Involvement of thioredoxin peroxidase type II (Ahp1p) of Saccharomyces cerevisiae in Mn2+ homeostasis

To identify new proteins involved in Mn2+ homeostasis, we isolated Mn(2+)-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (delta cmp1 delta cmp2). The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca(2+)-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a delta cmp1 delta cmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane.     

nhaG Na(+)/H(+) antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of strain 168, and properties of the antiporter.

We cloned a gene which enabled Escherichia coli mutant host cells lacking all of the major Na(+)/H(+) antiporters to grow in the presence of 0.2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372. An Na(+)/H(+) antiport activity was observed with membrane vesicles prepared from E. coli cells possessing the cloned gene, but not with vesicles from the host cells. Lithium ion was also a substrate for the antiporter. We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence. The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58.1 kDa. None of the bacterial Na(+)/H(+) antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynNhaP (NhaS1) of Synechocystis sp., showed significant sequence similarity with the NhaG. However, the NhaP, the SynNhaP, animal NHEs (Na(+)/H(+) exchangers), and some hypothetical Na(+)/H(+) antiporters of several organisms showed significant sequence similarities with the NhaG. Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B. subtilis strain 168. We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B. subtilis ATCC9372 but not with that from strain 168. The missing DNA region (1,774 base pairs) is sandwiched by two identical sequences, TTTTCTT.     

ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils.

We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394]. In the present study, we obtained evidence that chicken non-muscle beta/gamma-actin, skeletal muscle alpha-actin and smooth-muscle gamma-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP-ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP-ribose/mol of beta/gamma-actin, alpha-actin and gamma-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 microM with beta/gamma-actin, 35 microM with alpha-actin and 20 microM with gamma-actin. The Km values for the actin isoforms were 15 microM for beta/gamma-actin, 2.5 microM for alpha-actin and 10 microM for gamma-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluorescence intensity with N-(1-pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of beta/gamma-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.     

Nucleotide sequence and copy control function of the extension of the incI region (incI-b) of Rts 1

An Rts1 derivative, pTW20, contains three incompatibility (inc) regions, incI-a (incI in previous studies), incII, and newly determined incI-b loci. By restriction analysis, we have located the incI-b adjacent to the incI-a region on the pTW20 map. Nucleotide sequence analysis of the minimal incI-b region revealed the presence of four repeated sequences, each consisting of 18 bp, which is similar to the incI-a and incII repeats existing on mini-Rts1. All four repeating units were required for expression of a strong incompatibility. In addition, RepA protein, essential for the replication of Rts1, bound specifically to the repeated sequences, suggesting that the repeats would titrate out RepA protein as do incI-a and IncII. Insertion of the incI-b to a mini-Rts1 plasmid in a natural arrangement decreases the copy number of mini-Rts1 to the same level as that of mini-F. The incI-a and incI-b might be a single constituent in incompatibility and copy number control of Rts1.     

A respiratory-driven and an artificially driven ATP synthesis in mutants of Vibrio parahaemolyticus lacking H+-translocating ATPase

Mutants of Vibrio parahaemolyticus lacking the H+-translocating ATPase were isolated to evaluate both the role of this enzyme and the possibility of the involvement of other cation-translocating ATPase in the energy transduction in this organism. Dicyclohexylcarbodiimide-sensitive ATPase activity which represents the H+-translocating ATPase was not detected either in the membrane vesicles or in the cytosol of the mutants. Three major subunits, alpha, beta and gamma, of the H+-translocating ATPase were missing in the membranes of the mutants. Although ATP was synthesized in wild type cells when an artificial H+ gradient was imposed, little ATP was synthesized in the mutants. However, we observed a large ATP synthesis driven by the respiration not only in the wild type but also in the mutants. The respiratory-driven ATP synthesis in wild type was inhibited by an H+ conductor, carbonylcyanide m-chlorophenylhydrazone, by about 50%. On the other hand, the ATP synthesis in the mutants was not affected by the H+ conductor. Since this organism possesses a respiratory Na+ pump, Na+-coupled ATP synthesis might take place. In fact, we observed some ATP synthesis driven by an artificially imposed Na+ gradient both in the wild type and the mutant.     

Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides.

The role of Ca2+ for the signaling of rhodotorucine A, a mating pheromone of Rhodosporidium toruloides, was investigated. The efficiency with which the target cells responded to the mating pheromone was dependent on the Ca2+ concentration in the medium. The pheromone induced a very rapid and transient increase of Ca2+ uptake in the recipient cell. We concluded that the transient increase in the intracellular Ca2+ concentration could play an essential role in the control of differentiation by the pheromone.     

Characterization of a novel L-serine transport system in Escherichia coli.

A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture. This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system. Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine. This system was induced by L-leucine, but not by L-serine. The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein. Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor. Uptake of H+ was induced by L-serine influx. These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.     

Reconstitution of the melibiose carrier of Escherichia coli

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.     

Biological half time of deposited nickel oxide aerosol in rat lung by inhalation

Wistar male rats were exposed to nickel oxide (NiO) aerosols (mass median aerodynamic diameter, 1.2 and 4.0 μm). The average exposure concentration was controlled from a low level of 0.6 mg/m³ to a high level of 70 mg/m³ and total exposure time was 140 h. Some rats were sacrificed just after the exposure, whereas others were exposed for 1 mo and kept for 12 and 20 mo clearance periods before sacrifice. There were no differences in body weight gain between NiO exposure groups and controls. Nickel concentrations in lungs of exposure groups were much higher than those of controls and decreased with the passing of the clearance time. No apparent deposition of nickel was observed in the liver, kidney, spleen, and blood immediately after the exposure, but in the case of the high exposure groups, the nickel concentration in the liver, spleen, and blood slightly increased with the increasing time of clearance. The biological half time of NiO deposited in the lungs was estimated by the assumption that the amount of the clearance is proportional to the amount of the NiO deposited. This resulted in a biological half time of 11.5 and 21 mo for 1.2 and 4.0 μm, respectively.