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The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.
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Tamaño de la Célula , Proteína Quinasa Deficiente en Lisina WNK 1 , Humanos , Animales , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Evolución Molecular , Células HEK293 , Unión Proteica , Familia de Multigenes , Presión OsmóticaRESUMEN
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.
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Poliubiquitina , Proteínas Ribosómicas , Ribosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Poliubiquitina/metabolismo , Poliubiquitina/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteostasis , Núcleo Celular/metabolismoRESUMEN
Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenomics approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued growth inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in promoting G1 progression.
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We previously constructed TheCellVision.org, a central repository for visualizing and mining data from yeast high-content imaging projects. At its inception, TheCellVision.org housed two high-content screening (HCS) projects providing genome-scale protein abundance and localization information for the budding yeast Saccharomyces cerevisiae, as well as a comprehensive analysis of the morphology of its endocytic compartments upon systematic genetic perturbation of each yeast gene. Here, we report on the expansion of TheCellVision.org by the addition of two new HCS projects and the incorporation of new global functionalities. Specifically, TheCellVision.org now hosts images from the Cell Cycle Omics project, which describes genome-scale cell cycle-resolved dynamics in protein localization, protein concentration, gene expression, and translational efficiency in budding yeast. Moreover, it hosts PIFiA, a computational tool for image-based predictions of protein functional annotations. Across all its projects, TheCellVision.org now houses >800,000 microscopy images along with computational tools for exploring both the images and their associated datasets. Together with the newly added global functionalities, which include the ability to query genes in any of the hosted projects using either yeast or human gene names, TheCellVision.org provides an expanding resource for single-cell eukaryotic biology.
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Minería de Datos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Minería de Datos/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo CelularRESUMEN
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of â¼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucariotas/metabolismo , Redes Neurales de la Computación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.
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Longevidad , Procesamiento Proteico-Postraduccional , Masculino , Humanos , Secuencia de Aminoácidos , Acetilación , Longevidad/genética , Ubiquitinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with Ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs), Ubp2 and Ubp14, and E3 ligases, Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the Ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the Intranuclear Quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with Ribosomopathies.
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CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background, or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal. A record of this paper's transparent peer review process is included in the supplemental information.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Reproducibilidad de los Resultados , Fenotipo , Línea CelularRESUMEN
A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells.
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Genes cdc , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Mitosis/genética , Fosforilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphological profiling using drug-hypersensitive strains to minimize compound use, HT microscopy to speed up data generation and analysis, and a generalized linear model to predict targets with high reliability. We first conducted a proof-of-concept study using six compounds with known targets: bortezomib, hydroxyurea, methyl methanesulfonate, benomyl, tunicamycin, and echinocandin B. Then we applied our platform to predict the mechanism of action of a novel diferulate-derived compound, poacidiene. Morphological profiling of poacidiene implied that it affects the DNA damage response, which genetic analysis confirmed. Furthermore, we found that poacidiene inhibits the growth of phytopathogenic fungi, implying applications as an effective antifungal agent. Thus, our platform is a new whole-cell target prediction tool for drug discovery.
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Descubrimiento de Drogas , Saccharomyces cerevisiae , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/genéticaRESUMEN
Single-cell image analysis provides a powerful approach for studying cell-to-cell heterogeneity, which is an important attribute of isogenic cell populations, from microbial cultures to individual cells in multicellular organisms. This phenotypic variability must be explained at a mechanistic level if biologists are to fully understand cellular function and address the genotype-to-phenotype relationship. Variability in single-cell phenotypes is obscured by bulk readouts or averaging of phenotypes from individual cells in a sample; thus, single-cell image analysis enables a higher resolution view of cellular function. Here, we consider examples of both small- and large-scale studies carried out with isogenic cell populations assessed by fluorescence microscopy, and we illustrate the advantages, challenges, and the promise of quantitative single-cell image analysis.
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Variación Biológica Poblacional , Análisis de la Célula Individual , Microscopía Fluorescente , FenotipoRESUMEN
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
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Genes Esenciales , Saccharomyces cerevisiae , Biblioteca de Genes , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
We describe a protocol for live-cell high-throughput (HTP) screening of yeast mutant strains carrying fluorescent protein markers for subcellular compartments of choice using automated confocal microscopy. This procedure, which combines HTP genetics and microscopy, results in the acquisition of thousands of images that can be analyzed in a systematic and quantitative way to identify morphology defects in the tagged subcellular compartments. This HTP protocol is readily adapted for screening any combination of markers and can be expanded to different growth conditions or higher order mutant genetic backgrounds.
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Colorantes Fluorescentes/química , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ensayos Analíticos de Alto Rendimiento , Microscopía Confocal , Microscopía Fluorescente/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.
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Redes Reguladoras de Genes , Pruebas Genéticas/métodos , Mitocondrias/genética , Biología de Sistemas/métodos , Algoritmos , Benchmarking , Sesgo , Sistemas CRISPR-Cas , Línea Celular , Células HEK293 , HumanosRESUMEN
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
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Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Proteolisis , Proteómica/métodos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/clasificación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína Fluorescente RojaRESUMEN
Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.
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Epistasis Genética , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Genes Fúngicos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genética , Alelos , Genes Esenciales , Genes Sintéticos , Genotipo , Haploidia , Mutación , Fenotipo , Saccharomyces cerevisiae/metabolismoRESUMEN
Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The protocol instructs users on the trigenic synthetic genetic array analysis technique, τ-SGA, for high-throughput screens. The steps describe construction of the double-mutant query strains and the corresponding single-mutant control query strains, which are screened in parallel in two replicates. The screening experimental set-up consists of sequential replica-pinning steps that enable automated mating, meiotic recombination and successive haploid selection steps for the generation of triple mutants, which are scored for colony size as a proxy for fitness, which enables the calculation of trigenic interactions. The procedure described here was used to conduct 422 trigenic interaction screens, which generated ~460,000 yeast triple mutants for trigenic interaction analysis. Users should be familiar with robotic equipment required for high-throughput genetic interaction screens and be proficient at the command line to execute the scoring pipeline. Large-scale screen computational analysis is achieved by using MATLAB pipelines that score raw colony size data to produce τ-SGA interaction scores. Additional recommendations are included for optimizing experimental design and analysis of smaller-scale trigenic interaction screens by using a web-based analysis system, SGAtools. This protocol provides a resource for those who would like to gain a deeper, more practical understanding of trigenic interaction screening and quantification methodology.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Levaduras/genética , Alelos , Biología Computacional/métodos , Redes Reguladoras de Genes/genética , Técnicas Genéticas , Pruebas Genéticas/métodos , Haploidia , Meiosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genéticaRESUMEN
Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.
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Transporte Activo de Núcleo Celular/genética , Señales de Exportación Nuclear/genética , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Perfil Genético , Genómica , Proteínas Fluorescentes Verdes , Mutación , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project.