Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
SHIGEHIKO TAMURA Last modified date:2023.11.28

Professor / Division for Experimental Natural Science / Faculty of Arts and Science


Papers
1. Yuichi Abe, Shigehiko Tamura, Masanori Honsho, Yukio Fujiki, A Mouse Model System to Study Peroxisomal Roles in Neurodegeneration of Peroxisome Biogenesis Disorders., Adv Exp Med Biol., doi: 10.1007/978-3-030-60204-8_10., 1299, 119-143, 2020.11.
2. Kanji Okumoto, Shigehiko Tamura, Masanori Honsho, Yukio Fujiki, Peroxisome: Metabolic Functions and Biogenesis., Adv Exp Med Biol., doi: 10.1007/978-3-030-60204-8_1., 1299, 3-17, 2020.11.
3. Masanori Honsho, Kanji Okumoto, Shigehiko Tamura, Yukio Fujiki, Peroxisome Biogenesis Disorders., Adv Exp Med Biol., doi: 10.1007/978-3-030-60204-8_4., 1299, 45-54, 2020.11.
4. Koichiro Yamashita, Shigehiko Tamura, Masanori Honsho, Hiroto Yada, Yuichi Yagita, Hidetaka Kosako, Yukio Fujiki, Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery, Journal of Cell Biology, 10.1083/JCB.202001003, 219, 10, 2020.10, © 2020 Yamashita et al. Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis..
5. Yuichi Abe, Yoshiki Nishimura, Kaori Nakamura, Shigehiko Tamura, Masanori Honsho, Hiroshi Udo, Toshihide Yamashita and Yukio Fujiki, Peroxisome Deficiency Impairs BDNF Signaling and Memory, Front. Cell Dev. Biol., 10.3389/fcell.2020.567017, 8, 1-15, 2020.10.
6. Yukio Fujiki, Yuichi Abe, Yuuta Imoto, Akemi J. Tanaka, Kanji Okumoto, Masanori Honsho, Shigehiko Tamura, Non Miyata, Toshihide Yamashita, Wendy K. Chung, Tsuneyoshi Kuroiwa, Recent insights into peroxisome biogenesis and associated diseases, Journal of Cell Science, 10.1242/jcs.236943, 133, 9, 2020.05, © 2020. Published by The Company of Biologists Ltd. Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs. A number of recent findings have advanced our understanding of the biology, physiology and consequences of functional defects in peroxisomes. In this Review, we discuss a cooperative cell defense mechanisms against oxidative stress that involves the localization of BAK (also known as BAK1) to peroxisomes, which alters peroxisomal membrane permeability, resulting in the export of catalase, a peroxisomal enzyme. Another important recent finding is the discovery of a nucleoside diphosphate kinase-like protein that has been shown to be essential for how the energy GTP is generated and provided for the fission of peroxisomes. With regard to PBDs, we newly identified a mild mutation, Pex26-F51L that causes only hearing loss. We will also discuss findings from a new PBD model mouse defective in Pex14, which manifested dysregulation of the BDNF-TrkB pathway, an essential signaling pathway in cerebellar morphogenesis. Here, we thus aim to provide a current view of peroxisome biogenesis and the molecular pathogenesis of PBDs..
7. Shigehiko Tamura, Naomi Matsumoto, Ryota Takeba, Yukio Fujiki, AAA Peroxins and Their Recruiter Pex26p Modulate the Interactions of Peroxins Involved in Peroxisomal Protein Import, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M114.588038, 289, 35, 24336-24346, 2014.08, Background: Pex26p and AAA peroxins are required for the peroxisome biogenesis. Results: Pex26p directly binds to Pex14p and acts as a scaffold protein to specify the molecular target of AAA peroxins. Conclusion: AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane. Significance: Functional modulation by Pex26p and AAA peroxins of the interaction between peroxins makes important contribution to peroxisome biogenesis.
Pex1p and Pex6p are required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We herein show that mammalian Pex26p directly binds to Pex14p, the initial docking receptor of Pex5p, and interacts with Pex5p via Pex14p. The binding affinity of Pex26p to Pex14p is altered by Pex5p. Further evidence suggests that the N-terminal region in Pex26p acts as a scaffold protein to recruit Pex14pPex5p complex together with Pex1pPex6p complexes on peroxisomes. Pex26p binding to Pex14p was suppressed by overexpression of Pex1p and Pex6p in an ATP-dependent manner, whereas Pex14p was not competed out by Pex1p and Pex6p from Pex26p mutant defective in peroxisomal matrix protein import. These results suggested that peroxisome biogenesis requires Pex1p- and Pex6p-regulated dissociation of Pex14p from Pex26p. Pex1p homo-oligomer directly binds to Pex5p as assessed by a surface plasmon resonance-based assay. Moreover, cytosolic Pex1p is likely to maintain the functional oligomer of Pex5p. Taken together, in the peroxisomal protein import, AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane as well as Pex5p oligomer in the cytosol..
