Mitochondrial protein synthesis in a mammalian cell-line with a temperature-sensitive leucyl-tRNA synthetase.

The temperature-sensitive Chinese hamster ovary cell mutant tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 degrees C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 degrees C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34 degrees C is not inhibied by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 degrees C is inhibited by tevenel, (b) Protein synthesis by isolated mitochondria from tsH1 cells is not significantly inhibited at 40 degrees C. (c) At 40 degrees C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [14C]leucine at 40 degrees C into mitochondrial proteins of tsH1 cells is inh-bited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsH1 cells at 40 degrees C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40 degrees C and at 34 degrees C in the presence of cycloheximide have been compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40,000 to 20,000 and a second with an apparent molecular weight range from 20,000 to 10,000.     

Nuclear protein synthesis at permissive and non-permissive temperatures in a Chinese hamster ovary cell line with a temperature-sensitive defect in cytoplasmic non-mitochondrial protein synthesis.

A Chinese hamster ovary cell line with a temperature-sensitive defect in cytoplasmic non-mitochondrial protein synthesis was used to examine protein synthesis thought to be intrinsic to nuclei. Nuclear fractions did not contain whole cells, endoplasmic reticulum or mitochondria, as judged by light and electron microscopy and contaminating microorganisms and PPLO were absent. When cytoplasmic protein synthesis was almost totally suppressed at 40 °C, in some experiments in the presence of cycloheximide, nuclear and mitochondrial proteins continued to be labelled with radioactive leucine for 15–30 min. Nuclear incorporation at 40 °C was suppressed by puromycin and partially inhibited by 225 μg of chloramphenicol per ml. Most of the nuclear proteins labelled at 40 °C, which included a majority of recovered radioactive proteins, were soluble in 1 N NaOH, and can be classified as acidic nuclear proteins. The majority of radioactive leucine was incorporated internally into nuclear proteins, as judged by lack of reactivity with 2,4-dinitrofluorobenzene. Preliminary studies with SDS polyacrylamide gel electrophoresis suggest that some of the radioactive proteins present in the nuclear extract differed from those of the cytosol and mitochondrial fractions. Provided whole cells, mitochondria and endoplasmic reticulum neither contaminated the nuclear pellet nor transferred proteins to that site before or during nuclear isolation, that microorganisms, including PPLO and possibly even viruses capable of causing artefactual incorporation are absent, and that nuclei contain a leucyl tRNA synthetase able to function at 40 °C, the tsHl CHO cell line should provide a valuable experimental system with which to examine the properties of protein synthesis intrinsic to cell nuclei and to elucidate its functions.     

Rapidly reversible enzyme inhibition in a temperature-sensitive mammalian cell mutant lacks thermotolerance

The temperature-sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsH1 contains a thermolabile leucyl-tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5 degrees C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45 degrees C was identical to that of wild-type (SC) cells. In addition, the heat-induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45 degrees C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 45 degrees C, at 39.5 degrees C, neither the inhibition of leucyl-tRNA synthetase activity nor the killing of tsH1 expressed thermotolerance. Also, treatment of tsH1 at 39.5 degrees C did not induce thermotolerance to killing at 45 degrees C. The inhibition of leucyl-tRNA synthetase activity in tsH1 at 39.5 degrees C was further distinguished from the 45 degrees C-induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl-tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45 degrees C in SC cells was decreased by increased heat dose. Such was not true for the 39.5 degrees C inhibition of leucyl-tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition--one slowly reversible type which was observed during and after heating at 45 degrees C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsH1 at 39.5 degrees C and neither induced nor expressed thermotolerance.     

