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Volume 129 (2001)
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- Issue 2 Pages 185-
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Volume 129, Issue 2
Displaying 1-20 of 20 articles from this issue
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Yuki Yamaguchi, Takashi Narita, Naoto Inukai, Tadashi Wada, Hiroshi Ha ...2001 Volume 129 Issue 2 Pages 185-191
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS_??_u_??_pressor of _??_y (SPT) genes were originally identified through a genetic screen for mutations in the yeast Saccharomyces cerevisiae that restore gene expression disrupted by the insertion of the transposon Ty. Classic members of the SPT gene family, SPT11, SPT12, and SPT15, encode for the histones H2A and H2B, and for _??_ATA-_??_inding _??_rotein (TBP), respectively. Over the past few years, molecular complexes and cellular functions in which other SPT gene products involve have been discovered through genetic and biochemical studies in yeast and several other organisms: Key regulators of transcription and chromatin structure, such as DSIF, SAGA, and FACT, all contain SPT gene products as essential subunits. In addition, accumulating evidence suggests that SPT gene products play more diverse roles, including roles in DNA replication, DNA recombination and developmental regulation. Here we review the current understanding of the functions and roles of the SPT genes, with special emphasis on the role of SPT5 in transcript elongation and in neuronal development in vertebrates.View full abstractDownload PDF (1353K) -
Hitoshi Fujisawa2001 Volume 129 Issue 2 Pages 193-199
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+/calmodulin-dependent protein kinases (CaM-kinases) II, IV, and I play important roles as Ca2+responsive multifunctional protein kinases in controlling a variety of cellular functions in response to an increase in intracellular Ca2+, and hence regulation of their activities is very important. CaM-kinase II is activated through autophosphorylation of threonine-286 (in the case of α isoform), and CaM-kinases IV and I are activated through phosphorylation of threonine-196 and 177, respectively, by CaM-kinase kinase. After activation, CaM-kinases II and IV lose their Ca2+/calmodulin-dependent activity upon autophosphorylation of threonine-305 and serine-332, respectively, in the absence of Ca2+, becoming Ca2+/cahnodulin-independent forms. The activated CaM-kinases II, IV, and I are deactivated upon dephosphorylation of phosphothreonine-286, 196, and 177, respectively, by CaM-kinase phosphatase or other multifunctional protein phosphatases and restored to the original ground states. Thus, the activities of the three multifunctional CaM-kinases are regulated by phosphorylation and dephosphorylation.View full abstractDownload PDF (2782K) -
Takuo Yasunaga, Takeyuki Wakabayashi2001 Volume 129 Issue 2 Pages 201-204
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCys374 is an important landmark of actin, because its sulfhydryl group is reactive and can be labeled with various reagents. The atomic coordinates of actin Cys374 have been determined by X-ray crystallography of co-crystals of actin with either profilin or gelsolin. However, the positions of Cys374 in the crystals determined were not consistent with the data obtained through fluorescence resonance energy transfer. Here, we examined its position by means of probabilistic distance geometry using published fluorescence resonance transfer data and found that Cys374 of actin was relocated when Cys374 was labeled with fluorescent dyes that caused steric hindrance. The atomic coordinates of Cys374 after heavy atom labeling have been found to be different from those for the native crystal. This is consistent with our results. Therefore, the position of Cys374 is sensitive to chemical modification that introduces a bulky reagent. This also suggests that the conformation of the C-terminal region of actin could also be sensitive to the environment.View full abstractDownload PDF (942K) -
Masato Otagiri, Genji Kurisu, Sadaharu Ui, Yusuke Takusagawa, Moriya O ...2001 Volume 129 Issue 2 Pages 205-208
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of a ternary complex of meso-2, 3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 Å resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2, 3-butanediol dehydrogenase. Two residues around the active site, G1n140 and G1y183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.View full abstractDownload PDF (1587K) -
Michihiro Nakamura, Hideki Watanabe, Yoshiyuki Nishimiya, Kouhei Tsumo ...2001 Volume 129 Issue 2 Pages 209-212
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VII genes of these phages were cloned and expressed as VII-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies.View full abstractDownload PDF (973K) -
Shoji Odani, Junko Nakamura, Tetsuro Sato, Hiroshi Fujii2001 Volume 129 Issue 2 Pages 213-219
