Yeast Genetics Research Articles

Four decades of Eukaryotic DNA replication: From yeast genetics to high-resolution cryo-EM structures of the replisome

I had my eyes set on DNA replication research when I took my first molecular biology course in graduate school. My election to the National Academy of Sciences came just when I was retiring from active research. It gives me an opportunity to reflect on my personal journey in eukaryotic DNA replication research, which started as a thought experiment and culminated in witnessing the determination of the cryoelectron microscopic structure of the yeast replisome in the act of transferring histone-encoded epigenetic information at the replication fork. I would like to dedicate this inaugural article to my talented trainees and valuable collaborators in gratitude for the joy they gave me in this journey. I also want to thank my mentors who instilled in me the purpose of science. I hope junior scientists will not be disheartened by the marathon nature of research, but mindful enough to integrate and pause for other equally fun and meaningful activities of life into the marathon.

The conserved protein adaptors CALM/AP180 and FCHo1/2 cooperatively recruit Eps15 to promote the initiation of clathrin-mediated endocytosis in yeast.

Clathrin-mediated endocytosis (CME) is a critical trafficking process that begins when an elaborate endocytic protein network is established at the plasma membrane. Interaction of early endocytic proteins with anionic phospholipids and/or cargo has been suggested to trigger CME initiation. However, the exact mechanism by which CME sites are initiated has not been fully elucidated. In the budding yeast Saccharomyces cerevisiae, higher levels of anionic phospholipids and cargo molecules exist in the newly formed daughter cell compared to the levels in the mother cell during polarized growth. Taking advantage of this asymmetry, we quantitatively compared CME proteins in S. cerevisiae mother versus daughter cells, observing differences in the dynamics and composition of key endocytic proteins. Our results show that CME site initiation occurs preferentially on regions of the plasma membrane with a relatively higher density of endocytic cargo and/or acidic phospholipids. Furthermore, our combined live cell-imaging and yeast genetics analysis provided evidence for a molecular mechanism in which CME sites are initiated when Yap1801 and Yap1802 (yeast CALM/AP180) and Syp1 (yeast FCHo1/2) coordinate with anionic phospholipids and cargo molecules to trigger Ede1 (yeast Eps15)-centric CME initiation complex assembly at the plasma membrane.

On the Anniversary of the Honorary President of the Vavilov Society of Geneticists and Breeders, Academician Sergei Georgievich Inge-Vechtomov

On April 4, 2024, the outstanding geneticist and teacher, Honorary President of the Vavilov Society of Geneticists and Breeders, Honorary Professor of St. Petersburg State University, Member of the Academic Council of VIR, Academician of the Russian Academy of Sciences Sergei Georgievich Inge-Vechtomov turned 85 years old. S.G. Inge-Vechtomov is known as an eminent scientist in the field of Saccharomyces yeast genetics; he has made a significant contribution to the study of the regulation of translation termination in eukaryotes and the phenomenon of protein inheritance. Sergei Georgievich is the author of a number of textbooks on genetics that have received significant recognition. For more than forty years he was the Head of the oldest department of genetics in our country – the Department of Genetics and Biotechnology of St. Petersburg State University, established a leading scientific and pedagogical school, trained a number of disciples who now lead scientific teams not only in Russia, but also abroad. S.G. Inge-Vechtomov does a lot of organizational work, he made a significant contribution to the creation of the Vavilov Society of Geneticists and Breeders, to the organization and holding of a number of Vavilov Society congresses, and is currently its Honorary President. S.G. Inge-Vechtomov is a member of editorial boards of a number of journals, but the most important result of his editorial activities was the creation of the journal “Ecological Genetics”, which is currently actively developing. Colleagues and friends, members of the genetic scientific community, researchers, students and professors wish Sergei Georgievich inexhaustible energy, health, good mood and new achievements in science and educational activities.

Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how cell size influences physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be compositionally different. In the present study, we utilize the natural heterogeneity of hepatocyte ploidy and yeast genetics to establish that the ploidy-to-cell size ratio is a highly conserved determinant of proteome composition. In both mammalian and yeast cells, genome dilution by cell growth elicits a starvation-like phenotype, suggesting that growth in large cells is restricted by genome concentration in a manner that mimics a limiting nutrient. Moreover, genome dilution explains some proteomic changes ascribed to yeast aging. Overall, our data indicate that genome concentration drives changes in cell composition independently of external environmental cues.

