Regulatory mechanism of cold-inducible diapause in Caenorhabditis elegans.

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.

The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

Importance and mechanisms of S-adenosylmethionine and folate accumulation in sake yeast.

Sake yeasts have a range of brewing characteristics that are particularly beneficial for sake making including high ethanol fermentability, high proliferative capacity at low temperatures, lactic acid tolerance, and high ester productivity. On the other hand, sake yeasts also accumulate a diverse range of functional components. For example, significantly greater accumulation of S-adenosylmethionine (SAM), a compound that plays important regulatory roles in a range of biological processes as a major donor of methyl groups, occurs in sake yeasts compared to other microorganisms. Significantly greater accumulation of folate, a bioactive water-soluble vitamin (vitamin B9), also occurs in sake yeasts compared to laboratory yeasts, and the methyl group on SAM is supplied by folate. Accordingly, fully characterizing 'sake yeast identity' requires detailed understanding of the mechanisms underlying both the nutritional characteristics (functional components) and the brewing characteristics in sake yeasts. Therefore, this mini-review focuses on the accumulation of SAM and folate in sake yeast including descriptions of the genes known to contribute to SAM and folate accumulation and the underlying mechanisms.

Multifarious Translational Regulation during Replicative Aging in Yeast.

Protein synthesis is strictly regulated during replicative aging in yeast, but global translational regulation during replicative aging is poorly characterized. To conduct ribosome profiling during replicative aging, we collected a large number of dividing aged cells using a miniature chemostat aging device. Translational efficiency, defined as the number of ribosome footprints normalized to transcript abundance, was compared between young and aged cells for each gene. We identified more than 700 genes with changes greater than twofold during replicative aging. Increased translational efficiency was observed in genes involved in DNA repair and chromosome organization. Decreased translational efficiency was observed in genes encoding ribosome components, transposon Ty1 and Ty2 genes, transcription factor HAC1 gene associated with the unfolded protein response, genes involved in cell wall synthesis and assembly, and ammonium permease genes. Our results provide a global view of translational regulation during replicative aging, in which the pathways involved in various cell functions are translationally regulated and cause diverse phenotypic changes.

S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects.

Methionine restriction (MetR) can extend lifespan and delay the onset of aging-associated pathologies in most model organisms. Previously, we showed that supplementation with the metabolite S-adenosyl-L-homocysteine (SAH) extends lifespan and activates the energy sensor AMP-activated protein kinase (AMPK) in the budding yeast Saccharomyces cerevisiae. However, the mechanism involved and whether SAH can extend metazoan lifespan have remained unknown. Here, we show that SAH supplementation reduces Met levels and recapitulates many physiological and molecular effects of MetR. In yeast, SAH supplementation leads to inhibition of the target of rapamycin complex 1 (TORC1) and activation of autophagy. Furthermore, in Caenorhabditis elegans SAH treatment extends lifespan by activating AMPK and providing benefits of MetR. Therefore, we propose that SAH can be used as an intervention to lower intracellular Met and confer benefits of MetR.

The sake yeast YHR032W/ERC1 allele contributes to the regulation of the tetrahydrofolate content in the folate synthetic pathway in sake yeast strains.

To elucidate the mechanism underlying tetrahydrofolate (THF) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed a quantitative trait locus (QTL) analysis. The results revealed that the sake yeast ERC1 allele contributes to an increase in the ratio of THF to the total folate content in sake yeast.

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae.

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.

Breeding of a cordycepin-resistant and adenosine kinase-deficient sake yeast strain that accumulates high levels of S-adenosylmethionine.

Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.

Implications of maintenance of mother-bud neck size in diverse vital processes of Saccharomyces cerevisiae.

The mother-bud neck is defined as the boundary between the mother cell and bud in budding microorganisms, wherein sequential morphological events occur throughout the cell cycle. This study was designed to quantitatively investigate the morphology of the mother-bud neck in budding yeast Saccharomyces cerevisiae. Observation of yeast cells with time-lapse microscopy revealed an increase of mother-bud neck size through the cell cycle. After screening of yeast non-essential gene-deletion mutants with the image processing software CalMorph, we comprehensively identified 274 mutants with broader necks during S/G2 phase. Among these yeasts, we extensively analyzed 19 representative deletion mutants with defects in genes annotated to six gene ontology terms (polarisome, actin reorganization, endosomal tethering complex, carboxy-terminal domain protein kinase complex, DNA replication, and maintenance of DNA trinucleotide repeats). The representative broad-necked mutants exhibited calcofluor white sensitivity, suggesting defects in their cell walls. Correlation analysis indicated that maintenance of mother-bud neck size is important for cellular processes such as cell growth, system robustness, and replicative lifespan. We conclude that neck-size maintenance in budding yeast is regulated by numerous genes and has several aspects that are physiologically significant.

Role of nucleocytoplasmic transport in interphase microtubule organization in fission yeast.

