KAGIWADA Satoshi

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• Name (Japanese)

  Kagiwada

• Name (Kana)

  Satoshi

Research Interests

• biomembrane, phospholipid, membrane traffic, protein transport, yeast, endoplasmic reticulum, Golgi, lipid transfer protein, phospholipid metabolism, gene expression,

Research Areas

• Life sciences, Cell biology
• Life sciences, Molecular biology

Research Experience

• Jun. 2016, 奈良女子大学研究院自然科学系・教授
• Apr. 2006, May 2016, 奈良女子大学研究院自然科学系・准教授
• Apr. 1998, Mar. 2006, 奈良女子大学理学部・助教授
• Apr. 1997, Mar. 1998, 科学技術振興事業団研究員
• May 1995, Mar. 1997, 理化学研究所基礎科学特別研究員
• Apr. 1993, May 1993, 日本学術振興会特別研究員
• ヒューマンフロンティアサイエンスプログラム長期研究員

Education

• 1993, Kyoto University, Graduate School, Division of Natural Science, Biophysics
• 1988, Kyoto University, Faculty of Science

Association Memberships

• 日本生化学会
• 日本分子生物学会
• American Society for Microbiology
• 酵母研究会

Ⅱ．研究活動実績

Published Papers

• Nov. 2020, 6, 4, 280, https://doi.org/10.3390/jof6040284
• Refereed, JOURNAL OF BIOCHEMISTRY, OXFORD UNIV PRESS, Induction of intranuclear membranes by overproduction of Opi1p and Scs2p, regulators for yeast phospholipid biosynthesis, suggests a mechanism for Opi1p nuclear translocation, Miki Masuda; Ayaka Oshima; Tetsuko Noguchi; Satoshi Kagiwada, In the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes is suppressed by the Opi1p negative regulator. Opi1p enters into the nucleoplasm from the nuclear membrane to suppress the gene expression under repressing conditions. The binding of Opi1p to the nuclear membrane requires an integral membrane protein, Scs2p and phosphatidic acid (PA). Although it is demonstrated that the association of Opi1p with membranes is affected by PA levels, how Opi1p dissociates from Scs2p is unknown. Here, we found that fluorescently labelled Opi1p accumulated on a perinuclear region in an Scs2p-dependent manner. Electron microscopic analyses indicated that the perinuclear region consists of intranuclear membranes, which may be formed by the invagination of the nuclear membrane due to the accumulation of Opi1p and Scs2p in a restricted area. As expected, localization of Opi1p and Scs2p in the intranuclear membranes was detected by immunoelectron microscopy. Biochemical analysis showed that Opi1p recovered in the membrane fraction was detergent insoluble while Scs2p was soluble, implying that Opi1p behaves differently from Scs2p in the fraction. We hypothesize that Opi1p dissociates from Scs2p after targeting to the nuclear membrane, making it possible to be released from the membrane quickly when PA levels decrease., Mar. 2016, 159, 3, 351, 361, Scientific journal, 10.1093/jb/mvv105

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• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast, Yuta Tsukamoto; Satoshi Kagiwada; Sayuri Shimazu; Kaoru Takegawa; Tetsuko Noguchi; Masaaki Miyamoto, The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guaninenucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Delta vps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Delta vps901 Delta vps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Delta vps901 Delta vps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. (C) 2015 Elsevier Inc. All rights reserved., Mar. 2015, 458, 4, 802, 809, Scientific journal, 10.1016/j.bbrc.2015.02.031

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• Refereed, Algal Research, Colony sheath formation is accompanied by shell formation and release in the green alga Botryococcus braunii (race B), KAGIWADA Satoshi; Uno, Y; Nishii, I; Noguchi, T, Feb. 2015, 8, 214-223
• Refereed, Experimental Eye Research, Academic Press, Immediate differentiation of neuronal cells from stem/progenitor-like cells in the avian iris tissues, Tamami Matsushita; Ai Fujihara; Lars Royall; Satoshi Kagiwada; Mitsuko Kosaka; Masasuke Araki, A simple culture method that was recently developed in our laboratory was applied to the chick iris tissues to characterize neural stem/progenitor-like cells. Iris tissue is a non-neuronal tissue and does not contain any neuronal cells. In the present study we isolated iris tissues from chick embryos just prior to hatching. The isolated iris pigmented epithelium (IPE) or the stroma was embedded in Matrigel and cultured in Dulbecco's MEM supplemented with either fetal bovine serum or the synthetic serum replacement solution B27. Within 24h of culture, elongated cells with long processes extended out from the explants of both tissues and were positively stained for various neuronal markers such as transitin, Tuj-1 and acetylated tubulin. After a longer culture period, cells positive for photoreceptor markers like rhodopsin, iodopsin and visinin were found, suggesting that the iris tissues contain retinal stem/progenitor-like cells. Several growth factors were examined to determine their effects on neuronal differentiation. EGF was shown to dramatically enhance neuronal cell differentiation, particularly the elongation of neuronal fibers. The addition of exogenous FGF2, however, did not show any positive effects on neuronal differentiation, although FGF signaling inhibitor, SU5402, suppressed neuronal differentiation. The results show that neuronal stem/progenitor-like cells can differentiate into neuronal cells immediately after they are transferred into an appropriate environment. This process did not require any exogenous factors, suggesting that neural stem/progenitor-like cells are simply suppressed from neuronal differentiation within the tissue, and isolation from the tissue releases the cells from the suppression mechanism. © 2014 Elsevier Ltd., 2014, 123, 16, 26, Scientific journal, 10.1016/j.exer.2014.04.007