8. Y. Fujiki, K. Okumoto, S. Mukai, S. Tamura, Molecular basis for peroxisome biogenesis disorders, Molecular Machines Involved in Peroxisome Biogenesis and Maintenance, 10.1007/978-3-7091-1788-0_5, 91-110, 91-110, 2014.02, © 2014 Springer-Verlag Wien. All rights reserved. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS), autosomal recessive, and progressive disorders characterized by loss of multiple peroxisomal metabolic functions and defects in peroxisome assembly, consisting of 13 complementation groups (CGs). Two mutually distinct but complementary approaches, forward genetic approach using more than a dozen CGs of peroxisome-deficient Chinese hamster ovary (CHO) cell mutants and the homology search by screening the human expressed sequence tag (EST) database using yeast peroxin (PEX) genes, have been taken in order to isolate mammalian PEX genes. Search for pathogenic genes responsible for PBDs of all 13 CGs is now accomplished. Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins, Pex1p, Pex2p, Pex5p, Pex6p, Pex7p, Pex10p, Pex12p, Pex13p, Pex14p, and Pex26p, for 10 CGs of PBDs, are required for matrix protein import; three, Pex3p, Pex16p, and Pex19p, are essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three CGs, 12, 9, and 14, respectively; PEX11β mutation causes dysmorphogenesis of peroxisomes in ZS-like phenotype of CG16. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts, and deletions. Prenatal DNA diagnosis using PEX genes is now possible for PBDs of all 13 CGs..
9. Y. Fujiki, K. Okumoto, S. Mukai, S. Tamura, Molecular basis for peroxisome biogenesis disorders, Molecular Machines Involved in Peroxisome Biogenesis and Maintenance, 10.1007/978-3-7091-1788-0_5, 91-110, 2014.02.
10. Yukio Fujiki, Kanji Okumoto, Satoru Mukai, Masanori Honsho, Shigehiko Tamura, Peroxisome biogenesis in mammalian cells, Frontiers in Physiology, 10.3389/fphys.2014.00307, 5 AUG, 2014.01.
11. New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis..
12. Chika Nashiro, Astuko Kashiwagi, Takashi Matsuzaki, Shigehiko Tamura, Yukio Fujiki, Recruiting Mechanism of the AAA Peroxins, Pex1p and Pex6p, to Pex26p on the Peroxisomal Membrane, TRAFFIC, 10.1111/j.1600-0854.2011.01182.x, 12, 6, 774-788, 2011.06, A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26p delta 33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle..
13. Jian-Rong Su, Kazuki Takeda, Shigehiko Tamura, Yukio Fujiki, Kunio Miki, Monomer-dimer transition of the conserved N-terminal domain of the mammalian peroxisomal matrix protein import receptor, Pex14p, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2010.02.160, 394, 1, 217-221, 2010.03, Pex14p is a central component of the peroxisomal matrix protein import machinery. In the recently determined crystal structure, a characteristic face consisting of conserved residues was found on a side of the conserved N-terminal domain of the protein. The face is highly hydrophobic, and is also the binding site for the WXXXF/Y motif of Pex5p. We report herein the dimerization of the domain in the isolated state. The homo-dimers are in equilibrium with the monomers. The homo-dimers are completely dissociated into monomers by complex formation with the WXXXF/Y motif peptide of Pex5p. A putative dimer model shows the interaction between the conserved face and the PXXP motif of another protomer. The model allows us to discuss the mechanism of the oligomeric transition of the full-length Pex14p modulated by the binding of other peroxins. (C) 2010 Elsevier Inc. All rights reserved..
14. Jian-Rong Su, Kazuki Takeda, Shigehiko Tamura, Yukio Fujiki, Kunio Miki, Crystal structure of the conserved N-terminal domain of the peroxisomal matrix protein import receptor, Pex14p, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.0808681106, 106, 2, 417-421, 2009.01, Pex14p is a central component of the peroxisomal protein import machinery, in which the conserved N-terminal domain mediates dynamic interactions with other peroxins including Pex5p, Pex13p, and Pex19p. Here, we report the crystal structure of the conserved N-terminal domain of Pex14p with a three-helix bundle. A hydrophobic surface is composed of the conserved residues, of which two phenylalanine residues (Phe-35 and Phe-52) protrude to the solvent. Consequently, two putative binding pockets suitable for recognizing the helical WXXXF/Y motif of Pex5p are formed on the surface by the two phenylalanine residues accompanying with positively charged residues. The structural feature agrees well with our earlier findings where F35A/L36A and F52A/L53A mutants were impaired in the interactions with other peroxins such as Pex5p and Pex13p. Pex14p variants each with Phe-to-Ala mutation at positions 35, 52, and 35/52, respectively, were defective in restoring the impaired peroxisomal protein import in pex14 Chinese hamster ovary mutant ZP161 cells. Moreover, in GST pull-down assays His6-Pex5pL bound only to GST-Pex14p(25-70), not to any of GST-Pex14p(25-70) F35A, GST-Pex14p(25-70) F52A, and GST-Pex14p(25-70) F35A/F52A. Endogenous Pex5p was recruited to FLAG-Pex14p on peroxisomes in vivo but barely to FLAG-Pex14pF35A, FLAG-Pex14pF52A, and FLAG-Pex14pF35A/F52A. Collectively, Phe-35 and Phe-52 are essential for the Pex14p functions, including the interaction between Pex14p and Pex5p..
