[Show abstract][Hide abstract] ABSTRACT: Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1γ, Cki3 is a critical determinant of NETO onset. Not only did cki3∆ cells undergo NETO during G1- or S-phase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinase-dead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3∆ cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for protein-protein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner.
Genes to Cells 11/2015; DOI:10.1111/gtc.12309 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.
[Show abstract][Hide abstract] ABSTRACT: In the fermentation industry, the traceability of microorganisms during the process is important to ensure safety and efficacy. Ethyl carbamate, a group-2A carcinogen, is produced from ethanol and urea during the storage of food/alcoholic beverages. We isolated non-urea-producing sake yeast car1 mutants carrying a discriminable molecular marker, and demonstrated, by the use of PCR assays, that these mutants are useful for traceability analysis and identification during the sake brewing process.
[Show abstract][Hide abstract] ABSTRACT: The Japanese high-quality sake Daiginjo-shu is made from highly polished rice (polishing ratio, less than 50%). Here we showed that the sake rice Koshitanrei (KOS) has an excellent polishing property. Rice grains of KOS had the same lined white-core region as the sake rice Yamadanishiki (YAM). The grain rigidity/hardness of KOS was higher than that of the sake rice Gohyakumangoku (GOM). The loss ratio of KOS after high polishing by an industrial polishing machine was lower than that of GOM. Further, a clear taste of sake produced from KOS was confirmed by sensory evaluation.
[Show abstract][Hide abstract] ABSTRACT: Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.
[Show abstract][Hide abstract] ABSTRACT: The Start/G1 phase in the cell cycle is an important period during which cells determine the fate, onset of mitotic progression or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell-size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here we show that Whi3 was phosphorylated by the RAS/cAMP-dependent protein kinase (PKA) and that the phosphorylation of Ser-568 in Whi3 by PKA played an inhibitory role in the Whi3 function. Phosphorylation of Whi3 by PKA led to decreased interaction of it with CLN3 G1 cyclin mRNA and was required for the promotion of the G1/S progression. Further, we demonstrate that the phospho-mimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events.
[Show abstract][Hide abstract] ABSTRACT: We show that the concentration of total free fatty acids (FFAs) in sake produced by yeast with high productivity of ethyl caproate could be approximated by the concentration of 2 FFAs, caproic and caprylic acids. Measurement of the total FFAs concentration by an enzymatic method proved useful for both estimating the ethyl caproate concentration in sake and also for yeast breeding.
[Show abstract][Hide abstract] ABSTRACT: We conducted a mitotic localization study on gene products encoded by 56 uncharacterized fission yeast ORFs that were transcriptionally up-regulated during meiotic division. Despite meiotic gene induction, these genes were expressed during mitosis as well. Seven gene products were localized in the nucleus and/or chromatin; another one was a mitosis-specific spindle pole body component and, intriguingly, its human homologue was also localized in the centrosome of cultured HeLa cells. Two products appeared to be localized in cytoplasmic microtubules, whereas four were mitochondrial proteins. Three other proteins were found in the medial ring upon cytokinesis and another was localized on the entire cell periphery. The remaining 38 proteins were detected in the cytoplasm and showed varied spatial patterns. This systematic study helps our integrated understanding of all the protein functions in the fission yeast as a eukaryotic model.
[Show abstract][Hide abstract] ABSTRACT: To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions.
FEMS Yeast Research 11/2011; 12(3):293-304. DOI:10.1111/j.1567-1364.2011.00770.x · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, calcium (Ca2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved
Ca2+/calmodulin (CaM)-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms
ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of lifespan, as a suppressor of the Ca2+ sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca2+-signaling and lifespan in yeast. Here we show that Ca2+ affected the replicative lifespan (RLS) of yeast. Increased external and intracellular Ca2+ levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted ageing without impairing
the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a
regulation of RLS even under normal growth condition. Thus, our results indicate that Ca2+ homeostasis/Ca2+-signaling is required to regulate longevity in budding yeast.
Journal of Biological Chemistry 06/2011; · 4.57 Impact Factor