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• Refereed, JOURNAL OF CELL SCIENCE, COMPANY OF BIOLOGISTS LTD, Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast, Kentaro Kajiwara; Atsuko Ikeda; Auxiliadora Aguilera-Romero; Guillaume A. Castillon; Satoshi Kagiwada; Kentaro Hanada; Howard Riezman; Manuel Muniz; Kouichi Funato, Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments., Jan. 2014, 127, 2, 376, 387, Scientific journal, 10.1242/jcs.132001

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• Refereed, PLoS ONE, Transformation of lipid bodies related to hydrocarbon accumulation in a green alga, Botryococcus braunii (race B), Reiko Suzuki; Naoko Ito; Yuki Uno; Ichiro Nishii; Satoshi Kagiwada; Sigeru Okada; Tetsuko Noguchi, The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells. Copyright © 2013 Suzuki et al., 05 Dec. 2013, 8, 12, e81626, Scientific journal, 10.1371/journal.pone.0081626

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• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S), Shoko Nakamichi; Kumiko Yamanaka; Mai Suzuki; Toshio Watanabe; Satoshi Kagiwada, A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAR Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAR activities. (C) 2010 Elsevier Inc. All rights reserved., Jan. 2011, 404, 2, 605, 609, Scientific journal, 10.1016/j.bbrc.2010.12.011

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• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, The yeast VAP homolog Scs2p has that is correlated a phosphoinositide-binding ability with its activity, Satoshi Kagiwada; Misa Hashimoto, The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol, monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition. (C) 2007 Elsevier Inc. All rights reserved., Dec. 2007, 364, 4, 870, 876, Scientific journal, 10.1016/j.bbrc.2007.10.079

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• Refereed, Marine Biotechnology, Docosahexaenoic acid production and lipid-body formation in Schizochytrium limacinum SR21., KAGIWADA Satoshi; Morita E; Kumon Y; Nakahara T; Noguchi T, May 2006, 8, 3, 319-327, 327
• Refereed, PLANT SCIENCE, ELSEVIER IRELAND LTD, Characterization of coat proteins of COPI- and clathrin-coated vesicles in the unicellular green alga Botryococcus braunii, S Kagiwada; S Sugita; Y Masaike; S Kato; T Noguchi, Unicellular green algae are a useful system for studying intracellular protein transport in plant cells because they possess various plantspecific features such as cell wall formation and isolated Golgi bodies with distinct multilayered cisternae. In this study, two genes encoding the P-COP subunit of COPI-coated vesicles and the sigma 1 subunit of the AP-1 complex of clathrin-coated vesicles were cloned from the green alga Botryococcus braunii. The beta-COP gene contains an open reading frame (ORF) that codes for a 947-amino acid protein with a deduced molecular mass of 104 kDa, while the sigma 1 gene contains an ORF for a 159-amino acid protein with a deduced molecular mass of 19 kDa. Polyclonal antibodies raised against these proteins recognized proteins with predicted molecular masses in cell lysates prepared from B. braunii and the green alga Chlamydomonas reinhardtii. Cell fractionation studies revealed that both beta-COP and sigma 1 were associated with membranous compartments. By immunoelectron microscopy, beta-COP was found mainly on Golgi cisternae and to a lesser extent on Golgi-associated vesicles at all levels of the Golgi bodies, consistent with a proposed role of COPI-coated vesicles in inter-compartmental transport between Golgi cisternae. (c) 2005 Elsevier Ireland Ltd. All rights reserved., Oct. 2005, 169, 4, 668, 679, Scientific journal, 10.1016/j.plantsci.2005.05.027

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• Refereed, PLANT SCIENCE, ELSEVIER SCI IRELAND LTD, Cytoskeleton-dependent polarized secretion of arylsulfatase in the unicellular green alga, Chlamydomonas reinhardtii, S Kagiwada; Nakamae, I; M Kayukawa; S Kato, In the green alga Chlamydomonas reinhardtii, protein secretion has not been well investigated at the molecular level although the process plays important roles in cell wall biogenesis, the mating process, and stress response. Here, we studied the cytoskeleton dependence of Chlamydomonas protein secretion by examining the effects of cytoskeleton-disrupting reagents. Protein transport activity was monitored by the secretion of arylsulfatase (ARS), a glycoprotein which is synthesized in response to sulfur deprivation and transported to the periplasmic space. Microtubule-disrupting reagents, oryzalin and nocodazole, inhibited ARS secretion in a dose-dependent manner, while actin-disrupting reagents, cytochalasins B and D, induced up to a 1.4-fold increase in ARS secretion. The effect of oryzatin on the intracellular ARS distribution was different from that of brefeldin A (BFA), which disrupts the ER to Golgi protein transport. Immunofluorescence analysis using an anti-ARS polyclonal antibody revealed specific punctate staining on the anterior side of the cell surface of cells cultured in sulfur-depleted medium. This staining was not observed when the cells were treated with oryzalin. These results suggest that Chlamydomonas protein secretion is dependent on microtubules and inhibited by actin-related cytoskeletons. This cytoskeleton dependence is similar to that found in mammalian cells rather than that found in plant cells. (C) 2004 Elsevier Ireland Ltd. All rights reserved., Jun. 2004, 166, 6, 1515, 1524, Scientific journal, 10.1016/j.plantsci.2004.02.003

