基本情報

所属

• 遺伝子病制御研究所　疾患制御研究部門

職名

• 教授

学位

• 工学博士（論文博士）（広島大学）

ホームページURL

http://www.igm.hokudai.ac.jp/molint/index.html

J-Global ID

• 200901098699348935

研究キーワード

• 生化学   分子生物学   細胞生物学   Genetics   Biochemistry   Molecular biology   Cell biology   

研究分野

• ライフサイエンス / 分子生物学
• ライフサイエンス / 細胞生物学

担当教育組織

•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2000年 - 遺伝子病制御研究所 分子間情報分野 教授
• 2000年 - Professor

学歴

•         - 1980年   大阪大学   工学部
•         - 1980年   大阪大学

研究活動情報

論文

• Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

  Kaori Kanemaru, Makoto Shimozawa, Manabu Kitamata, Rikuto Furuishi, Hinako Kayano, Yui Sukawa, Yuuki Chiba, Takatsugu Fukuyama, Junya Hasegawa, Hiroki Nakanishi, Takuma Kishimoto, Kazuya Tsujita, Kazuma Tanaka, Toshiki Itoh, Junko Sasaki, Takehiko Sasaki, Kiyoko Fukami, Yoshikazu Nakamura

  Nature communications 13 1 2347 - 2347 2022年05月09日 [査読有り]
   

  Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.
• Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells.

  Tetsuo Mioka, Tian Guo, Shiyao Wang, Takuma Tsuji, Takuma Kishimoto, Toyoshi Fujimoto, Kazuma Tanaka

  Journal of cell science 135 5 2022年03月01日 [査読有り]
   

  Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.
• Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane.

  Takuma Kishimoto, Tetsuo Mioka, Eriko Itoh, David E Williams, Raymond J Andersen, Kazuma Tanaka

  Molecular biology of the cell 32 15 1374 - 1392 2021年07月15日 [査読有り]
   

  Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.
• An optogenetic system to control membrane phospholipid asymmetry through flippase activation in budding yeast

  Tomomi Suzuki, Tetsuo Mioka, Kazuma Tanaka, Akira Nagatani

  Scientific Reports 10 1 2020年12月 [査読有り][通常論文]
  
• A complex genetic interaction implicates that phospholipid asymmetry and phosphate homeostasis regulate Golgi functions.

  Mamoru Miyasaka, Tetsuo Mioka, Takuma Kishimoto, Eriko Itoh, Kazuma Tanaka

  PloS one 15 7 e0236520  2020年 [査読有り][通常論文]
   

  In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.
• Comparative Glycomic Analysis of Sialyl Linkage Isomers by Sialic Acid Linkage-Specific Alkylamidation in Combination with Stable Isotope Labeling of α2,3-Linked Sialic Acid Residues.

  Hanamatsu H, Nishikaze T, Tsumoto H, Ogawa K, Kobayashi T, Yokota I, Morikawa K, Suda G, Sho T, Nakai M, Miura N, Higashino K, Sekiya S, Iwamoto S, Miura Y, Furukawa JI, Tanaka K, Sakamoto N

  Analytical chemistry 91 21 13343 - 13348 2019年11月05日 [査読有り][通常論文]
   

  Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.
• Sialic Acid Linkage-Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones.

  Hanamatsu H, Nishikaze T, Miura N, Piao J, Okada K, Sekiya S, Iwamoto S, Sakamoto N, Tanaka K, Furukawa JI

  Analytical chemistry 90 22 13193 - 13199 2018年10月 [査読有り][通常論文]
  
• Synthesis of omega-3 long-chain polyunsaturated fatty acid-rich triacylglycerols in an endemic goby, Gymnogobius isaza, from Lake Biwa, Japan

  Takuto Suito, Kohjiro Nagao, Masataka Hatano, Kenichi Kohashi, Aiko Tanabe, Hiromichi Ozaki, Jun Kawamoto, Tatsuo Kurihara, Tetsuo Mioka, Kazuma Tanaka, Yuji Hara, Masato Umeda

  JOURNAL OF BIOCHEMISTRY 164 2 127 - 140 OXFORD UNIV PRESS 2018年08月 [査読有り][通常論文]
  
• Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane

  Tetsuo Mioka, Konomi Fujimura-Kamada, Nahiro Mizugaki, Takuma Kishimoto, Takamitsu Sano, Hitoshi Nunome, David E. Williams, Raymond J. Andersen, Kazuma Tanaka

  Molecular Biology of the Cell 29 10 1203 - 1218 2018年05月15日 [査読有り][通常論文]
   

  Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.
• Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

  Takaharu Yamamoto, Konomi Fujimura-Kamada, Eno Shioji, Risa Suzuki, Kazuma Tanaka

  G3-GENES GENOMES GENETICS 7 1 179 - 192 2017年01月 [査読有り][通常論文]
   

  Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50 Delta mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1 Delta mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1 Delta mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.
• Inositol Depletion Restores Vesicle Transport in Yeast Phospholipid Flippase Mutants

  Kanako Yamagami, Takaharu Yamamoto, Shota Sakai, Tetsuo Mioka, Takamitsu Sano, Yasuyuki Igarashi, Kazuma Tanaka

  PLOS ONE 10 3 e0120108  2015年03月 [査読有り][通常論文]
   

  In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.
• Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

