Fission yeast tor1 functions in response to various stresses including nitrogen starvation, high osmolarity, and high temperature.

A target of rapamycin (TOR) protein is a protein kinase that exerts cellular signal transduction to regulate cell growth in response to extracellular nutrient conditions. In the Schizosaccharomyces pombe genome database, there are two genes encoding TOR-related proteins, but their functions have not been analyzed. Here we report that one of the genes, referred to as tor1+, is required for sexual development induced by nitrogen starvation. Ste11 is a key transcription factor for the initiation of sexual development. The expression of ste11+ is normally regulated in tor1- cells; and overexpression of ste11+ hardly rescues the defect in fertility in tor1-. Upon nitrogen starvation, tor1+ cells promote two rounds of the cell cycle to become arrested at the G1 phase before initiation of sexual development. The tor1- cells do not promote such a cell cycle, suggesting that Tor1 is necessary for the response to nitrogen starvation. The tor1- cells show no growth or very slow growth under various stress conditions, including external high pH, high concentrations of salts or sorbitol, and high temperature. These results suggest that Tor1 is necessary for any response to a wide range of stresses. The vegetative growth of tor1- cells is inhibited by rapamycin, although tor1+ cells are resistant to the drug. The tor1- cells are hypersensitive to fluphenazine and cyclosporin A, which specifically inhibit calmodulin and calcineurin, respectively.

Schizosaccharomyces pombe taf1+ is required for nitrogen starvation-induced sexual development and for entering the dormant GO state.

Environmental change, such as nutritional starvation, induces physiological and morphological alterations that enable fission yeast cells to survive. We isolated a novel gene, taf1+, required for the response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. taf1 disruptants could not mate upon nitrogen starvation, but could upon carbon starvation. taf1 disruptants had a defect in inducing stell+ expression under nitrogen starvation conditions. Furthermore, they lost viability quickly in nitrogen-depleted medium. Unlike wild-type cells, starved taf1-cells had nuclear chromatin that were flat and adhered to the cell periphery. These results indicate that tqf1+ is required for nitrogen starvation-induced sexual development and entering the dormant G0 state.

Detection and analysis of translation elongation factor 3 genes from various yeasts.

Yeast translation requires a unique elongation factor, EF-3. However, information about EF-3 genes has been limited to only a few yeast species. Here, we developed a PCR-based system to detect the EF-3 genes specifically, and identified EF-3 gene fragments from various yeast species in which EF-3 genes have not yet been found.

Activation of the 20S proteasome of Xenopus oocytes by cardiolipin: blockage of the activation of trypsin-like activity by the substrate.

The effects of an activator, cardiolipin, on the three peptidase activities of the 20S proteasome of Xenopus oocytes were examined. The trypsin-like activity was activated when the enzyme was treated with cardiolipin before the addition of the substrate, but there was no appreciable activation when cardiolipin was added concomitantly with the substrate. On the other hand, the chymotrypsin-like peptidase and peptidylglutamylpeptide hydrolase (PGPH) were activated regardless of the sequence of addition. When very low concentrations of the substrate (e.g. 0.1-0.5 microM; about 1/100 of the K(m)) were used, cardiolipin strongly activated trypsin-like peptidase by the simultaneous addition but not after substrate addition. These results suggest that the trypsin-type substrate produces a conformational change in the enzyme in a concentration-dependent manner which makes the activator sites inaccessible to cardiolipin.

Protection by trehalose of DNA from radiation damage.

The most serious damage to cells exposed to radiation is attributed mostly to effects on the structure of cellular DNA. We found that trehalose protects DNA from irradiation. In the presence of 10 mM trehalose, DNA can be protected from about 4 times higher doses of beta- and gamma-ray irradiation. The protective effect increases with the amount of the sugar. Other disaccharides, sucrose, and maltose had similar effects.

Protective effect of disaccharides on restriction endonucleases during drying under vacuum.

Desiccation by vacuum-drying inactivates the restriction endonuclease HindIII completely. However, when dried in the presence of a disaccharide such as trehalose, maltose, or sucrose, the endonuclease retains its lambda DNA-cleaving activity and produces the same digestive fragments as does the intact enzyme. Thus, the disaccharides are effective in protecting the restriction enzyme in terms of both recognition and accurate cleavage of the substrate. Among the disaccharides, trehalose protects the enzyme most effectively; and it also stabilizes the enzyme during dilution in aqueous solution. The restriction enzyme dried with trehalose maintains its activity without detectable loss for at least 4 days at 37 degrees C, but it shows reduced activity after 30-day storage at either 4 degrees C or room temperature. Trehalose also protects other restriction endonucleases, EcoRI and BamHI, from inactivation during vacuum-drying, whereas drying them alone leads to severe loss of their activity. The restriction endonucleases dried with trehalose retain their activities for at least 20 days at 4 degrees C and for 7 days at room temperature.

Polyamino acids that inhibit the interaction of yeast translational elongation factor-3 (EF-3) with ribosomes.

EF-3 is a translational elongation factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.

Purification and characterization of RNases from fission yeast under nitrogen-starvation.

Two RNases were purified from nitrogen-starved fission yeast cells in which cellular RNA was being degraded drastically. The two RNases showed similar properties. Their molecular weight in native form was about 170kDa. They were endoRNases which required divalent cations.

The difference in the type of codon-anticodon base pairing at the ribosomal P-site is one of the determinants of the translational rate.

By utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNA(Phe) (anticodon; GmAA) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U.S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.

The yeast peptide elongation factor 3 (EF-3) carries an active site for ATP hydrolysis which can interact with various nucleoside triphosphates in the absence of ribosomes.

