Molecular characterization of Vig4/Vrg4 GDP-mannose transporter of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae Vig4/Vrg4 protein is a Golgi membrane protein which has multiple transmembrane domains and is essential for transport of GDP-mannose across the Golgi membrane. By immunoprecipitation of detergent-solubilized tagged protein, we found that this protein exists as oligomer. Two mutants vig4-1 and vig4-2 had amino acid substitutions in the C-terminal region, Ala286Val and Ser278Cys, respectively. In accord with these mutations, trimming of the C-terminal hydrophobic part close to the region impaired the function and traffic of the proteins from the endoplasmic reticulum to the Golgi compartments.     

Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.     

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.     

Distribution of Prx-linked hydroperoxide reductase activity among microorganisms

Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.     

The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of β-glucanase

To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method. Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2. Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large. A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced. This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C. Complementation tests with pkc1Δ showed that hpo2 is allelic to pkc1. To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed. The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell. Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level. This 33 kDa protein was identified as a β-glucanase, encoded by BGL2. Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect. This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the β-glucanase is partially responsible for the growth defect. Since the bgl2 disruption did not rescue the hypo-osmolarty-sensitive phenotype of the hpo2 mutant, PKC1 must negatively regulate other enzymes involved in the biosynthesis and metabolism of the cell wall.     

Induction of differentiation in cultured mouse neuroblastoma cells by N-methyl-N'-nitro-N-nitrosoguanidine.

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent mutagen and carcinogen, induces differentiation uniquely in N-18 mouse neuroblastoma cells, when compared with that of dibutyryl cyclic adenosine monophosphate. After treatment with 10 μM MNNG for only 2 h, RNA and protein synthesis are stimulated together with neurite formation, while DNA synthesis and growth of the cells are inhibited indefinitely. Induction of neurite formation by MNNG is irreversible, being inhibited by actinomycin D or cycloheximide but recovering after withdrawal of these inhibitors.     

Properties of proteins produced after damage to deoxyribonucleic acid of Escherichia coli.

Large amounts of extra proteins, X (in the envelope fraction) and X' (in the cytoplasmic fraction) were detected by SDS-polyacrylamide gel electrophoresis when DNA of Escherichia coli was damaged. These two proteins had the same apparent molecular weight (appproximately 40,000) and were produced under identical conditions, including requirement for the recA" and lexA+ genotype. Sucrose density gradient centrifugation revealed that protein X' consisted of relatively large and heterogeneous aggregates in the cytoplasmic fraction; the distribution of protein X in the envelope was not determined. As proteins X and X' were shown to be equivalent, it is suggested that they are identical. Co-precipitation of the aggregates of protein X' with the envelope led to the appearance of protein X in the envelope fraction.