Biomedical subjects

Y Anraku

Publications and source records attributed to Y Anraku.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1995

Cyanide-binding site of bd-type ubiquinol oxidase from Escherichia coli.

We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of cyanide to the air-oxidized O2-bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm-1 and concomitant disappearance of the 647 nm absorption band of the cytochrome d (Fe2+)-O2 species. Addition of cyanide to the air-oxidized CO-bound enzyme also resulted in disappearance of the 635 nm absorption band and the 1983.4 cm-1 C-O infrared band of the cytochrome d (Fe2+)-CO species. The resulting species had a derivative-shaped electron paramagnetic resonance signal at g = 3.15. Upon partial reduction with sodium dithionite, this species was converted partly to a transient heme d (Fe3+)-C = N species having an electron paramagnetic resonance signal at gz = 2.96 and a C-N infrared band at 2138 cm-1. These observations suggest that the active site of the enzyme has a heme-heme binuclear metal center distinct from that of the heme-copper terminal oxidase and that the treatment of the air-oxidized enzyme with cyanide resulted in a cyanide-bridging species with "heme d(Fe3+)-C = N-heme b595(Fe3+)" structure.

Binding Sites↗

PubMed1995

Requirement of Saccharomyces cerevisiae Ras for completion of mitosis.

In the yeast Saccharomyces cerevisiae, Ras regulates adenylate cyclase, which is essential for progression through the G1 phase of the cell cycle. However, even when the adenosine 3',5'-monophosphate (cAMP) pathway was bypassed, the double disruption of RAS1 and RAS2 resulted in defects in growth at both low and high temperatures. Furthermore, the simultaneous disruption of RAS1, RAS2, and the RAS-related gene RSR1 was lethal at any temperature. The triple-disrupted cells were arrested late in the mitotic (M) phase, which was accompanied by an accumulation of cells with divided chromosomes and sustained histone H1 kinase activity. The lethality of the triple disruption was suppressed by the multicopies of CDC5, CDC15, DBF2, SPO12, and TEM1, all of which function in the completion of the M phase. Mammalian ras also suppressed the lethality, which suggests that a similar signaling pathway exists in higher eukaryotes. These results demonstrate that S. cerevisiae Ras functions in the completion of the M phase in a manner independent of the Ras-cAMP pathway.

Adenylyl Cyclases↗

PubMed1995

Stabilization of a semiquinone radical at the high-affinity quinone-binding site (QH) of the Escherichia coli bo-type ubiquinol oxidase.

Reaction of ubiquinone in the high-affinity quinone-binding site (QH) in bo-type ubiquinol oxidase from Escherichia coli was revealed by EPR and optical studies. In the QH site, ubiquinol was shown to be oxidized to ubisemiquinone and to ubiquinone, while no semiquinone signal was detected in the oxidase isolated from mutant cells that cannot synthesize ubiquinone. The QH site highly stabilized ubisemiquinone radical with a stability constant of 1-4 at pH 8.5 and the stability became lower at the lower pH. Midpoint potential of QH2/Q couple was -2 mV at pH 8.5 and showed -60 mV/pH dependence indicative of 2H+/2e- reaction. The Em was more negative than that of low-spin heme b above pH 7.0. We conclude that the QH mediates intramolecular electron transfer from ubiquinol in the low-affinity quinol oxidation site (QL) to low-spin heme b. Unique roles of the quinone-binding sites in the bacterial ubiquinol oxidase are discussed.

Benzoquinones↗

PubMed1995

STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast.

Mutations of genes involved in the STT1/PKC1 pathway in yeast show staurosporine and temperature sensitivities (stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis elegans and mouse mandibular condyle (about 80% homologous and 60% identical). Unlike the STT1/PKC1 gene, STT3 is essential for cell growth irrespective of osmotic support. Pulse-chase experiments show that the sst3 mutants are defective in protein glycosylation. The stt3 mutants are sensitive to hygromycin B and resistant to sodium orthovanadate, whose phenotypes are common to those defective in protein glycosylation.

