A novel function of the human chaperonin CCT epsilon subunit in yeast.

A malfunction in the yeast HAC1 causes the unfolding-protein response in the endoplasmic reticulum, resulting in stress-sensitive and inositol auxotrophic phenotypes. Chaperonin-containing TCP1 (CCT) is necessary for the folding of actin and tubulin in the cytosol. The introduction of the truncated human CCT epsilon subunit into yeast cells of which hac1 was disrupted clearly suppressed not only its inositol auxotrophic phenotype but also its stress-sensitive phenotype.

Functional analyses of immediate early gene ETR101 expressed in yeast.

ETR101, a human homolog of rat pip92, is a cellular immediate early gene induced by extracellular stimuli such as serum growth factors. ETR101 encodes a short-lived, proline-rich protein (ETR101) exhibiting no significant sequence similarity to any other known protein, and little is known about its function. We investigated the functioning of ETR101 as a transcriptional activator for the gene ISYNA1, which encodes human inositol 1-phosphate synthase.

Erythropoietin-producing hepatocyte B6 variant-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2+ glioma patients.

We recently cloned a variant form of erythropoietin-producing hepatocyte (Eph)B6, a member of the Eph receptor tyrosine kinase family. In the present study, we examined the expression of the EphB6 variant (EphB6v) in a panel of brain tumor cell lines and glioblastoma tissues and we found that EphB6v was preferentially expressed in malignant brain tumors, such as glioblastomas and anaplastic astrocytomas. The EphB6v has a unique 54 amino acid sequence at the C-terminal that is not found in normal EphB6.

Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae.

Pseudomonas putida benF, benK, benE1, and benE2 genes encode proteins belonging to benzoate transporter super family, but those functions have not yet been elucidated. In this study we analyzed the functions of these gene products using the yeast Saccharomyces cerevisiae. P.

Identification of EphB6 variant-derived epitope peptides recognized by cytotoxic T-lymphocytes from HLA-A24+ malignant glioma patients.

We found previously that EphB6, a member of the erythropoietin-producing hepatocyte (Eph) receptor tyrosine kinase family, was preferentially expressed in malignant gliomas. In the present study, RT-PCR revealed a putative secretory variant form of human EphB6 that was expressed in the majority of glioma cell lines, though not in normal tissues. The variant has a unique 54 amino acid sequence that is not found in the normal EphB6.

Methionine aminopeptidase II: A molecular chaperone for sarcoplasmic reticulum calcium ATPase.

The monoclonal antibody to the beta-subunit of H(+)/K(+)-ATPase (mAbHKbeta) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca(2+)-ATPase. We partially purified a mAbHKbeta-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2).

Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant.

Ternary complex formation of Ino2p-Ino4p transcription factors and Apl2p adaptin beta subunit in yeast.

Yeast Ino2p-Ino4p heterodimeric complex is well known as a transcriptional activator for the genes regulated by inositol and choline, such as the INO1 gene. Apl2p is a large subunit of the yeast adaptin complex, an adaptor complex required for the clathrin coat to bind to the membrane. We found that Ino2p, Ino4p, and Apl2p form a ternary complex.

The promoter of the yeast OPI1 regulatory gene.

In Saccharomyces cerevisiae, the expression of several genes encoding enzymes involved in lipid metabolism is regulated by inositol and choline. The transcriptional heterodimeric complex composed of the gene products of INO2 and INO4 binds to a conserved cis-acting upstream activating sequence designated as the inositol-choline responsive element (ICRE), and activates the expression of these genes. In the presence of inositol and choline, the expression of these genes is downregulated and a functional OPI1 gene product is necessary for this repression.

Mutational analysis and localization of the inositol transporters of Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses two inositol transport proteins, Itr1p and Itr2p, encoded by the ITR1 and ITR2 genes, respectively. Itr1p and Itr2p are high and low affinity transporters, respectively. Eight out of nine cysteine residues in Itr1p, which are common in two transporters, were converted to serine residues by site directed mutagenesis.

Effects of N-glycosylation and inositol on the ER stress response in yeast Saccharomyces cerevisiae.

IRE1 and HAC1 are essential for the unfolded protein response in the endoplasmic reticulum (ER). IRE1- and HAC1-disruptants require high concentrations of inositol for its normal growth. The ALG6, ALG8, and ALG10 genes encode the glucosyltransferases necessary for the completion of the synthesis of the lipid-linked oligosaccharide used for the asparagine-linked glycosylation of proteins in that order.

A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer.

We have identified an enhancer responsible for induction by 3-methylcholanthrene in the upstream region of the CYP1A2 gene. The enhancer does not contain the invariant core sequence of XREs that are binding sites for the Ah receptor (AhR) and Arnt heterodimer. The enhancer did not show any inducible expression in Hepa-1-derived cell lines, C4 and C12, deficient of Arnt and AhR, respectively.

Characterization of the products of the genes SNO1 and SNZ1 involved in pyridoxine synthesis in Saccharomyces cerevisiae.

Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity.

Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase.

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no.