Kazuhiro Mitsui

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.

Valproic acid induces apoptosis dependent of Yca1p at concentrations that mildly affect the proliferation of yeast.

Valproic acid (VPA) inhibited the growth of yeast in a dose‐dependent manner with complete inhibition attained at 100 mM. When cells were exposed to 25 mM VPA, the wild‐type died showing apoptotic markers, while yca1Δ deleted of YCA1 encoding yeast caspase 1 survived. On the other hand, when cells were exposed to 50 mM VPA, both the wild‐type and yca1Δ died showing morphological features similar to those of the autophagic death of cdc28 which was also independent of YCA1. Thus, these results suggested that yeast cells die via YCA1‐dependent apoptosis when their proliferative activity is mildly impaired.

An ARL1 mutation affected autophagic cell death in yeast, causing a defect in central vacuole formation.

When the cdc28 strain of Saccharomyces cerevisiae is incubated at restrictive temperatures, the yeasts digest themselves in 7 days by activating autophagic machinery. In parallel, the cell-proliferative activity decreases progressively after about 48 h. We have previously referred to this phenomenon as autophagic death. In the present study, we isolated and characterized a recessive mutant strain, dlp2, which delays the progression toward autophagic death. The cdc28 dlp2 cells contain many small vesicles instead of the large central vacuoles that are usually found in parental cdc28 cells. We showed that the dlp2 phenotype results from the presence of a single mutation in the gene ARL1 (ADP-ribosylation factor-like protein 1). Morphological and biochemical analyses of cdc28 dlp2 suggested that a defect in central vacuole formation is caused by aberrant membrane trafficking, although the protein-sorting to vacuoles is not affected. After a shift to a restrictive temperature, the components of the cytoplasm and nucleus of cdc28 dlp2 were condensed, with an accompanying formation of vesicles in the periphery (epiplasm) of the cells rather than an activation of the autophagic machinery. Introducing this ARL1 mutation into the normal ARL1 locus of the wild-type W303 strain again inhibited the progression of apoptotic cell death due to a defect in vacuole formation, which in this case was induced by the proapoptotic protein Bax. Thus, the ARL1 gene plays an important role in the formation of central vacuoles and in the progression of programmed cell death induced by cell-cycle arrest or Bax. These results suggested the presence of a programmed-cell death machinery in yeast that is similar to that related to the Type II cell death of mammalian cells characterized by autophagocytosis.

Evidence for the involvement of the GTS1 gene product in the regulation of biological rhythms in the continuous culture of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, ultradian oscillations of energy metabolism have been observed in continuous cultures. Here, we found that the level of the GTS1 gene product oscillated in concert with the ultradian rhythm of energy metabolism. When GTS1 was inactivated by gene disruption, the metabolic oscillation was affected severely, mostly disappearing within a day, in the absence of synchronized stress‐response oscillations throughout the continuous culture. The disappearance of biological rhythms in the GTS1‐deleted mutant was substantially rescued by transformation with chimera plasmids carrying GTS1 under the control of GTS1s own promoter. On the other hand, this disappearance was not rescued by constitutive expression of GTS1 under the control of the triose phosphate isomerase promoter.

Shear stress-induced collagen XII expression is associated with atherogenesis

Fluid shear stress has been shown to modulate various endothelial functions. We selected a shear stress-specific clone, identified as collagen XII, from a bovine aortic endothelial cell (BAEC) cDNA library. We confirmed that shear stress induces collagen XII expression at both the mRNA and protein levels in cultured BAECs and human umbilical vein ECs (HUVECs) by stimulating transcription. When HUVECs were exposed to shear stress, they secreted collagen XII protein and it was deposited underneath them. Strong expression of collagen XII was found in the intima of human aortic wall lacking atherosclerotic lesions, whereas weak expression was seen in the intima of atherosclerotic plagues. Furthermore, the downstream portion of atherosclerotic plaques showed apparently weak collagen XII expression compared with the upstream portion. These results suggest that collagen XII expression induced by fluid shear stress may play a role in stabilizing the vascular structure and preventing the formation of atherosclerotic lesions.