Alkaloids↗

PubMed1995

Purification and characterization of 3-isopropylmalate dehydrogenase from a thermoacidophilic archaebacterium Sulfolobus sp. strain 7.

3-Isopropylmalate dehydrogenase was purified (about 2000-fold) to homogeneity for the first time from an archaebacterium, Sulfolobus sp. strain 7. The enzyme showed an apparent molecular mass of about 110 kDa by gel filtration and a single 36-kDa polypeptide band on SDS-PAGE, suggesting tri- or tetrameric structure. The pI value was 6.9. The N-terminal amino acid sequence was similar to enzymes from other sources. The enzyme activity was greatly stimulated by the presence of Mn2+, Cd2+, Mg2+, or Co2+. In contrast to 3-isopropylmalate dehydrogenase from other sources, monovalent cations such as K+ and Na+ were neither essential for activity nor stability of the protein. The enzyme was extraordinarily thermostable.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

CuB promotes both binding and reduction of dioxygen at the heme-copper binuclear center in the Escherichia coli bo-type ubiquinol oxidase.

A CuB-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of CuB seems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that CuB plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.

Carbon Monoxide↗

PubMed1995

STT10, a novel class-D VPS yeast gene required for osmotic integrity related to the PKC1/STT1 protein kinase pathway.

We report the genetic and biochemical properties of a staurosporine (ST)- and temperature-sensitive mutant, stt10, of Saccharomyces cerevisiae. The stt10 mutant shows an osmoremedial phenotype in a medium with 1 M sorbitol. ST sensitivity of the stt10 mutant was suppressed by overexpression of PKC1/STT1, showing the genetic interactions of STT10 with the PKC1/STT1 pathway. The nucleotide sequence of STT10 predicts a hydrophilic protein composed of 577 amino acids that possesses 20-25% sequence similarity with yeast Slp1/Vam5p, Sec1p and Sly1p, and nematode Unc-18. The stt10 deletion mutant is viable and shows a typical class-D vacuolar protein sorting defective (vps) phenotype. Vacuoles from stt10 cells have a normal vacuolar H(+)-ATPase activity, but are defective in vacuolar acidification. Genetic studies of yeast mutants carrying delta stt10, delta bck1, stt1/pkc1 or stt4 have revealed that their functions are phenotypically related to maintenance of cellular osmotic integrity.

Alkaloids↗

PubMed1995

Cooperation of calcineurin and vacuolar H(+)-ATPase in intracellular Ca2+ homeostasis of yeast cells.

Saccharomyces cerevisiae VMA genes, encoding essential components for the expression of vacuolar membrane H(+)-ATPase activity, are involved in intracellular ionic homeostasis and vacuolar biogenesis. We report here that the immunosuppressants FK506 and cyclosporin A cause general growth inhibition of the vma3 mutant. Upon addition of the drugs, the mutant grew neither in the presence of more than 5 mM Ca2+ nor above pH 6.0. The action of the immunosuppressants is dependent on their binding proteins and ascribable to inhibition of calcineurin activity; a mutation of a calcineurin subunit (cnb1) shows synthetic lethal interaction with the vma mutation. The addition of FK506 decreases the cytosolic free concentration of Ca2+ in the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of a nonexchangeable Ca2+ pool. These results suggest that calcineurin controls calcium homeostasis by repression of Ca2+ flux into a cellular compartment(s) and that the vacuolar H(+)-ATPase is essential for cell growth cooperating with calcineurin to regulate the cytosolic free concentration of Ca2+.

Base Sequence↗

PubMed1995

The CLS2 gene encodes a protein with multiple membrane-spanning domains that is important Ca2+ tolerance in yeast.