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• Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, Role of the yeast VAP homolog, Scs2p, in INO1 expression and phospholipid metabolism, S Kagiwada; R Zen, In the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes, including the INO1 gene (encoding inositol-1-phosphate synthase), is coordinately regulated by a cis-acting transcriptional element, UAS(INO) (inositol-sensitive upstream activating sequence). For this paper we studied the effect of SCS2 disruption on INO1 expression. SCS2 encodes a type II membrane protein and its deletion leads to inositol auxotrophy at temperatures above 34degreesC. We found that the expression of the INO1 gene was reduced in the scs2Delta strain even when the cells were cultured under derepressing conditions for INO1 expression. However, the beta-galactosidase gene fused with the INO1 promoter region was expressed normally in the scs2Delta strain. The phospholipid composition of scs2Delta cells was not dramatically changed compared with wild-type cells at 28degreesC, but the phosphatidylinositol level was reduced in scs2Delta cells cultured at 34degreesC. In addition, elevated phosphatidylcholine synthesis through the CDP-choline pathway was observed in the scs2Delta strain, and the disruption of genes involved in the CDP-choline pathway rescued the INO1 expression defect of the scs2Delta strain. These results indicate that Scs2p can contribute to coordinated phospholipid metabolism including INO1 expression by regulating phosphatidylcholine synthesis through the CDP-choline pathway., Apr. 2003, 133, 4, 515, 522, Scientific journal, 10.1093/jb/mvg068

