The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

Summary The effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 g/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of ¹⁴C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts^- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts^- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.     

The interaction between newly-formed mRNA and ribosomal particles during retarded translation]

The formation of polyribosomes in mouse liver cells at the reduced-rate translation was studied by treatment with cycloheximide (CHI) and aurintricarboxylic (ATA) acid. An increase of polypeptide synthesis time by 1.7-2.7 times (0.5 mg CHI per 25 g of weight or 15 mg ATA per 25 g) leads to a delay of the entrance of newly formed cytoplasmic D-RNA into polyribosomes. These results are in agreement with the model of polyribosome formation from ribonucleoprotein precursors containing cytoplasmic D-RNA. On the other hand, in the presence of a CHI dose (5 mg/25 g) causing a dramatic (240-fold) increase of polypeptide synthesis time, the kinetics of entrance of newly formed D-RNA into polyribosomes does not differ from the normal one, and amount of the incorporated mRNA is even somewhat higher than under normal conditions. It is suggested that in this situation ribosomes are moving along the newly formed mRNA, and their movement is not accompanied by the synthesis of completed polypeptide chain.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max

Proliferative growth in cultured soybean cells (Glycine max cv. Sodifuri, of cotyledenary origin) is regulated to a substantial degree by the plant hormone, cytokinin. Previously we reported that stationary phase soybean cells exhibit a rapid increase in polyribosome formation upon transfer to fresh cytokinin-containing medium. Cytokinin plays a significant role in this expansion of the cell's protein synthetic capacity through a heretofore unidentified effect on protein synthesis at the translational level. The present study was undertaken to identify the deficiency in the translational process of stationary phase cells which is overcome by cytokinin treatment. We have demonstrated that cytokinin-induced polyribosome formation was brought about by the recruitment of existing monoribosomes into polyribosomal aggregates. Three hours of cytokinin stimulation nearly doubled the total amount of [³H]leucine incorporated into nascent polypeptides. Average polyribosome size was nearly identical in stationary phase cells and in cells which were induced to grow by cytokinin. Cytokinin-enhanced protein synthetic capacity was not accompanied by changes in polypeptide chain elongation or termination rates. Partial cycloheximide inhibition of elongation was used to demonstrate that initiation of polypeptide synthesis was limiting to a slight degree both before and after cytokinin stimulation. These results suggest that cytokinin stimulates protein synthesis by making more message available for translation. We obtained direct evidence to support this hypothesis by showing that labeled poly(A)-containing, nonpolyribosomal RNA, which was synthesized during stationary phase but not translated, moves into polyribosomes upon cytokinin treatment.     

Ayalysis of translational parameters in cultured cells

A method is described for the measurement of protein synthesis parameters in cultured cells. Ribosome transit times, polyribosome size distribution and relative synthetic rates are measured on individual cultures of cells. This method is applied to an analysis of cycloheximide (1 micrometer) inhibition of protein synthesis in cultured mouse hepatoma cells.     

Effects of Hypoglycemia on Rat Brain Polyribosome Sedimentation Pattern

Insulin-induced hypoglycemia provokes polyribosome disaggregation and accumulation of monomeric ribosomes in the brain of rats with hypoglycemic paresis and coma. The extent of brain polyribosome disaggregation depends on the decrease of blood glucose concentration, and in comatose animals on the duration of hypoglycemia. Cycloheximide prevents the disaggregation of brain polyribosomes induced by hypoglycemia, indicating that hypoglycemia affects brain protein synthesis, decreasing the rate of initiation relative to the rate of elongation of polypeptide chain synthesis.     

Determination of the time of polypeptide chain synthesis in animal experiments]

A method is worked out for the estimation of the time of polypeptide chain synthesis in animals in vivo. The time of the synthesis of "middle" polypeptide was found to be 1.45 min. in mouse liver cells under normal conditions, while in the presence of translation inhibitors, cycloheximide (20 mg/kg) and aurintricarboxilic acid (1 g/mg) the time of the synthesis increased in 2.7 and 2.5 times respectively. The time of polypeptide synthesis linearly increased with the increase of aurintricarboxilic acid dose.     