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunoblot analysis with polyclonal antibodies raised against a recombinant rat cutaneous fatty acid-binding protein revealed a 30-kDa protein other than the 15-kDa fatty acid-binding protein in rat skin cytosol. This protein was present in a number of rat organs and in mouse 3T3 L1 cells. The amino acid sequences of the enzymatic peptides of the 30-kDa protein extracted from SDS-PAGE gels suggested that it was a mixture of the subunits of the eukaryotic signaling molecule, 14-3-3 protein. Glutathione S-transferase fusion proteins of 14-3-3 protein subunits were examined for cross-reaction by Western blotting, and the ε-subunit alone was found to be immunoreactive, so far as tested. It is likely that the 30-kDa protein detected in the rat tissues by the antibodies is the 14-3-3 protein ε-subunit. Although there is no apparent sequence similarity between the fatty acid-binding protein and the 14-3-3 protein subunit, they appear to share a common structural element recognized by the antibodies. Since 14-3-3 proteins and fatty acid-binding proteins are known to interact with a wide variety of cellular proteins, the presence of a common local structure might mutually modulate such interactions.View full abstractDownload PDF (2217K) -
Naoki Tani, Genji Kamada, Koji Ochiai, Minoru Isobe, Sathorn Suwan, Hi ...2001 Volume 129 Issue 2 Pages 221-227
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ATPase called EA4 seems to measure time as a diapause-duration timer in the seasonal cycle of the silkworm, Bombyx mori. A peptide named PIN seems to regulate the time measurement of EA4. We characterize the EA4 as the first step to analyse its interaction with PIN. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry shows EA4 forms an equimolar complex with PIN. The binding affinity of EA4 for PIN is about 460nM, as measured by surface plasmon resonance. Western blot analysis of EA4 with a variety of biotinylated lectins suggests that EA4 is a glycoprotein containing N-linked oligosaccharide. On enzymatic cleavage of the glycosyl chain, the carbohydrate is revealed to be essential for the regulation of EA4-time measurement through the interaction with PIN. PIN holds the timer by binding to EA4, and the dissociation of the complex could constitute the cue for the time measurement.View full abstractDownload PDF (1853K) -
Natsuko Goto, Shinsaku Muto, Isamu Sugimoto, Kaori Ikawa-Kitayama, Kaz ...2001 Volume 129 Issue 2 Pages 229-236
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to get some clue as to the function of Mr 25, 000 protein, a protein Ser/Thr kinase substrate detected in Xenopus laevis oocytes [Hashimoto, E. et al. (1995) J. Biochem. 118, 453-460], the binding protein was surveyed using the 32P-labeled protein by casein kinase II as a screening probe. When the cytosolic proteins from oocytes were transferred to a polyvinylidene fluoride membrane and incubated with the labeled protein, only one protein with Mr 43, 000 was visualized on autoradiography. This protein was purified to a nearly homogeneous state through several column chromatography steps. The amino acid sequence of the amino-terminal region of this protein identified it as a kind of serine protease inhibitor (serpin) [Holland, L. J. et al. (1992) J. Biol. Chem. 267, 7053-7059]. However, the Mr 25, 000 protein did not have any effect on the inhibitory action of this serpin on α-chymotrypsin. In addition, several binding proteins were also detected in the particulate fraction of oocytes, although the exact identity of these proteins is not clear at this time. These results suggest that the Mr 25, 000 protein may play some role (s) by interacting with these binding proteins in Xenopus oocytes.View full abstractDownload PDF (3926K) -
Masakazu Kikuchi, Chikako Takeda, Yoshiyuki Tsujimoto, Shinichi Asada, ...2001 Volume 129 Issue 2 Pages 237-242
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated from a human synthetic phage display library a clone, 2A3, which discriminates native lysozyme from denatured forms. Binding of single-chain Fv fragments (scFvs) of the clone to native hen egg white lysozyme was competitively inhibited by native hen egg white (hew) and human (h) lysozymes. Dot blotting analysis indicated that scFv of the clone did not react with denatured lysozymes. The Kd values for scFv of 2A3 binding to native hew- and h-lysozymes were 3.78×10-9 and 9.31×10-9 M, respectively, indicating that 2A3 binds more strongly to native hew-lysozyme than to native h-lysozyme. The deduced amino acid sequence of the VH chain-CDR3 region of 2A3 was RRYALDY, of which the Arg residues at positions 1 and 2 of the CDR3 region were observed to be extremely rare in other antibodies by homology analysis. Based on these observations, site-directed mutagenesis of the RRYALDY-coding region was carried out. The results, combined with biomolecular analyses, demonstrated that Arg residues at positions 1 and 2 of this region were important for native lysozyme-binding.View full abstractDownload PDF (1191K) -