15. Yukio Fujiki, Non Miyata, Naomi Matsumoto, Shigehiko Tamura, Dynamic and functional assembly of the AAA peroxins,, Pex1p and Pex6p, and their membrane receptor Pex26p involved in shuttling of the PTS1 receptor Pex5p in peroxisome biogenesis, BIOCHEMICAL SOCIETY TRANSACTIONS, 10.1042/BST0360109, 36, Pt 1, 109-113, 2008.02, The peroxisome is a single-membrane-bound organelle found in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient PBDs (peroxisome biogenesis disorders), such as Zellweger syndrome. Two AAA (ATPase associated with various cellular activities) peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for CG (complementation group) 1 and I PBDs respectively. PEX26, which is responsible for I PBDs, codes for Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. We recently assigned the binding regions between human Pex1p and Pex6p and elucidated the pivotal roles that the AAA cassettes, D1 and D2 domains, play in Pex1p-Pex6p interaction and in peroxisome biogenesis. ATP binding to both AAA cassettes of Pex1p and Pex6p was a prerequisite for the Pex1p-Pex6p interaction and peroxisomal localization, but ATP hydrolysis by the D2 domains was not required. Pex1p exists in two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, with these possibly having distinct functions in peroxisome biogenesis. AAA peroxins are involved in the export from peroxisomes of Pex5p, the PTS1 (peroxisome-targeting signal type 1) receptor..
16. Shigehiko Tamura, A pathogenic gene in an inherited peroxisome disorder and its cellular dysfunction, Seikagaku, 79, 4, 329-339, 2007.12.
17. Shigehiko Tamura, Shinobu Yasutake, Naomi Matsumoto, Yukio Fujiki, Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M605159200, 281, 38, 27693-27704, 2006.09, Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for peroxisome biogenesis disorders of complementation group 1 (CG1) and CG4, respectively. PEX26 responsible for peroxisome biogenesis disorders of CG8 encodes Pex26p, the recruiter of Pex1p (.) Pex6p complexes to peroxisomes. We herein assigned the binding regions between human Pex1p and Pex6p and elucidated pivotal roles of the AAA cassettes, called D1 and D2 domains, in Pex1p - Pex6p interaction and peroxisome biogenesis. ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p - Pex6p interaction and their peroxisomal localization. The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p. In HEK293 cells, endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm, while Pex6p and Pex26p were predominantly localized on peroxisomes. Interaction of Pex1p with Pex6p conferred a conformational change and dissociation of the Pex1p oligomer. These results suggested that Pex1p possesses two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, possibly playing distinct functions in peroxisome biogenesis..
18. Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxisomal localization, and interaction with the Pex1p x Pex6p complex..
19. N Matsumoto, S Tamura, S Furuki, N Miyata, A Moser, N Shimozawa, HW Moser, Y Suzuki, N Kondo, Y Fujiki, Mutations in novel peroxin gene PEX26 that cause peroxisome-biogenesis disorders of complementation group 8 provide a genotype-phenotype correlation, AMERICAN JOURNAL OF HUMAN GENETICS, 10.1086/377004, 73, 2, 233-246, 2003.08, The human disorders of peroxisome biogenesis (PBDs) are subdivided into 12 complementation groups (CGs). CG8 is one of the more common of these and is associated with varying phenotypes, ranging from the most severe, Zellweger syndrome (ZS), to the milder neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). PEX26, encoding the 305-amino-acid membrane peroxin, has been shown to be deficient in CG8. We studied the PEX26 genotype in fibroblasts of eight CG8 patients - four with the ZS phenotype, two with NALD, and two with IRD. Catalase was mostly cytosolic in all these cell lines, but import of the proteins that contained PTS1, the SKL peroxisome targeting sequence, was normal. Expression of PEX26 reestablished peroxisomes in all eight cell lines, confirming that PEX26 defects are pathogenic in CG8 patients. When cells were cultured at 30 degreesC, catalase import was restored in the cell lines from patients with the NALD and IRD phenotypes, but to a much lesser extent in those with the ZS phenotype, indicating that temperature sensitivity varied inversely with the severity of the clinical phenotype. Several types of mutations were identified, including homozygous G89R mutations in two patients with ZS. Expression of these PEX26 mutations in pex26 Chinese hamster ovary cells resulted in cell phenotypes similar to those in the human cell lines. These findings confirm that the degree of temperature sensitivity in pex26 cell lines is predictive of the clinical phenotype in patients with PEX26 deficiency..
