs2 is also thought to promote synthesis-dependent strand annealing by unwinding D-loops. However, it has not been possible to evaluate the relative contributions of these two distinct activities to any aspect of recombination. Here, we used a structure-based approach to design an Srs2 separation-of-function mutant that can dismantle Rad51-ssDNA filaments but is incapable of disrupting D-loops, allowing us to assess the relative contributions of these pro- and anti-recombinogenic functions. We show that this separation-of-function mutant phenocopies wild-type SRS2 in vivo, suggesting that the ability of Srs2 to remove Rad51 from ssDNA is its primary role during HR.

Gross chromosomal rearrangements (GCRs) play an important role in human diseases, including cancer. The identity of all Genome Instability Suppressing (GIS) genes is not currently known. Here multiple Saccharomyces cerevisiae GCR assays and query mutations were crossed into arrays of mutants to identify progeny with increased GCR rates. One hundred eighty two GIS genes were identified that suppressed GCR formation. Another 438 cooperatively acting GIS genes were identified that were not GIS genes, but suppressed the increased genome instability caused by individual query mutations. Analysis of TCGA data using the human genes predicted to act in GIS pathways revealed that a minimum of 93% of ovarian and 66% of colorectal cancer cases had defects affecting one or more predicted GIS gene. These defects included loss-of-function mutations, copy-number changes associated with reduced expression, and silencing. In contrast, acute myeloid leukaemia cases did not appear to have defects affecting the predicted GIS genes.

Sulfate is a well-established sulfur source for fungi; however, in soils sulfonates and sulfate esters, especially choline sulfate, are often much more prominent. Here we show that Saccharomyces cerevisiae YIL166C(SOA1) encodes an inorganic sulfur (sulfate, sulfite and thiosulfate) transporter that also catalyses sulfonate and choline sulfate uptake. Phylogenetic analysis of fungal SOA1 orthologues and expression of 20 members in the sul1Δ sul2Δ soa1Δ strain, which is deficient in inorganic and organic sulfur compound uptake, reveals that these transporters have diverse substrate preferences for sulfur compounds. We further show that SOA2, a S. cerevisiae SOA1 paralogue found in S. uvarum, S. eubayanus and S. arboricola is likely to be an evolutionary remnant of the uncharacterized open reading frames YOL163W and YOL162W. Our work highlights the importance of sulfonates and choline sulfate as sulfur sources in the natural environment of S. cerevisiae and other fungi by identifying fungal transporters for these compounds.

Understanding how cellular functions emerge from the underlying molecular mechanisms is a key challenge in biology. This will require computational models, whose predictive power is expected to increase with coverage and precision of formulation. Genome-scale models revolutionised the metabolic field and made the first whole-cell model possible. However, the lack of genome-scale models of signalling networks blocks the development of eukaryotic whole-cell models. Here, we present a comprehensive mechanistic model of the molecular network that controls the cell division cycle in Saccharomyces cerevisiae. We use rxncon, the reaction-contingency language, to neutralise the scalability issues preventing formulation, visualisation and simulation of signalling networks at the genome-scale. We use parameter-free modelling to validate the network and to predict genotype-to-phenotype relationships down to residue resolution. This mechanistic genome-scale model offers a new perspective on eukaryotic cell cycle control, and opens up for similar models-and eventually whole-cell models-of human cells.

Triptolide is a valuable multipotent antitumor diterpenoid in Tripterygium wilfordii, and its C-14 hydroxyl group is often selected for modification to enhance both the bioavailability and antitumor efficacy. However, the mechanism for 14-hydroxylation formation remains unknown. Here, we discover 133 kb of tandem duplicated CYP82Ds encoding 11 genes on chromosome 12 and characterize CYP82D274 and CYP82D263 as 14-hydroxylases that catalyze the metabolic grid in triptolide biosynthesis. The two CYP82Ds catalyze the aromatization of miltiradiene, which has been repeatedly reported to be a spontaneous process. In vivo assays and evaluations of the kinetic parameters of CYP82Ds indicate the most significant affinity to dehydroabietic acid among multiple intermediates. The precursor 14-hydroxy-dehydroabietic acid is successfully produced by engineered Saccharomyces cerevisiae. Our study provides genetic elements for further elucidation of the downstream biosynthetic pathways and heterologous production of triptolide and of the currently intractable biosynthesis of other 14-hydroxyl labdane-type secondary metabolites.