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• Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, Effect of brefeldin A on melatonin secretion of chick pineal cells, T Hirota; S Kagiwada; T Kasahara; T Okano; M Murata; Y Fukada, Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity., Jan. 2001, 129, 1, 51, 59, Scientific journal
• Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: Relationship to "bypass sec14p" and inositol auxotrophy, MP Rivas; BG Kearns; ZG Xie; SL Guo; MC Sekar; K Hosaka; S Kagiwada; JD York; VA Bankaitis, SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect "bypass Sec14p" will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sad mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sad mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene., Jul. 1999, 10, 7, 2235, 2250, Scientific journal
• Refereed, JOURNAL OF BACTERIOLOGY, AMER SOC MICROBIOLOGY, The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism, S Kagiwada; K Hosaka; M Murata; J Nikawa; A Takatsuki, The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580-1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C, Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells., Apr. 1998, 180, 7, 1700, 1708, Scientific journal
• Refereed, NATURE, MACMILLAN MAGAZINES LTD, Essential role for diacylglycerol in protein transport from the yeast Golgi complex, BG Kearns; TP McGee; P Mayinger; A Gedvilaite; SE Phillips; S Kagiwada; VA Bankaitis, Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway(1-6)). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin(9), a protein found in rat brain, Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia call further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles., May 1997, 387, 6628, 101, 105, Scientific journal
• Refereed, EMBO JOURNAL, WILEY-BLACKWELL, Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis, M Fang; BG Kearns; A Gedvilaite; S Kagiwada; M Kearns; MKY Fung; VA Bankaitis, The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function, We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP), Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis., Dec. 1996, 15, 23, 6447, 6459, Scientific journal
• Refereed, GENETICS, GENETICS, The yeast BSD2-1 mutation influences both the requirement for phosphatidylinositol transfer protein function and derepression of phospholipid biosynthetic gene expression in yeast, S Kagiwada; BG Kearns; TP McGee; M Fang; K Hosaka; VA Bankaitis, The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino(-) phenotype is the inability of BSD2-1 strains to derepress transcription of lNO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the ''bypass Sec14p'' phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidylinositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed., Jun. 1996, 143, 2, 685, 697, Scientific journal
• Refereed, BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER SCIENCE BV, FUSION OF DIOLEOYLPHOSPHATIDYLCHOLINE VESICLES INDUCED BY AN AMPHIPHILIC CATIONIC PEPTIDE AND OLIGOPHOSPHATES AT NEUTRAL PH, M MURATA; Y SHIRAI; R ISHIGURO; S KAGIWADA; Y TAHARA; S OHNISHI; S TAKAHASHI, Peptide E5 is an analogue of the fusion peptide of influenza virus hemagglutinin and K5 is a cationic peptide which has an arrangement of electric charges complementary to that of E5. We reported that a stoichiometric mixture of E5 and K5 caused fusion of large unilamellar vesicles (LUV) of neutral phospholipids (Murata, M., Kagiwada, S., Takahashi, S. and Ohnishi, S. (1991) J. Biol. Chem. 266, 14353-14358). K5 caused fusion of LUV composed of dioleoylphosphatidylcholine (DOPC) at pH > 10, but not at neutral pH. In the presence of oligophosphates, such as 1 mM ATP, GTP, or polyphosphate, K5 caused rapid and efficient fusion of DOPC LUV at neutral pH without hydrolysis of oligophosphate groups, but another anions such as citrate, acetate, AMP, phosphate, or EDTA were ineffective. The peptide/oligophosphate-induced fusion behaviors have been investigated by a fluorescence resonance energy transfer assay for lipid mixing of LUV and negative staining electron microscopy. At higher ionic strengths (> 0.3 M KCl) or in the presence of 5.0 mM MgCl2, the fusion was inhibited. Even at the inhibitory conditions, the association of K5 with lipid vesicles at neutral pH was directly confirmed by the Ficoll gradient assay method and by blue shifts of the tryptophan fluorescence of the peptide. A nonhydrolyzable GTP analogue, GTPgammaS, also induced fusion. These observations suggested that the electrostatic interactions between the positive and negative charges of K5 and oligophosphate, respectively, induced complex formation, triggering membrane fusion., Oct. 1993, 1152, 1, 99, 108, Scientific journal
• Refereed, BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, SPECIFICITY OF AMPHIPHILIC ANIONIC PEPTIDES FOR FUSION OF PHOSPHOLIPID-VESICLES, M MURATA; S TAKAHASHI; Y SHIRAI; S KAGIWADA; R HISHIDA; S OHNISHI, We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues, We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion., Mar. 1993, 64, 3, 724, 734, Scientific journal
• Refereed, JOURNAL OF BIOLOGICAL CHEMISTRY, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, INVITRO FUSION OF RABBIT LIVER GOLGI MEMBRANES WITH LIPOSOMES, S KAGIWADA; M MURATA; R HISHIDA; M TAGAYA; S YAMASHINA; S OHNISHI, Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10-degrees-C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport., Jan. 1993, 268, 2, 1430, 1435, Scientific journal
• Refereed, BIOLOGY OF THE CELL, EDITIONS SCIENTIFIQUES ELSEVIER, INTERACTION OF THE GOLGI MEMBRANES ISOLATED FROM RABBIT LIVER WITH MICROTUBULES INVITRO, M MURATA; TJ ITOH; S KAGIWADA; R HISHIDA; H HOTANI; S OHNISHI, We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient, The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes., 1992, 75, 2, 127, 134, Scientific journal
• Refereed, BIOCHEMISTRY, AMER CHEMICAL SOC, PH-DEPENDENT MEMBRANE-FUSION AND VESICULATION OF PHOSPHOLIPID LARGE UNILAMELLAR VESICLES INDUCED BY AMPHIPHILIC ANIONIC AND CATIONIC PEPTIDES, M MURATA; S TAKAHASHI; S KAGIWADA; A SUZUKI; S OHNISHI, We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation., Feb. 1992, 31, 7, 1986, 1992, Scientific journal
• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, MODIFICATION OF THE N-TERMINUS OF MEMBRANE FUSION-ACTIVE PEPTIDES BLOCKS THE FUSION ACTIVITY, M MURATA; S KAGIWADA; R HISHIDA; R ISHIGURO; S OHNISHI; S TAKAHASHI, Sep. 1991, 179, 2, 1050, 1055, Scientific journal
• Refereed, JOURNAL OF BIOLOGICAL CHEMISTRY, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, MEMBRANE-FUSION INDUCED BY MUTUAL INTERACTION OF THE 2 CHARGE-REVERSED AMPHIPHILIC PEPTIDES AT NEUTRAL PH, M MURATA; S KAGIWADA; S TAKAHASHI; SI OHNISHI, An anionic amphiphilic peptide and the charge-reversed cationic peptide are synthesized. They contain 20 amino acids with the same sequence except for 5 Glu residues for the anionic versus 5 Lys residues for the cationic peptides. Fusion of egg phosphatidylcholine large unilamellar vesicles is assayed with the fluorescent probes by the lipid mixing and the internal content mixing at neutral pH. The peptide mixture causes a rapid and efficient membrane fusion, in spite of no fusions with each peptide by itself. Each peptide takes nearly random coils with a small amount of helix, but the peptide mixture has an ordered helical structure. The equimolar peptide mixture forms a much more hydrophobic complex than those of different molar ratios of peptides and also that of each peptide itself. The equimolar peptide mixture causes the most efficient fusion. Preincubations of two peptides before addition to vesicles cause the slower rates of fusion. The fusion is greatly reduced at higher ionic strength and nearly zero at 800 mM NaCl and 40 mM sodium phosphate. Each peptide and the peptide mixture show the same alpha-helical structure, interact with vesicles, but do not induce fusion at higher ionic strengths. These results suggest that the two peptides interact mutually through the electrostatic Coulombic interaction between the charged groups. The electrically neutralized hydrophobic complex aggregates the separate vesicles together and interacts with the hydrocarbon region of lipid bilayers to cause fusion., Aug. 1991, 266, 22, 14353, 14358, Scientific journal
• Refereed, BIOCHEMISTRY, AMER CHEMICAL SOC, DEVELOPMENT OF A STREAK-CAMERA-BASED TIME-RESOLVED MICROSCOPE FLUOROMETER AND ITS APPLICATION TO STUDIES OF MEMBRANE-FUSION IN SINGLE CELLS, A KUSUMI; A TSUJI; M MURATA; Y SAKO; AC YOSHIZAWA; S KAGIWADA; T HAYAKAWA; S OHNISHI, A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3-mu-m diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1-mu-m2 domain. A signal-to-noise ratio better than 30 was obtained for almost-equal-to 30 000 fluorescein-labeled band 3 molecules in a 2-mu-m2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues., Jul. 1991, 30, 26, 6517, 6527, Scientific journal
• Journal of Fungi, MDPI AG, The Fission Yeast RNA-Binding Protein Meu5 Is Involved in Outer Forespore Membrane Breakdown during Spore Formation, Bowen Zhang; Erika Teraguchi; Kazuki Imada; Yuhei O. Tahara; Shuko Nakamura; Makoto Miyata; Satoshi Kagiwada; Taro Nakamura, In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation., 13 Nov. 2020, 6, 4, 284, 284, Scientific journal, 10.3390/jof6040284

