The TOR‐dependent phosphoproteome and regulation of cellular protein synthesis

Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR‐dependent growth inhibition. Here, we present a resource providing an in‐depth characterisation of Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR‐dependent phospho‐regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR‐regulated targets both in fission yeast and other eukaryotes.     

Nucleo-cytoplasmic interactions in the petite negative yeast Schizosaccharomyces pombe

Summary In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4–6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.Dedicated to Prof. Dr. F. Kaudewitz on occasion of his 60th birthday     

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

Oxygen toxicity in a fission yeast

Continuous exposure of synchronous cultures of Schizosaccharomyces pombe to 2.0 atmospheres oxygen beginning at any point in the first two-thirds of the cell cycle prevented subsequent cell division. Similar exposure during the last one-third of the cell cycle did not prevent cell division. The inhibition of division was totally reversible. Exposure to 2.0 atmospheres oxygen for 2.5 hours did not affect oxygen consumption. Oxygen at 1.0 atmospheres reduced growth rate and protein synthesis by 44%. Similar exposure to 1.0 atmospheres reduced transport of glycine-¹⁴C, L-leucine-¹⁴C, and uracil-¹⁴C by 95%, 73%, and 89% respectively. Analysis of the kinetics of uptake of these materials showed noncompetitive inhibition of transport by oxygen. The primary effect in rapidly appearing oxygen toxicity apparently involved interference with the transport capabilities of the cell membrane.     

Erythromycin inhibition of cell proliferation and in vitro mitochondrial protein synthesis in human HeLa cells is pH dependent

The growth of HeLa cells in Hepes-buffered medium was significantly more sensitive to the inhibitory effects of erythromycin than in medium buffered by the more conventional bicarbonate-CO₂ system. Since growth inhibition by erythromycin became more pronounced as the pH of the medium was increased the difference in erythromycin sensitivity between the Hepes-buffered medium vs. the bicarbonate-CO₂-buffered medium is most likely due to pH effects. The relative growth sensitivity to erythromycin of ERY2301, an erythromycin-resistant mutant of HeLa, was also affected by elevated pH of the growth medium. However, ERY2301 cells were able to proliferate to a greater extent in the presence of erythromycin than HeLa cells grown under the same conditions. The selective growth advantage of ERY2301 (in the presence of erythromycin) is best seen in medium of pH 7.4, or in the Hepes-buffered medium. In vitro protein synthesis by intact mitochondria isolated from HeLa cells was relatively insensitive to erythromycin inhibition at pH 7.4 and 7.6, but at high pH values was inhibited approx. 50%. Although the erythromycin sensitivity of ERY2301 mitochondrial protein synthesis was also affected by increasing the pH, the incorporation of [³H]leucine was more resistant to erythromycin than that observed for HeLa mitochondria over the pH range tested. Increasing the concentration of erythromycin at a given pH did not result in a further increase in the inhibition of either HeLa or ERY2301 mitochondrial protein synthesis. When the mitochondrial membranes were disrupted by Triton X-100, erythromycin inhibition of HeLa mitochondrial protein synthesis was pH dependent and, at the lower pH values tested, greater inhibition was observed as the erythromycin concentration was increased. ERY2301 mitochondrial protein synthesis under the same conditions displayed a high level of erythromycin-resistant activity independent of both pH and erythromycin concentration. It is suggested that, as has been proposed for bacterial systems, only the non-protonated molecule of erythromycin is effective in inhibiting mitochondrial protein synthesis. The ability of erythromycin to permeate the mitochondrial membranes and the plasma membres may also be facilitated by a higher pH.     

Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

Cell wall formation in yeast

Summary Incorporation of tritiated glucose into cell walls of growingSaccharomyces cerevisiac andSchizosaccharomyces pombe was studied using electron microscopic autoradiography. The pattern and the extent of labelling ofS. cerevisiae cell walls depended on the cell stage in the cell cycle. Quantitative evaluation of autoradiographs showed that the highest rate of wall synthesis took place during bud growth. The incorporation of new material into the wall of growing bud showed an increasing rate with the magnitude of the bud. The incorporation into the mother cell wall was almost negligible during bud growth. The rate of wall synthesis in double cells decreased during cell division. This period and that before new bud initiation was found to be the time of substantially reduced rate of wall replication inS. cerevisiae. A significant random incorporation was observed into the walls of post-division adult cells, both parental and daughter. The cell walls ofS. pombe were labelled almost exclusively at growing tips. The incorporation of tritiated carbohydrates into non-extensile regions ofS. pombe cell walls was found to be only about 5% of the total wall labelling.     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Inhibitory action of erythromycin on bacteriophage SPO1 multiplication in sporulating cells of Bacillus subtilis 168

Summary Erythromycin (2–4 g/ml) was found to inhibit specifically multiplication of SPO1 in sporulating cells of an erythromycin-resistant, conditional asporogenous mutant of Bacillus subtilis 168 thy ^- trp ^-, Ery1040. In contrast, streptomycin (150–200 g/ml) which inhibits protein synthesis to a similar extent as erythromycin did not inhibit SPO1 multiplication severely, suggesting that the inhibition of SPO1 multiplication by erythromycin is not caused by an overall inhibition of protein synthesis. Neither phage DNA synthesis nor phage messenger RNA synthesis was affected appreciably under these conditions. However, the synthesis of three phage proteins that are synthesized 15 min after infection was preferentially inhibited by erythromycin. In addition, the inhibition of SPO1 multiplication has been correlated with the stimulation of host stable RNA synthesis exhibited by erythromycin. Possible mechanisms for the inhibition of SPO1 multiplication in Ery1040 cells are discussed.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells

The steroid transformation of hydrocortisone to prednisolone, combining the two techniques of immobilized whole cells and high steroid concentrations, was investigated and found to be a feasible process. Freeze-dried Corynebacterium simplex cells were immobilized in collagen, tanned with glutaraldehyde, and cast into a membrane. The reaction was studied at hydrocortisone concentrations ranging from 5 to 50 mg/ml. The following aspects of the system were examined: (1) the substrate concentration effect upon the reaction; (2) the effect of enzyme concentration; (3) the rate-concentration relationship; and (4) the product inhibition characteristics of the system. The optimal substrate concentration was found to be 15 mg/ml of a membrane concentration of 80 mg/ml. This reaction attained an 80% conversion in 48 hr. A liner relation was found between the initial reaction rate and membrane concentration. One can thus increase the net production of steroid per unit volume and time by increasing the membrane levels. A physical limit to this increase occurred at membrane concentrations greater than 125 mg/ml. The rate-concentration relationship was linear when graphed on a Line weaver-Burk plot: giving a K_m′ and V_m′ value of 5.39 mg/ml and 0.556 mg/ml/hr, respectively. When the data were tested for competitive product inhibition, the curves fitted the experimental points fairly well and produced K_m′ and V_m′ values of 4.52 mg/ml and 0.566 mg/ml/hr, respectively. Product inhibition experiments showed that the inhibition was not purely competitive. At low substrate concentrations, product inhibited the enzyme; at high substrate concentrations, the enzyme was first stimulated and then depressed by increasing levels of products. This behavior has been analyzed and shown to be possibly a result of the information of a tertiary intermediate produced during the reaction.     

Chemical genetic screen in fission yeast reveals roles for vacuolar acidification,mitochondrial fission,and cellular GMP levels in lifespan extension

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age‐related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti‐aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V‐ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti‐aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G‐protein‐coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti‐aging activities for several phytochemicals and open up opportunities for the development of novel anti‐aging therapies.     

Induction of the cytoplasmic petite mutation in Saccharomyces cerevisiae by the antibacterial antibiotics erythromycin and chloramphenicol

Summary Low concentrations of erythromycin and chloramphenicol (0.3 mg/ml) specifically affect intra-mitochondrial protein synthesis in most strains of Saccharomyces cerevisiae, thereby preventing growth on non-fermentable substrates. This effect is reversible, the genetic capacity for respiration in the absence of the drug being unaffected. However, we now show that exposure of growing cells to high concentrations (1.3–3.0 mg/ml) of either antibiotic generates a high frequency of cytoplasmic petite (respiratory-deficient) mutants with a concomitant loss of the cytoplasmic genetic determinant for respiration known as the rho factor. In one strain in which the effect of erythromycin was examined, the entire population abruptly underwent mutation but only after exposure to the drug for several generations. Mitochondrial DNA was synthesised normally during the silent pre-mutational period, but was rapidly lost, by a process partly dependent on degradation, at the time of the mutational event. Intra-mitochondrial protein synthesis was inhibited only about 67% by the lower levels of erythromycin but was completely (99%) inhibited by the higher petite-inducing levels. These results are interpreted as evidence that the normal maintenance of mitochondrial DNA in this organism requires a protein(s) whose assembly in the mitochondria is completely blocked only by high erythromycin concentrations. This protein is normally present in excess and on exposure to high drug levels replication of mitochondrial DNA is unaffected until the supply runs out. When this happens, replication ceases, existing molecules are degraded, and rho factors are destroyed.     

Continuous cultivation of fission yeast: Classical and flow microfluorometry observations

The fission yeast Schizosaccharomyces pombe was grown in glucose-limited medium in a steady-state continuous flow reactor. Changes in mean cell protein and RNA contents with growth rate are consistent with earlier observations under different conditions. Flow microfluorometry measurements of the frequency functions of DNA at different dilution rates show changes in coordination of DNA synthesis and cell separation. Shifting from batch growth to small dilution rates results in unusual cell aggregation which leads to multiple steady states at identical operating conditions.     

Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

Yeast strains for concentrated substrates

Summary The screening of twenty yeast strains for ethanol productivity at high osmotic pressure at temperatures ranging from 32°C to 45°C is described. Shake flask fermentations of 30°, 40°, and 50° Bx cane molasses were performed. The effect of temperature on productivity at a non-inhibitory ethanol level is weakly pronounced. Most strains fermented poorly at 50° Bx molasses but two Schizosaccharomyces pombe and one commercial baker's yeast, Saccharomyces cerevisiae performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomyces cerevisiae (SJAB, fresh yeast), simulating a continuous run it was shown that Schizosaccharomyces pombe was less sensitive to high DS than Saccharomyces cerevisiae. At 25% DS the productivity of Schizosaccharomyces pombe is almost twice that of Saccharomyces cerevisiae.