The proper organization of microtubules is essential for many cellular functions. Microtubule organization and reorganization are highly regulated during the cell cycle, but the underlying mechanisms remain elusive. Here we characterized unusual interphase microtubule organization in fission yeast nuclear export mutant crm1-124. The mutant cells have an intranuclear microtubule bundle during interphase that pushes the nuclear envelope to assume a protruding morphology. We showed that the formation of this protruding microtubule bundle requires the nuclear accumulation of two microtubule-associated proteins (MAPs), Alp14/TOG and Mal3/EB1. Interestingly, the forced accumulation of Alp14 in the nucleus of wild type cells is sufficient to form the intranuclear microtubule bundle. Furthermore, the frequency of the intranuclear microtubule formation by Alp14 accumulated in the nucleus is prominently increased by a reduction in the nucleation activity of interphase cytoplasmic microtubules. We propose that properly regulated nucleocytoplasmic transport and maintained activity of cytoplasmic microtubule nucleation during interphase are important for the proper organization of interphase cytoplasmic microtubules.

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast.

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca2+-CN signaling plays a central role in cell polarity control by checkpoint activation.

Evaluation of the comprehensive palatability of Japanese sake paired with dishes by multiple regression analysis based on subdomains.

Many factors contribute to palatability. In order to evaluate the palatability of Japanese alcohol sake paired with certain dishes by integrating multiple factors, here we applied an evaluation method previously reported for palatability of cheese by multiple regression analysis based on 3 subdomain factors (rewarding, cultural, and informational). We asked 94 Japanese participants/subjects to evaluate the palatability of sake (1st evaluation/E1 for the first cup, 2nd/E2 and 3rd/E3 for the palatability with aftertaste/afterglow of certain dishes) and to respond to a questionnaire related to 3 subdomains. In E1, 3 factors were extracted by a factor analysis, and the subsequent multiple regression analyses indicated that the palatability of sake was interpreted by mainly the rewarding. Further, the results of attribution-dissections in E1 indicated that 2 factors (rewarding and informational) contributed to the palatability. Finally, our results indicated that the palatability of sake was influenced by the dish eaten just before drinking.

A genetic method to enhance the accumulation of S-adenosylmethionine in yeast.

S-Adenosylmethionine (SAM) is a key component of sulphur amino acid metabolism in living organisms and is synthesised from methionine and adenosine triphosphate by methionine adenosyltransferase. This molecule serves as the main biological methyl donor due to its active methylthio ether group. Notably, SAM has shown beneficial effects in clinical trials for the treatment of alcoholic liver disease, depression and joint pain. Due to the high potential value of SAM, current research efforts are attempting to develop a more rapid, cost-effective and higher yielding SAM production method than the conventional production system. In this mini-review, we describe the previously reported yeast gene that contributes to SAM accumulation by overexpression, mutation or deletion and summarise the genetic approach for the production of SAM in large industrial quantities.

Sake yeast YHR032W/ERC1 haplotype contributes to high S-adenosylmethionine accumulation in sake yeast strains.

Sake yeasts are ideally suited for sake making, producing higher levels of ethanol, proliferating at lower temperatures, and producing greater levels of various aromatic components and nutrients than laboratory yeasts. To elucidate the mechanism underlying S-adenosylmethionine (SAM) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed quantitative trait locus (QTL) analysis and identified a significant QTL on chromosome VIII. Of the 165 genes mapped at 49.8 cM from the left-end DNA marker of chromosome VIII, we focused on the YHR032W/ERC1 gene, encoding a member of the multi-drug and toxin extrusion family having antiporter activity and involved in SAM accumulation and ethionine resistance. Expression of the sake yeast ERC1 haplotype (K7ERC1) in a low- and high-copy number plasmid BYΔerc1 resulted in intracellular SAM accumulation, whereas expression of the laboratory yeast ERC1 haplotype (XERC1) did not. Comparison between DNA sequences of K7ERC1 and XERC1 revealed three amino acid substitutions: S51N, V263I, and N545I. Site-directed mutagenesis revealed that the N545I frameshift mutation was responsible for the K7ERC1 phenotype. These results indicate that K7ERC1 contributes to SAM accumulation in sake yeast strains.

Stimulating S-adenosyl-l-methionine synthesis extends lifespan via activation of AMPK.

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801.

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity.

Natural thioallyl compounds increase oxidative stress resistance and lifespan in Caenorhabditis elegans by modulating SKN-1/Nrf.

Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.

Isolation of a spontaneous cerulenin-resistant sake yeast with both high ethyl caproate-producing ability and normal checkpoint integrity.

In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.

Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin.

Calcineurin, which is a Ca(2+)/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the ∆vrp1 cells. Finally, we demonstrated that the ∆vrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.

Late-maturing cooking rice Sensyuraku has excellent properties, equivalent to sake rice, for high-quality sake brewing.

Low protein content and sufficient grain rigidity are desired properties for the rice used in high-quality sake brewing such as Daiginjo-shu (polishing ratio of the rice, less than 50%). Two kinds of rice, sake rice (SR) and cooking rice (CR), have been used for sake brewing. Compared with those of SR, analyses of CR for high-quality sake brewing using highly polished rice have been limited. Here we described the original screening of late-maturing CR Sensyuraku (SEN) as rice with low protein content and characterization of its properties for high-quality sake brewing. The protein content of SEN was lower than those of SR Gohyakumangoku (GOM) and CR Yukinosei (YUK), and its grain rigidity was higher than that of GOM. The excellent properties of SEN with respect to both water-adsorption and enzyme digestibility were confirmed using a Rapid Visco Analyzer (RVA). Further, we confirmed a clear taste of sake produced from SEN by sensory evaluation. Thus, SEN has excellent properties, equivalent to those of SR, for high-quality sake brewing.