記述言語：英語   掲載種別：研究論文（学術雑誌）  

In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H+-coupled transporters in Saccharomyces cerevisiae. In addition to the H+-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.

DOI： 10.1038/s41598-019-51531-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In yeast cells growing under nutrient-rich condition approximately 50% of total amino acids are accumulated in the vacuoles; however, the composition of amino acids in the cytosol and in the vacuoles is quite different. The vacuoles, like lysosomes, degrade proteins transported into their lumen and produce amino acids. These amino acids should be quickly excreted to the cytosol under nutrient starvation condition and recycled for de novo protein synthesis. These suggest that specific machineries that transport amino acids into and out of the vacuoles operate at the vacuolar membrane. Several families of transporter involved in the vacuolar compartmentalization of amino acids have been identified and characterized using budding yeast Saccharomyces cerevisiae. In this review, we describe the vacuolar amino acid transporters identified so far and introduce recent findings on their activity and physiological function.

DOI： 10.1248/bpb.b18-00165

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Amino acids stored in the vacuoles are exported to the cytosol mainly for protein synthesis; however, the molecular identity of vacuolar amino acid exporters remains obscure in plants. Here, we demonstrate that the heterologous expression of AtAVT3 genes, Arabidopsis homologs of AVT3 and AVT4 encoding vacuolar amino acid exporters in yeast, reduces vacuolar amino acid levels in the avt3∆avt4∆ yeast cells. In vitro experiments revealed that 14 C-labeled Ala and Pro are exported from vacuolar membrane vesicles by AtAvt3A in an ATP-dependent manner. In Arabidopsis, AtAvt3A fused with green fluorescent protein localizes to the vacuolar membrane. We propose that AtAVT3 family represents the long sought-for vacuolar amino acid exporters in plants.

DOI： 10.1002/1873-3468.12507

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3(∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3+ but was not completely rescued by the expression of avt3(∆1-270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

DOI： 10.1080/09168451.2016.1220819

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

DOI： 10.1080/09168451.2016.1141041

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.

DOI： 10.1080/09168451.2015.1083401

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

DOI： 10.1080/09168451.2014.963501

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.

DOI： 10.1080/09168451.2014.998621

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

DOI： 10.1080/09168451.2015.1058703

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

DOI： 10.1371/journal.pone.0130542

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.

DOI： 10.1074/jbc.M113.528380

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

DOI： 10.2131/jts.39.311

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

DOI： 10.1080/09168451.2014.910095

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09168451.2014.918489

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

DOI： 10.1271/bbb.130387

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

L-hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α(4)β(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.

DOI： 10.1074/jbc.M112.374272

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.

DOI： 10.1128/JB.00714-12

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DOI： 10.2210/pdb2rsg/pdb

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その他リンク： http://orcid.org/0000-0002-6227-4627

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

DOI： 10.1271/bbb.120372

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Vba5p is closest to Vba3p in the vacuolar transporter for basic amino acids (VBA) family of Saccharomyces cerevisiae. We found that green fluorescence protein (GFP)-tagged Vba5p localized exclusively to the plasma membrane. The uptake of lysine and arginine by whole cells was little affected by deletion of the VBA5 gene, but was stimulated by overexpression of the VBA5 gene. The inhibitory effect of 4-nitroquinoline N-oxide on cell growth was accelerated by expression of the VBA5 gene, and was lessened by the addition of arginine. These results suggest that Vba5p is a plasma membrane protein involved in amino acid uptake and drug sensitivity.

DOI： 10.1271/bbb.120455

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.

DOI： 10.1128/JB.01537-10

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE SOC TOXICOLOGICAL SCIENCES  

ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfr1p, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC50) was 25 mu M for the parent strain and 10.2 mu m for the bfr1 Delta mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

DOI： 10.2131/jts.36.117

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22 Delta cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

DOI： 10.1271/bbb.100747

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

PagL, a lipid A deacylase, is unique in that it is latent in the outer membrane of Salmonella enterica serovar Typhimurium. Several point mutations in the extracellular loops of PagL, which do not affect its enzymatic activity, release it from this latency. Precipitation analysis revealed that latent wild-type PagL associated with lipopolysaccharide, but non-latent PagL mutants did not. In contrast, non-latent PagL mutants preferentially associated with some membrane proteins. Precipitation analysis using inactive PagL mutants demonstrated that membrane lipid A deacylation did not affect association. These results indicate that mutations in the lipid A deacylase PagL which relieve the enzyme from its latency affect the ability of PagL to interact with lipopolysaccharide. (c) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.04.153