  Tetsuo Mioka, Konomi Fujimura-Kamada, Kazuma Tanaka

  MICROBIOLOGYOPEN 3 5 803 - 821 2014年10月 [査読有り][通常論文]
   

  In eukaryotic cells, phosphatidylserine (PS) is predominantly located in the cytosolic leaflet of the plasma membrane; this asymmetry is generated by an unknown mechanism. In this study, we used the PS-specific probe mRFP-Lact-C2 to investigate the possible involvement of type 4 P-type ATPases, also called phospholipid flippases, in the generation of this asymmetry in Saccharomyces cerevisiae. PS was not found in the trans-Golgi Network in wild-type cells, but it became exposed when vesicle formation was compromised in the sec7 mutant, and it was also exposed on secretory vesicles (SVs), as reported previously. However, flippase mutations did not reduce the exposure of PS in either case, even at low levels that would only be detectable by quantitative analysis of mRFP-Lact-C2 fluorescence in isolated SVs. Furthermore, no reduction in the PS level was observed in a mutant with multiple flippase mutations. Because PS was not exposed in a mutant that accumulates ER or cis/medial-Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation. Our results suggest that an unknown mechanism, possibly a protein with flippase-like activity, acts in conjunction with known flippases to regulate PS translocation.
• Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

  Miyoko Takeda, Kanako Yamagami, Kazuma Tanaka

  EUKARYOTIC CELL 13 3 363 - 375 2014年03月 [査読有り][通常論文]
   

  Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.
• Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast

  Hisatoshi Hanamatsu, Konomi Fujimura-Kamada, Takaharu Yamamoto, Nobumichi Furuta, Kazuma Tanaka

  JOURNAL OF BIOCHEMISTRY 155 1 51 - 62 2014年01月 [査読有り][通常論文]
   

  Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.
• The roles of C-terminal helices of human apolipoprotein A-I in formation of higsih-denty lipoprotein particles

  Nagao K, Hata M, Tanaka K, Takechi Y, Nguyen D, Dhanasekaran P, Lund-Katz S, Phillips MC, Saito H

  Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1841 1 80 - 87 2014年 [査読無し][通常論文]
   

  Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.
• Identification of a second amphipathic lipid-packing sensor-like motif that contributes to Gcs1p function in the early endosome-to-TGN pathway

  Zahra Zendeh-boodi, Takaharu Yamamoto, Hiroshi Sakane, Kazuma Tanaka

  JOURNAL OF BIOCHEMISTRY 153 6 573 - 587 2013年06月 [査読有り][通常論文]
   

  ArfGAPs, GTPase-activating proteins for Arf small GTPases, are involved in multiple steps of vesicle formation of various transport pathways. Amphipathic lipid-packing sensor (ALPS) motif was first identified in the C-terminal regions of ArfGAP1 and its yeast homologue Gcs1p as a region that adsorbs preferentially onto highly curved membranes by folding into an amphipathic alpha-helix (AH). We previously showed that Gcs1p functionally interacted with the phospholipid flippase Cdc50p-Drs2p in the early endosome-to-TGN retrieval pathway. In this study, we performed functional analyses of the C-terminal region of Gcs1p containing ALPS. Hydrophobic cluster analysis suggested that there is another potential AH-forming region downstream of ALPS in Gcs1p. Mutational analysis suggested that the ALPS motif is important for the Gcs1p function in the Golgi-to-ER retrograde pathway, whereas ALPS and the predicted AH region redundantly function in the post-Golgi pathways including the early endosome-to-TGN pathway. Liposome flotation assay indicated that this downstream region preferentially interacted with liposomes of smaller size. The region containing the ALPS motif was also required for the interaction with SNARE proteins including Snc1p and Tlg1p. These results suggest that ALPS and the predicted AH region are involved in the regulation and function of Gcs1p by interacting with membrane phospholipids and vesicle proteins.
• Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

  Takeru Hachiro, Takaharu Yamamoto, Kenji Nakano, Kazuma Tanaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 288 5 3594 - 3608 2013年02月 [査読有り][通常論文]
   

  The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3 Delta mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans-Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3 Delta mutant. The NH2-terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH2-terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3 Delta mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.
• Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin

  Yusuke Aihara, Takaharu Yamamoto, Koji Okajima, Kazuhiko Yamamoto, Tomomi Suzuki, Satoru Tokutomi, Kazuma Tanaka, Akira Nagatani

  Journal of Biological Chemistry 287 13 9901 - 9909 2012年03月23日 [査読有り][通常論文]
   

  Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
• Essential Role of the NH2-Terminal Region of Cdc24 Guanine Nucleotide Exchange Factor in Its Initial Polarized Localization in Saccharomyces cerevisiae

  Konomi Fujimura-Kamada, Tomoe Hirai, Kazuma Tanaka

  EUKARYOTIC CELL 11 1 2 - 15 2012年01月 [査読有り][通常論文]
   