Inhibitor-“resistant” labelling of rat ventral prostate nuclear proteins : Further evidence consistent with protein synthesis associated with cell nuclei

When minced rat ventral prostate was incubated with labelled amino acids and cycloheximide or puromycin, the specific radioactivity of proteins associated with Triton X 100-washed nuclei exceeded that of the 105 000 g cytosol. The distribution of radioactive proteins from incubated mince, examined by SDS polyacrylamide gel electrophoresis was also consistent with labelling of some nuclear proteins that was resistant to inhibitors. Highly purified prostate nuclei, washed with detergent, labelled proteins of from 1–6 × 10⁴ D with radioactive amino acids. When these proteins were fractionated according to solubility, NaOH-soluble ‘acidic’ proteins, examined by SDS polyacrylamide gel electrophoresis, were highly labelled, with a distribution of radioactivity that differed from the patterns of 0.4 N H₂SO₄-soluble basic proteins (including histones), and proteins soluble in Krebs-Ringer-phosphate buffer. Although these results cannot be interpreted unambiguously, they are consistent with the synthesis of certain nuclear proteins at a site(s) sequestered from cycloheximide and puromycin. Nuclei may represent one such site.     

NUCLEAR-CYTOSOL INTERACTIONS THAT FACILITATE RELEASE OF RNA FROM MOUSE BRAIN NUCLEI

Abstract— Mouse brain nuclei were incubated in vitro under conditions that primarily lead to the synthesis of radioactive polydisperse and messengerlike nuclear RNA. After incubation the effects of Mg² concentrations, nucleoside triphosphate levels and brain cytosol were examined with regard to their ability to influence the release of RNA from brain nuclei. The presence of 8 mM -MgCl₂ and a total of 0.3 mM-nuclcoside triphosphates during the labelling procedure allowed only a minimal amount of RNA to be released. However, when the MgCl₂ was decreased to 2 mM and the nucleoside triphosphates were increased to 1 mM, a stimulation of RNA release was observed. The addition of unfractionated brain cytosol under these conditions resulted in an inhibition of RNA release.
G-100 Sephadex filtration removed detectable RNase activity from the cytosol preparations and allowed the identification of fractions that were able to facilitate nuclear RNA release by 3-fold. The fractions that stimulated release did not have detectable levels of RNase, protease or DNA-dependenl RNA polymerase. Under conditions that provided maximum nuclear RNA release by both labelled mouse brain and neuroblastoma nuclei, no release of DNA could be measured. The cytosol fractions that facilitated RNA release did not have a high affinity for nuclear RNA or an ability to stimulate nuclear RNA synthesis. However, other components in the cytosol were shown to stimulate RNA metabolism in isolated mouse brain nuclei and to have a relatively high binding affinity to nuclear RNA. Further purification of the RNA release components in the brain cytosol by DEAF. Sephadex chromatography allowed an increase in specific activity of at least 40-fold. The thermal lability, effective filtration size, and solubility in phenol suggested that the cytosol factors that facilitiated nuclear RNA release were associated with cellular proteins.     

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.     

Nature of the facilitated messenger ribonucleic acid transport from isolated nuclei.

Cytoplasmic macromolecules were previously identified which regulate both qualitatively and quantitatively the release of messenger-like RNA from isolated nuclei. These macromolecules are now shown to be denatured at 45-50 degrees C and their synthesis is sensitive to pactamycin or cycloheximide. The putative regulatory proteins are essentially quantitatively precipitated with high specificity from the cytosol by streptomycin at a concentration 10-fold higher than that used to precipitate RNA. The nuclear concentration-dependence of RNA transport from successive samples of nuclei strongly suggests that the regulatory factors are recycled. Quantitative changes in the sequences transported at various dilutions of the cytosol suggest that not all the different classes of the putative regulatory macromolecules are present in an effective concentration at any one dilution.     

Products of mitochondrial protein synthesis in Tetrahymena.

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57,500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform-methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.     

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.     

Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.     

Effect of cycloheximide on nonpermissive temperature killing in tsH1 mutant cells

1. 1.|We investigated the mechanism of cycloheximide-induced heat protection. We proposed a hypothesis to account for the mechanism [Lee and Dewey (1986) Radiat. Res. 106, 98–110].

