Effect of low pH on organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1Delta did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1Delta and slt2Delta mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1Delta or slt2Delta cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.

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.

Analysis of the upstream regulatory region of the GTS1 gene required for its oscillatory expression.

The protein level of the GTS1 gene product (Gts1p) fluctuated during the oscillation of energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. Here, we found that the GTS1 mRNA level oscillated with the same periodicity as the metabolic oscillation, suggesting that the expression of GTS1 was regulated at the transcriptional level. As the 5'-upstream sequence of GTS1 contains two short open-reading frames at -310 and -829 bp from the initiation codon, we determined the GTS1 promoter required for the oscillatory expression. The upstream sequence was truncated into fragments of 183, 355, 1,042, and 1,572 bp, named GTS1pr.183 and so on, and their effects on the expression of lacZ as a reporter gene and the GTS1 gene itself were examined. The beta-galactosidase activity and Gts1p level oscillated in the continuous cultures when genes were expressed under the control of GTS1pr.183 but not GTS1pr.355. The disappearance of the metabolic and cell-cycle oscillations in the GTS1-deleted mutant was rescued by the transformation with GTS1pr.183-GTS1 but not with GTS1pr.355-GTS1. However, the stress-resistance oscillations were not found in the cells transformed with GTS1pr.183-GTS1, differing from the case of GTS1pr.1042-GTS1 reported previously [Wang et al. (2001) FEBS Lett. 489, 81-86]. Thus, we suggest that the 183-bp upstream sequence of GTS1 is basically required for the metabolic oscillation, while the 1,042-bp upstream sequence is required for oscillations of stress resistance.

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.

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 GTS1's 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.

Pleiotropic effects of HTLV type 1 Tax protein on cellular metabolism: mitotic checkpoint abrogation and NF-kappaB activation.

Tax protein expressed by human T cell leukemia virus type 1 (HTLV-1) is a strong trans-activator of its own LTR promoter; it also affects the function of multiple cellular genes involved in cell cycle control and transcription. One way in which Tax exerts its pleiotropic effects is through protein-protein interaction with cellular cofactors. By using yeast two-hybrid technology, we have isolated several cellular proteins that bind to Tax. Two of these are MAD1, a mitotic checkpoint control protein, and TXBP151, a suppressor of tumor necrosis factor alpha-induced apoptosis. Here we discuss findings describing the role of MAD1 in exit of cells from mitosis and TXBP151 in NF-kappaB activation.

Cellular stress responses oscillate in synchronization with the ultradian oscillation of energy metabolism in the yeast Saccharomyces cerevisiae.

We investigated whether cellular responses to various stress conditions are regulated in synchronization with the ultradian rhythm of respiratory-fermentative metabolism which is coupled to the cell cycle rhythm in continuous cultures of the yeast Saccharomyces cerevisiae. The cellular resistance to heat oscillated with a peak at the late respiro-fermentative phase, which approximately corresponds to the unbudding period of the cell cycle. Cellular resistance to H(2)O(2) and that to the superoxide-generating agent menadione oscillated in the same phase as that of heat resistance. The resistance to cadmium and that to 1-chloro-2,4-dinitrobenzene, an uncoupler of energy metabolism in mitochondria, both oscillated with a peak advanced by about 80 degrees relative to that of heat resistance, approximately covering the respiro-fermentative phase. Thus, cellular resistance to various stresses in S. cerevisiae oscillated in synchronization with the metabolic oscillation in the continuous culture.

Phosphorylation of the GTS1 gene product of the yeast Saccharomyces cerevisiae and its effect on heat tolerance and flocculation.

The GTS1 gene from the yeast Saccharomyces cerevisiae showed pleiotropic effects on yeast phenotypes, including an increase of heat tolerance in stationary-phase cells and an induction of flocculation. Here, we found that the GTS1 product, Gts1p, was partially phosphorylated at some serine residue(s) in cells grown on glucose. Studies using mutants of protein kinase A (PKA) and CDC25, the Ras-GTP exchange activator, showed that PKA positively regulated the phosphorylation level of Gts1p. Overexpression of Gts1p in a mutant with attenuated PKA activity did not show any increase of heat tolerance and partially decreased flocculation inducibility, suggesting that phosphorylation of Gts1p is required for induction of these phenomena.

The DLP1 mutant of the yeast Saccharomyces cerevisiae with an increased copy number of the 2micron 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.

