酵母の多剤耐性における多様なABC蛋白の機能および発現制御ネットワーク

• Project/Area Number: 10217208
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Hiroshima University
• Principal Investigator: 宮川 都吉 広島大学, 大学院・先端物質科学研究科, 教授 (10116676)
• Co-Investigator(Kenkyū-buntansha): 平田 大 広島大学, 大学院・先端物質科学研究科, 助教授 (30243603)
• Project Period (FY): 1998 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥36,500,000 (Direct Cost: ¥36,500,000); Fiscal Year 2002: ¥4,900,000 (Direct Cost: ¥4,900,000); Fiscal Year 2001: ¥5,000,000 (Direct Cost: ¥5,000,000); Fiscal Year 2000: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 1999: ¥8,700,000 (Direct Cost: ¥8,700,000); Fiscal Year 1998: ¥9,400,000 (Direct Cost: ¥9,400,000)
• Keywords: ABCトランスポーター / 出芽酵母 / Ca^<2+>シグナル / 多剤耐性 / カルシウム

Research Abstract

ABCトランスポーターが構造的に関連性のない非常に広範な薬剤を認識して排出する機構は不明である。
出芽酵母においてフルサイズのABCトランスポーターは約15種類存在するが、多剤耐性現象においては、Pdr5 ABCトランスポーターが圧倒的に重要な役割を果たしている。Pdr5は、極めて多種薬剤を認識して細胞外排出する。酵母のPdr5はABCトランスポーター機能解析の良いモデルと考えられる。そこで、特定薬剤に対する特異性を喪失した変異pdr5遺伝子を取得し、遺伝学的に解析し、Pdr5による薬剤認識の機構を解析した。PDR5遺伝子ORFをヒドロキシルアミン処理したプラスミドにより、pdr5破壊株を形質転換した。シクロヘキシイミド、スタウロスポリン、トリフルオペラジンまたはセルレニンを含む培地で各薬剤に対する薬剤耐性を試験し、調べた薬剤のいずれかの薬剤に感受性を示す株をその薬剤に対する感受性を失った変異と予想し選抜した。合計7各PDR5遺伝子の変異点をシークエンスにより決定し、各種薬剤に対する耐性、感受性の試験をした。変異点はORF全体に分散しており、各薬剤に特定の部位で対応しているのではなく、ABCトランスポーターの全体構造で対応している可能性が示唆された。