20. N Matsumoto, S Tamura, Y Fujiki, The pathogenic peroxin Pex26p recruits the Pex1p-Pex6p AAA ATPase complexes to peroxisomes, NATURE CELL BIOLOGY, 10.1038/ncb982, 5, 5, 454-460, 2003.05, Peroxisomes are ubiquitous organelles with a single membrane that contain over 50 different enzymes that catalyse various metabolic pathways, including beta-oxidation and lipid synthesis(1). Peroxisome biogenesis disorders (PBDs), such as Zellweger syndrome and neonatal adrenoleukodystrophy, are fatal genetic diseases that are autosomal recessive(2,3). Among the PBDs of the 12 complementation groups (CGs)(4), 11 associated PEX genes have been isolated(4-7). Accordingly, only the PBD pathogenic gene for CG8 (also called CG-A) remains unidentified. Here we have isolated human PEX26 encoding a type II peroxisomal membrane protein of relative molecular mass 34,000 (M-r 34K) by using ZP167 cells, a Chinese hamster ovary (CHO) mutant cell line(5,8). Expression of PEX26 restores peroxisomal protein import in the fibroblasts of an individual with PBD of CG8. This individual possesses a homozygous, inactivating pathogenic point mutation, Arg98Trp, in Pex26. Pex6 and Pex1 of the AAA ATPase family co-immunoprecipitate with Pex26. Epitope-tagged Pex6 and Pex1 are discernible as puncta in normal CHO-K1 cells, but not in PEX26-defective cells. PEX26 expression in ZP167 cells re-establishes colocalization of Pex6 and Pex1 with Pex26, in a Pex6-dependent manner. Thus, Pex26 recruits Pex6-Pex1 complexes to peroxisomes..
21. S Tamura, N Matsumoto, A Imamura, N Shimozawa, Y Suzuki, N Kondo, Y Fujiki, Phenotype-genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p-Pex6p interaction, BIOCHEMICAL JOURNAL, 10.1042/0264-6021:3570417, 357, 2, 417-426, 2001.07, The peroxisome bioggenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of I RD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CGI in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin Pex1p of the AAA ATPase family. It has been also reported that Pex1p and Pex6p interact with each other. In the present study we investigated phenotype-genotype relationships of CGI PBDs. Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37 degreesC, whereas a normal level of Pex1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634-690, were stably present at both temperatures. Pex1p-GS43D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pex1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p-Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS..
22. N Matsumoto, S Tamura, A Moser, HW Moser, N Braverman, Y Suzuki, N Shimozawa, N Kondo, Y Fujiki, The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6, JOURNAL OF HUMAN GENETICS, 10.1007/s100380170078, 46, 5, 273-277, 2001.04, Human genetic peroxisomal biogenesis disorders (PBDs), such as Zellweger syndrome, comprise 13 different complementation groups (CGs). Eleven peroxin genes. termed PEXs, responsible for PBDs have been identified, whereas pathogenic genes for PBDs of 2CGs, CG-A (the same CG as CG8 in the United States and Europe) and CG6, remained unidentified. We herein provide several lines of novel evidence indicating chat PEX6, the pathogenic gene for CG4. is impaired in PBD of CG6. Expression of PEX6 restored peroxisome assembly in fibroblasts from a CG6 PBD patient. This patient was a compound heterozygote for PEX6 gene alleles, Accordingly, by merging CG6 with CG4, human PBDs are now classified into 12 CGs..
23. K Ghaedi, S Tamura, K Okumoto, Y Matsuzono, Y Fujiki, Pex3p is required for a membrane-assembly stage in peroxisome biogenesis, MOLECULAR BIOLOGY OF THE CELL, 11, 423A-423A, 2000.12.
24. K Ghaedi, S Tamura, K Okumoto, Y Matsuzono, Y Fujiki, The peroxin Pex3p initiates membrane assembly in peroxisome biogenesis, MOLECULAR BIOLOGY OF THE CELL, 11, 6, 2085-2102, 2000.06, Rat cDNA encoding a 372-amino-aeid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed similar to 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated..
25. Fujiki Y., Okumoto K., Otera H., Tamura S., Peroxisome biogenesis and molecular defects in peroxisome assembly disorders., Cell Biochem. Biophys., 10.1385/CBB:32:1-3:155, 32, 155-164, 32, Spring:155-164, 2000.01.
26. R Toyama, S Mukai, A Itagaki, S Tamura, N Shimozawa, Y Suzuki, N Kondo, RJA Wanders, Y Fujiki, Isolation, characterization and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants, HUMAN MOLECULAR GENETICS, 10.1093/hmg/8.9.1673, 8, 9, 1673-1681, 8 (9), 1673-1681, 1999.09, We isolated peroxisome biogenesis mutants ZP128 and ZP150 from rat PEX2-transformed Chinese hamster ovary (CHO) cells, by the 9-(1'-pyrene)nonanol/ultraviolet method. The mutants lacked morphologically recognizable peroxisomes and showed a typical peroxisome assembly-defective phenotype such as a high sensitivity to 12-(1'-pyrene)dodecanoic acid/UV treatment. By means of PEX cDNA transfection and cell fusion, ZP128 and ZP150 were found to belong to a recently identified complementation group H. Expression of human PEX13 cDNA restored peroxisome assembly in ZP128 and ZP150. CHO cell PEX13 was isolated; its deduced sequence comprises 405 amino acids with 93% identity to human Pex13p, Mutation in PEX13 of mutant ZP150 was determined by RT-PCR: G to A transition resulted in one amino acid substitution, Ser319Asn, in one allele and truncation of a 42 amino acid sequence from Asp265 to Lys306 in another allele, Therefore, ZP128 and ZP150 are CHO cell lines with a phenotype of impaired PEX13..