  The cortical recruitment and accumulation of the small GTPase Cdc42 are crucial steps in the establishment of polarity, but this process remains obscure. Cdc24 is an upstream regulator of budding yeast Cdc42 that accelerates the exchange of GDP for GTP in Cdc42 via its Dbl homology (DH) domain. Here, we isolated five novel temperature-sensitive (ts) cdc24 mutants, the green fluorescent protein (GFP)-fused proteins of which lose their polarized localization at the nonpermissive temperature. All amino acid substitutions in the mutants were mapped to the NH2-terminal region of Cdc24, including the calponin homology (CH) domain. These Cdc24-ts mutant proteins did not interact with Bem1 at the COOH-terminal PB1 domain, suggesting a lack of exposure of the PB1 domain in the mutant proteins. The cdc24-ts mutants were also defective in polarization in the absence of Bem1. It was previously reported that a fusion protein containing Cdc24 and the p21-activated kinase (PAK)-like kinase Cla4 could bypass the requirement for Bem1 in polarity cue-independent budding (i.e., symmetry breaking). Cdc24-ts-Cla4 fusion proteins also showed ts localization at the polarity site. We propose that the NH2-terminal region unmasks the DH and PB1 domains, leading to the activation of Cdc42 and interaction with Bem1, respectively, to initiate cell polarization.
• 出芽酵母の細胞極性形成におけるエンドサイトーシス-リサイクリング経路の役割 : アクチンケーブルが形成されない変異株からわかったこと

  山本 隆晴, 田中 一馬

  化学と生物 49 4 231 - 233 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2011年04月01日
• Isolation and characterization of novel mutations in CDC50, the non-catalytic subunit of the Drs2p phospholipid flippase

  Yasuhiro Takahashi, Konomi Fujimura-Kamada, Satoshi Kondo, Kazuma Tanaka

  JOURNAL OF BIOCHEMISTRY 149 4 423 - 432 2011年 [査読有り][通常論文]
   

  Flippases (type 4 P-type ATPases) are believed to translocate phospholipids from the exoplasmic to the cytoplasmic leaflet in bilayer membranes. Since flippases are structurally similar to ion-transporting P-type ATPases such as the Ca2+ ATPase, one important question is how flippases have evolved to transport phospholipids instead of ions. We previously showed that a conserved membrane protein, Cdc50p, is required for the endoplasmic reticulum exit of the Drs2p flippase in yeast. However, Cdc50p is still associated with Drs2p after its transport to the endosomal/trans-Golgi network (TGN) membranes, and its function in the complex with Drs2p is unknown. In this study, we isolated novel temperature-sensitive (ts) cdc50 mutants whose products were still localized to endosomal/TGN compartments at the non-permissive temperature. Mutant Cdc50 proteins colocalized with Drs2p in endosomal/TGN compartments, and they co-immunoprecipitated with Drs2p. These cdc50-ts mutants exhibited defects in vesicle transport from early endosomes to the TGN as the cdc50 deletion mutant did. These results suggest that mutant Cdc50 proteins could be complexed with Drs2p, but the resulting Cdc50p-Drs2p complex is functionally defective at the non-permissive temperature. Cdc50p may play an important role for phospholipid translocation by Drs2p.
• Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable-dependent Vesicle Transport in Yeast

  Takaharu Yamamoto, Junko Mochida, Jun Kadota, Miyoko Takeda, Erfei Bi, Kazuma Tanaka

  MOLECULAR BIOLOGY OF THE CELL 21 7 1237 - 1252 2010年04月 [査読無し][通常論文]
   

  The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.
• Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry

  Kenzi Nakano, Takaharu Yamamoto, Takuma Kishimoto, Takehiro Noji, Kazuma Tanaka

  MOLECULAR BIOLOGY OF THE CELL 19 4 1783 - 1797 2008年04月 [査読無し][通常論文]
   

  Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50 Delta mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3 Delta/dnf1 Delta dnf2 Delta mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1 Delta fpk2 Delta mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.
• Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins

  Koji Saito, Konomi Fujimura-Kamada, Hisatoshi Hanamatsu, Utako Kato, Masato Umeda, Keith G. Kozminski, Kazuma Tanaka

  DEVELOPMENTAL CELL 13 5 743 - 751 2007年11月 [査読無し][通常論文]
   

  An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. Here, we report that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3 Delta mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3 Delta cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rgalp and Rga2p toward Cdc42p, whereas Pl(4,5)P-2 inhibits it. We propose that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs.
• Endocytic recycling in yeast is regulated by putative phospholipid translocases and the Ypt31p/32p-Rcy1p pathway

  Nobumichi Furuta, Konomi Fujimura-Kamada, Koji Saito, Takaharu Yamamoto, Kazuma Tanaka

  MOLECULAR BIOLOGY OF THE CELL 18 1 295 - 312 2007年01月 [査読無し][通常論文]
   

  Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3 Delta crf1 Delta background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1 Delta mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p-Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.
• Identification of novel mutations in ACT1 and SLA2 that suppress the actin-cable-overproducing phenotype caused by overexpression of a dominant active form of Bni1p in Saccharomyces cerevisiae

  S Yoshiuchi, T Yamamoto, H Sakane, J Kadota, J Mochida, M Asaka, K Tanaka

  GENETICS 173 2 527 - 539 2006年06月 [査読無し][通常論文]
   

  A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1 Delta N). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1 Delta. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PRF1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1 Delta sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1 Delta N phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.
• Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Letn3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

  T Noji, T Yamamoto, K Saito, K Fujimura-Kamada, S Kondo, K Tanaka

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 344 1 323 - 331 2006年05月 [査読無し][通常論文]
   

  Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBD-labeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50 Delta background. Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it Substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments. (c) 2006 Elsevier Inc. All rights reserved.
• [Actin organization regulated by lipids].

