-
Nature Dec 2023Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification...
Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal ( www.yeast-interactome.org ) enables extensive exploration of the interactome dataset.
Topics: Mass Spectrometry; Protein Interaction Mapping; Proteome; Reproducibility of Results; Saccharomyces cerevisiae; Protein Interaction Maps; Saccharomyces cerevisiae Proteins; Epigenesis, Genetic; Databases, Factual
PubMed: 37968396
DOI: 10.1038/s41586-023-06739-5 -
Cell Mar 2024Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural...
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 ∼20 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.
Topics: Eukaryotic Cells; Neural Networks, Computer; Proteome; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38452761
DOI: 10.1016/j.cell.2024.02.014 -
Nature Sep 2023Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical...
Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.
Topics: Cryoelectron Microscopy; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mitochondria
PubMed: 37344598
DOI: 10.1038/s41586-023-06239-6 -
Molecular Cell Jul 2023The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire...
The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.
Topics: Humans; Spliceosomes; Epoxy Compounds; Macrolides; RNA Splicing; Saccharomyces cerevisiae; Mutagenesis
PubMed: 37402368
DOI: 10.1016/j.molcel.2023.06.003 -
The Journal of Biological Chemistry Aug 2023The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes...
The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.
Topics: Anti-Infective Agents; Drug Resistance, Fungal; Endoplasmic Reticulum-Associated Degradation; Hygromycin B; Lipids; Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37331602
DOI: 10.1016/j.jbc.2023.104939 -
World Journal of Microbiology &... Sep 2023Saccharomyces cerevisiae is a health microorganism closely related to human life, especially in food and pharmaceutical industries. S. cerevisiae W303a and CEN.PK2-1C...
Saccharomyces cerevisiae is a health microorganism closely related to human life, especially in food and pharmaceutical industries. S. cerevisiae W303a and CEN.PK2-1C are two commonly used strains for synthetic biology-based natural product production. Yet, the metabolomic and transcriptomic differences between these two strains have not been compared. In this study, metabolomics and transcriptomics were applied to analyze the differential metabolites and differential expression genes (DEGs) between W303a and CEN.PK2-1C cultured in YPD and SD media. The growth rate of W303a in YPD medium was the lowest compared with other groups. When cultured in YPD medium, CEN.PK2-1C produced more phenylalanine than W303a; when cultured in SD medium, W303a produced more phospholipids than CEN.PK2-1C. Transcriptomic analysis revealed that 19 out of 22 genes in glycolysis pathway were expressed at higher levels in CEN.PK2-1C than that in W303a no matter which media were used, and three key genes related to phenylalanine biosynthesis including ARO9, ARO7 and PHA2 were up-regulated in CEN.PK2-1C compared with W303a when cultured in YPD medium, whereas seven DEGs associated with phospholipid biosynthesis were up-regulated in W303a compared with CEN.PK2-1C when cultured in SD medium. The high phenylalanine produced by CEN.PK2-1C and high phospholipids produced by W303a indicated that CEN.PK2-1C may be more suitable for synthesis of natural products with phenylalanine as precursor, whereas W303a may be more appropriate for synthesis of phospholipid metabolites. This finding provides primary information for strain selection between W303a and CEN.PK2-1C for synthetic biology-based natural product production.
Topics: Humans; Saccharomyces cerevisiae; Transcriptome; Metabolomics; Biological Products; Phenylalanine; Phospholipids
PubMed: 37661201
DOI: 10.1007/s11274-023-03736-8 -
Nature Communications Dec 2023Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S...
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.
Topics: Nucleosomes; Saccharomyces cerevisiae; Histones; Saccharomyces cerevisiae Proteins; Methylation; Acetylation; Acetyltransferases
PubMed: 38065958
DOI: 10.1038/s41467-023-43968-8 -
Seminars in Cell & Developmental Biology 2024The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to... (Review)
Review
The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to fundamental metabolic processes, with significant impact on genome integrity and aging. The repetitive nature of the rDNA gene units forces the locus to maintain sequence homogeneity through recombination processes that are closely related to genomic stability. The co-presence of basic DNA transactions, such as replication, transcription by major RNA polymerases, and recombination, in a defined and restricted area of the genome is of particular relevance as it affects the stability of the rDNA locus by both direct and indirect mechanisms. This condition is well exemplified by the rDNA of Saccharomyces cerevisiae. In this review we summarize essential knowledge on how the complexity and overlap of different processes contribute to the control of rDNA and genomic stability in this model organism.
Topics: Humans; Saccharomyces cerevisiae; DNA, Ribosomal; Saccharomyces cerevisiae Proteins; Genomic Instability; DNA Replication
PubMed: 38244478
DOI: 10.1016/j.semcdb.2024.01.004 -
Science (New York, N.Y.) Jul 2023Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains...
Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains unclear. Here, we report structures of INO80 bound to a hexasome or a nucleosome. INO80 binds the two substrates in substantially different orientations. On a hexasome, INO80 places its ATPase subunit, Ino80, at superhelical location -2 (SHL -2), in contrast to SHL -6 and SHL -7, as previously seen on nucleosomes. Our results suggest that INO80 action on hexasomes resembles action by other remodelers on nucleosomes such that Ino80 is maximally active near SHL -2. The SHL -2 position also plays a critical role for nucleosome remodeling by INO80. Overall, the mechanistic adaptations used by INO80 for preferential hexasome sliding imply that subnucleosomal particles play considerable regulatory roles.
Topics: Chromatin; Chromatin Assembly and Disassembly; Histones; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37384669
DOI: 10.1126/science.adf4197 -
Essays in Biochemistry Sep 2023Branched-chain amino acids (BCAAs)-isoleucine, leucine, and valine-are synthesized by fungi. These amino acids are important components of proteins and secondary...
Branched-chain amino acids (BCAAs)-isoleucine, leucine, and valine-are synthesized by fungi. These amino acids are important components of proteins and secondary metabolites. The biochemical pathway for BCAA biosynthesis is well-characterized in the yeast Saccharomyces cerevisiae. The biosynthesis of these three amino acids is interconnected. Different precursors are metabolized in multiple steps through shared enzymes to produce isoleucine and valine, and the valine biosynthesis pathway branches before the penultimate step to a series of leucine biosynthesis-specific steps to produce leucine. Recent efforts have made advances toward characterization of the BCAA biosynthesis pathway in several fungi, revealing diversity in gene duplication and functional divergence in the genes for these enzymatic steps in different fungi. The BCAA biosynthesis pathway is regulated by the transcription factor LEU3 in S. cerevisiae, and LeuB in Aspergillus nidulans and Aspergillus fumigatus, and the activity of these transcription factors is modulated by the leucine biosynthesis pathway intermediate α-isopropylmalate. Herein, we discuss recent advances in our understanding of the BCAA pathway and its regulation, focusing on filamentous ascomycete fungi and comparison with the well-established process in yeast.
Topics: Leucine; Isoleucine; Saccharomyces cerevisiae; Amino Acids, Branched-Chain; Valine; Transcription Factors; Trans-Activators; Saccharomyces cerevisiae Proteins
PubMed: 37455545
DOI: 10.1042/EBC20230003