27. K Ghaedi, A Kawai, K Okumoto, S Tamura, N Shimozawa, Y Suzuki, N Kondo, Y Fujiki, Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein, EXPERIMENTAL CELL RESEARCH, 10.1006/excr.1999.4413, 248, 2, 489-497, 1999.05, We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CG;s. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG. (C) 1999 Academic Press..
28. K Ghaedi, A Itagaki, R Toyama, S Tamura, T Matsumura, A Kawai, N Shimozawa, Y Suzuki, N Kondo, Y Fujiki, Newly identified Chinese hamster ovary cell mutants defective in peroxisome assembly represent complementation group A of human peroxisome biogenesis disorders and one novel group in mammals, EXPERIMENTAL CELL RESEARCH, 10.1006/excr.1999.4412, 248, 2, 482-488, 248 (2), 482-488, 1999.05, We isolated peroxisome biogenesis-defective mutants from rat PEX2-transformed Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet method. A total of 18 mutant cell clones showing cytosolic localization of catalase were isolated. By complementation group (CG;) analysis by means of PEX cDNA transfection and cell fusion, cell mutants, ZP124 and ZP126, were found to belong to two novel CGs of CHO mutants. Mutants, ZP135 and ZP167, mere also classified to the same CG as ZP124. Further cell fusion analysis using 12 CGs fibroblasts from patients with peroxisome deficiency disorders such as Zellweger syndrome revealed that ZP124 belonged to human CG-A, the same group as CC-WI in the United States. ZP126 could not be classified to any of human and CHO CGs. These mutants also showed typical peroxisome assembly-defective phenotypes such as severe loss of catalase latency and impaired biogenesis of peroxisomal enzymes. Collectively, ZP124 represents CG-A, and ZP126 is in a newly identified CG distinct from the 14 mammalian CGs previously characterized, (C) 1999 Academic Press..
29. N Shimizu, R Itoh, Y Hirono, H Otera, K Ghaedi, K Tateishi, S Tamura, K Okumoto, T Harano, S Mukai, Y Fujiki, The peroxin Pex14p - cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.274.18.12593, 274, 18, 12593-12604, 1999.04, Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14), The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14..
30. Y Matsuzono, N Kinoshita, S Tamura, N Shimozawa, M Hamasaki, K Ghaed, RJA Wanders, Y Suzuki, N Kondo, Y Fujiki, Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.96.5.2116, 96, 5, 2116-2121, 1999.03, At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A(764), in a codon for Met(255), resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein..
31. Ghaedi, K., Itagaki, A., Toyama, R., Tamura, S., Matsumura, T., Kawai, A., Shimozawa, N., Suzuki, Y., Kondo, N., and Fujiki, Y., Newly identified Chinese hamster ovary cell mutants defective in peroxisome assembly represent complementation group A of human peroxisome biogenesis disorders and one novel group in mammals., Exp. Cell Res., 10.1006/excr.1999.4412, 248, 2, 482-488, 248, 482-488, 1999.01.
32. Toyama, R., Mukai, S., Itagaki, A., Tamura, S., Shimozawa, N., Suzuki, Y., Kondo, N., Wanders, R. J. A., and Fujiki, Y., Isolation, characterization, and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants., Hum. Mol. Genet., 10.1093/hmg/8.9.1673, 8, 9, 1673-1681, 8, 1673-1681, 1999.01.
33. M Honsho, S Tamura, N Shimozawa, Y Suzuki, N Kondo, Y Fujiki, Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D, AMERICAN JOURNAL OF HUMAN GENETICS, 10.1086/302161, 63, 6, 1622-1630, 1998.12, Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which >10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 ina codon ((C) under bar GA) for (176)Arg, that resulted in a termination codon ((T) under bar GA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CC-D PBDs..