Genetic screening of Saccharomyces cerevisiae mutants defective in Ca2+ homeostasis identified cls2, which exhibits a specific Ca(2+)-sensitive growth phenotype. We describe here the CLS2 gene and a multicopy suppressor (named BCL21, for bypass of CLS2) of the cls2 mutation. The CLS2 gene encodes a polypeptide of 410 amino acid residues, and its hydropathy profile indicates that the predicted Cls2 protein (Cls2p) contains ten putative membrane spanning regions. Immunofluorescent staining of the yeast cells expressing epitope-tagged Cls2p suggests that Cls2p is localized to endoplasmatic reticulum (ER) membrane. A cls2 disruption strain is viable, but shows a Ca(2+)-sensitive phenotype like the original cls2 mutants. BCL21 suppresses the cls2 disruption mutation, indicating that the multicopy suppression does not require the Cls2p. Suppression of cls2 was observed even after introduction of a single-copy plasmid harboring BCL21. The BCL21 gene encodes a protein of 382 amino acid residues and is identical to the SUR1 gene. sur1 was originally isolated as a suppressor of rvs161, which has reduced viability in nutrient starvation conditions. Possible mechanisms of the multicopy suppression are discussed.

Amino Acid Sequence↗

PubMed1995

Facilitated intramolecular electron transfer in the Escherichia coli bo-type ubiquinol oxidase requires chloride.

Previous flow-flash measurements using the bo-type ubiquinol oxidase of Escherichia coli have revealed that facilitated heme B-heme O intramolecular electron transfer initiated upon reaction of the fully-reduced enzyme with dioxygen proceeds with a rate constant higher than 5 x 10(4) s-1 at pH 7.4 and 20 degrees C. Depletion of chloride anions from the enzyme by HPLC performed in the present study considerably decreased the rate constant to approximately 700 s-1, but the reaction of either dioxygen or carbon monoxide at the binuclear center was not affected at all kinetically. These results strongly suggest that Cl- is essential in maintaining a subtle molecular structure around the heme B and heme O that enables facilitated intramolecular electron transfer. Furthermore, a series of absorption spectra of the enzyme collected on time scales from microseconds to milliseconds during its single turnover indicate that as heme-heme intramolecular electron transfer is retarded by depletion of Cl-, an alternative electron transfer pathway is invoked. We discuss a possible role of novel bound Cl- in electron transfer from bound quinol to the binuclear center to accomplish dioxygen reduction.

Carbon Monoxide↗

PubMed1995

Calmodulin-dependent protein kinase II and calmodulin are required for induced thermotolerance in Saccharomyces cerevisiae.

We show here that yeast mutants lacking calmodulin-dependent protein kinase II fail to fully acquire induced thermotolerance. A similar result was also obtained with mutants depending solely on either the N-terminal half or the C-terminal half of calmodulin. These findings indicate that both calmodulin-dependent protein kinase II and calmodulin are required for induced thermotolerance.

Calcium↗

PubMed1995

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated.

Amino Acid Sequence↗

PubMed1994

The MID2 gene encodes a putative integral membrane protein with a Ca(2+)-binding domain and shows mating pheromone-stimulated expression in Saccharomyces cerevisiae.

The MID2 gene whose defect (the mid2-1 mutation) results in mating-pheromone-induced death in Saccharomyces cerevisiae was cloned and its nucleotide (nt) sequence determined. The sequence showed an open reading frame (ORF) coding for a 376-amino-acid (aa) protein with an estimated M(r) of 39,104, and six potential TATA boxes and two pheromone-response elements in its 5'-upstream region. The deduced aa sequence showed that the MID2 product (Mid2p) contains a putative N-terminal signal sequence followed by a long Ser-rich region that could contain O-glycosylation sites, a potential transmembrane domain and a conserved Ca(2+)-binding domain, with the latter two located in the C-terminal half. Northern blot analysis showed that the expression of MID2 is stimulated threefold by mating pheromone. Cells that lack MID2 were able to grow normally, but died when exposed to mating pheromone, like the original mid2-1 mutant.

Amino Acid Sequence↗

PubMed1994

Molecular structure of redox metal centers of the cytochrome bo complex from Escherichia coli. Spectroscopic characterizations of the subunit I histidine mutant oxidases.

