Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia

The ribosomes extracted from the mitochondria of the ciliate, Paramecium aurelia, have been shown to sediment at 80S in sucrose gradients. The cytoplasmic ribosomes also sediment at 80S but can be distinguished from their mitochondrial counterparts by a number of criteria. Lowering of the Mg++ concentration, addition of EDTA, or high KCl concentrations results in the dissociation of the cytoplasmic ribosomes into 60S and 40S subunits, whereas the mitochondrial ribosomes dissociate into a single sedimentation class at 55S. Furthermore, the relative sensitivity of the two types of ribosome to dissociating conditions can be distinguished. Electron microscopy of negatively stained 80S particles from both sources has also shown that the two types can be differentiated. The cytoplasmic particles show dimensions of 270 X 220 A whereas the mitochondrial particles are larger (330 X 240 A). In addition, there are several distinctive morphological features. The incorporation of [14C]leucine into nascent polypeptides associated with both mitochondrial and cytoplasmic ribosomes has been shown: the incorporation into cytoplasmic 80S particles is resistant to erythromycin and chloramphenicol but sensitive to cycloheximide, whereas incorporation into the mitochondrial particles is sensitive to erythromycin and chloramphenicol but resistant to cycloheximide.     

A gene coding for a ribosomal protein L41 in cycloheximide-resistant ribosomes has a promoter which is upregulated under the growth-inhibitory conditions in yeast, Candida maltosa.

We previously found by using yeast, Candida maltosa, that cycloheximide (CYH) sensitivity of ribosomes is dependent on the 56th amino acid residues of a ribosomal protein, L413 (proline in sensitive and glutamine in resistant ribosomes). We also revealed that in this yeast, which has both L41-P type and L41-Q type genes, the expression of the latter type genes is induced by the addition of CYH in the medium to make the cells inducibly resistant to CYH. In this paper, we analyzed the promoter region of L41-Q2a, one of the CYH-inducible L41-Q type genes and found two elements required for the induction of expression: one was a GCRE (Gcn4p-responsive element of Saccharomyces cerevisiae)-like element and the other was a GT-rich element. This promoter region was also required for its expression under some other growth inhibitory conditions. Furthermore, it was suggested that Q-type ribosomes synthesized under these conditions are more resistant to these inhibitory conditions.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

Role of ribosomes in cycloheximide resistance of Neurospora mutants

Summary In Neurospora crassa, mutants resistant against cycloheximide appear with a marked time lag after mutation induction. We have suggested (Neuhäuser et al., 1970) that this lag indicates the time needed for the synthesis of altered ribosomes (phenotypic lag), that the drug in the wildtype acts upon the ribosomes, and that resistance is due to alterations in them.By measurements of poly-U directed poly-Phe synthesis on ribosomes of the wildtype and two different cycloheximide resistant mutants in a cell free system it is shown here that mutant ribosomes indeed differ from those of the wildtype. Poly-Phe synthesis on mutant ribosomes proceeds in the presence of the drug, whereas that on wildtype ribosomes is inhibited. This means that the earlier suggestions are correct.Abbreviation CHX cycloheximide     

Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei

Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.     

Cloning in Saccharomyces cerevisiae of a cycloheximide resistance gene from the Candida maltosa genome which modifies ribosomes.

We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae.     

An in vitro polypeptide synthesizing system from methanogenic bacteria: Sensitivity to antibiotics

Summary An in vitro polypeptide synthesis system was set up for three methanogenic bacteria, Methanococcus vannielii, Methanobacterium formicicum and Methanosarcina barkeri, and the effect of classical 70S and 80S protein synthesis inhibitors studied. The following results were obtained: (i) The activity of ribosomes from all three methanogens was unaffected by a number of 70S inhibitors such as tetracycline, chloramphenicol, streptomycin, tiamulin and, probably, erythromycin as well; (ii) However, the ribosomes were sensitive to thiostrepton, virginiamycin and, to varying degrees, to those aminoglycosides containing a 2-deoxystreptamine moiety. Among the aminoglycosides examined, streptomycin induced no translational misreading. The compounds containing 2-deoxystreptamine stimulated misreading, albeit only at high concentrations (neomycin being an exception); (iii) Ribosomes from all three organisms were insensitive to the 80S inhibitors cycloheximide and ricin, but those from Methanobacterium formicicum were highly sensitive to anisomycin and moderately sensitive to verrucarin. The results support those of in vivo studies and provide conclusive evidence that archaebacterial ribosomes despite being 70S ribosomes lack binding sites for many classical eubacterial ribosome inhibitors. At the same time they possess sites for others, as well as for some inhibitors of 80S ribosomes.     

Binding of amphipathic drugs and probes to biological membranes

Bouvardain is an antitumor drug that inhibits protein synthesis in intact eukaryotic cells and cell-free systems. Our present studies have shown that bouvardin acts at the level of the 80 S ribosome in a site somehow involved with the interaction of EF1 and EF2. Indeed bouvardin inhibits EF1-dependent binding of aminoacyl-tRNA and EF2-dependent translocation of peptidyl-tRNA but does not affect the non-enzymic translocation since this relation does not require EF2. The site of the 80 S ribosome involved in the interaction with bouvardin appears to be independent from the cycloheximide and the cryptopleurine binding sites since yeast mutants resistant to cycloheximide or cryptopleurine are sensitive to bouvardin.     