34. A Imamura, S Tamura, N Shimozawa, Y Suzuki, ZY Zhang, T Tsukamoto, T Orii, N Kondo, T Osumi, Y Fujiki, Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders, HUMAN MOLECULAR GENETICS, 7, 13, 2089-2094, 1998.12, The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), are autosomal recessive diseases caused by deficiency of peroxisome assembly as well as malfunction of peroxisomes, where >10 genotypes have been reported. ZS patients manifest the most severe clinical and biochemical abnormalities, while those with NALD and IRD show the least severity and the mildest features, respectively. PEX1 is the causative gene for PBDs of complementation group I (CG1), the highest incidence PBD, and encodes the peroxin, Pex1p, a member of the AAA ATPase family. In the present work, we found that peroxisomes were morphologically and biochemically formed at 30 but not 37 degrees C, in the fibroblasts from all CG1 IRD patients examined, whereas almost no peroxisomes were seen in ZS and NALD cells, even at 30 degrees C. A point missense mutation, G843D, was identified in the PEX1 allele of most CG1 IRD patients. The mutant PEX1, termed HsPEX1G843D, gave rise to the same temperature-sensitive phenotype on CG1 CHO cell mutants upon transfection. Collectively, these results demonstrate temperature-sensitive peroxisome assembly to be responsible for the mildness of the clinical features of PEX1-defective IRD of CG1..
35. K Okumoto, R Itoh, N Shimozawa, Y Suzuki, S Tamura, N Kondo, Y Fujiki, Mutations in PEX10 is the cause of Zellweger peroxisome deficiency syndrome of complementation group B, HUMAN MOLECULAR GENETICS, 10.1093/hmg/7.9.1399, 7, 9, 1399-1405, 1998.09, Peroxisome biogenesis disorders (PBD), such as Zellweger syndrome, are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as a malfunction of the peroxisomes, where at least 10 genotypes have been reported. We have isolated a human PEX10 cDNA (HsPEX10) by an expressed sequence tag homology search on a human DNA database using yeast PEX10 from Hansenula polymorpha, followed by screening of a human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex10p) comprising 326 amino acids, with two putative transmembrane segments and a C3HC4 zinc finger RING motif, Both the N- and C-terminal regions of Pex10p are exposed to the cytosol, as assessed by an expression study of epitope-tagged Pex10p. HsPEX10 expression morphologically and biochemically restored peroxisome biogenesis in fibroblasts from Zellweger patients of complementation group B in Japan (complementation group VII in the USA). One patient (PBDB-01) possessed a homozygous, inactivating mutation, a 2 bp deletion immediately upstream of the RING motif, which resulted in a frameshift,;altering 65 amino acids from the normal. This implies that the C-terminal part, including the RING finger, is required for biological function of Pex10p, PEX10 cDNA derived from patient PBDB-01 was defective in peroxisome-restoring activity when expressed in patient fibroblasts, These results demonstrate that mutation in PEX10 is the genetic cause of complementation group B PBD..
36. Abe, I, K Okumoto, S Tamura, Y Fujiki, Clofibrate-inducible, 2X-kDa peroxisomal integral membrane protein is encoded by PEX11, FEBS LETTERS, 10.1016/S0014-5793(98)00815-1, 431, 3, 468-472, 1998.07, We cloned a human PEX11 cDNA by expressed sequence tag homology search using yeast Candida boininii PEX11, followed by screening of human liver cDNA library. PEX11 encoded a peroxisomal protein Pex11p comprising 247 amino acids, with two transmembrane segments and a dilysine motif at the C-terminus, Pex11p comigrated in SDS-PAGE with a 28-kDa peroxisomal integral membrane protein (PMP28) isolated from the liver of clofibrate-treated rats and was crossreactive to anti-PMP28 antibody, thereby indicating PEX11 to encode PMP28. Pex11p exposes both N- and C-terminal parts to the cytosol, PEX11 mas not responsible for ten complementation groups of human peroxisome deficiency disorders. (C) 1998 Federation of European Biochemical Societies..
37. K Okumoto, N Shimozawa, A Kawai, S Tamura, T Tsukamoto, T Osumi, H Moser, RJA Wanders, Y Suzuki, N Kondo, Y Fujiki, PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of Pex12p, MOLECULAR AND CELLULAR BIOLOGY, 18, 7, 4324-4336, 1998.07, Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group LII (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for (291)Asn and (292)Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-m peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function..
38. S Tamura, N Shimozawa, Y Suzuki, T Tsukamoto, T Osumi, Y Fujiki, A cytoplasmic AAA family peroxin, Pex1p, interacts with Pex6p, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.1998.8522, 245, 3, 883-886, 1998.04, Human PEX1 (HsPEX1) is the causative gene for peroxisome-deficiency disorders such as Zellweger syndrome of complementation group I, encoding the peroxin, Pex1p, a member of AAA family. Pex1p tagged with an epitope flag was expressed in wild-type Chinese hamster ovary (CHO) cell, CHO-K1. Pex1p was localized in the cytoplasm, as assessed by immunofluorescent microscopy. Cell-lysate of HsPEX1-transfected CHO-K1 was incubated with in vitro synthesized S-35-labelled Pex6p, an AAA family peroxin. Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of S-35-Pex6p. Conversely, S-35-Pex1p was obtained in immunoprecipitate from CHO-K1 expressing human Pex6p, using anti-Pex6p antibody. These results strongly suggest that Pex1p and Pex6p interact with each other, (C) 1998 Academic Press..