2. 2.|Cycloheximide protects cells from hyperthermic killing by means of protecting thermolabile proteins from denaturation.

3. 3.|For this study, we have employed temperature-sensitive mutant tsH1 which contains a thermolabile leucyl-tRNA synthetase.

4. 4.|By 15 h of incubation at the nonpermissive temperature of 39.5 or 40°C, 40 or 93% of mutant cells respectively, were killed. In contrast, wild type SC cells did not lose viability after this same incubation.

5. 5.|Although killing of tsH1 by incubation at the nonpermissive temperatures was mainly due to denaturation of a thermolabile leucyl-tRNA synthetase, cycloheximide did not protect mutant cells from killing. However, tsH1 and SC cells exhibited similar sensitivities to killing at 43°C and above. Furthermore, cycloheximide protected both cell types from hyperthermic killing.

6. 6.|There was a 200- or 700-fold increase in survival after 2.5 h at 43°C by treatment with cycloheximide in tsH1 or SC cell type, respectively. Thus, the cellular target(s) for hyperthermic killing at this temperature apparently are similar in both types of cells.

7. 7.|The data indicate that the mechanism behind cycloheximide-induced heat protection is probably not the prevention of protein denaturation.

Synthesis of mitochondrial protein in the flight muscles of the Colorado beetle. Differentiation in vivo between extra- and intra-mitochondrial contributions to the amino acid incorporation into mitochondrial protein

By using cycloheximide, an inhibitor of cytoplasmic protein synthesis, conditions were investigated to estimate in vivo the extra- and intra-mitochondrial contributions to the synthesis of organelle protein in the flight muscles of Colorado beetles. With 4-day-old beetles about 15% of the [(14)C]leucine incorporation into mitochondrial protein is resistant to the action of cycloheximide. The incorporation into cytosol protein is inhibited by more than 99.5% with cycloheximide. During the first hour after precursor administration the incorporation into mitochondrial protein proceeds, in both the presence and the absence of cycloheximide, at a more-or-less linear rate with time. The cycloheximide-resistant amino acid incorporation is sensitive to the inhibitor of mitochondrial protein synthesis, chloramphenicol. The uncertainties inherent in the use of cycloheximide were discussed in arriving at the conclusion that about 15% of the mitochondrial protein is formed inside the organelle.     

Alteration of heat sensitivity by treatment with nonpermissive temperature or cycloheximide in temperature-sensitive CHO mutant tsH1 cell

1. 1.|The temperature-sensitive mutant CHO-tsH1 and wild type (CHO-SC) cells became thermal resistant when cells were treated for either 2 h at 39.5°C before heating at 43°C or 2 h with 10 μg/ml cycloheximide (CHM) before and during heating at 43°C.

2. 2.|There was a 2000-fold increase in survival after 2.5 h at 43°C by preincubation at 39.5°C in both cell types. There was also a 200- or 700-fold increase in survival after 2.5 h at 43°C by treatment with CHM in tsH1 or SC cell type respectively.

3. 3.|In contrast to the effects at 43°C, at 41.8°C these protective effects were not evident in tsH1 cells. In wild type, however, there was an 800- or 1800-fold increase in survival after 8 h at 41.8°C by preincubation at the temperature of 39.5°C or treatment with CHM, respectively.

4. 4.|Therefore, these results suggest that killing of tsH1 at low temperature hyperthermia (41.8°C) is probably due to denaturation of thermolabile leucyl-tRNA synthetase.

5. 5.|The denaturation of this enzyme may not be protected by inhibition of protein synthesis by preincubation at the nonpermissive temperature of 39.5°C or by CHM.

Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1.

Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Pleiotropic changes in cycloheximide-resistant insect cell clones

Summary Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. FromAedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5°C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%. This investigation was supported by grant AI20385 from the National Institutes of Health, Bethesda, MD and by a Basil O’Connor Starter Research Grant (5–415) from the March of Dimes Birth Defects Foundation.     

Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7- 1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.