So-ichi Yaguchi

The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

Gts1p stabilizes oscillations in energy metabolism by activating the transcription of TPS1 encoding trehalose-6-phosphate synthase 1 in the yeast Saccharomyces cerevisiae

We reported previously that Gts1p regulates oscillations of heat resistance in concert with those of energy metabolism in continuous cultures of the yeast Saccharomyces cerevisiae by inducing fluctuations in the levels of trehalose, but not in those of Hsp104 (heat shock protein 104). Further, the expression of TPS1, encoding trehalose-6-phosphate synthase 1, and HSP104 was activated by Gts1p in combination with Snf1 kinase, a transcriptional activator of glucose-repressible genes, in batch cultures under derepressed conditions. Here we show that, in continuous cultures, the mRNA level of TPS1 increased 6-fold in the early respiro-fermentative phase, while that of HSP104 did not change. The expression of SUC2, a representative glucose-repressible gene encoding invertase, also fluctuated, suggesting the involvement of the Snf1 kinase in the periodic activation of these genes. However, this possibility was proven to be unlikely, since the oscillations in both TPS1 and SUC2 mRNA expression were reduced by approx. 3-fold during the transient oscillation in gts1Delta (GTS1-deleted) cells, in which the energy state determined by extracellular glucose and intracellular adenine nucleotide levels was comparable with that in wild-type cells. Furthermore, neither the mRNA level nor the phosphorylation status of Snf1p changed significantly during the oscillation. Thus we suggest that Gts1p plays a major role in the oscillatory expression of TPS1 and SUC2 in continuous cultures of Saccharomyces cerevisiae, and hypothesized that Gts1p stabilizes oscillations in energy metabolism by activating trehalose synthesis to facilitate glycolysis at the shift from the respiratory to the respiro-fermentative phase.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix‐loop‐helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant‐negative HLH (dnHLH) proteins lack DNA‐binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho‐physiological processes. We report the first structure of a free‐standing human dnHLH protein, HHM (Human homologue of murine maternal Id‐like molecule). HHM adopts a V‐shaped conformation, with N‐terminal and C‐terminal five‐helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N‐ and C‐terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V‐shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V‐shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V‐shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Functional Correlation between the Nuclear Localization of Fht1p and Its Flocculation and Heat Tolerance Activities in Budding Yeast Saccharomyces cerevisiae

Fht1p is involved in the flocculation and heat tolerance machinery of budding yeast Saccharomyces cerevisiae. Despite knowledge of its involvement in those phenotypes, a precise mechanism has yet to be discovered. To this end, we monitored the relationship between subcellular localization of Fht1p and its flocculation or heat tolerance function using newly developed expression vectors with a recombinant green fluorescent protein (GFP; S65T/S147P) of Aequorea victoria added at both the N- and C-terminus of Fht1p. The main fluorescent signal of the GFP tagged with either a wild-type Fht1p or mutants which preserve their flocculation function was detected in the nucleus, whereas signals of functionless mutants were dispersed to the cytoplasm.

The DLP1 mutant of the yeast Saccharomyces cerevisiae with an increased copy number of the 2μ plasmid shows a shortened lifespan

We isolated and characterized a recessive mutant, named dlp1, which shows the Dlp phenotype (delayed loss of proliferation activity) during the autophagic death of cdc28. The dip1 mutant was found to consist of two subtypes of cells based on colony morphology. One subtype with the Dlp phenotype, named dlp1-1, became large, red, and nibbled during the incubation, suggesting that the cells on the surface of the colonies were dying. The other without the Dlp phenotype, named dlp1-s, retained small, white colonies even after a prolonged incubation and was found to be a petite mutant. The change from dlp1-1 to dlp1-s (petite) occurred much more frequently (about 15%) than that from the wild-type to petite mutant (less than 1%). The lifespan of both subtypes of cells was severely shortened. The copy number of the endogenous 2micron plasmid of dlp1-1 was 68-fold that of the original cdc28, and decreased by half after the conversion to dlp1-s (petite). A 4.0-kbp fragment of the 2micron plasmid containing REP2 decreased the copy number of the endogenous 2micron plasmid to 8-fold that of the original cdc28 cells and partially rescued the shortened lifespan, in addition to resulting in the complete complementation of the Dlp and nibbled-colony phenotypes. These results suggest that DLP1 is a chromosomal gene that regulates the copy number of the 2micron plasmid, and that the shortening of the lifespan and other effects of the dlp1 mutation are likely caused by the increased copy number of the endogenous 2micron plasmid.