  Kishimoto T, Yamamoto T, Tanaka K

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 6 Suppl 767 - 775 2006年05月 [査読有り][通常論文]
  
• The functional relationship between the Cdc50p-Drs2p putative aminophospholipid translocase and the arf GAP Gcs1p in vesicle formation in the retrieval pathway from yeast early endosomes to the TGN

  Hiroshi Sakane, Takaharu Yamamoto, Kazuma Tanaka

  CELL STRUCTURE AND FUNCTION 31 2 87 - 108 2006年 [査読無し][通常論文]
   

  Drs2p, the catalytic subunit of the Cdc50p-Drs2p putative aminophospholipid translocase, has been implicated in conjunction with the Arf1 signaling pathway in the formation of clathrin-coated vesicles (CCVs) from the TGN. Herein, we searched for Arf regulator genes whose mutations were synthetically lethal with cdc50 Delta, and identified the Arf GAP gene GCS1. Most of the examined transport pathways in the Cdc50p-depleted gcs1 Delta mutant were nearly normal, including endocytic transport to vacuoles, carboxypeptidase Y sorting, and the processing and secretion of invertase. In contrast, this mutant exhibited severe defects in the early endosome-to-TGN transport pathway; proteins that are transported via this pathway, such as the v-SNARE Snc1p, the t-SNARE Tlg1p, and the chitin synthase III subunit Chs3p, accumulated in TGN-independent aberrant membrane structures. We extended our analyses to clathrin adaptors, and found that Gga1p/Gga2p and AP-1 were also involved in this pathway. The Cdc50p-depleted ggaI Delta gga2 Delta mutant and the gcs1 Delta apl2 Delta (the beta 1 subunit of AP-1) mutant exhibited growth defects and intracellular Snc1p-containing membranes accumulated in these cells. These results suggest that Cdc50p-Drs2p plays an important role in the Arf1p-mediated formation of CCVs for the retrieval pathway from early endosomes to the TGN.
• Defects in structural integrity of ergosterol and the Cdc50p-Drs2p putative phospholipid translocase cause accumulation of endocytic membrances, onto which actin patches are assembled in yeast

  T Kishimoto, T Yamamoto, K Tanaka

  MOLECULAR BIOLOGY OF THE CELL 16 12 5592 - 5609 2005年12月 [査読無し][通常論文]
   

  Specific changes in membrane lipid composition are implicated in actin cytoskeletal organization, vesicle formation, and control of cell polarity. Cdc50p, a membrane protein in the endosomal/trans-Golgi network compartments, is a noncatalytic subunit of Drs2p, which is implicated in translocation of phospholipids across lipid bilayers. We found that the cdc50 Delta mutation is synthetically lethal with mutations affecting the late steps of ergosterol synthesis (erg2 to erg6). Defects in cell polarity and actin organization were observed in the cdc50 Delta erg3 Delta mutant. In particular, actin patches, which are normally found at cortical sites, were assembled intracellularly along with their assembly factors, including Las17p, Abp1p, and Sla2p. The exocytic SNARE Snc1p, which is recycled by an endocytic route, was also intracellularly accumulated, and inhibition of endocytic internalization suppressed the cytoplasmic accumulation of both Las17p and Snc1p. Simultaneous loss of both phospholipid asymmetry and sterol structural integrity could lead to accumulation of endocytic intermediates capable of initiating assembly of actin patches in the cytoplasm.
• 3SF53 膜脂質の非対称性と細胞極性形成(バイオとナノの融合 : 生物物理学からのアプローチ)(北海道大学21世紀COE共催シンポジウム)

  田中 一馬, 鎌田 このみ, 山本 隆晴

  生物物理 45 S30  一般社団法人 日本生物物理学会 2005年
• Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae

  J Kadota, T Yamamoto, S Yoshiuchi, EF Bi, K Tanaka

  MOLECULAR BIOLOGY OF THE CELL 15 12 5329 - 5345 2004年12月 [査読無し][通常論文]
   

  Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta, cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.
• Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae

  K Saito, K Fujimura-Kamada, N Furuta, U Kato, M Umeda, K Tanaka

  MOLECULAR BIOLOGY OF THE CELL 15 7 3418 - 3432 2004年07月 [査読無し][通常論文]
   

  Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc5op-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.
• She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae

  Hirofumi Toi, Konomi Fujimura-Kamada, Kenji Irie, Yoshimi Takai, Satoru Todo, Kazuma Tanaka

  Molecular Biology of the Cell 14 6 2237 - 2249 2003年06月01日 [査読無し][通常論文]
   

  She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain. She4p/Dim1p was required for Myo5p localization to cortical patch-like structures. Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant. All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well. She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA. Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.
• The upstream regulator, Rsr1p, and downstream effectors, Gic1p and Gic2p, of the Cdc42p small GTPase coordinately regulate initiation of budding in Saccharomyces cerevisiae

  R Kawasaki, K Fujimura-Kamada, H Toi, H Kato, K Tanaka

  GENES TO CELLS 8 3 235 - 250 2003年03月 [査読無し][通常論文]
   

