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Y Anraku

Publications and source records attributed to Y Anraku.

At least 37 records · Page 2Linked to original sources
PubMed1997

Protein splicing: its chemistry and biology.

Protein splicing is a chemical reaction in which a spliced intervening polypeptide is excised from a precursor protein and the flanking N- and C-terminal regions are ligated with the peptide bond to produce two mature proteins. This unique autocatalytic reaction was first discovered in the yeast VMA1 protein, a 120kDa spliced polypeptide encoded by the VMA1 gene of Saccharomyces cerevisiae. The VMA1 protein catalyses a self protein splicing post-translationally to yield the 70 kDa catalytic subunit of the vacuolar H+-ATPase and the 50 kDa DNA endonuclease. Accumulating evidence has indicated that splicing precursors distribute widely in many organisms covering eukarya, bacteria and archaea. This article argues and summarizes current chemical and biological views on protein splicing.

Amino Acid Sequence↗
PubMed1997

Probing novel elements for protein splicing in the yeast Vma1 protozyme: a study of replacement mutagenesis and intragenic suppression.

Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions. This unique autocatalytic reaction was first discovered in the yeast Vma1p protozyme where the 50-kD site-specific endonuclease (VDE) is excised from the 120-kD precursor containing the N- and G-terminal regions of the catalytic subunit of the vacuolar H(+)-ATPase. In this work, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the Vma1 protozyme and found that these site-specific random mutations interfere with normal protein splicing to different extents. Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal splicing junction genetically interacts with three hydrophobic residues preceding the N-terminal splicing junction. This is the first evidence showing that the N-terminal portion of the V-ATPase subunit is involved in protein splicing. Our genetic evidence is consistent with a structural model that correctly aligns two parallel beta-strands ascribed to the triplets. This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions.

Amino Acid Sequence↗
PubMed1997

Assignment and functional roles of the cyoABCDE gene products required for the Escherichia coli bo-type quinol oxidase.

Cytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump. In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo. We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33,500, 75,000, 20,500, 12,000, and 28,000, respectively. The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex. To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor. Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes. Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products. However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I.

Alkyl and Aryl Transferases↗
PubMed1997

Substitutions of charged amino acid residues conserved in subunit I perturb the redox metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu(B), and serves as a reaction center of the oxidase complex. This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases. Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes. In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes. The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum. The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center. The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the Cu(B) center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive. Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases.

Amino Acid Sequence↗
PubMed1997

A novel chloride-binding site modulates the heme-copper binuclear center of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping. Previous studies [Y. Orii, T. Mogi, M. Sato-Watanabe, T. Hirano, and Y. Anraku (1995) Biochemistry 34, 1127-1132] suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer. To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties. UQO-412, which exhibits the Soret peak at 412 nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift. UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center. Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a K(d) value of 3.5 mM. Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable. The present studies demonstrated that the E. coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center. Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion.

Binding Sites↗
PubMed1997

Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase.

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.

Bacillus↗
PubMed1996

Probing substrate binding site of the Escherichia coli quinol oxidases using synthetic ubiquinol analogues.

Substrate binding sites of the Escherichia coli bo- and bd-type quinol oxidases were probed with systematically synthesized ubiquinol analogues. The apparent Km values of ubiquinol-2 derivatives to the bo-type enzyme were much lower than that of the corresponding 6-n-decyl derivatives. The isoprenoid structure is less hydrophobic than the saturated n-alkyl group with the same carbon number; therefore, the native isoprenoid side chain appears to play a specific role in quinol binding besides simply increasing hydrophobicity of the molecule. The Vmax values of 2-methoxy-3-ethoxy analogues were greater than that of 2-ethoxy-3-methoxy analogues irrespective of the side chain structure. This result indicates not only that a methoxy group in the 2-position is recognized more strictly than the 3-position by the binding site but also that the side chain structure does not affect binding of the quinol ring moiety. Systematic analysis of the electron-donating activities of the analogues with different substituents in the 5-position revealed that the 5-methyl group is important for the activity. In the parallel studies with the bd-type enzyme, we obtained similar observations except that almost all quinol analogues, but not ubiquinol-1, elicited a remarkable substrate inhibition at higher concentrations. These results indicate that the two structurally unrelated terminal oxidases share common structural properties for the quinol-oxidation site.