Endogenous ADP-ribosylation of elongation factor 2 in polyribosome fraction of rabbit reticulocytes

Several polypeptides of about 120, 96, 85, 60 and 38 kDa are shown to be radiolabeled during incubation of the mono- and polyribosome fraction of rabbit reticulocytes with [32P]NAD. Among them is a polypeptide coinciding with elongation factor 2 (EF-2) in its electrophoretic mobility in SDS-polyacrylamide gel. The addition of pure EF-2 to the polyribosome fraction results in an increase of the radioactive label in this polypeptide band. From this it is concluded that both endogenous and added EF-2 is ADP-ribosylated by an enzyme associated with polyribosomes. A possibility of regulation of protein synthesis through endogenous ADP-ribosylation in vivo is considered.     

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.     

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.     

Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.     

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.     

EFFECT OF THE ANTIBIOTIC,CYCLOHEXIMIDE, ON THE METABOLISM AND GROWTH OF SACCHAROMYCES PASTORIANUS

Coursen, B. W., and H. D. Sisler (U. Maryland, College Park.) Effect of the antibiotic, cycloheximide, on the metabolism and growth of Saccharomyces pastorianus. Amer. Jour. Bot. 47(7): 541–549. Illus. 1960.—Studies were made of the toxicity of cycloheximide and certain of its derivatives to Saccharomyces pastorianus Hansen. The ED₅₀ values for cycloheximide, its semicarbazone derivative and its oxime derivative are 0.018, 0.37, and 12.0.p.p.m., respectively. In auxanographic and liquid culture tests involving 160 organic and biochemicals, only certain methylated ring ketones and vitamin A alcohol or acetate showed appreciable antagonistic activity to the toxicity of cycloheximide. Yeast cells exposed to 3.16 p.p.m. of cycloheximide and incubated for 30 min. with uniformly labeled ¹⁴C glucose remove about 10% less activity from the medium than untreated cells. Measurements of radioactivity in compounds extracted from cells with 80% ethanol showed the presence of appreciable activity in the glutamine from untreated cells but no measurable activity in this compound from treated cells. Activity in glutamic acid from treated cells was reduced while activity in alanine and aspartic acid was increased when compared with the activity in these compounds from untreated cells. There were other differences, also, especially in the levels of activity in organic phosphorus compounds, but, in many cases, the activity in compounds from treated cells was similar to that in the corresponding compounds from untreated cells. It is possible that the antibiotic interferes with the metals involved in the enzymatic reaction leading to the synthesis of glutamic acid and glutamine or it may act as an inhibitory analog in the synthesis of these or similar compounds. The apparent interference of cycloheximide with the formation of a CO-NH bond in the synthesis of glutamine suggests also that peptide bond formation in protein synthesis may be similarly affected. A block of glutamine synthesis by cycloheximide may be sufficient to account for the toxicily of the antibiotic, but the failure of exogenous sources of glutamine to reverse the toxicity indicates that other reactions in cell metabolism may be as sensitive to cycloheximide as the synthesis of glutamine.     

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

Effect of metabolic inhibitors on formed novocaine and neutral red segregation zones in frog erythrocytes

Effects of some metabolic inhibitors, as well as of biologically active compounds (diakarb, ethidium bromide and a phenanthridine alkaloid sanguinarine) on the formed novocaine and neutral red segregation zones were studied. The volume of granules diminished under the influence of a glycolytic inhibitor iodoacetate, uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonyl cyanide trifluoromethoxyphenylhydrozone), and respiratory inhibitors (antimycin A and rotenone), as well as under the influence of cycloheximide - an inhibitor of protein synthesis. Diakarb, ethidium bromide or sanguinarine also provoked a regression of the segregation zones. It has been found that all these compounds are inhibitors of ATPase activity of the isolated segregation zones. A possible mechanism of volume decreasing in segregation zones under the influence of both the metabolic inhibitors and diakarb, ethidium bromide and sanguinarine is discussed.     

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.     

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.     

Gastric antisecretory activity of cycloheximide due to inhibition of protein synthesis

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.     

Stimulating effect of histones on DNA synthesis in the regenerating rat liver]

Effect of exogenous histones, nuclear globulins and acid proteins on DNA synthesis is studied in regenerating liver of rats in which the synthesis of their own proteins and thus DNA replication are inhibited by cycloheximide. In these conditions histones from regenerating rat liver are found to stimulate 3H-thymidine incorporation into DNA of hepatectomyzed rat liver. Nuclear globulins and acid proteins from regenerating liver, and histones from intact liver produced no stimulating effect on DNA sythesis.