Research Products

• [Publications] Yuji Miyamoto et al.: "Identification of Saccharomyces cerevisiae isoeucyl tRNA synthetase as target of G_1-specific inhibitor reveromycin A"J. Biol. Chem.. 277. 28810-28814 (2002)
• [Publications] Dai Hirata et al.: "Fission yeast Mor2/Cps12, a protein similar to Drosophyla Furry, is essential for cell morophogenesis and its mutation induces Weel-dependent G_2 delay"EMBO J.. 21. 4863-4874 (2002)
• [Publications] Daisuke Morita et al.: "Rpf2p, an evolutionarily conserved protein, interacts with ribosomal protein L11 and is essential for the processing of 27 SB pre-rRNA to 25 SrRNA"J. Biol. Chem.. 277. 28780-28786 (2002)
• [Publications] Koji Kakugawa et al.: "Cloning, characterization, and expression of cDNA encoding a lipase from Kurtzumanomyces sp. I-11"Biosci. Biotechnol. Biochem.. 66. 1328-1336 (2002)
• [Publications] Koji Kakugawa et al.: "Purification and characterization of a lipase from the glycolipid-producing yeast Kurtzumanomyces sp. I-11"Biosci. Biotechnol. Biochem.. 66. 978-985 (2002)
• [Publications] Koji Kakugawa et al.: "Isolation of yeast Kurtzumanomyces sp. I-11, novel producer of mannosylerythritol lipid"Biosci. Biotechnol. Biochem.. 66. 188-191 (2002)
• [Publications] 下向敦範 ほか: "清酒酵母の研究(分担執筆)"清酒酵母研究会. 8 (2003)
• [Publications] Masaki Mizunuma et al.: "GSK-3 Kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca^<2+> in budding yeast"ENBO.J. 20. 1074-1085 (2001)
• [Publications] X.Zhang et al.: "Crosstalk between transcriptional regulators in multidrug resistance in Sacccharomyces cerevisae"J.Biol.Chem.. 276. 8812-8819 (2001)
• [Publications] Osamu Tanabe et al.: "Fission yeast homologues of the B subunit of protein phosphatase 2A : multiple roles in mitotic cell division and functional interaction with calcineurin"Gene Cells. 6. 455-473 (2001)
• [Publications] Tomoki Hosotani et al.: "PKC1, a protein kinase C homologue of Saccharomyces cerevisiase, participates in microtubule function through the yeast EB1 homologue BIM1"Gene Cells. 6. 775-788 (2001)
• [Publications] Eiko Tsuchiya et al.: "Borrelidin inhibits a cyclin-dependent kinase (CDK), Cdc28/Cln2, of Saccharomyces cerevisiae"J.Antibiotics. 54. 84-90 (2001)
• [Publications] A.Shitamukai: "A positive screening for drugs that specifically inhibits the Ca^<2+> signaling acitivity on the basis of the growth promoting effect on a yeast mutant with a peculiar phenotype"Biosci.Biotechnol.Biocehm.. 64. 1942-1946 (2000)
• [Publications] A.Tsuno: "RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces serevisiae"Mol.Cell.Biol.. 20. 2066-2074 (2000)
• [Publications] M.Suda: "Regulation of Wee1 kinase in response to protein synthesis inhibition"FEBS Letters. 486. 305-309 (2000)
• [Publications] M.Mizunuma: "GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down regulation by Ca^<2+> in budding yeast"EMBO J.. 20(印刷中). (2001)
• [Publications] Zhang,X.: "Crosstalk between transcriptional regulations for multidrug resistance in yeast"J.Biol.Chem.. (印刷中). (2001)
• [Publications] 水沼正樹: "Ca^<2+>シグナルによる細胞周期制御機構"実験医学. 18. 840-845 (2000)
• [Publications] 宮川都吉: "真核微生物の多剤耐性"バイオサイエンスとバイオインダストリー. 2000. 17-20
• [Publications] Z.Cui: "Functional analysis of the yeast SNQ2 gene encoding a yeast multidrug resistance transporter Phat confers resistaine to 4NQC"Biosci.Biotechnol.Biochem.. 63(1). 162-167 (1999)
• [Publications] I.C.Farcasanu: "Involvement of thioredoxin peroxidase typeII(Ahp1) of S.cerevisiae in Mn^<2+> homeostasis"Biosci.Biotechnol.Biochem.. 63(11). 1871-1881 (1999)
• [Publications] M.Suda: "Overproduction of EF1α, an esseutial translational componennt, causes abberant cell merphology in yeast"Genes to Cells. 4. 517-527 (1999)
• [Publications] M.Yukawa: "Nps1/Sth1, a component of chromatin-remodeling complex of S.cerevisiae, is required for meiotic genes"Genes to Cells. 4. 99-110 (1999)
• [Publications] 平田大: "新規の細胞周期チェックポイント制御とMAPキナーゼ"実験医学. 17. 124-129 (1999)
• [Publications] M.Mizunuma: "Role of calcineurin in regulating the onset of mitosis in budding yeast" Nature. 392. 303-306 (1998)
• [Publications] K.Miyahara: "Functional interaction of IsR1,a predicted protein Rinase with the Pkc1 pathway in Saccharowyces cerevisial." Biosci.Biotechnol.Biochem.62(6). 1376-1380 (1998)
• [Publications] Z.Cui: "Yeast gene YRRI,which is required for the resistance to 4-NQO,mediates transcriptional activation of SNQ2 gene." Mol.Microbiol.29(5). 1307-1315 (1998)
• [Publications] E.Tsuchiya: "A mutation in NPS1,an essential gene encoding a conponent of RSC1,alters the chromatin structure of S.cerevisiae" Nucleic Acids Research.26(13). 3286-3292 (1998)
• [Publications] I.C.Farcasanu: "Involvement of histidine permease(Hip1p)in manganese transport in Saccharowyces cerevisial." Mol.Gen.Genet.259(5). 541-548 (1998)
• [Publications] Z.Cui: "Functional analysis of the promoter of the yeast SNQ2 gene encoding multidrug resistance transporter." Biosci.Biotechnol.Biochem.63(1). 162-167 (1999)