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• Seibutsu Butsuri, The Biophysical Society of Japan General Incorporated Association, Inhibition of metatonin synthesis in chick pineal cells by pulse treatment with brefeldin A, Hirota T.; Kagiwada S.; Kasahara T.; Okano T.; Fukada Y.; Murata M., 1999, 39, S48, 10.2142/biophys.39.S48_1

  DOI URL
• Refereed, TIME-RESOLVED LASER SPECTROSCOPY IN BIOCHEMISTRY II, PTS 1 AND 2, SPIE - INT SOC OPTICAL ENGINEERING, DEVELOPMENT OF TIME-RESOLVED MICROFLUORIMETRY AND ITS APPLICATION TO STUDIES OF CELLULAR MEMBRANES, A KUSUMI; A TSUJI; M MURATA; Y SAKO; S KAGIWADA; T HAYAKAWA; SI OHNISHI, 1990, 1204, 776, 783, International conference proceedings, 10.1117/12.17720

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MISC

• Not Refereed, 蛋白質核酸酵素, 共立出版, 酵母におけるイノシトールリン脂質の機能, KAGIWADA Satoshi, Jun. 1999, 44, 8, 1194-1199, 1199

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• Not Refereed, KAGAKU TO SEIBUTSU, Japan Society for Bioscience, Biotechnology, and Agrochemistry, リン脂質輸送タンパク質の新しい機能, KAGIWADA Satoshi, Oct. 1996, 34, 7, 433-439, 439, 10.1271/kagakutoseibutsu1962.34.433

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• Not Refereed, 実験医学, 細胞内蛋白質輸送におけるリン脂質輸送タンパク質の新しい機能, KAGIWADA Satoshi, Aug. 1996, 14, 14, 2058-2064, 2064

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• Not Refereed, 生物物理, 細胞内の膜小胞形成に関与する蛋白質, KAGIWADA Satoshi, Oct. 1992, 32, 5, 268-272
• Not Refereed, 細胞, 細胞内小胞輸送を制御する蛋白質, KAGIWADA Satoshi, Dec. 1991, 23, 12, 475-479
• Not Refereed, 組織培養, 新しい物理化学的手法によるエンドサイトーシス機構の解析, KAGIWADA Satoshi, Jul. 1991, 17, 7, 220-224
• Not Refereed, Seibutsu Butsuri, The Biophysical Society of Japan General Incorporated Association, 膜融合活性ペプチドのマイクロマニュピレーションとその応用, KAGIWADA Satoshi, Dec. 1989, 29, 6, 316-319, 319, 10.2142/biophys.29.6_316

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• Not Refereed, 細胞, 膜小胞化と膜融合, KAGIWADA Satoshi, Nov. 1989, 21, 11, 416-420
• Not Refereed, PHYCOLOGIA, INT PHYCOLOGICAL SOC, BIOSYNTHESIS AND ACCUMULATION OF HYDROCARBONS AND POLYSACCHARIDES IN A COLONIAL GREEN ALGA, BOTRYOCOCCUS BRAUNII RACE B, R. Suzuki; Y. Uno; I. Nishii; S. Kagiwada; T. Noguchi, Jul. 2013, 52, 4, 109, 109, Summary international conference
• Not Refereed, 生化学, (公社)日本生化学会, ブレフェルジンAを用いたニワトリ松果体細胞のメラトニン分泌機構の解析, 広田 毅; 鍵和田 聡; 笠原 和起; 岡野 俊行; 村田 昌之; 深田 吉孝, Aug. 2000, 72, 8, 1016, 1016

Books etc

• Phospholipid transfer protein function in the yeast Saccharomyces cerevisiae., Plenum Press, KAGIWADA Satoshi; M.A.Kearns; M.Fang; M.Rivas; B.G.Kearns; V.A.Bankaitis, 分担, Aug. 1996, 83-91, Not Refereed
• Phospholipid transfer proteins: emerging roles in vesicle trafficking, signal transduction, and metabolic regulation, Springer-Verlag, KAGIWADA Satoshi, 1996, Not Refereed

