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.

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.

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity.

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.

Preferential biosynthesis of ribosomal structural proteins by free and loosely bound polysomes from regenerating rat liver.

Homologous cell-free systems were prepared using free, total bound, tightly bound or KCl-sensitive loosely bound (KCl-sensitive) polysomes from regenerating rat liver. [14C]Leucine was incubated with one kind of polysomes and [3H]leucine with another kind. The reaction mixtures were then combined, and ribosomal structural proteins were purified as described previously [4], using two-dimensional polyacrylamide gel electrophoresis as the final step [5]. The 3H to 14C ratios of the purified fractions were estimated to compare the activities of the two kinds of polysomes for biosynthesis of ribosomal structural proteins. The following results were obtained: (1) The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 3.6 or 2.4 times higher than that of total bound polysomes in two experiments in which 14C and 3H labeling was reversed. The radioactivities incorporated by free polysomes into most of the proteins separated on two-dimensional gel were found to be definitely higher than those in the surrounding areas, suggesting that most of the ribosomal structural proteins were synthesized by free polysomes. The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 7 times higher than that of tightly bound polysomes, which were prepared by washing the microsomal membrane fraction with 0.5 M KCl. The radioactivities incorporated by tightly bound polysomes into the proteins separated on two-dimensional gel were only slightly higher than those in the surrounding areas, indicating that these polysomes had very low synthetic activity. (2) Preferential synthesis of histones by free polysomes was also shown using the same procedures. (3) KCl-sensitive polysomes which were released by washing the microsomal membrane fraction with 0.5 M KCl, were shown to have definitely higher activity than tightly bound polysomes for biosynthesis of ribosomal structural proteins. (4) From these results, it is concluded that most of the ribosomal structural proteins are preferentially synthesized by free and KCl-sensitive polysomes in regenerating rat liver.

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.

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.

Thyrotropin stimulates the expression of an acidic ribosomal protein, P0, messenger ribonucleic acid in cultured rat thyroid (FRTL) cells.

Acidic ribosomal proteins, P0, P1, and P2 in eukaryotic 60S subunits play an important role in polypeptide chain elongation during the translational step. To investigate the role of TSH in protein synthesis in the thyroid, we examined the effect of TSH on ribosomal P-protein biogenesis in FRTL cells. First, we investigated the influence of TSH on P0-protein gene expression. RNA slot blot hybridization revealed that the effect of TSH on P0-protein mRNA accumulation in the quiescent FRTL cells was time- and dose-related. This stimulatory effect of TSH was mimicked by (Bu)2cAMP. Nuclear run-off transcription assays revealed that TSH increased the transcriptional activity of the P0-protein mRNA without an increase in beta-actin transcriptional activity. On the contrary, the stability of P0-protein mRNA decreased after the addition of TSH. Cycloheximide markedly inhibited TSH-induced P0-protein mRNA accumulation in FRTL cells. Second, using a synthetic oligonucleotide probe, we have shown that TSH also increased P2-protein mRNA levels in FRTL cells. Furthermore, immunodetection of P-proteins using anti-P-protein antibody showed that TSH significantly increased the amount of P-proteins in the cells. These results suggest that TSH can increase the amount of acidic ribosomal P-proteins at least in part through an increase in the level of P-protein gene transcripts. This effect occurs via a transcriptional mechanism and requires ongoing protein synthesis. Thus, TSH might play an important role in ribosome biogenesis in FRTL cells.

The site of action of the A-chain of mistletoe lectin I on eukaryotic ribosomes. The RNA N-glycosidase activity of the protein.

The site of action of the A-chain of mistletoe lectin (ML-A) from Viscum album on eukaryotic ribosomes was studied. Treatment of rat liver ribosomes with ML-A, followed by treatment of the isolated rRNA with aniline, caused the release of a fragment with about 450 nucleotides from 28 S rRNA. Further analysis of nucleotide sequences of this fragment revealed that the aniline-sensitive site of phosphodiester bond was between positions A-4324 and G-4325 in 28 S rRNA. These results indicate that ML-A inactivates the ribosomes by cleaving a N-glycosidic bond at A-4324 of 28 S rRNA in the ribosomes as ricin A-chain does.