Jau-Song Yu, Sheng-Hung Chang, Wen-Hsiung Chan, Hua-Chien Chen2001 Volume 129 Issue 2 Pages 243-251
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the crosslinked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP•Mg2+, and detecting peptide phosphorylation using the phosphoepitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC50 value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.View full abstractDownload PDF (2118K) -
Kenichiro Ishii, Shigeyuki Usui, Hajime Yamamoto, Yoshiki Sugimura, Ma ...2001 Volume 129 Issue 2 Pages 253-258
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGood molecular markers for investigating the biochemical differences between renal cancer and surrounding tissues have not yet been developed. Sixteen kidney samples (clear cell RCC) were investigated to determine the differences in the protein components between renal cancer and surrounding tissues, using HPLC analysis. The metallothionein (MT) and zinc levels were consistently lower in renal cancer tissues compared with in surrounding tissues. The mean concentration of MT in normal tissues surrounding renal tumors was about 15 times higher than that in cancer tissues. An immunohistochemical study confirmed that the expression of MT in renal cancer tissues was lower than that in adjacent normal tissues. The activities of aminopeptidases (APs) were significantly decreased in renal cancer tissues compared with in adjacent normal tissues. An immunohistochemical study and Western blot analysis confirmed that the expression of AP-N in renal cancer tissues was also lower than in adjacent normal tis-sues. These results suggest that the immunohistochemical detection of MT and AP-N could provide useful information as a pathological diagnostic tool for classifying renal cancer and surrounding tissues.View full abstractDownload PDF (1802K) -
Kazutomi Kusano, Masao Sakaguehi, Norio Kagawa, Michael R. Waterman, T ...2001 Volume 129 Issue 2 Pages 259-269
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino-terminal region of microsomal P450s contains three distinct sequence motifs, the signal-anchor sequence (SA), the basic sequence (BS), and the proline-rich sequence (PR). Studies with two P450s of the CYP2C subfamily, P4502C11 (CYP2C11) and P4502C2 (CYP2C2), have indicated that upon expression in eukaryotic cells (yeast, COS cells, and insect cells), specific proline residues in PR are important for proper folding. In the present study, we have established that the PR region in a very different CYP gene family, P450c17 (CYP17), is also important for efficient folding. These studies have been carried out using expression in Escherichia coli. Using P4502C11, we have established that the folding requirements for P450s in bacteria are very similar to those in eukaryotic cells. Interestingly, when the PR from P450c17 is swapped for that of P4502C11 and visa versa, complete misfolding is observed. However, both the BS and SA can be swapped between these P450s without affecting folding. After proper folding of P450c17, removal of the PR by factor Xa protease has no effect on the maintenance of the P450 structure. Inspection of the sequences of many different CYP gene families indicates that the PR sequence is conserved within a gene family but varies considerably between families. We conclude that PR is important for directing the folding pathway leading to the functional P450, but not for maintaining the functional form.View full abstractDownload PDF (3638K) -
Kazutomi Kusano, Norio Kagawa, Masao Sakaguchi, Tsuneo Omura, Michael ...2001 Volume 129 Issue 2 Pages 271-277
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAll microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M. R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.View full abstractDownload PDF (2642K) -
Hisazumi Araki, Yao-Hua Li, Yoshio Yamamoto, Masakazu Haneda, Katsuji ...2001 Volume 129 Issue 2 Pages 279-288
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1, 720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of eDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP 11 vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP H vs. mouse QPP) and 79.4% (rat DPP 11 vs. human QPP). The predicted amino acid sequences of rat DPP H and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP H is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney _??_testis≥heart>brain≥lung>spleen>skeletal muscle≥liver. In parallel with Northern blot analysis, the DPP H antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.View full abstractDownload PDF (3667K) -
Yoshiaki Kin, Yoshio Misumi, Yukio Ikehara2001 Volume 129 Issue 2 Pages 289-295