  Background: Cdc42p, a Rho family small GTPase, is essential for budding initiation in the yeast Saccharomyces cerevisiae. The homologous proteins Gic1p and Gic2p (Gic1/2p) are effectors of Cdc42p, but their precise functions remain unknown. Rsr1p/Bud1p is a Ras family small GTPase that controls the selection of the budding site. Previous observations suggested that Rsr1p-GTP recruits Cdc24p, a GDP/GTP exchange factor for Cdc42p, at the incipient bud site. However, this model only addresses how Rsr1p determines the budding site, because the rsr1 mutant normally initiates budding. Results: Here we show that a rsr1 gic1 gic2 mutant fails to initiate budding, resulting in unbudded, large, and multinucleated cells. Expression of a dominant active or dominant negative mutant of RSR1 also inhibited the growth of the gic1 gic2 mutant, suggesting that cycling of Rsr1p between the GTP- and GDP-bound forms is required for budding initiation in the gic1 gic2 mutant. Among the mutations in effectors of CDC42, only the gic1 gic2 mutation demonstrated a synthetic lethal interaction with rsr1. Increased gene dosage of CDC42 suppressed defects in budding initiation of rsr1 gic1 gic2 mutants containing additional mutations in other effectors of CDC42, including BNI1, CLA4 or STE20. The polarized localization of Bni1p-GFP (green fluorescent protein) and Cla4p-GFP was lost after depletion of Gic1p in the rsr1 gic2 mutant. Conclusion: We propose that Gic1/2p may stabilize or maintain a complex consisting of Cdc42p-GTP and its effectors at the budding site, which are assembled by the action of the Rsr1p-Cdc24p system.
• Cdc50p, a conserved endosomal membrane protein, controls polarized growth in Saccharomyces cerevisiae

  K Misu, K Fujimura-Kamada, T Ueda, A Nakano, H Katoh, K Tanaka

  MOLECULAR BIOLOGY OF THE CELL 14 2 730 - 747 2003年02月 [査読無し][通常論文]
   

  During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.
• The novel adaptor protein, Mti1p, and Vrp1p, a homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may antagonistically regulate type I myosins in Saccharomyces cerevisiae

  J Mochida, T Yamamoto, K Fujimura-Kamada, K Tanaka

  GENETICS 160 3 923 - 934 2002年03月 [査読無し][通常論文]
   

  Typo I invosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like stnictures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mtilp-GFP co-localized with cortical actin patches. A null mutation of MTH exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed upsilonrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.
• Complementing yeast rho1 mutation groups with distinct functional defects

  A Saka, M Abe, H Okano, M Minemura, H Qadota, T Utsugi, A Mino, K Tanaka, Y Takai, Y Ohya

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 49 46165 - 46171 2001年12月 [査読無し][通常論文]
   

  Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta -glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta -glucan synthase activity.
• Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae

  T Fujiwara, K Tanaka, E Inoue, M Kikyo, Y Takai

  MOLECULAR AND CELLULAR BIOLOGY 19 12 8016 - 8027 1999年12月 [査読無し][通常論文]
   

  The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated tao genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pad or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin path but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pad act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pad bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.
• An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae

  M Kikyo, K Tanaka, T Kamei, K Ozaki, T Fujiwara, E Inoue, Y Takita, Y Ohya, Y Takai

  ONCOGENE 18 50 7046 - 7054 1999年11月 [査読無し][通常論文]
   

  Proteins containing formin homology domains, FH1 and FH2, are involved in cytokinesis or establishment of cell polarity in a variety of organisms. Bni1p and Bnr1p are FPF proteins and potential targets of the Rho family small GTP-binding proteins in S. cerevisiae. We have shown that Bnr1p is localized at the bud neck to interact with Hof1p, involved in cytokinesis. We report here that the overexpression of BNR1 causes a cytokinesis deficiency which is similar to the phenotypes of the septin mutants, including cdc3, cdc10, cdc11, and cdc12. The region required for the septin mutant phenotypes was mapped to Bnr1p (35-500), which coincided,vith the region required for the bud-neck localization. To further isolate a gene interacting with BNI1 or BNR1, a multicopy suppressor of the bni1 bnr1 mutant was isolated. This gene encoded Smy1p, a kinesin-related protein. Bnr1p, but not Bni1p, directly interacted with the C-terminal region of Smy1p. The Smy1p-interacting region of Bnr1p was mapped to a region containing the FH2 domain. Bnr1p also directly interacted with Bud6p, a novel actin-binding protein. Bnr1p is thus a multifunctional protein which interacts with the septin system, a microtubule-dependent motor protein, and the actin system, to regulate cytoskeletal functions in S. cerevisiae.
• 低分子量GTP結合タンパク質の機能 低分子量G蛋白質Rhoによる細胞骨格の制御

  藤原 武志, 尾崎 公美, 田中 一馬, 高井 義美

  生化学 71 8 689 - 689 (公社)日本生化学会 1999年08月
• 細胞骨格系のオーガナイザーとしてのRHO4標的蛋白質BNR1

  桔梗 充博, 田中 一馬, 亀井 尚, 尾崎 公美, 藤原 武志, 井上 英二, 滝田 陽子, 大矢 禎一, 高井 義美

  生化学 71 8 1063 - 1063 (公社)日本生化学会 1999年08月
• Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p - Implication in cytokinesis in Saccharomyces cerevisiae

  T Kamei, K Tanaka, T Hihara, M Umikawa, H Imamura, M Kikyo, K Ozaki, Y Takai

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 43 28341 - 28345 1998年10月 [査読無し][通常論文]
   

  Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnrlp and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both Bnrlp and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnrlp directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae.
• Shs1p: A novel member of septin that interacts with Spa2p, involved in polarized growth in Saccharomyces cerevisiae

  A Mino, K Tanaka, T Kamei, M Umikawa, T Fujiwara, Y Takai

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251 3 732 - 736 1998年10月 [査読無し][通常論文]
   