A site-directed mutagenesis study on the conserved subunit I histidines of the cytochrome bo complex in Escherichia coli identified ligands of the low spin heme B and CuB centers; however, the assignment of the proximal ligand of the high spin heme O was ambiguous (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). We have extended this work and characterized the metal centers in the purified histidine mutant oxidases by optical, EPR, and resonance Raman spectroscopies and by biochemical analysis. We found that the H284A and H333A oxidases contain two heme B molecules, which exhibit the gz = 2.99 low spin and cyanide-sensitive g perpendicular = 6 high-spin EPR signals, whereas the H419A oxidase contains only low spin heme B, which shows the gz component in a considerably higher magnetic field at g = 2.92. The CuB center was partially retained in the H284A oxidase but was almost completely lost in the H333A and H419A oxidases. Thus, we concluded that His419 is the proximal ligand of the high spin heme O and that His284 is located at the distal side of the high spin heme O. His284 plays an indispensable role in maintaining the structure of the Feo-CuB binuclear site suitable for the exogenous ligand bindings in the reduced state, since its substitution eliminated the CO binding activity. In addition, we found that His106 and His421 are in fact the axial ligands of the low spin heme B, although the H421A mutation perturbed the binuclear metal center seriously. Based upon experimental results with isotopic substitutions on iron in the oxidases, we assigned the Raman band at 208 cm-1 to the iron-histidine stretching mode (vFc(2+)-N(His)) of the wild-type ferrous cytochrome o, which was upshifted slightly by a loss of the CuB center in the H333A oxidase. A molecular structure of the metal centers and a possible mechanism of the electron transfer-coupled proton pumping in the cytochrome bo complex are proposed on the basis of our present findings.

Copper↗

PubMed1994

Structure-function studies on the ubiquinol oxidation site of the cytochrome bo complex from Escherichia coli using p-benzoquinones and substituted phenols.

To characterize the structural features of the quinol oxidation site (the QL site) of the cytochrome bo complex, a heme-copper respiratory oxidase in Escherichia coli, we carried out structure-inhibitory potency analyses using 7 p-benzoquinones and 33 substituted phenols. Their effects on its ubiquinol-1 oxidase activity were compared with those on the cytochrome bd complex in E. coli and on cytochromes o and alpha 1 in Acetobacter aceti. They showed similar structural properties of the QL site, although cytochrome o was more sensitive to 4-cyanophenols, suggesting a specific interaction of the hydrogen bond-accepting cyano group with the binding pocket. Replacing one of the methyl groups of 2,6-dimethyl-p-benzoquinone, which is the most potent competitive inhibitor, with an ethyl group markedly decreased the inhibitory activity, indicating that the QL site specifically recognizes one C = O group with two methyl groups as the ortho-substituents. In substituted phenols, ortho-chlorine substituents were the most effective in recognition, and the electron-withdrawing ability of the para-substituent determined an inhibitory potency, probably by stabilizing an anionic form. Based on these observations, we postulate that the QL site of the cytochrome bo complex asymmetrically recognizes exogenous ligands and that this property accounts for the sequential electron transfer from ubiquinols to the low-spin heme.

Benzoquinones↗

PubMed1994

Identification of a novel quinone-binding site in the cytochrome bo complex from Escherichia coli.

The cytochrome bo complex is a heme BO-type heme-copper quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as an electron transfer-linked proton pump. To study the protein-mediated electron transfer from substrates to metal centers, we carried out quantitative and qualitative analyses of a bound quinone in the purified oxidase and found that it has a novel high affinity ubiquinone-binding site distinct from the quinol oxidation site. Enzymatic and spectroscopic studies suggest that the quinone-binding site is located close to both the quinol oxidation site in subunit II and low-spin heme B in subunit I. The quinone-binding site of a bound ubiquinone-free oxidase was reconstituted with the potent quinol oxidation site inhibitor 2,6-dichloro-4-nitrophenol, which decreased the Vmax value of the ubiquinol-1 oxidase activity to one-fourth of the control activity. These results indicate that the quinone-binding site is essential for the catalytic functions of the cytochrome bo complex and mediates electron transfer from the quinol oxidation site to the low-spin heme.

Air↗