39. S Tamura, K Okumoto, R Toyama, N Shimozawa, T Tsukamoto, Y Suzuki, T Osumi, N Kondo, Y Fujiki, Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.95.8.4350, 95, 8, 4350-4355, 1998.04, The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PE,YI cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family, ri stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well, as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders..
40. S TAMURA, H NELSON, A TAMURA, N NELSON, SHORT EXTERNAL LOOPS AS POTENTIAL SUBSTRATE-BINDING SITE OF GAMMA-AMINOBUTYRIC-ACID TRANSPORTERS, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.270.48.28712, 270, 48, 28712-28715, 1995.12, While the gamma-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, -3, and 4 also transport beta-alanine, Cross-mutations in the external loops IV, V, and VI among the various GABA transporters were performed by site-directed mutagenesis. The affinity of GABA transport as well as inhibitor sensitivity of the modified transporters was analyzed, Kinetic analysis revealed that a cross-mutation in which loop IV of GAT1 was modified to resemble GAT4 resulted in increased affinity to GABA from K-m = 8.7 to 2.0 mu M without changing the V-max. A cross mutation in loop VI, which swapped the amino acid sequence of GATE for GAT1, decreased the affinity to GABA (K-m, 35 mu M). These results suggest that loops TV and VI contribute to the binding affinity of GABA transporters, A substitution of three amino acids in loop V of GAT1 by the corresponding sequence of GAT3 resulted in beta-alanine sensitivity of its GABA uptake activity, These three amino acids in loop V seem to participate in the beta-alanine binding domain of GAT3. It is suggested that those three external loops (IV, V, and VI) form a pocket in which the substrate binds to the GABA transporters..
41. F JURSKY, S TAMURA, A TAMURA, S MANDIYAN, H NELSON, N NELSON, STRUCTURE, FUNCTION AND BRAIN LOCALIZATION OF NEUROTRANSMITTER TRANSPORTERS, JOURNAL OF EXPERIMENTAL BIOLOGY, 196, 283-295, 1994.11, We studied four different cDNAs encoding GABA transporters and three different cDNAs encoding glycine transporters in mouse and rat brains. A genomic clone of two of the glycine transporters (GLYT1a and GLYT1b) revealed that they derive from differential splicing of a single gene. The third glycine transporter (GLYT2) is encoded by a separate gene. Antibodies were raised against seven of these neurotransmitter transporters and their cytochemical localization in the mouse brain was studied. In general, we observed a deviation from the classical separation of neuronal and glial transporters. It seems that each of the neurotransmitter transporters is present in specific places in the brain and is expressed in a different way in very specific areas. For example, the GABA transporter GAT4, which also transports beta-alanine, was localized to neurons. However, GAT1, which is specific for GABA, was localized not only to neurons but also to glial cells. The recently discovered glycine transporter GLYT2 was of particular interest because of its deviation from the general structure by a very extended N terminus containing multiple potential phosphorylation sites. Western analysis and immunocytochemistry in frozen sections of mouse brain demonstrated a clear caudal-rostral gradient of GLYT2 distribution, with massive accumulation in the spinal cord and brainstem and less in the cerebellum. Its distribution is typically neuronal and it is present in processes with varicosities. A correlation was observed between the pattern we obtained and that observed previously from strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission at the classical inhibitory system in the hindbrain. The availability of four different GABA transporters made it possible to look for specific binding sites upon the neurotransmitter transporters. An extensive program of site-directed mutagenesis led us to identify a potential neurotransmitter binding site on the GABA transporters..
42. S TAMURA, XH WANG, M MAEDA, M FUTAI, GASTRIC DNA-BINDING PROTEINS RECOGNIZE UPSTREAM SEQUENCE MOTIFS OF PARIETAL CELL-SPECIFIC GENES, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.90.22.10876, 90, 22, 10876-10880, 1993.11, Polymerase chain reaction amplification of cDNA from pig gastric mucosa demonstrated the presence of zinc-finger proteins called GATA-GT1, GATA-GT2, and GATA-GT3, each having zinc-finger sequences similar to previously characterized GATA-binding proteins. Subsequently, full-length cDNAs of GATA-GT1 and GATA-GT2 were obtained from rat stomach. The zinc-finger domains of GATA-GT1 and -GT2 were 66-86% identical on the amino acid level with each other and with other GATA-binding proteins. Potential protein kinase phosphorylation sites were present in the zinc-finger region. In contrast, regions outside the zinc fingers shared significantly lower similarities. GATA-GT2 was found to bind to the upstream sequence of the H+/K+-ATPase beta gene and to a sequence containing the GATA motif. GATA-GT1 and -GT2 were expressed predominantly in the gastric mucosa and at much lower levels in the intestine (GATA-GT2, also in testis), their tissue distributions being distinct from those of GATA-1, -2, or -3. These results clearly suggest that GATA-GT1 and GATA-GT2 are involved in gene regulation specifically in the gastric epithelium and represent two additional members of the GATA-binding protein family..