  The Rho family small G proteins regulate various cell functions including cytokinesis. We have shown that Bni1p, a potential target of Rho1p, interacts with Spa2p and that Spa2p is required for the localization of Bni1p at the growth sites in Saccharomyces cerevisiae. We isolated here a novel member of the septin family, implicated in cytokinesis, as a Spa2p-binding protein by the yeast two-hybrid method. We named this gene SHS1 (Seventh Homolog of Septin). The shs1 mutant cells showed cytokinesis deficiency and Shs1p was localized at the bud neck in budded cells. The Spa2p-Shs1p interactions may play an important role in cytokinesis. (C) 1998 Academic Press.
• Rho1p-Bni1p-Spa2p interactions: Implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae

  T Fujiwara, K Tanaka, A Mino, M Kikyo, K Takahashi, K Shimizu, Y Takai

  MOLECULAR BIOLOGY OF THE CELL 9 5 1221 - 1233 1998年05月 [査読無し][通常論文]
   

  Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.
• Interaction of Rho1p target Bni1p with F-actin-binding elongation factor 1 alpha: implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae

  M Umikawa, K Tanaka, T Kamei, K Shimizu, H Imamura, T Sasaki, Y Takai

  ONCOGENE 16 15 2011 - 2016 1998年04月 [査読無し][通常論文]
   

  The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1 alpha (EF1 alpha). EF1 alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)binding and -bundling activities. EF1 alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1 alpha inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1 alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1 alpha and profilin.
• Control of reorganization of the actin cytoskeleton by Rho family small GTP-binding proteins in yeast

  K Tanaka, Y Takai

  CURRENT OPINION IN CELL BIOLOGY 10 1 112 - 116 1998年02月 [査読無し][通常論文]
   

  Accumulating evidence indicates that Rho family small GTP-binding proteins regulate reorganization of the actin cytoskeleton. There are members of the Rho family in the budding yeast Saccharomyces cerevisiae, in which powerful molecular genetical approaches are applicable. Recent identification of regulators and targets of the Rho family members has enhanced our understanding of the regulation and modes of action of Rho family members in reorganization of the actin cytoskeleton.
• Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae

  T Asakura, T Sasaki, F Nagano, A Satoh, H Obaishi, H Nishioka, H Imamura, K Hotta, K Tanaka, H Nakanishi, Y Takai

  ONCOGENE 16 1 121 - 130 1998年01月 [査読無し][通常論文]
   

  We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the I-125-labeled F-actin blot overlay method. The purified protein showed a minimum M-r of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated M-r of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.
• Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae

  G Koch, K Tanaka, T Masuda, W Yamochi, H Nonaka, Y Takai

  ONCOGENE 15 4 417 - 422 1997年07月 [査読有り][通常論文]
   

  The small GTP-binding proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility and cytokinesis, through reorganization of actin cytoskeleton. Rho GDI is a general regulator which forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation. We have purified Rho GDI from the yeast Saccharomyces cerevisiae, cloned its gene, and named it RD11 (Rho GD). In this study, we have further characterized Beast Rho GDI, Rho GDI was found in the cytosol by immunoblot and immunofluorescence microscopic analyses. Rho1p and Cdc42p were co-immunoprecipitated with Rho GDI from the cytosol. This immunoprecipitated Rho1p was mainly bound to GDP. In the disruption mutant of Rho GDI, which did not show any apparent phenotype, both Rho1p and Cdc42p were also present in the cytosol. These results indicate that yeast Rho GDI possesses properties similar to those of mammalian Rho GDI, and that there is a cytosolic factor which functionally substitutes for Rho GDI in yeast.
• Bni1p and Bnr1p: Downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae

  H Imamura, K Tanaka, T Hihara, M Umikawa, T Kamei, K Takahashi, T Sasaki, Y Takai

  EMBO JOURNAL 16 10 2745 - 2755 1997年05月 [査読有り][通常論文]
   

  The RHO1 gene encodes a homologue of mammalian RhoA small G-protein in the yeast Saccharomyces cerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. We have recently shown that Bni1p is a potential target of Rho1p., Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity, In S.cerevisiae, there is an open reading frame (YIL159W) which encodes another protein having the FH1 and FH2 domains and we have named this gene BNR1 (BNI1 Related). Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p. Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature-sensitive growth phenotype. Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate. These phenotypes are similar to those of the mutant of PFY1, which encodes profilin, an actin-binding protein. Moreover, yeast two-hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains. These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton.
• Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae

  H Kohno, K Tanaka, A Mino, M Umikawa, H Imamura, T Fujiwara, Y Fujita, K Hotta, H Qadota, T Watanabe, Y Ohya, Y Takai

  EMBO JOURNAL 15 22 6060 - 6068 1996年11月 [査読有り][通常論文]
   

  The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae, Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation, We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p, Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p, This gene was identified as BN11, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis, Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase, These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.
• Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS

  C Ueguchi, T Suzuki, T Yoshida, K Tanaka, T Mizuno

  JOURNAL OF MOLECULAR BIOLOGY 263 2 149 - 162 1996年10月 [査読有り][通常論文]
   

  The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hits mutations which resulted in derepression of the proV WX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by on:ly a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS. (C) 1996 Academic Press Limited
• ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae

  H Hirano, K Tanaka, K Ozaki, H Imamura, H Kohno, T Hihara, T Kameyama, K Hotta, M Arisawa, T Watanabe, H Qadota, Y Ohya, Y Takai

  MOLECULAR AND CELLULAR BIOLOGY 16 8 4396 - 4403 1996年08月 [査読有り][通常論文]
   

  The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the Delta bem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the Delta bem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.
• Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor

  K Hotta, K Tanaka, A Mino, H Kohno, Y Takai

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225 1 69 - 74 1996年08月 [査読有り][通常論文]
   

  The Rho family small G proteins are implicated in various cell functions, such as cell morphological change, cell motility, and cytokinesis. However, their modes of action in regulating these cell functions remain to be clarified. In the present study, we have isolated a cDNA encoding a protein which interacts with the GTP-bound form, but not with the GDP bound form, of the Rho family members, including RhoA, Rad, and Cdc42, by the yeast two-hybrid method. This protein is kinectin, known to be a vesicle membrane anchoring protein of kinesin, which is an ATPase motor transporting vesicles along microtubules. (C) 1996 Academic Press, Inc.
• Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae

  K Ozaki, K Tanaka, H Imamura, T Hihara, T Kameyama, H Nonaka, H Hirano, Y Matsuura, Y Takai

  EMBO JOURNAL 15 9 2196 - 2207 1996年05月 [査読有り][通常論文]
   

  The RNO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1 (G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins, Disruption of ROM2 resulted in a temperature-sensitiv:e growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of Delta rom1 Delta rom2 cells were similar to those of Delta rho1 cells, including growth arrest with a small bud and cell lysis, Moreover, the temperature-sensitive growth phenotype of Delta rom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rho1p-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor, These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.
• A DOWNSTREAM TARGET OF RHO1 SMALL GTP-BINDING PROTEIN IS PKC1, A HOMOLOG OF PROTEIN-KINASE-C, WHICH LEADS TO ACTIVATION OF THE MAP KINASE CASCADE IN SACCHAROMYCES-CEREVISIAE

  H NONAKA, K TANAKA, H HIRANO, T FUJIWARA, H KOHNO, M UMIKAWA, A MINO, Y TAKAI

  EMBO JOURNAL 14 23 5931 - 5938 1995年12月 [査読有り][通常論文]
   

  The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell. functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseudosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain, MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant, The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E45I), but not that of rho1(V43T), These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.
• Rho as a regulator of the cytoskeleton

  Takai Y, Sasaki T, Tanaka K, Nakanishi H

  Trends in Biochemical Sciences 20 6 227 - 231 1995年 [査読有り][通常論文]
  
• MOLECULAR-CLONING AND CHARACTERIZATION OF YEAST-RHO GDP DISSOCIATION INHIBITOR

  T MASUDA, K TANAKA, H NONAKA, W YAMOCHI, A MAEDA, Y TAKAI

  JOURNAL OF BIOLOGICAL CHEMISTRY 269 31 19713 - 19718 1994年08月 [査読有り][通常論文]
   

  We have previously isolated rho GDP dissociation inhibitor (rho GDI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids, rho GDI inhibits the GDP/GTP exchange reaction of post translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.
• GROWTH SITE LOCALIZATION OF RHO1 SMALL GTP-BINDING PROTEIN AND ITS INVOLVEMENT IN BUD FORMATION IN SACCHAROMYCES-CEREVISIAE

  W YAMOCHI, K TANAKA, H NONAKA, A MAEDA, T MUSHA, Y TAKAI

  JOURNAL OF CELL BIOLOGY 125 5 1077 - 1093 1994年06月 [査読有り][通常論文]
   

  The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the motherbud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.
• THE REGULATORY AND TARGET PROTEINS FOR SMALL G-PROTEINS

  Y TAKAI, K KAIBUCHI, T SASAKI, H SHIRATAKI, K TANAKA, H NAKANISHI

  GTPASE-CONTROLLED MOLECULAR MACHINES 6 221 - 233 1994年 [査読有り][通常論文]
  
• Rho small G protein and cytoskeletal control.

  Takai Y, Kaibuchi K, Sasaki T, Tanaka K, Shirataki H, Nakanishi H

  Princess Takamatsu symposia 24 338 - 350 1994年 [査読有り][通常論文]
  

書籍

• 酵母のすべて 系統、細胞から分子まで 大隅良 典・下田親編

  シュプリンガー・ジャパン 2007年
• バイオとナノの融合。新生命科学の基礎』北海道大学 COE研究成果編集委員会編

  北海道大学出版会 2007年
• 「酵母ラボマニュアル」<山本正幸・大矢禎一編>

  シュプリンガー・フェアラーク東京（株） 1998年

その他活動・業績

• ホスファチジルセリンが酵母におけるタンパク質を含まない巨大形質膜ドメインの生成を阻害する

  Tetsuo Mioka, Guo Tian, Wang Shiyao, Takuma Tsuji, Takuma Kishimoto, Toyoshi Fujimoto, Kazuma Tanaka bioRxiv 2020年08月11日 [査読無し][通常論文]
   

  Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the liquid-ordered domain caused by phase separation. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.
• Yeast DRAM1/TMEM150 family protein Sfk1p, a potential regulator of the phospholipid asymmetry, is involved in the permeability of the plasma membrane.