Human trehalase is a stress responsive protein in Saccharomyces cerevisiae.

Three trehalases ATH1, NTH1, and NTH2 have been identified in Saccharomyces cerevisiae. ATH1, and NTH1 hydrolyze trehalose to glucose to provide energy and assist in recovery from stress. Human trehalase (TREH) is expressed in the intestine and kidney and probably hydrolyzes ingested trehalose in the intestine and acts as marker of renal tubular damage in kidney. Since trehalose is not present in circulation or kidney tubules, its renal effect suggests it has other yet unidentified actions. Here we examined the function of human trehalase in budding yeast. We constructed three yeast trehalase mutants (NTH1Delta, NTH2Delta, and ATH1Delta) and then transformed TREH into these mutants. NTH1Delta did not grow on media containing trehalose as the carbon source, and TREH did not rectify NTH1Delta dysfunction and also did not grow on trehalose medium, suggesting that TREH is not responsible for utilization of exogenous trehalose in yeast. In experiments involving exposure to heat, osmotic and oxidative stresses, NTH1Delta showed no recovery. Interestingly, ATH1Delta-TREH showed high sensitivity to all three stressors. ATH1Delta and NTH2Delta showed very low neutral trehalase activity and NTH1Delta did not show any neutral trehalase activity, and trehalose concentrations were higher. Increased neutral trehalase activity (equivalent to the wild type), reduction of trehalose content and brisk sensitivity to stressors were noted in TREH-ATH1Delta strain, but not in TREH-NTH1Delta or -NTH2Delta. Our results suggest that TREH acts as a stress-response protein in the kidney rather than involved in utilization of exogenous trehalose.

Valproate induces apoptosis by inducing accumulation of neutral lipids which was prevented by disruption of the SIR2 gene in Saccharomyces cerevisiae

We investigated the participation of HDACs in VPA induced apoptosis in Saccharomyces cerevisiae. VPA (20 mM) induced apoptosis in several HDAC mutants, including PRD3 and HDA1‐disrupted cells and SIR2 over expressing cells, as well as in wild‐type cells but not SIR2‐disrupted cells. Intracellular reactive oxygen species and neutral lipid content increased markedly in all kinds of HDAC mutant cells tested except for SIR2‐disrupted cells. Thus, these results suggest that 20 mM VPA induces neutral lipid accumulation and apoptosis‐like features in S. cerevisiae, and that VPA‐induced apoptosis was evaded by deletion of SIR2.

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.

PSK2 coordinates glucose metabolism and utilization to maintain ultradian clock-coupled respiratory oscillation in Saccharomyces cerevisiae yeast.

Ultradian clock-coupled respiratory oscillation (UCRO) in an aerobic continuous culture of Saccharomyces cerevisiae S288C is principally regulated by control of certain redox reactions of energy metabolism. It is also modulated by the metabolism of storage carbohydrates during adaptation to environmental change. However, the mechanism of cell sensing and response to environmental nutrients in UCRO is unknown. The purpose of the present study was to determine the role of PSK2 kinase in UCRO in yeast. S. cerevisiae in culture showed oscillation in PSK2 mRNA levels with a definite phase relationship to the respiratory oscillation. Furthermore, inactivation of Psk2 by gene disruption severely affected UCRO and its decline to undetectable levels within 2days. In addition, the extracellular and intracellular glucose concentrations of PSK2 deletion mutants in culture were higher and lower, respectively, than those of the wild type. PSK2 mutant cells showed no alteration in redox state. Furthermore, the levels of storage carbohydrates such as glycogen and trehalose fluctuated in PSK2 mutants with attenuated amplitudes comparable to those in the wild type. The results indicated that PSK2 kinase is important for the uptake of glucose and regulation of storage-carbohydrate synthesis and hence the maintenance of an unperturbed continuously oscillating state.