43. S TAMURA, KI OSHIMAN, T NISHI, M MORI, M MAEDA, M FUTAI, SEQUENCE MOTIF IN CONTROL REGIONS OF THE H+/K+ ATPASE ALPHA-SUBUNIT AND BETA-SUBUNIT GENES RECOGNIZED BY GASTRIC SPECIFIC NUCLEAR PROTEIN(S), FEBS LETTERS, 298, 2-3, 137-141, 1992.02, A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K+-ATPase alpha-subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta-subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor)..
44. M MAEDA, KI OSHIMAN, S TAMURA, S KAYA, S MAHMOOD, MA REUBEN, LS LASATER, G SACHS, M FUTAI, THE RAT H+ K+-ATPASE BETA-SUBUNIT GENE AND RECOGNITION OF ITS CONTROL REGION BY GASTRIC DNA-BINDING PROTEIN, JOURNAL OF BIOLOGICAL CHEMISTRY, 266, 32, 21584-21588, 1991.11, The rat gastric H+/K+-ATPase beta-subunit gene was cloned, and its nucleotide sequence was determined. The coding region is separated by 6 introns, whereas the related human Na+/K+-ATPase beta-subunit gene was shown to have 5 introns (Lane, L. K., Shull, M. M., Whitmer, K. R., and Lingrel, J. B. (1989) Genomics 5, 445-453). The positions of introns 1, 2, and 5 of the two genes were the same. The similarities in intron/ exon organizations and primary structures (30-40% identical residues) suggest that the beta-subunit genes for H+/K+- and Na+/K+-ATPases were derived from a common ancestor. The upstream region of the rat H+/K+-ATPase beta-subunit gene contains direct repeat sequences and palindromes, potential binding sites for RNA polymerase II and E4TF1, and CACCC box sequences. Gel retardation assay demonstrated that the stomach, but not other tissues (liver, brain, kidney, spleen, and lung), has a nuclear protein(s) capable of binding to the regions upstream of the potential RNA polymerase II binding sites (TATA box). The nuclear protein(s) are suggested to recognize three tandem GATAGC sequences and may be important for controlled transcription of the H+/K+-ATPase beta-subunit gene in gastric parietal cells..
45. KI OSHIMAN, K MOTOJIMA, S MAHMOOD, A SHIMADA, S TAMURA, M MAEDA, M FUTAI, CONTROL REGION AND GASTRIC SPECIFIC TRANSCRIPTION OF THE RAT H+,K+-ATPASE ALPHA-SUBUNIT GENE, FEBS LETTERS, 10.1016/0014-5793(91)80404-Q, 281, 1-2, 250-254, 1991.04, The rat gastric H+,K+-ATPase alpha-subunit gene was cloned and the nucleotide sequence of its 5'-upstream region was determined. Sequence comparison with the corresponding part of the human gene indicated the presence of highly conserved regions which may be important for specific transcription of the alpha-subunit in gastric parietal cells. The amino-terminal sequence (Met-Gly-Lys-Ala-Glu-) of the rat enzyme was similar to those of the pig and human enzymes. The gene organization of the rat enzyme was also similar to that of the human gene: introns 1, 2 and 9 were located in exactly the same positions as those in the human gene, and, as in the latter, exon 6 was not separated by an intron. The sequences of introns 1 and 2 were highly conserved among the rat, human and pig genes, but were entirely different from those of Na+,K+-ATPase catalytic subunit genes. Northern blot hybridization indicated that the gene was transcribed only in gastric mucosa..
46. M MAEDA, KI OSHIMAN, S TAMURA, M FUTAI, HUMAN GASTRIC (H+ + K+)-ATPASE GENE - SIMILARITY TO (NA+ + K+)-ATPASE GENES IN EXON INTRON ORGANIZATION BUT DIFFERENCE IN CONTROL REGION, JOURNAL OF BIOLOGICAL CHEMISTRY, 265, 16, 9027-9032, 1990.06.
47. M MAEDA, S TAMURA, M FUTAI, STRUCTURE AND CHEMICAL MODIFICATION OF PIG GASTRIC (H++K+)-ATPASE, BIOENERGETICS, 217-225, 1990.05.
48. S TAMURA, M TAGAYA, M MAEDA, M FUTAI, PIG GASTRIC (H+ + K+)-ATPASE - LYS-497 CONSERVED IN CATION TRANSPORTING ATPASES IS MODIFIED WITH PYRIDOXAL 5'-PHOSPHATE, JOURNAL OF BIOLOGICAL CHEMISTRY, 264, 15, 8580-8584, 1989.05.
49. F TOKUNAGA, K NAGASAWA, S TAMURA, T MIYATA, S IWANAGA, W KISIEL, THE FACTOR-V-ACTIVATING ENZYME (RVV-V) FROM RUSSELLS VIPER VENOM - IDENTIFICATION OF ISOPROTEINS RVV-V-ALPHA, RVV-V-BETA, AND RVV-V-GAMMA AND THEIR COMPLETE AMINO-ACID SEQUENCES, JOURNAL OF BIOLOGICAL CHEMISTRY, 263, 33, 17471-17481, 1988.11.