Electrons↗
PubMed1996

Mutational analysis of the beta-subunit of yeast geranylgeranyl transferase I.

The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

Alkyl and Aryl Transferases↗
PubMed1996

Probing a role of subunit IV of the Escherichia coli bo-type ubiquinol oxidase by deletion and cross-linking analyses.

Subunit IV of the Escherichia coli bo-type ubiquinol oxidase is a 12-kDa membrane protein encoded by the cyoD gene and is conserved in the bacterial heme-copper terminal oxidases. To probe the functional role of subunit IV, we carried out deletion analysis and chemical cross-linking experiments with a homobifunctional and cleavable reagent. Spectroscopic properties of the mutant oxidases suggest that the C-terminal two-third (Val45 to His109) containing helices II and III is essential for the functional expression of the oxidase complex and for the CuB binding to the heme-copper binuclear center in subunit I. Cross-linking studies indicate that subunit IV is in close vicinity to subunit III. Based on these observations, we propose that subunit IV is present in a cleft formed by subunits I and III and assists the CuB binding to subunit I during biosynthesis or assembly of the oxidase complex.

Amino Acid Sequence↗
PubMed1996

Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme.

VMA1 translational product undergoes excision of a 50-kDa intervening segment (VDE: VMA1-derived endonuclease) and religation of the flanking regions to create a 69-kDa catalytic subunit of vacuolar membrane H+-ATPase. VDEs conjugated with polypeptides at both N- and C-terminal ends were expressed in Escherichia coli and examined for their ability to catalyze self-splicing. Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies. We demonstrate in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction efficiently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed.

Amino Acid Sequence↗
PubMed1996

Activation of yeast protein kinase C by Rho1 GTPase.

We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.

Cell Cycle Proteins↗
PubMed1996

EPR study of NO complex of bd-type ubiquinol oxidase from Escherichia coli.

The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex.

Binding Sites↗
PubMed1996

Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase.

1,3-beta-D-glucan synthase [also known as beta(1-->3) glucan synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1p. Glucan synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with beta(1-->3)glucan synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of beta(1-->3)glucan synthase.

GTP Phosphohydrolases↗
PubMed1996

Infrared and EPR studies on cyanide binding to the heme-copper binuclear center of cytochrome bo-type ubiquinol oxidase from Escherichia coli. Release of a CuB-cyano complex in the partially reduced state.

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding.

Anaerobiosis↗
PubMed1996

Yeast Cls2p/Csg2p localized on the endoplasmic reticulum membrane regulates a non-exchangeable intracellular Ca2+ pool cooperatively with calcineurin.

Saccharromyces cerevisiae CLS2 gene product (Cls2p) that is localized on the endoplasmic reticulum is important for the regulation of intracellular Ca2+ in a compartment distinct from the vacuole. Using a vma3 mutation that impairs the Ca2+ sequestering activity into the vacuole, we have shown that the cls2 mutation results in 3.4-fold increase in the Ca2+ pool that is not exchangeable with extracellular Ca2+. Accumulation of Ca2+ within the cls2 cells is synergistically elevated by the addition of immunosuppressant, FK506. Moreover, in the vma3 background, toxicity caused by the cls2 mutation is greatly enhanced by FK506. Given that FK506 inhibits the calcineurin activity, Cls2p likely functions in releasing Ca2+ flux from the endoplasmic reticulum, somehow cooperating with calcineurin.

Calcineurin↗
PubMed1996

Signaling toward yeast 1,3-beta-glucan synthesis.

1,3-beta-glucan synthase catalyzes the synthesis of a 1,3-beta-linked glucan polymer which produces the main rigidity of the yeast cell wall. Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall. This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase. In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme. Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth. In this article, we will focus on biochemistry of 1,3-beta-glucan synthase and signaling through rho type GTPase.

Fungal Proteins↗
PubMed1995

Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3.

We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties. Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H. salinarium cytochrome aa3 showed the spectral properties similar to those of the native E. coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I. However, CuB was absent in the chimera. Helix VII of subunit I of the H. salinarium cytochrome aa3 is 70% similar to the counterpart of the E. coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands. These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.

Amino Acid Sequence↗