Presentations

• KAGIWADA Satoshi; Izumi Mizutani; Shigeru Okada; Satoshi Kagiwada, 日本農芸化学会2019年度大会, Excretion of triterpenes overproduced in the yeast Saccharomyces cerevisiae into the cell wall fraction, Mar. 2019, 東京, False
• KAGIWADA Satoshi; Shuko Nakamura; Satoshi Kagiwada, 日本農芸化学会2019年度大会, Morphological analysis of a nuclear membrane domain necessary for the Opi1p-induced membrane invagination in the yeast Saccharomyces cerevisiae., Mar. 2019, 東京, False
• KAGIWADA Satoshi; Tsou ChungYau; Victor Ferriols; Satoshi Kagiwada; Shigeki Matsunaga; Shigeru Okada, 平成29年度日本水産学会春季大会, Functional characterization of a unique squalene epoxidase from the freshwater alga Botryococcus braunii, race B, Mar. 2018, 東京, False
• KAGIWADA Satoshi, 日本農芸化学会2017年度大会, 緑藻Botryococcus brauniiのトリテルペン炭化水素合成酵素の出芽酵母Saccharomyces cerevisiaeを用いた機能解析, Mar. 2017, 京都女子大学, False
• KAGIWADA Satoshi; Tsou ChungYau; Victor Ferriols; Satoshi Kagiwada; Shigeki Matsunaga; Shigeru Okada, The 2017 Spring Meeting of JSFS, cDNA cloning, yeast expression, and functional characterization of 10,11-squalene epoxidase from the freshwater alga Botryococcus braunii, race B, Mar. 2017, 東京海洋大学品川キャンパス, False
• KAGIWADA Satoshi, 第38回日本分子生物学会 第88回日本生化学会大会 共同年会, 出芽酵母Saccharomyces cerevisiaeにおけるリン脂質生合成遺伝子発\n現の負の調節因子Opi1pは正の調節因子Ino2pを分解することで遺伝子発現を抑制\nする, Dec. 2015, 神戸市, False
• KAGIWADA Satoshi, 第33回植物細胞分子生物学会, Botryococcus braunii B品種におけるスクアレンエポキシダーゼ遺伝子群の\n単離と機能解析, Aug. 2015, 東京, False
• KAGIWADA Satoshi, 日本植物学会第78回年会, 緑藻Botryococcus由来炭化水素（Botryococcene）生産酵母での細胞内脂質蓄積部位の形態, Sep. 2014, 川崎市, False
• KAGIWADA Satoshi, 日本植物学会第77回大会, 緑藻Botryococcus brauniiのトリテルペン合成酵素群の出芽酵母での発現と細胞内局在, Sep. 2013, 札幌, False
• KAGIWADA Satoshi; Suzuki, R; Uno, Y; Nishii, I; Kagiwada, S; Noguchi, T, 10th International Phycological Congress Annual Meeting, Biosynthesis and accumulation of hydrocarbons and polysaccharides in a colonial green alga, Botryococcus braunii race B, Aug. 2013, Orlando, USA, True
• KAGIWADA Satoshi; Yuki Uno; Reiko Suzuki; Naoko Ito; nSatoshi Kagiwada; Ichiro Nishii; Tetsuko Noguchi, 第54回日本植物生理学会年会, Biosynthesis and accumulation of lipids and polysaccharides in a colonial\ngreen alga, Botryococcus braunii race B, Mar. 2013, False
• KAGIWADA Satoshi; Ayaka Oshima; Risa Yokoi; Miki Masuda; Satoshi Kagiwada, 第35回日本分子生物学会年会, Abundance of the Opi1p transcriptional factor involved in the transcriptional regulation of phospholipid biosynthetic genes is regulated by its binding to membranes in Saccharomyces cerevisiae, Dec. 2012, 福岡, False
• KAGIWADA Satoshi; Nodoka Miyake; Shoko Nakamichi; Satoshi Kagiwada, 第35回日本分子生物学会年会, Multi-layered ER induced by ALS-8 type Scs2p can be dissolved by modulating phospholipid metabolism in the yeast Saccharomyces cerevisiae, Dec. 2012, 福岡, False
• KAGIWADA Satoshi, 日本植物学会第76回大会, 緑藻Botryococcus braunii Ｂ品種における脂質の分泌に伴うオイルボディの動態, Sep. 2012, 兵庫県姫路市, False
• KAGIWADA Satoshi; Mana Hirose; Satoshi Kagiwada; Ichiro Nishii; Tetsuko Noguchi, The 2nd International Conference on Algal Biomass, Biofuels and Bioproducts, Hydrocarbon biosynthesis and secretion in a green alga, Botryococcus braunii, Jun. 2012, San Diego, USA, True
• KAGIWADA Satoshi; Miki Masuda; Shoko Nakamichi; Satoshi Kagiwada, 第34回日本分子生物学会年会, Localization of Opi1p, a negative transcriptional factor for the inositol biosynthetic gene INO1, in Saccharomyces cerevisiae., Dec. 2011, 横浜市みなとみらい, False
• KAGIWADA Satoshi; Ayaka Oshima; Shoko Nakamichi; Risa Yokoi; Satoshi Kagiwada, 第34回日本分子生物学会年会, The expression of the OPI1 negative transcriptional factor for phospholipid synthetic genes is regulated by the Opi1p-associated nuclear/ER membrane protein Scs2p in Saccharomyces cerevisiae., Dec. 2011, 横浜市みとみらい
• KAGIWADA Satoshi, 日本植物形態学会第23回大会, 緑藻Botryococcus braunii B品種における炭化水素生産について, Sep. 2011
• Nanasa Ieda; Satoshi Kagiwada, 日本分子生物学会, Pil1, 03 Dec. 2021, 01 Dec. 2021, 03 Dec. 2021
• 坂巻美羽; 鍵和田聡, 日本農芸化学会2023年度大会, 緑藻Botryococcus braunii由来ボツリオコッセン合成酵素Ssl- 3pの酵素活性向上, 14 Mar. 2023, 14 Mar. 2023, 17 Mar. 2023
• 池田亜衣梨; 鍵和田聡, 第46回日本分子生物学会年会, 出芽酵母 Saccharomyces cerevisiae の スクアレン 合成活性 が 膜接触部位 に 与える 影響, 01 Dec. 2022
• 中池由美; 鍵和田聡, 2023年度 実験・実習技術研究会, ガラスビーズ破砕法を用いた パン酵母由来アルコールデヒドロゲナーゼの 抽出方法の改善, 15 Mar. 2024
• 城寳愛結; 池田亜衣梨; 鍵和田聡, 第96回日本生化学会大会, スクアレンの大量生産が出芽酵母Saccharomyces cerevisiaeの形質膜の堅牢性に与える影響, 01 Nov. 2023