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDipeptidyl peptidase IV-related protein (DPPX) was found to be preferentially expressed in the brain tissue. We isolated two rat cDNA clones encoding DPPX-S and DPPX-L from a brain cDNA library, of which DPPX-L had a longer sequence at the NH2 terminus. The biosynthesis of DPPXs was examined in both in vitro and in vivo systems. In the cell-free translation system, DPPX-S and DPPX-L were synthesized as 93-kDa and 97-kDa forms, respectively, which are in good agreement with the molecular masses estimated from their primary structure. In COS-1 cells transfected with the cDNAs, DPPX-S and DPPX-L were initially synthesized as 113-kDa and 117-kDa forms, respectively, with high-mannose type oligosaccharides, which were then converted to 115-kDa and 120-kDa forms, mostly with the complex-type sugar chains. Immunofluoreseence-microscopic observations confirmed that both DPPXs were expressed on the cell surface. DPPXs were found to have no enzyme activity of DPPIV, even when they were mutated to have the consensus active-site sequence Gly-X-Ser-X-Gly for serine proteases. Immunoblot analysis of samples prepared from various rat tissues demonstrated that DPPX-S, but not DPPX-L, was detectable only in the brain tissue. These results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present.View full abstractDownload PDF (2463K) -
Seiji Tateyama, Hiroshi Sagami2001 Volume 129 Issue 2 Pages 297-302
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C55 polyprenal (BP55), and biotinylated C80-polyprenol (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.View full abstractDownload PDF (1989K) -
Keizo Yamamoto, Genji Kurisu, Masami Kusunoki, Shiro Tabata, Itaru Ura ...2001 Volume 129 Issue 2 Pages 303-312
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of glucose dehydrogenase (GIcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 Å resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP_??_-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-interface. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.View full abstractDownload PDF (3321K) -
Norihiko Furuya, Takumi Kanazawa, Shinobu Fujimura, Takashi Ueno, Eiki ...2001 Volume 129 Issue 2 Pages 313-320
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cytosolic enzyme, betaine homocysteine methyltransferase (BHMT), and its partial fragments were discovered as autolysosomal membrane proteins from rat liver in the presence of leupeptin [Ueno et al. (1999) J. Biol. Chem. 274, 15222-15229]. The present study was undertaken to further characterize the transport and processing of BHMT from cytosol to autolysosome and to test if the fragment can be used as an in vitro probe for the maturation step of macroautophagy. Upon subcellular fractionation, BHMT (p44) was found in all fractions, while its 32-kDa fragment (p32) was found only in the mitochondrial-lysosomal (ML) fraction. Incubation of isolated hepatocytes with leupeptin induced time-dependent accumulation of p32 in the ML fraction from 30 to 90min after the start of incubation. However, chloroquine completely inhibited the appearance of p32, indicating that the processing from p44 to p32 is lysosomal. Incubation with Bafilomycin A1, a vacuolar H+-ATPase inhibitor, together with leupeptin, led to linear accumulation of p44, but not of p32. The p44 accumulation rate was calculated to be 4.9%/h, which was comparable to autophagic sequestration rate. The distribution of p44 within the ML fraction turned out to be dual, i.e., the membrane-surface attached and luminall sedimentable forms. Amino acids and 3-methyladenine, both of which specifically suppress macroautophagy, inhibited the accumulation of p32 as well as of p44. Finally, energy-dependent appearance of p32 was demonstrated during incubation of postnucler supernatant fractions, making it possible to establish an in vitro assay system. All the results strongly support the idea that BHMT is taken up and degraded to p32 through the macroautophagic pathway, and that p32 could be a novel probe for the maturation of macroautophagy.View full abstractDownload PDF (3139K) -
Masayo Sakaki, Hisashi Koike, Nobuhiro Takahashi, Noboru Sasagawa, Shi ...2001 Volume 129 Issue 2 Pages 321-327
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEmerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD). Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C. Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically. We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A. Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD). These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C. The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C. This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin. These data also suggest that lamin A is important for nuclear membrane integrity.View full abstractDownload PDF (1682K) -
Atsushi Nakamura, Daisuke Kojima, Toshiyuki Okano, Hiroo Imai, Akihisa ...2001 Volume 129 Issue 2 Pages 329-334
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta H stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.View full abstractDownload PDF (1132K)
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