Lipid-binding and antimicrobial properties of synthetic peptides of bovine apolipoprotein A-II.

We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu(49)-Thr(76) exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae. Experiments using amino-acid-substituted peptides indicated that the residues Phe(52)-Phe(53)-Lys(54)-Lys(55) are required for the activity. Peptide Leu(49)-Thr(76) induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu(49)-Thr(76) can destabilize the E. coli membrane. CD measurements showed that the alpha-helicity of peptide Leu(49)-Thr(76) increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu(49)-Thr(76), some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu(49)-Thr(76), transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe(52)Phe(53)-->AlaAla (Phe(52, 53)-->Ala) in peptide Leu(49)-Thr(76) caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu(49)-Thr(76)(Phe(52,53)-->Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu(49)-Thr(76) appears to primarily target the cytoplasm rather than the membrane of E. coli cells.

Protein interactions of Gts1p of Saccharomyces cerevisiae throughout a region similar to a cytoplasmic portion of some ATP-binding cassette transporters.

The GTS1 gene product, Gts1p, has pleiotropic effects on the timing of budding, cell size, heat tolerance, sporulation and the lifespan of the yeast Saccharomyces cerevisiae. In this study, we found (using the yeast two-hybrid system) that Gts1p forms homodimers throughout the 18-amino acid region 296-313 which has considerable similarity to a region downstream of the Walker nucleotide-binding motif A of some ATP-binding cassette (ABC) transporters. The region contains two aspartic acid residues at 301 and 310 preceded by hydrophobic amino acid residues, and Gts1p with an Asp310 to Ala substitution showed considerably reduced homodimerization, as shown by the two-hybrid assay. Overexpression of the point-mutated Gts1p did not efficiently induce the Gts1p-related phenotypes described above, suggesting that the homodimerization of Gts1p is required for it to function in vivo. The C-terminal cytoplasmic domain of the yeast ABC transporters Mdl1p (multidrug resistance-like transporter) and Ycf1p (yeast cadmium factor or glutathione S-conjugate pump) bound to Gts1p in the two-hybrid system, and the heterodimerization activity of the Gts1p with the Asp301 to Ala substitution was more affected than the Gts1p with the Asp310 to Ala substitution. Overexpression of GTS1 considerably reduced, and disruption of GTS1 slightly decreased, cellular resistance to cycloheximide, cadmium, cisplatin and 1-chloro-2,4-dinitrophenol, which (except for cycloheximide) are all substrates of Ycf1p. These results suggest that Gts1p interacts with some ABC transporters through the binding site overlapping that of homodimerization and modulates their activity.

Purification, primary structure, and antimicrobial activities of bovine apolipoprotein A-II.

We purified an antimicrobial protein of 76 residues, denoted bovine antimicrobial protein-1 (BAMP-1), from fetal calf serum using hydrophobic chromatography, gel filtration, and reverse-phase high-performance liquid chromatography. The amino acid sequence of BAMP-1 was similar to that of human apolipoprotein A-II (apo A-II), a major component of high-density lipoprotein (HDL), and the amino acid composition was almost identical to that of a previously reported candidate for bovine apo A-II. BAMP-1 was recovered from the post-HDL fraction, but not from the HDL fraction of the serum and was associated with a small amount of triglycerides (5%, w/w). These results suggest that BAMP-1 is the bovine homologue of apo A-II and is present in almost free form in serum. BAMP-1 showed a weak growth-inhibitory activity against Escherichia coli and yeasts tested in phosphate-buffered saline (PBS).

The GTS1 gene product influences the ultradian oscillation of glycolysis in cell suspension of the yeast Saccharomyces cerevisiae.

We tested the effect of the GTS1 gene of the yeast Saccharomyces cerevisiae on the cyanide-induced ultradian oscillation of the glycolytic metabolite NADH in cell suspension of strains with different copy numbers of the gene, that is, the wild-type, GTS1-disrupted and GTS1-overexpressing strains. The cells showed long-lasting oscillations when harvested between 1 and 2 hours after the diauxic shift from glucose to ethanol as a growth substrate. The frequencies of oscillation did not vary very much among the three strains tested. However, the amplitudes and durations of the oscillation were changed significantly as a function of the GTS1 gene-dosage. The effect of GTS1 on the amplitude was not caused by changing rates of glucose incorporation into cells as the rates were the same among the three strains during the macroscopic oscillation.

The pleiotropic effect of the GTS1 gene product on heat tolerance, sporulation and the life span of Saccharomyces cerevisiae.