  共同研究・競争的資金等URL

Research Projects

• 生体膜の構造と機能に関する分子生物学的研究, 0, 0, 0, Competitive research funding
• Structure and function of biomembrane, 0, 0, 0, Competitive research funding
• 基盤研究(C), Apr. 2022, Mar. 2025, 22K04842, 細胞壁自己消化性酵母を用いたスクアレン回収システムの構築, 鍵和田 聡, 日本学術振興会, 科学研究費助成事業 基盤研究(C), 奈良女子大学, 4290000, 3300000, 990000, kaken;rm:presentations

  対象リソースIRI
• 萌芽研究, 2003, 2004, 15657016, 海洋微生物ラビリンチュラのドコサヘキサエン酸生成に関する細胞学的解析, 野口 哲子; 鍵和田 聡, 日本学術振興会, 科学研究費助成事業, 奈良女子大学, 3200000, 3200000, ラビリンチュラ類は卵菌と不等毛植物の特徴を持つ汽水域に生息する単細胞生物である。Schizochytrium limacinum 21株(SR21株)は多量の脂肪酸を細胞内に蓄積し、なかでも高度不飽和脂肪酸のドコサヘキサエン酸(DHA)を多量に生成する微生物として、近年注目されている。本研究では、SR21株のDHA生成を細胞学的に解析した。まず、SR21株の同調培養系を確立し、100%の遊走子群を得た。この系を用い、遊走子及び遊走子着床後4、8、12、24時間、10日目の栄養細胞で、オイルボディーの形成過程を形態学的に観察し、それに伴う脂質量、及び脂肪酸組成の変化を解析した。 Nile red染色によって生活史の全ての時期にオイルドロップが観察されたが、オイルドロップは遊走子着床後の細胞成長に伴って大きくなり、細胞当たりの数も増加した。電子顕微鏡観察では、パルミチン酸を1、3位に、DHAを2位に持つトリアシルグリセロール(TG)の結晶様構造による縞模様を呈するオイルボディーが観察された。このタイプのオイルボディーは遊走子では余り観察されず、成長中の細胞で出現し、12時間目で細胞質の1/2以上を占めた。成長中の細胞では、小さなオイルボディーが小胞体に接し、小胞体内には観察されないため、オイルボディーは小胞体上で合成されると考えられた。 細胞生長に伴い細胞当たりの脂質量も増加したが、総脂肪酸中のDHAの比率は24時間目以降低下した。そこで脂質を、主にオイルボディーの構成成分であるTGと生体膜成分であるホスファチジルコリン(PC)に分画し、それぞれの脂肪酸組成を解析した。その結果、遊走子及び着床後24時間目の細胞では、PCにおけるDHAの脂肪酸組成がおよそ60%であったのに対し、10日後には10%以下に低下した。一方、TGではDHAの脂肪酸組成に大きな変動は認められなかった。, kaken

  対象リソースIRI
• Grant-in-Aid for Scientific Research (C), 2001, 2002, 13640665, Cytological studies on the mechanism of hydrocarbon synthesis by a green alga, Botryococcus braunii, NOGUCHI Tetsuko; KAGIWADA Satoshi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara women's University, 3400000, 3400000, The colonial green alga Botryococcus braunii produces unusually high levels of hydrocarbons, amounting to 30%-60% of its dry weight, and accumulates them around the cell surfaces. Usually, the hydrocarbon synthesis occurrs just after cell division but before the formation of polysaccharide cell wall. In this study, we developed the induction-system of interphase cells into an active hydrocarbon synthesis. Active hydrocarbon synthesis occurred after the digestion of cell walls in interphase cells (spheroplasts), but didn't occur by the removal of already accumulated hydrocarbons. Maximally, we could induce 12 times of hydrocarbon synthesis by spheroplasts than those by normal interphase cells. The morphologies of Golgi bodies and the trans-Golgi networks (TGN) in spheroplasts were not similar to those in normal interphase cells, but those in cells just after cell division. The result strongly supports our hypothesis that the TGN is participated in the hydrocarbon synthesis. Then, we have isolated a cDNA coding for β-COP that is a coat-protein of transport vesicles mediating the vesicular transport through the Golgi apparatus and the TGN. The cDNA encodes a protein of 947 amino acids with a predicted molecular mass of 109 kDa. Immunoelectron microscopic analysis was also carried out with the anti-body against β-COP. To produce spheroplasts of B. braunii, we examined the polysaccharide and sugar compositions of the cell wall, and it was clarified that B.braunii had a characteristic one., kaken

