JaponicusDB: rapid deployment of a model organism database for an emerging model species

The fission yeast Schizosaccharomyces japonicus has recently emerged as a powerful system for studying the evolution of essential cellular processes, drawing on similarities as well as key differences between S. japonicus and the related, well-established model Schizosaccharomyces pombe. We have deployed the open-source, modular code and tools originally developed for PomBase, the S. pombe model organism database (MOD), to create JaponicusDB (www.japonicusdb.org), a new MOD dedicated to S. japonicus. By providing a central resource with ready access to a growing body of experimental data, ontology-based curation, seamless browsing and querying, and the ability to integrate new data with existing knowledge, JaponicusDB supports fission yeast biologists to a far greater extent than any other source of S. japonicus data. JaponicusDB thus enables S. japonicus researchers to realize the full potential of studying a newly emerging model species and illustrates the widely applicable power and utility of harnessing reusable PomBase code to build a comprehensive, community-maintainable repository of species-relevant knowledge.     

Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G₁, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A^Rts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.     

Sporadic Distribution of Prion-Forming Ability of Sup35p from Yeasts and Fungi

Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p’s from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function.     

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.     

DNA Replication Error-Induced Extinction of Diploid Yeast

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)–treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L–like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease–causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms.     

Stimulation of Chromosomal Rearrangements by Ribonucleotides

We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase ε that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201Δ, rnh202Δ, and rnh203Δ), topoisomerase 1 (TOP1Δ), and/or carrying mutant alleles of DNA polymerases ε, α, and δ. We observed an ∼7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201Δ. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases α and δ, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication.     

The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our two-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5^CA) and proteasome (rpn10∆) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5^CA, and apc10∆ cells, and suppressed apc5^CA cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5^CA RLS, suggesting an epistatic interaction between apc5^CA and fob1∆. Mutation to a putative L-Box (Fob1^E420V), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.     

Autophagy Competes for a Common Phosphatidylethanolamine Pool with Major Cellular PE-Consuming Pathways in Saccharomyces cerevisiae

Autophagy is a highly regulated pathway that selectively degrades cellular constituents such as protein aggregates and excessive or damaged organelles. This transport route is characterized by engulfment of the targeted cargo by autophagosomes. The formation of these double-membrane vesicles requires the covalent conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE). However, the origin of PE and the regulation of lipid flux required for autophagy remain poorly understood. Using a genetic screen, we found that the temperature-sensitive growth and intracellular membrane organization defects of mcd4-174 and mcd4-P301L mutants are suppressed by deletion of essential autophagy genes such as ATG1 or ATG7. MCD4 encodes an ethanolamine phosphate transferase that uses PE as a precursor for an essential step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor used to link a subset of plasma membrane proteins to lipid bilayers. Similar to the deletion of CHO2, a gene encoding the enzyme converting PE to phosphatidylcholine (PC), deletion of ATG7 was able to restore lipidation and plasma membrane localization of the GPI-anchored protein Gas1 and normal organization of intracellular membranes. Conversely, overexpression of Cho2 was lethal in mcd4-174 cells grown at restrictive temperature. Quantitative lipid analysis revealed that PE levels are substantially reduced in the mcd4-174 mutant but can be restored by deletion of ATG7 or CHO2. Taken together, these data suggest that autophagy competes for a common PE pool with major cellular PE-consuming pathways such as the GPI anchor and PC synthesis, highlighting the possible interplay between these pathways and the existence of signals that may coordinate PE flux.     

WormBase in 2022—data,processes, and tools for analyzing Caenorhabditis elegans

WormBase (www.wormbase.org) is the central repository for the genetics and genomics of the nematode Caenorhabditis elegans. We provide the research community with data and tools to facilitate the use of C. elegans and related nematodes as model organisms for studying human health, development, and many aspects of fundamental biology. Throughout our 22-year history, we have continued to evolve to reflect progress and innovation in the science and technologies involved in the study of C. elegans. We strive to incorporate new data types and richer data sets, and to provide integrated displays and services that avail the knowledge generated by the published nematode genetics literature. Here, we provide a broad overview of the current state of WormBase in terms of data type, curation workflows, analysis, and tools, including exciting new advances for analysis of single-cell data, text mining and visualization, and the new community collaboration forum. Concurrently, we continue the integration and harmonization of infrastructure, processes, and tools with the Alliance of Genome Resources, of which WormBase is a founding member.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.