The RNA Helicase Ded1 from Yeast Is Associated with the Signal Recognition Particle and Is Regulated by SRP21.

The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.

The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage.

The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

Abstract 5902: Targeting MAPK-driven tumors via inhibition of MAP2K4

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination.

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans.

Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.

Systematic profiling of ale yeast protein dynamics across fermentation and repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is among the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout 2 fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

Collective production of hydrogen sulfide gas enables budding yeast lacking MET17 to overcome their metabolic defect.

Assimilation of sulfur is vital to all organisms. In S. cerevisiae, inorganic sulfate is first reduced to sulfide, which is then affixed to an organic carbon backbone by the Met17 enzyme. The resulting homocysteine can then be converted to all other essential organosulfurs such as methionine, cysteine, and glutathione. This pathway has been known for nearly half a century, and met17 mutants have long been classified as organosulfur auxotrophs, which are unable to grow on sulfate as their sole sulfur source. Surprisingly, we found that met17Δ could grow on sulfate, albeit only at sufficiently high cell densities. We show that the accumulation of hydrogen sulfide gas underpins this density-dependent growth of met17Δ on sulfate and that the locus YLL058W (HSU1) enables met17Δ cells to assimilate hydrogen sulfide. Hsu1 protein is induced during sulfur starvation and under exposure to high sulfide concentrations in wild-type cells, and the gene has a pleiotropic role in sulfur assimilation. In a mathematical model, the low efficiency of sulfide assimilation in met17Δ can explain the observed density-dependent growth of met17Δ on sulfate. Thus, having uncovered and explained the paradoxical growth of a commonly used "auxotroph," our findings may impact the design of future studies in yeast genetics, metabolism, and volatile-mediated microbial interactions.

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head.

Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. A combination of data from yeast genetics, mass spectrometry, DMS probing and biochemical experiments demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

Pdr5: A master of asymmetry

Pdr5 is a founding member of a large (pdr) subfamily of clinically and agriculturally significant fungal ABC transporters. The tremendous power of yeast genetics combined with biochemical and structural approaches revealed the astonishing asymmetry of this efflux pump. Asymmetry is manifested in Pdr5′s ATP-binding sites, drug binding sites, signal transformation interface, and molecular exit gate. Even its mode of conformational switching is asymmetric with one half of the protein remaining nearly stationary. In the case of its ATP-binding sites, asymmetry is created by replacing a set of highly conserved residues with a characteristic set of deviant ones. This contrasts with the asymmetry of the molecular gate. There, a full complement of canonical residues is present, but structural features in the vicinity prevent some of these from forming a molecular plug during closure. Compared to their canonical-functioning counterparts, the deviant ATP site and these gating residues have different, essential functions. In addition to its remarkable asymmetry, the surprising observation that Pdr5 is a drug / proton co-transporter shines a new light on this remarkable protein.

A Zpr1 co-chaperone mediates folding of eukaryotic translation elongation factor 1A via a GTPase cycle

General protein folding is mediated by chaperones that utilize ATP hydrolysis to regulate client binding and release. Zinc-finger protein 1 (Zpr1) is an essential ATP-independent chaperone dedicated to the biogenesis of eukaryotic translation elongation factor 1A (eEF1A), a highly abundant GTP-binding protein. How Zpr1-mediated folding is regulated to ensure rapid Zpr1 recycling remains an unanswered question. Here, we use yeast genetics and microscopy analysis, biochemical reconstitution, and structural modeling to reveal that folding of eEF1A by Zpr1 requires GTP hydrolysis. Furthermore, we identify the highly conserved altered inheritance of mitochondria 29 (Aim29) protein as a Zpr1 co-chaperone that recognizes eEF1A in the GTP-bound, pre-hydrolysis conformation. This interaction dampens Zpr1⋅eEF1A GTPase activity and facilitates client exit from the folding cycle. Our work reveals that a bespoke ATP-independent chaperone system has mechanistic similarity to ATPase chaperones but unexpectedly relies on client GTP hydrolysis to regulate the chaperone-client interaction.