  対象リソースIRI
• 奨励研究(A), 2000, 2001, 12780474, 酵母のイノシトールリン脂質の低温耐性および細胞内膜小胞輸送における役割の解明, 鍵和田 聡, 日本学術振興会, 科学研究費助成事業, 奈良女子大学, 2400000, 2400000, 酵母(Saccharomyces cerevisiae)のSCS2遺伝子産物は、細胞内膜小胞輸送に関与する膜蛋白質であるシナプトブレビンに結合する蛋白質としてラットやマウスなどで発見されたVAP-33と相同性がある。一方、この遺伝子を破壊した酵母はイノシトール要求性や低温耐性を示す。これらの表現型と他の遺伝子との遺伝学的相互作用の結果から、SCS2遺伝子破壊株ではイノシトールリン脂質の代謝に異常があり、それが結果として低温耐性を引き起こすと考え、この関係の分子的要因を明らかにするためにSCS2遺伝子産物の機能解明を試みた。 本研究ではまずSCS2破壊株のイノシトール要求性を与える要因を明らかにしようとし、SCS2破壊株では酵母のイノシトール生合成に必須の遺伝子であるINO1遺伝子の発現量が野性株に比べて明らかに低下していたこと、しかし、外来プラスミドに挿入したINO1プロモーターからの遺伝子発現はほぼ正常であったことを明らかにした。このことから、SCS2遺伝子産物は染色体に働きかけてINO1プロモーターからの遺伝子発現を正に調節するタンパク質であることが示唆された。一方、SCS2遺伝子破壊株のイノシトール要求性は、ホスファチジルコリン生合成のためのCDPコリン経路に位置する遺伝子を破壊すると回復するが、もうひとつのPC生合成経路であるPEメチル化経路を破壊しても回復しないことを明らかにした。 また、SCS2破壊株のリン脂質と中性脂質組成を調べたところ、この破壊株には他のリン脂質代謝変異株に見られたような野性株との大きな相違は観察されなかった。ただし、34℃で生育させたときにイノシトールの代謝が盛んになることを明らかにしたことから、SCS2岐壊株でみられた高温感受性のイノシトール要求性は高温でのイノシトール代謝昂進にイノシトール生合成が追いつかなくなったためと推測された。, kaken

  対象リソースIRI
• Grant-in-Aid for Scientific Research (C), 1999, 2000, 11640664, Hydrocarbon synthesis in a green alga, Botryococcus braunii., NOGUCHI Tetuko; KAGIWADA Satoshi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara Women's University, 3000000, 3000000, The green alga Botryococcus. braunii produce unusually high levels of hydrocarbons, 15-75% of the cell dry weight It makes B.braunii as excellent candidate for a renewable source of liquid hydrocarbon fuel as a "solar oil". In this study, the mechanism of hydrocarbon synthesis was studied cytologically. The structure of hydrocarbons was examined by gas chromatography mass spectrometer. They were straight odd number_7 C_<29> (35%) and C_<31> (26%) chains with two double bonds. The stage of hydrocarbon synthesis during the cell cycle was determined by the incorporation of [1-^<14>C] acetate into hydrocarbons and the observations on ultrastructures of cell organelle by the cryo-freezing/freeze-substitution for electron microscopy. The results showed that the hydrocarbon synthesis occurred just after cell division but before cell wall formation. When the hydrocarbon synthesis occurs, the trans-Golgi networks produced special very large vesicles that contained fine fibrils and several masses of electron-dense substance. Cytochalasin B treatment results the appearance of hydrocarbon droplets outside the plasma membrane of cells just after cell division, which showed that the hydrocarbon synthesis occurs on the plasma membrane., kaken

  対象リソースIRI
• Grant-in-Aid for Challenging Exploratory Research, 01 Apr. 2012, 31 Mar. 2014, 24658289, Engineering of a hydrocarbon producing yeast for biofuel production, KAGIWADA Satoshi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara Women's University, 3380000, 2600000, 780000, A unicellular green algae, Botryococcus braunii, secretes a hydrocarbon called botryococcene, which is very suitable for biofuel production, into its environment. However, Botryococcus do not grow fast and is not subjected to biotechnological engineering, making it difficult to improve this organism for more efficient biofuel production. In this study, genes required for botryococcene synthesis are incorporated into the budding yeast Saccharomyces cerevisiae, which is very suitable for biotechnological engineering, to make a yeast strain which can prouduce large amounts of botryococcene and secrete them., kaken

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