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Modification of lipid A is essential for bacterial adaptation to its host. Salmonella Typhimurium LpxR potentially detoxifies lipid A by 3'-O-deacylation; however, the involvement of deacylation in its adaptation remains unclear. LpxR-dependent 3'-O-deacylation was observed in the stationary phase. When macrophages were infected with stationary phase bacteria, the intracellular growth of the lpxR-null strain was lower than that of the wild-type strain. Furthermore, the expression level of inducible nitric oxide synthase was higher in the cells infected with the lpxR-null strain than in the cells infected with the wild-type strain. These results indicate that lipid A 3'-O-deacylation is beneficial for intracellular growth. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2009.11.062

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C-5-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (1R, 3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

DOI： 10.1074/jbc.M800799200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Protein phosphatase 2C epsilon(PP2C epsilon), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2C epsilon is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2C epsilon as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2C epsilon resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2C epsilon also enhanced the association between CERT and VAPA. In addition, knockdown of PP2C epsilon expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2C epsilon and that dephosphorylation of CERT by PP2C epsilon may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.

DOI： 10.1074/jbc.M707691200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositidebinding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.

DOI： 10.1074/jbc.M702291200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbalip.2007.01.009

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ceramide is synthesized at the endoplasmic reticulum ( ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleckstrin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.

DOI： 10.1074/jbc.M605032200

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：Springer Japan  

Synthesis and sorting of lipids are essential events for membrane biogenesis and its homeostasis. The endoplasmic reticulum (ER) is the center of the de novo synthesis of various lipid types. Trafficking of various lipids from the ER to other organelles has been suggested to occur by mechanisms different from the vesicle-mediated mechanism for protein trafficking. However, molecular mechanisms underlying intracellular trafficking of lipids remain poorly understood. Ceramide is synthesised at the ER, and translocated to the Golgi compartment for conversion to sphingomyelin (SM). We previously isolated a mammalian cultured cell mutant defective in ceramide trafficking, and have recently identified a key factor (named CERT) for ceramide trafficking in functional rescue experiments and proposed a molecular lipid extraction and transfer model for the non-vesicular mechanism of CERT-mediated trafficking of ceramide. © Springer-Verlag Tokyo 2006. All rights reserved.

DOI： 10.1007/4-431-34200-1_8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate- binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

DOI： 10.1038/nature02188

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We here isolated an Enterococcus hirae mutant unable to grow well at pH 10. The influx rate calculated from steady-state K-42(+)/K+ exchange and the intracellular K+ concentration of the mutant were reduced to 53 and 55% of those of the wild-type, respectively. The activities of two high-affinity K+ uptake systems, KtrI and KtrII, were normal in the mutant, but the kinetics of net K+ uptake at pH 10 indicated that a low-affinity K+ uptake with a K-m of about 20 mM (Kawano, M, Abuki, R, Igarashi, K, Kakinuma, Y. (2001) Arch. Microbiol. 175: 41-45), which were seen in the wild-type, was deficient in this mutant.

DOI： 10.1271/bbb.66.1597

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The 76-kDa NtpI subunit constitutes the membrane-embedded V-0 moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, 1. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K, the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 +/- 0.03 and 0.40 +/- 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.

DOI： 10.1074/jbc.M200973200

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DOI： 10.1016/S0005-2728(00)00278-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER-VERLAG  

Two high-affinity K+ uptake systems, KtrI and KtrII, have been reported in Enterococcus hirae. A mutant, JEMK1, defective in these two systems did not grow at pH 10 in low-K+ medium (less than 1 mM K+), but grew well when supplemented with 10 mM KCl. In this mutant. we found an energy-dependent K+ uptake at pH 10 with a low affinity for K+ (K-m of similar to 20 mM) and an extremely high rate [V-max of 1.6 mu mol min(-1) (mg protein)(-1)]. Rb+ uptake [K-m of similar to 40 mM and V-max of 0.5 mu mol min(-1) (mg protein)(-1), which was inhibited competitively by Ki and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.

