Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe.

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival.

Polarized exocytosis induced by local Cdc42 GTPase activity results in membrane flows that deplete low-mobility membrane-associated proteins. A reaction-diffusion particle model comprising Cdc42 positive feedback activation, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis shows that flow-induced depletion of low mobility GAPs promotes polarization. We modified Cdc42 mobility in Schizosaccharomyces pombe by replacing its prenylation site with 1, 2 or 3 repeats of the Rit C-terminal membrane-binding domain (ritC), yielding alleles with progressively lower mobility and increased flow-coupling. While Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC are inviable, in agreement with model's predictions. Deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells, verifying the model's prediction that GAP deletion increases Cdc42 activity at the expense of polarization. Our work demonstrates how membrane flows are an integral part of Cdc42-driven pattern formation and require Cdc42-GTP to turn over faster than the surface on which it forms.

Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1.

Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

3,3'-Diindolylmethane disrupts the endoplasmic reticulum and nuclear envelope in Schizosaccharomyces pombe.

3,3'-Diindolylmethane is recognized for its anti-cancer activities in various pathways, though its mechanism remains to be fully elucidated. Previous studies have shown that 3,3'-Diindolylmethane disturbed the localization of Cut11, a nuclear pore complex subunit in Schizosaccharomyces pombe. This study further reveals that in Schizosaccharomyces pombe, 3,3'-Diindolylmethane also disrupts other components of nuclear envelope, causing GFP-NLS leakage, making it evident that 3,3'-Diindolylmethane disrupts the nuclear envelope. 3,3'-Diindolylmethane also disturbs the localization of GFP-ADEL and Ost4, which are endoplasmic reticulum lumen proteins and membrane proteins respectively, suggesting the function of 3,3'-Diindolylmethane on endoplasmic reticulum disturbance. The nuclear envelope repairment, normal nuclear envelope physical properties, and lipid metabolism homeostasis are crucial for cell survival in the presence of 3,3'-Diindolylmethane. These findings provide new insights into the understanding and development of 3,3'-Diindolylmethane as an anti-cancer agent.

Cross-regulations of two connected domains form a mechanical circuit for steady force transmission during clathrin-mediated endocytosis.

Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin. We also characterized a domain in End4p, Rend (rod domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15 pN terminates the transmission of force. We show that the mechanical properties (mechanical stability, unfolding extension, hysteresis) of Rend and THATCH are tuned to form a circuit for the initiation, transmission, and termination of force between the actin cytoskeleton and membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.

Differential Cytoophidium Assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by the enzyme CTP synthase (CTPS), which is known to form cytoophidia across all three domains of life. In this study, we use the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as model organisms to compare cytoophidium assembly under external environmental and intracellular CTPS alterations. We observe that under low and high temperature conditions, cytoophidia in fission yeast gradually disassemble, while cytoophidia in budding yeast remain unaffected. The effect of pH changes on cytoophidia maintenance in the two yeast species is different. When cultured in the yeast-saturated cultured medium, cytoophidia in fission yeast disassemble, while cytoophidia in budding yeast gradually form. Overexpression of CTPS results in the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demonstrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.

Uridylation regulates mRNA decay directionality in fission yeast.

Cytoplasmic mRNA decay is effected by exonucleolytic degradation in either the 5' to 3' or 3' to 5' direction. Pervasive terminal uridylation is implicated in mRNA degradation, however, its functional relevance for bulk mRNA turnover remains poorly understood. In this study, we employ genome-wide 3'-RACE (gw3'-RACE) in the model system fission yeast to elucidate the role of uridylation in mRNA turnover. We observe widespread uridylation of shortened poly(A) tails, promoting efficient 5' to 3' mRNA decay and ensuring timely and controlled mRNA degradation. Inhibition of this uridylation process leads to excessive deadenylation and enhanced 3' to 5' mRNA decay accompanied by oligouridylation. Strikingly we found that uridylation of poly(A) tails and oligouridylation of non-polyadenylated substrates are catalysed by different terminal uridyltransferases Cid1 and Cid16 respectively. Our study sheds new light on the intricate regulatory mechanisms underlying bulk mRNA turnover, demonstrating the role of uridylation in modulating mRNA decay pathways.

Fission yeast Duc1 links to ER-PM contact sites and influences PM lipid composition and cytokinetic ring anchoring.

Cytokinesis is the final stage of the cell cycle that results in the physical separation of daughter cells. To accomplish cytokinesis, many organisms build an actin- and myosin-based cytokinetic ring (CR) that is anchored to the plasma membrane (PM). Defects in CR-PM anchoring can arise when the PM lipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is depleted. In Schizosaccharomyces pombe, reduced PM PI(4,5)P2 results in a CR that cannot maintain a medial position and slides toward one cell end, resulting in two differently sized daughter cells. S. pombe PM PI(4,5)P2 is synthesized by the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, but what regulates this enzyme to maintain appropriate PM PI(4,5)P2 levels in S. pombe is not known. To identify Its3 regulators, we used proximity-based biotinylation, and the uncharacterized protein Duc1 was specifically detected. We discovered that Duc1 decorates the PM except at the cell division site and that its unique localization pattern is dictated by binding to the endoplasmic reticulum (ER)-PM contact site proteins Scs2 and Scs22. Our evidence suggests that Duc1 also binds PI(4,5)P2 and helps enrich Its3 at the lateral PM, thereby promoting PM PI(4,5)P2 synthesis and robust CR-PM anchoring.