Protein synthesis by brain-cortex mitochondria. Characterization of a 55S mitochondrial ribosome as the functional unit in protein synthesis by cortex mitochondria and its distinction from a contaminant cytoplasmic protein-synthesizing system

Homogenates of rat brain cortex were fractionated by conventional methods of velocity sedimentation and separated into a microsomal and a washed mitochondrial fraction. By electron microscopy the mitochondrial fraction was shown to be rich in synaptosomes. The mitochondria-synaptosome fraction synthesized protein in vitro by a route that was partially inhibited by cycloheximide and partly by chloramphenicol. The relative effectiveness of the two inhibitors varied greatly with the medium used. In the mitochondria-synaptosome fraction active 80S cytoplasmic ribosomes and active 55S mitochondrial ribosomes were detected; these were also seen in the electron microscope. Mild osmotic shock of the mitochondria-synaptosome fraction followed by velocity sedimentation in sucrose-EDTA allowed isolation of a mitochondrial fraction free of synaptosomes. Protein synthesis in this fraction was entirely inhibited by chloramphenicol, but was completely resistant to cycloheximide both in a medium promoting oxidative phosphorylation and in ATP-generating medium. Ouabain had no inhibitory effect on protein synthesis in a purified mitochondrial preparation. It is concluded that brain-cortex mitochondria synthesize protein entirely on 55S mitochondrial ribosomes.     

The L7/L12 proteins change their conformation upon interaction of EF-G with ribosomes

The different functional complexes of ribosomes with elongation factor F (EF-G) were studied by digestion experiments with trypsin. It was found that upon interaction of EF-G with ribosomes the L7/L12 proteins are sensitive to trypsin and are trypsin resistant after dissociation of EF-G from ribosomes. The significance of conformational alterations in the L7/L12 and also in the other proteins in the translation process is discussed.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

Biochemical genetic studies of cycloheximide resistance in Neurospora crassa

Genetic analysis of a number of cycloheximide-resistant mutants of Neurospora crassa has shown that resistance is controlled by several genes. Two of these appear to be located on linkage group V. Resistance to the antibiotic is dominant in wild-type-mutant heterokaryons. Two types of cycloheximide-resistant mutants were isolated: one type exhibited colonial morphology only when grown in the presence of cycloheximide and the other type maintained normal morphology even at high concentrations of the antibiotic. Reconstitution experiments with supernatant solutions and 80S monosomes prepared from wild-type and resistant mutant strains indicated that the property of cycloheximide resistance most likely is associated with the ribosomes. No electrophoretic or serological differences were found between the ribosomal proteins of the wild-type and resistant mutants.     

Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts.

Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.     

An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae.

Summary Mutants of Schizosaccharomyces pombe were isolated as resistant either to trichodermin or to anisomycin. Growth tests showed that the majority of mutants isolated were cross resistant to both drugs and also to cycloheximide. A limited genetic analysis showed that mutants at least four loci, tri3, tri4, ani1 and ani2, had this phenotype as was also the case for mutants at three cycloheximide resistant loci, cyh2, cyh3 and cyh4 reported previously (Ibrahim and Coddington, 1976). Allelism tests showed that the tri3, ani2 and cyh4 strains were allelic. A mutant at another trichodermin resistant locus, tri5, was cross resistant to anisomycin but sensitive to cycloheximide.Ribosomes from wild type and selected strains were analysed in a poly U directed cell free protein synthesising system. Three strains, cyh1-C7, ani1-F1 and tri-N15 (probably a tri5 allele) possessed ribosomes which were more resistant than the wild type to the drugs used in their isolation. In each case the site of the resistance was in the 60S subunit. Ribosomes from the cyh2, cyh3 and cyh4 strains were as sensitive to cycloheximide as those from wild type.     

Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline.     

Modification of Ribosomes in Cryptopleurine-Resistant Mutants of Yeast

Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.     

Distinct response of yeast ribosomes to a miscoding event during translation

Numerous mechanisms have evolved to control the accuracy of translation, including a recently discovered retrospective quality control mechanism in bacteria. This quality control mechanism is sensitive to perturbations in the codon:anticodon interaction in the P site of the ribosome that trigger a dramatic loss of fidelity in subsequent tRNA and release factor selection events in the A site. These events ultimately lead to premature termination of translation in response to an initial miscoding error. In this work, we extend our investigations of this mechanism to an in vitro reconstituted Saccharomyces cerevisiae translation system. We report that yeast ribosomes do not respond to mismatches in the P site by loss of fidelity in subsequent substrate recognition events. We conclude that retrospective editing, as initially characterized in Escherichia coli, does not occur in S. cerevisiae. These results highlight potential mechanistic differences in the functional core of highly conserved ribosomes.     

Impact of mitochondrial function on yeast susceptibility to antifungal compounds

Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.     

A possible ribosomal-directed regulatory system in Euglena gracilis. Chlorophyll synthesis

Cycloheximide at concentrations of 0.1-100mum stimulated chlorophyll synthesis when dark-grown cells of Euglena were illuminated. Chloramphenicol (1-4mm) inhibited chlorophyll synthesis. The effect of cycloheximide on the incorporation of [(14)C]leucine into material insoluble in trichloroacetic acid, and its failure to affect the incorporation of [(32)P]orthophosphate into such material in short incubations, are interpreted as evidence that cycloheximide specifically inhibits protein synthesis by 80S ribosomes. Since the inhibitory effect of chloramphenicol on chlorophyll synthesis is counteracted by the presence of cycloheximide, it is suggested that chlorophyll synthesis is subject to control by a cytoplasmic repressor synthesized on 80S ribosomes, and to a de-repressor synthesized on 70S ribosomes.