  T. Mioka, K. Fujimura-Kamada, N. Mizugaki, T. Sano, T. Yamamoto, K. Tanaka MOLECULAR BIOLOGY OF THE CELL 26 2015年 [査読無し][通常論文]
  
• Functions of phospholipid flippases

  Kazuma Tanaka, Konomi Fujimura-Kamada, Takaharu Yamamoto JOURNAL OF BIOCHEMISTRY 149 (2) 131 -143 2011年02月 [査読無し][通常論文]
   

  Asymmetrical distribution of phospholipids is generally observed in the eukaryotic plasma membrane. Maintenance and changes of this phospholipid asymmetry are regulated by ATP-driven phospholipid translocases. Accumulating evidence indicates that type 4 P-type ATPases (P4-ATPases, also called flippases) translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the plasma membrane and internal membranes. Among P-type ATPases, P4-ATPases are unique in that they are associated with a conserved membrane protein of the Cdc50 family as a non-catalytic subunit. Recent studies indicate that flippases are involved in various cellular functions, including transport vesicle formation and cell polarity. In this review, we will focus on the functional aspect of phospholipid flippases.
• 細胞極性形成におけるアクチン細胞骨格再編成の膜脂質による制御 (細胞骨格と接着) -- (細胞極性)

  岸本 拓磨, 山本 隆晴, 田中 一馬 蛋白質核酸酵素 51 (6) 767 -775 2006年05月
• 膜脂質二重層の内外のリン脂質輸送を制御するDnfl-Lem3複合体の生理機能の解明

  野路 武寛, 山本 隆晴, 近藤 哲, 田中 一馬 日本外科学会雑誌 107 (2) 484 -484 2006年03月05日
• The Cdc50p family is required for recycling via early endosome in Saccharomyces cerevisiae

  Nobumichi Furuta, Konomi Fuiimura-Kamada, Takaharu Yamamoto, Koji Saito, Kazuma Tanaka CELL STRUCTURE AND FUNCTION 30 70 -70 2005年06月 [査読無し][通常論文]
  
• Cdc50p, a protein required for polarized growth, is a potential subunit of the Drs2p aminophospholipid translocase in Saccharomyces cerevisiae

  K Saito, K Fujimura-Kamada, N Furuta, U Kato, M Umeda, K Tanaka MOLECULAR BIOLOGY OF THE CELL 15 319A -320A 2004年11月 [査読無し][通常論文]
  
• Regulation of the initial septin ring assembly by polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in budding yeast

  J Kadota, T Yamamoto, S Yoshiuchi, E Bi, K Tanaka MOLECULAR BIOLOGY OF THE CELL 15 45A -45A 2004年11月 [査読無し][通常論文]
  
• Phytochrome B-GFP expressed in mesophyll cells of cotyledons controls flowering by the FT gene expression

  M Endo, S Nakamura, T Araki, M Rai, T Masumura, K Tanaka, N Mochizuki, A Nagatani PLANT AND CELL PHYSIOLOGY 45 S46 -S46 2004年 [査読無し][通常論文]
  
• 細胞の極性形成に関与するSPA2と相互作用する新しいセプチン, SHS1の単離と解析

  三野 彰久, 田中 一馬, 海川 正人, 藤原 武志, 高井 義美 日本分子生物学会年会プログラム・講演要旨集 21 483 -483 1998年12月01日
• RasとRho低分子量G蛋白質の機能と作用機構 (がんと遺伝子--機能から診断・治療まで) -- (がん遺伝子とシグナル伝達)

  日原 太郎, 田中 一馬, 高井 義美 蛋白質核酸酵素 42 (10) 1501 -1508 1997年07月
• Dynamic activation and action of Rho in regulation of actin cytoskeleton

  Y Takai, K Tanaka, T Sasaki, K Takaishi, K Takahashi FASEB JOURNAL 11 (9) A994 -A994 1997年07月 [査読無し][通常論文]
  
• PURIFICATION AND PROPERTIES OF RECOMBINANT RHO-GDP DISSOCIATION INHIBITOR

  K TANAKA, T SASAKI, Y TAKAI SMALL GTPASES AND THEIR REGULATORS, PT B 256 41 -49 1995年 [査読無し][通常論文]
  
• 小胞輸送と低分子量GTP結合タンパク質

  佐々木卓也, 白瀧博通, 田中一馬, 中西宏之, 高井義美 日本細胞生物学会大会講演要旨集 47th 55 1994年 [査読無し][通常論文]
  
• REQUIREMENT OF PRENYLATION OF SMALL GTP-BINDING PROTEINS FOR THEIR ACTIVATION AND FUNCTIONS

  Y TAKAI, K KAIBUCHI, A KIKUCHI, T SASAKI, H SHIRATAKI, K TANAKA FASEB JOURNAL 7 (7) A1039 -A1039 1993年04月 [査読無し][通常論文]
  

共同研究・競争的資金等の研究課題

• 生体膜リン脂質非対称性の変化が制御する細胞機能の解析

  基盤研究 (B)

  研究期間 : 2009年04月 -2013年03月 

  代表者 : 田中一馬
• Molecular mechanisms of the formation of cell polarity

  研究期間 : 2000年

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大学運営

学内役職歴

• 2020年4月1日 - 2022年3月31日 遺伝子病制御研究所長
• 2012年4月1日 - 2014年3月31日 遺伝子病制御研究所附属感染癌研究センター長
• 2020年4月1日 - 2022年3月31日 教育研究評議会評議員