Functional roles of the interaction of Moa1 with CENP-C and Rec8 in meiosis of Schizosaccharomyces pombe.

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.

VAP-mediated membrane-tethering mechanisms implicate ER-PM contact function in pH homeostasis.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are highly conserved endoplasmic reticulum (ER)-resident proteins that establish ER contacts with multiple membrane compartments in many eukaryotes. However, VAP-mediated membrane-tethering mechanisms remain ambiguous. Here, focusing on fission yeast ER-plasma membrane (PM) contact formation, using systematic interactome analyses and quantitative microscopy, we predict a non-VAP-protein direct binding-based ER-PM coupling. We further reveal that VAP-anionic phospholipid interactions may underlie ER-PM association and define the pH-responsive nature of VAP-tethered membrane contacts. Such conserved interactions with anionic phospholipids are generally defective in amyotrophic lateral sclerosis-associated human VAPB mutant. Moreover, we identify a conserved FFAT-like motif locating at the autoinhibitory hotspot of the essential PM proton pump Pma1. This modulatory VAP-Pma1 interaction appears crucial for pH homeostasis. We thus propose an ingenious strategy for maintaining intracellular pH by coupling Pma1 modulation with pH-sensory ER-PM contacts via VAP-mediated interactions.

DEAD-box ATPase Dbp2 is the key enzyme in an mRNP assembly checkpoint at the 3'-end of genes and involved in the recycling of cleavage factors.

mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are tightly coordinated to ensure that only fully processed transcripts are released from chromatin for export from the nucleus. Here, we present the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is the key enzyme in an RNP assembly checkpoint at the 3'-end of genes. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localises to cleavage bodies, which are enriched for 3'-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3'-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, and CPAC components are depleted from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites and a delayed transcription termination, suggesting that levels of free CPAC components are insufficient to maintain normal levels of 3'-end processing. Our data support a model in which Dbp2 is the active component of an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release.

Mapping the dynamics of epigenetic adaptation in S. pombe during heterochromatin misregulation.

Epigenetic mechanisms enable cells to develop novel adaptive phenotypes without altering their genetic blueprint. Recent studies show histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), can be redistributed to establish adaptive phenotypes. We developed a precision-engineered genetic approach to trigger heterochromatin misregulation on-demand in fission yeast. This enabled us to trace genome-scale RNA and H3K9me changes over time in long-term, continuous cultures. Adaptive H3K9me establishes over remarkably slow timescales relative to the initiating stress. We captured dynamic H3K9me redistribution events which depend on an RNA binding complex MTREC, ultimately leading to cells converging on an optimal adaptive solution. Upon stress removal, cells relax to new transcriptional and chromatin states, establishing memory that is tunable and primed for future adaptive epigenetic responses. Collectively, we identify the slow kinetics of epigenetic adaptation that allow cells to discover and heritably encode novel adaptive solutions, with implications for drug resistance and response to infection.

A novel transcription factor Sdr1 involving sulfur depletion response in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the response to sulfur depletion has been less studied compared to the response to nitrogen depletion. Our study reveals that the fission yeast gene, SPCC417.09c, plays a significant role in the sulfur depletion response. This gene encodes a protein with a Zn2Cys6 fungal-type DNA-binding domain and a transcription factor domain, and we have named it sdr1+ (sulfur depletion response 1). Interestingly, while sulfur depletion typically induces autophagy akin to nitrogen depletion, we found that autophagy was not induced under sulfur depletion in the absence of sdr1+. This suggests that sdr1+ is necessary for the induction of autophagy under conditions of sulfur depletion. Although sdr1+ is not essential for the growth of fission yeast, its overexpression, driven by the nmt1 promoter, inhibits growth. This implies that Sdr1 may possess cell growth-inhibitory capabilities. In addition, our analysis of Δsdr1 cells revealed that sdr1+ also plays a role in regulating the expression of genes associated with the phosphate depletion response. In conclusion, our study introduces Sdr1 as a novel transcription factor that contributes to an appropriate cellular nutrient starvation response. It does so by inhibiting inappropriate cell growth and inducing autophagy in response to sulfur depletion.

The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Mrc1 regulates parental histone segregation and heterochromatin inheritance.

Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1's involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.

Creating Meiotic Recombination-Regulating DNA Sites by SpEDIT in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions.

Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.

Structural basis for transthiolation intermediates in the ubiquitin pathway.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins1-3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters4-6. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.

CDK phosphorylation of Sfr1 downregulates Rad51 function in late-meiotic homolog invasions.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved.

The effect of centromere protein Fta2 phosphorylation during meiosis.

During meiosis, defects in cohesin localization within the centromere region can result in various diseases. Accurate cohesin localization depends on the Mis4-Ssl3 loading complex. Although it is known that cohesin completes the loading process with the help of the loading complex, the mechanisms underlying its localization in the centromere region remain unclear. Previous studies suggest cohesin localization in the centromere is mediated by phosphorylation of centromeric proteins. In this study, we focused on the Fta2 protein, a component of the Sim4 centromere protein complex. Using bioinformatics methods, potential phosphorylation sites were identified, and fta2-9A and fta2-9D mutants were constructed in Schizosaccharomyces pombe. The phenotypes of these mutants were characterized through testing thiabendazole (TBZ) sensitivity and fluorescent microscopy localization. Results indicated that Fta2 phosphorylation did not impact mitosis but affected chromosome segregation during meiosis. This study suggests that Fta2 phosphorylation is vital for meiosis and may be related to the specific localization of cohesin during this process.