We investigated whether or not the potential clock gene, GTS1, of the yeast Saccharomyces cerevisiae, shows pleiotropic effects on the yeast cellular processes. We tested the efect of the Gts1 protein on heat tolerance, sporulation and life-span, by characterizing the phenotypes of transformants with different copy numbers of the gene. We found that the Gts1 protein affects the capacity of heat tolerance in the stationary phase and the speed leading to sporulation in a gene-dose dependent manner, and that both inactivation and overexpression of the gene shortened the life-span of yeast. These results supported the notion that GTS1 affects the biological clock of the yeast S. cerevisiae, although this cannot be definitively concluded because the strain cannot be synchronized with circadian or ultradian rhythms.

Autophagic death after cell cycle arrest at the restrictive temperature in temperature-sensitive cell division cycle and secretory mutants of the yeast Saccharomyces cerevisiae.

When virgin temperature-sensitive mutant cdc28 cells of the yeast Saccharomyces cerevisiae were incubated at restrictive temperatures, they continued to grow, reaching a maximum of 3.3-fold the original size after 24 h. The protein and RNA levels increased during the first 24 h, then gradually decreased. The cells that reached the maximal size lost proliferative activity and synthesized less protein. After a 72-h incubation, cellular components, protein, RNA and DNA, were progressively degraded, resulting in extensive fragmentation within 7 days. Light and electron microscopic observation revealed that cdc28 cells incubated at the restrictive temperature for 24 h were enriched with double-unit membranes in the cytoplasm, and the vacuoles were filled with autophagic body-like structures. After 7 days most cellular contents were lost, and the membrane systems were fragmented. The protein synthesis inhibitor cycloheximide added at 24 h inhibited degradation of protein for at least 7 days suggesting that protein synthesis was involved in the activation of autophagic death. All other temperature-sensitive cdc and secretory (sec) mutants tested showed similar morphological changes when arrested in the cell division cycle at the restrictive temperature. In contrast, the temperature-insensitive wild-type cells grew normally at 38 degrees C and only a few percent of them underwent autolysis 7 days after transfer to the stationary phase. These results suggested that the yeast cells underwent autophagic death when arrested at any stage of the cell division cycle, whereas those arrested at the stationary phase rarely underwent autophagic death.

Dystrophin localization at presynapse in rat retina revealed by immunoelectron microscopy.

PURPOSE: It has been accepted that retinal dystrophin is localized in the outer plexiform layer (OPL) of the retina, but no electron microscopic evidence has been reported until now. Using immunoelectron microscopy, the authors investigate whether retinal dystrophin localizes at the presynaptic or postsynaptic membrane of synaptic regions. METHODS: Monoclonal antibody raised against human dystrophin C-terminus was used. Immunoblotting analysis was used for testing dystrophin protein in retinal tissues. Tissue preparations were stained with the immunofluorescence or immunoperoxidase method and were observed with light and electron microscopy. RESULTS: Immunoblotting analysis showed a molecular weight of band 420 kDa in the sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the retinal tissues. Immunoelectron microscopy disclosed that retinal dystrophin was localized at the presynaptic membrane of synaptic regions in the OPL. CONCLUSIONS: Rat retinal dystrophin localizes at the presynaptic membrane in the OPL, suggesting that it may play some role in the neurotransmitter release of the photoreceptor cell.

Increased expression of acidic ribosomal protein (P0) mRNA after phorbol ester treatment of cultured rat thyroid (FRTL-5) cells.

P0, an acidic protein component of the ribosomal protein in eukaryotic 60 S ribosomal subunit, plays an important role in polypeptide chain elongation during translation. To investigate the role of protein kinase C in thyroid cell protein synthesis, we examined the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of P0 mRNA and protein. RNA slot blot hybridization revealed that TPA induced the accumulation of P0 mRNA in FRTL-5 cells in a time- and dose-dependent manner. A maximal increase of 2-fold was observed 18 h after addition of TPA. Cycloheximide markedly inhibited the TPA-induced accumulation of P0 mRNA. Nuclear runoff transcription assays using nuclei prepared from TPA-treated FRTL-5 cells revealed that TPA increased the transcription of P0 mRNA but not of beta-actin. Immunoblotting experiments using anti-P protein antibody showed that TPA also increased the protein amount of P0. These results suggest that TPA activates protein synthesis in thyroid cells by inducing the expression of ribosomal proteins.

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.