A set of vectors and strains for chromosomal integration in fission yeast

The expression of heterologous genes is an important technique in yeast genetics. In fission yeast, the leu1 and ura4 genes have been used mainly as selectable markers for heterologous expression. To expand the repertoire of selection markers available for heterologous expression of genes, here we developed new host-vector systems employing lys1 and arg3. By employing genome editing with the CRISPR/Cas9 system, we isolated several alleles of lys1 and arg3, each having a critical mutation in the ORF region. In parallel, we developed a set of vectors that complement the amino acid auxotrophy of lys1 and arg3 mutants when integrated into each locus. Using these vectors in combination with the previously developed integration vector pDUAL, we successfully observed the localization of three proteins in a cell simultaneously by fusing them with different fluorescent proteins. Thus, these vectors enable combinatorial expression of heterologous genes, which addresses increasingly diverse experimental challenges.

Transcriptional Regulation Technology for Gene Perturbation in Fission Yeast.

Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3' untranslated region (3'UTR) disruption, are available for yeast systems, CRISPR-Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR-Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.

Network Analysis Reveals the Molecular Bases of Statin Pleiotropy That Vary with Genetic Background.

Many approved drugs are pleiotropic: for example, statins, whose main cholesterol-lowering activity is complemented by anticancer and prodiabetogenic mechanisms involving poorly characterized genetic interaction networks. We investigated these using the Saccharomyces cerevisiae genetic model, where most genetic interactions known are limited to the statin-sensitive S288C genetic background. We therefore broadened our approach by investigating gene interactions to include two statin-resistant genetic backgrounds: UWOPS87-2421 and Y55. Networks were functionally focused by selection of HMG1 and BTS1 mevalonate pathway genes for detection of genetic interactions. Networks, multilayered by genetic background, were analyzed for key genes using network centrality (degree, betweenness, and closeness), pathway enrichment, functional community modules, and Gene Ontology. Specifically, we found modification genes related to dysregulated endocytosis and autophagic cell death. To translate results to human cells, human orthologues were searched for other drug targets, thus identifying candidates for synergistic anticancer bioactivity. IMPORTANCE Atorvastatin is a highly successful drug prescribed to lower cholesterol and prevent cardiovascular disease in millions of people. Though much of its effect comes from inhibiting a key enzyme in the cholesterol biosynthetic pathway, genes in this pathway interact with genes in other pathways, resulting in 15% of patients suffering painful muscular side effects and 50% having inadequate responses. Such multigenic complexity may be unraveled using gene networks assembled from overlapping pairs of genes that complement each other. We used the unique power of yeast genetics to construct genome-wide networks specific to atorvastatin bioactivity in three genetic backgrounds to represent the genetic variation and varying response to atorvastatin in human individuals. We then used algorithms to identify key genes and their associated FDA-approved drugs in the networks, which resulted in the distinction of drugs that may synergistically enhance the known anticancer activity of atorvastatin.

An unknown essential function of tRNA splicing endonuclease is linked to the integrated stress response and intron debranching.

tRNA splicing endonuclease (TSEN) has a well-characterized role in transfer RNA (tRNA) splicing but also other functions. For yeast TSEN, these other functions include degradation of a subset of mRNAs that encode mitochondrial proteins and an unknown essential function. In this study, we use yeast genetics to characterize the unknown tRNA-independent function(s) of TSEN. Using a high-copy suppressor screen, we found that sen2 mutants can be suppressed by overexpression of SEN54. This effect was seen both for tRNA-dependent and tRNA-independent functions indicating that SEN54 is a general suppressor of sen2, likely through structural stabilization. A spontaneous suppressor screen identified mutations in the intron-debranching enzyme, Dbr1, as tRNA splicing-independent suppressors. Transcriptome analysis showed that sen2 mutation activates the Gcn4 stress response. These Gcn4 target transcripts decreased considerably in the sen2 dbr1 double mutant. We propose that Dbr1 and TSEN may compete for a shared substrate, which TSEN normally processes into an essential RNA, while Dbr1 initiates its degradation. These data provide further insight into the essential function(s) of TSEN. Importantly, single amino acid mutations in TSEN cause the generally fatal neuronal disease pontocerebellar hypoplasia (PCH). The mechanism by which defects in TSEN cause this disease is unknown, and our results reveal new possible mechanisms.

Cis-Golgi phosphate transporters harboring an EXS domain are essential for plant growth and development.

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate-sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Renewing Lost Genetic Variability with a Classical Yeast Genetics Approach.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.