DOI： 10.1007/s002030000234

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na+ -ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K+ uptake system. We found that K+ accumulation in the ntpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10, Studies of the membrane potential (Delta psi; inside negative) by 3,3'-dihexyloxacarbocyanine iodide fluorescence revealed that the Delta psi was hyperpolarized at pH 10 in JEM2; the Delta psi values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K+ or Rb+ in the presence of valinomycin, were -118 and -160 mV, respectively. Delta psi generation at pH 10 was accomplished by an electrogenic Na+ efflux via the Na+-ATPase, whose levels in the two strains were quite similar. Na+ uptake driven by an artificially imposed Delta psi (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na+ movement in addition to K+ movement. Finally, the growth of JEM2 arrested in K+-Iimited high-Na+ medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K+ as well as Na+, that it likely mediates K+ and Na+ cotransport, and that Na+ movement via NtpJ is the major Na+ reentry pathway at high pH values.

DOI： 10.1128/JB.182.9.2507-2512.2000

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The Enterococcus hirae ntp operon encodes both a vacuolar ATPase, which transports Na+ as well as Li+, and the KtrII K+ transporter. A plasmid, in which the chloramphenicol acetyltransferase gene (CAT) was placed downstream of the ntp promoter, was introduced into a mutant totally defective in Na+ extrusion, The CAT activity of this transformant was increased preferentially by addition of NaCl, but not by LiCl, in the media or by elevating the medium pH, correlating well with the increase in amounts of the ATPase subunits observed by Western blotting. The physiological significance of these responses of the ntp promoter is discussed, (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00776-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

We isolated a mutant, JEMK1, which did not grow at pH 6.0 in medium containing 0.5 mM KCl, derived from a Enterococcus hirae mutant deficient in the KtrII K+ uptake system. This mutant showed an impairment in the proton potential-dependent K+ uptake, the activity of the KtrI K+ uptake system, and did not grow well in K+-poor media in a wide pH range from 6 to 10, suggesting that KtrI and KtrII are the main systems for potassium accumulation of this bacterium. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Enterococcus hirae has two sodium extrusion systems: the NapA Na+/H+ antiporter and the vacuolar Na+-ATPase. We found that a NapA mutant, WD4, which is deficient in Na+/H+ antiporter activity, grew well in the pH range of 6 to 10 up to 200 mM sodium. This was due to active, potential-independent sodium extrusion by the Na+-ATPase, which was induced under these conditions. The NapA Na+/H+ antiporter is thus not a prerequisite for growth of E. hirae in the presence of sodium, but plays a supplementary role in sodium extrusion at acidic pH.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Enterococcus hirae ATCC 9790 grew well in Na+-deficient, low-K+ medium, but growth was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth inhibition and decrease of cellular K+ levels in the presence of CCCP were relieved by the addition of Na+ and a high concentration of K+. In contrast, in the mutant defective in Na+-ATPase or the NtpJ component of the KtrII K+ uptake system, CCCP-induced growth inhibition was rescued by a high concentration of K+ but not of Na+. These transporters are thus indispensable for homeostatis of K+ and Na+ at low proton potential.

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記述言語：日本語   出版者・発行元：エリザベス・アーノルド富士財団  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：NATURE PUBLISHING GROUP  

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：ELSEVIER SCIENCE BV  

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT has been identified as a key factor for the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains including (i) a START domain capable of catalyzing inter-membrane transfer of ceramide, (ii) a pleckstrin homology domain, which serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monophosphate, and (iii) a short peptide motif named FIAT motif which interacts with the ER-resident membrane protein VAP. CERT is preferentially distributed to the Golgi region in cells, and Golgi-targeted CERT appears to retain the activity to interact with VAP. On the basis of these results, it has been proposed that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that a particularly efficient cycle of CERT movement for trafficking of ceramide may proceed at membrane contact sites between the ER and the Golgi apparatus. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbalip.2007.01.009

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：ELSEVIER SCIENCE BV  

V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na+-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na+-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at the transcriptional level by intracellular Na+ as the signal. Structural aspects and catalytic properties of purified Na+-ATPase closely resembled those of other V-type H+-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na+ at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na+ binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na+ binding sites of the enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.

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