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. We constructed a yeast strain in which the chromosomal region of the PSS1 promoter was replaced with the human ISYNA1 promoter. Using this yeast strain, we screened human cDNAs, which activated the ISYNA1 promoter, and thus expressed the PSS1 as a reporter gene. We obtained two types of cDNA, E2F1, known as a gene encoding Rb-binding protein, and ETR101. The E2F1 gene product (E2F1) is known to bind to and activate the ISYNA1 promoter. In a manner similar to E2F1, ETR101 binds to and activates the ISYNA1 promoter.

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. putida gene products expressed in yeast cells were localized to the yeast plasma membrane and were involved in taking up benzoate into the cells. According to the sensitivity of yeast cell-growth to benzoate, it is proposed that benK, benE1, and benE2 gene products function as transporters, that take up benzoate into the cells, whereas the benF gene product functions as an efflux pump of benzoate.

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). The effects of MetAP2 on SR Ca(2+)-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca(2+)-ATPase. Synthesis of functional SR Ca(2+)-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca(2+)-ATPase synthesis.

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. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.

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. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation. Ino2p and Ino4p bind to Apl2p in the same region of Apl2p, viz., at the middle part and the C-terminal part. Ino2p and Ino4p bind to Apl2p independently, but more strongly when both are present. Furthermore, a disruption of APL2 together with INO2 or INO4 rendered yeast cells sensitive to oxidative stress. INO2-APL2 double disruptants also showed growth inability in non-fermentable carbon sources, such as glycerol. These results indicate a genetic interaction between APL2, INO2 and INO4 and uncovere novel functions of the Ino2p-Ino4p-Apl2p complex in yeast.

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. Here we show that, given a combination of the hac1 defect with a disruption of ALG6, ALG8, and ALG10, no strains grow on inositol-free medium. However, the growth defect of the hac1-alg10 double disrupted was partially, but significantly, suppressed by the addition of inositol to the medium. These results indicate that inositol, according to the numbers of glucose residues in the oligosaccharide, plays an important role in the stress response and quality control of glycoproteins in the ER.

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. On the other hand, bacterially expressed AhR-Arnt heterodimer could not bind to the enhancer. Mutational analysis of the enhancer revealed that a repeated sequence separated by six nucleotides is important for expression. A factor binding specifically to the enhancer was found by using gel shift assays. Bacterially expressed AhR-Arnt heterodimer interacted with the factor. A dominant negative mutant of the AhR to XRE activated the enhancer. Collectively, these results demonstrate that a novel induction mechanism is present in which the AhR-Arnt heterodimer functions as a coactivator.

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. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol.mg(-1).min(-1) as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo-L-norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified.

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. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189-->Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

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. The promoter region of OPI1 contains one copy of ICRE, and here we analyzed the involvement of ICRE in the inositol-choline-mediated gene regulation of OPI1. Deletion analysis of the OPI1 promoter region, disruption of ICRE in it, and its activity in ino2- and ino4-disrupted strains showed that ICRE is essential for the expression of OPI1, and that the expression of OPI1 is dependent on the INO2 and INO4 gene products. Disruption of OPI1 resulted in the derepressed expression of OPI1 itself and no response to inositol-choline, showing that OPI1 is regulated in the same manner as the phospholipid biosynthetic genes. These results revealed the regulatory circuit of the expression of the positive regulatory gene INO2 and the negative regulatory gene OPI1.

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. All mutant genes suppressed the growth defect caused by itr1 disruption, indicating that cysteine residues are not essential for its function. Chimeric genes that express Itr1p and Itr2p fused to the green fluorescent protein (GFP) under the control of the ADH1 promoter were constructed. Both genes were functional. Fluorescence microscopy analysis indicated that both GFP-fused Itr1p and Itr2p are localized to the plasma membrane. A multi-copy plasmid that expresses GFP-fused Itr1p under the control of the original ITR1 promoter was constructed. Under inositol-free culture conditions, GFP-fused Itr1p appeared and was localized to the plasma membrane. When the cells were cultured in the presence of inositol, GFP-fused Itr1p gradually disappeared from the plasma membrane, the fluorescence being redistributed within the cell. Prolonged culture of the cells also caused the relocalization of transporter proteins. These results clearly indicate that the cellular relocalization of transport proteins is responsible for a reduction of inositol transport activity, which is caused by the presence of inositol in the medium or culturing of cells to the stationary phase.