The mechanism of action of the cytotoxic lectin from Phoradendron californicum: the RNA N-glycosidase activity of the protein.

A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline-sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N-glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A-chain.

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.

The effect of aging on protein synthesis in the yeast Saccharomyces cerevisiae.

The protein synthetic rate in the yeast S. cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times. Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells. However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes. Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.

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.

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.

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.

Effects of DNA and urea on the specificity for H1 histone of the neutral protease B partially-purified from rat liver chromatin.

A neutral protease, named protease B in the previous report (Tsurugi, K. & Ogata, K. (1982) J. Biochem. 92, 1369-1381), was partially purified from rat liver chromatin by gel filtration through Sepharose 6B followed by DE-Sephadex column chromatography. The proteolytic activity on total histones of the partially-purified protease B was increased about two fold by addition of DNA and again increased by further addition of 2 M urea. Analysis of the hydrolysed products showed that out of five species of histones, only H1 was degraded in the presence of an amount of DNA equivalent to the amount of histones, whereas core histones were also degraded in the absence or presence of one-tenth amount of DNA. Urea accelerated the selective degradation of H1 histone because H1 histone was preferentially degraded in the presence of even a low amount of DNA. In contrast, core histones became resistant to the protease B in the presence of DNA and/or urea. Heat-denatured DNA stimulated the degradation of H1 histone even in the absence of urea to almost the same extent that native DNA did in the presence of urea. Thus, protease B efficiently degrades H1 histone when its association with DNA is destabilized by either addition of urea or pretreatment of DNA with heat.

Studies on the serine proteases associated with rat liver chromatin.

The chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained. 1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52,000, 25,000, and 15,000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin. 2. Proteins A and B were extracted from the chromatin fraction by 5 M urea and 0.7 M NaCl, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B. 3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin. 4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor(s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor(s). 5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs. These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.

Evidence for the exchangeability of acidic ribosomal proteins on cytoplasmic ribosomes in regenerating rat liver.

We purified acidic ribosomal proteins (P1 and P2) in good yield from rat liver ribosomes by precipitation of ribosomes with MgCl2 prior to ethanol extraction and chromatography of the extract on a column of CM-cellulose at pH 4.8. The newly-synthesized acidic ribosomal proteins in regenerating rat liver, labeled in vivo with [3H]leucine, were rapidly incorporated into cytoplasmic ribosomes without any detectable time lag and, after reaching a maximum at 30 min, they gradually disappeared from the ribosomes, suggesting a short metabolic-life. However, it was found later that they were re-incorporated slowly when newly-labeled proteins were "chased" by an injection of a large amount of cold leucine intraperitoneally at 15 min after the injection of [3H]leucine. Furthermore, in a long-term experiment, acidic ribosomal proteins were found to disappear with a half-life of 100 h from the ribosomes. Thus, these results suggest that acidic ribosomal proteins have a long metabolic life and are exchangeable on cytoplasmic ribosomes in regenerating rat liver.

On the size and the role of a free cytosolic pool of acidic ribosomal proteins in yeast Saccharomyces cerevisiae.

A small but distinct amount of yeast acidic ribosomal proteins A1/A2 was detected in cytosol by immunoblotting on a two-dimensional gel electrophoretogram, while 38 kDa acidic protein A0 was not detected. The free forms of A1/A2 in the cytosol were eluted in gel filtration at the molecular mass of about 30 kDa under non-denaturation conditions, suggesting that they exist as a dimer or timer without association with A0. The amount of free A1/A2 was determined by immunoblotting to be 0.3% of the ribosome-bound A1/A2 in yeast. The time course of incorporation of radioactive amino acid showed that the cytosolic free A1/A2 are labeled more rapidly with high specific radioactivity than the ribosome-bound A1/A2. This result suggested that some of the cytosolic A1/A2, if not all, are newly-synthesized proteins which are ready for incorporation into cytoplasmic ribosomes.

A study on the identities of the three species of chromatin-associated proteinases in a mutant of Saccharomyces cerevisiae which lacks four major vacuolar proteinases.

Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins.

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.