ATP (GTP) hydrolysis was clearly demonstrated by using at most 16 pmol of yeast peptide elongation factor 3 (EF-3) in the absence of ribosomes. However, the highly active yeast ribosomes (up to 48 pmol) displayed virtually no ATPase (or GTPase) activity in the absence of EF-3. Several lines of evidence indicated that both the catalytic and binding sites of the ATPase reside in the elongation factor itself, not on the ribosomes. The patterns of protection by various nucleoside triphosphates against tryptic digestion of EF-3, reflecting the wide substrate specificity of the ATPase, confirmed that the active center of the endogenous ATPase is located on the factor itself and not on contaminants. The intrinsic activity was stimulated up to two orders of magnitude by the presence of the yeast ribosomes fully active in polyphenylalanine synthesis. The activation was achieved by enhancing the catalytic activity (kcat) to a much greater extent than the binding affinity (Km). On the other hand, the ribosome-activated ATPase activity was revealed to inherit its wide substrate specificity from the intrinsic property of EF-3, which shows an affinity to various XTPs, including pyrimidine- and purine-nucleoside triphosphates, irrespective of 2'-hydroxylation of the sugar moiety. From experiments on protection against tryptic digestion, we determined that intricate conformational changes of the factor molecule occur upon interaction with the substrate XTP and ribosomes.

Role of yeast peptide elongation factor 3 (EF-3) at the AA-tRNA binding step.

The stimulatory effect of peptide elongation factor 3 (EF-3), which is uniquely required for the yeast elongation cycle, on the step of binding of aminoacyl-tRNA (AA-tRNA) to ribosomes has been investigated in detail. Yeast EF-1 alpha apparently functions in a stoichiometric manner in the binding reaction of AA-tRNA to the ribosomes. The addition of EF-3 and ATP to this binding system strikingly stimulated the binding reaction, and the stimulated reaction proceeded catalytically with respect to both EF-1 alpha and EF-3, accompanied by ATP hydrolysis, indicating that EF-3 stimulated the AA-tRNA binding reaction by releasing EF-1 alpha from the ribosomal complex, thus recycling it. This binding stimulation by EF-3 was in many respects distinct from that by EF-1 beta gamma. The idea that EF-3 may participate in the regeneration of GTP from ATP and the formed GDP, as indicated by the findings that the addition of EF-3 along with ATP allowed the AA-tRNA binding and Phe polymerization reactions to proceed even in the presence of GDP in place of GTP, was not verified by the results of direct measurement of [32P]GTP formation from [gamma-32P]ATP and GDP under various conditions. Examination of the stability of the bound AA-tRNA disclosed the different binding states of AA-tRNA on ribosomes between in the cases of the complexes formed with EF-1 alpha alone, or factor-independently, and with EF-1 alpha and EF-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Changes in ribosomal properties during adenylate deprivation in the cells of Kluyveromyces lactis.

In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis, the intracellular content of ATP is one-third to one-fifth that in a prototrophic wild strain under growing conditions. The quantitative differences becomes rather small in resting stationary-phase cells. Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content is lowest during the transition phase of growth. The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours. Identification of ribosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits. The mutant ribosomes prepared from the transition-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resting stationary-phase cells. Thus, changes in ribosomal components associated with the differences in ribosome activity in a cell-free system were noted in the adenylate-deprived cells of K. lactis.

Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts.

Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo.

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.

Intrinsic ATPase activity of yeast peptide chain elongation factor 3(EF-3) and its direct interaction with various nucleotides.

Whereas the ribosome-dependent ATPase activity of EF-3 required highly active ribosomes for its full activity, a catalytic site for ATP hydrolysis may reside in the EF-3 as being supported by the activity-EF-3/ribosome amount profiles. The direct interaction of EF-3 with various nucleotides such as GTP, UTP, CTP, dATP, ADP and AMPPNP as well as ATP was analyzed by protection experiments against trypsin digestion of the factor according to SDS-gel electrophoresis. The protection effect varied with the used nucleotides roughly in accordance with the inhibitory effect of those on the ribosome-dependent ATPase. The ATPase activity of EF-3 alone in the absence of ribosome was observed by using large amounts of the factor and the rate was two orders of magnitude lower than that of the ribosome-dependent.

Interaction of yeast polypeptide chain elongation factor-3 (EF-3) with different nucleotides.

ATPase and GTPase activities of EF-3 were similarly inhibited by various nucleotides including CTP, UTP and four dNTP's. The low specificity of EF-3 was in remarkable contrast with the high specificity of EF-1 alpha and EF-2 directed only to quanine nucleotides. The pH-activity and salt concentration-activity profiles as well as the above inhibition experiments coincidently supported that the same active site functions for ATPase and GTPase of EF-3. The stimulation of poly(Phe) synthesis was not observed with AMPPNP in place of ATP. The stimulation required ATP hydrolysis, probably catalyzed by ATPase of EF-3. Reflecting the low specificity of the ATPase, UTP, dTTP, dATP and dGTP stimulated the poly(Phe) synthesis. EF-3 appears to drive yeast elongation cycle using the energy from ATP hydrolysis by its ATPase without serving for GTP regeneration.

Poly(U)-dependent polyphenylalanine and polytyrosine synthesis in vitro by a tRNATyr variant with an enzymatically altered anticodon, G-A-A.

A variant of T. utilis tRNATyr containing a base substitution (psi----A) in the middle position of the anticodon has been constructed by enzymatic procedures in vitro. This variant is unique in that it can accept both tyrosine and phenylalanine. This tRNA was shown to be active in transferring both tyrosine and phenylalanine into polypeptides in a cell-free, poly (U)-directed translation system from yeast. This result gives further support to the adapter hypothesis since tyrosine, attached to the variant tRNATyr with an anticodon G-A-A, is incorporated into polypeptides in response to poly (U) message.