The regulation of the decay of nitrate reductase Evidence for the existence of at least two mechanisms of decay

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37°C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.     

Sexual agglutinin of mating-type minus gametes in Chlamydomonas reinhardii : I. Loss and recovery of agglutinability of gametes treated with EDTA

The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin.     

Characterization of the translation-dependent step during iron-regulated decay of transferrin receptor mRNA.

Iron regulates the stability of the mRNA encoding the transferrin receptor (TfR). When iron is scarce, iron regulatory proteins (IRPs) stabilize TfR mRNA by binding to the 3'-untranslated region. High levels of iron induce degradation of TfR mRNA; the translation inhibitor cycloheximide prevents this. To distinguish between cotranslational mRNA decay and a trans effect of translation inhibitors, we designed a reporter system exploiting the properties of the selectable marker gene thymidine kinase (TK). The 3'-untranslated region of human transferrin receptor, which contains all elements necessary for iron-dependent regulation of mRNA stability, was fused to the TK cDNA. In stably transfected mouse fibroblasts, the expression of the reporter gene was perfectly regulated by iron. Introduction of stop codons in the TK coding sequence or insertion of stable stem-loop structures in the leader sequence did not affect on the iron-dependent regulation of the reporter mRNA. This implies that global translation inhibitors stabilize TfR mRNA in trans. Cycloheximide prevented the destabilization of TfR mRNA only in the presence of active IRPs. Inhibition of IRP inactivation by cycloheximide or by the specific proteasome inhibitor MG132 correlated with the stabilization of TfR mRNA. These observations suggest that inhibition of translation by cycloheximide interferes with the rate-limiting step of iron-induced TfR mRNA decay in a trans-acting mechanism by blocking IRP inactivation.     

The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA.

The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.     

Protein synthesis inhibition stabilizes urokinase-type plasminogen activator mRNA. Studies in vivo and in cell-free decay reactions

Inhibition of protein synthesis stabilizes a number of mRNAs, but little is known about the mechanism. To understand the relationship between protein synthesis and mRNA stability, we studied the degradation of calcitonin-induced urokinase-type plasminogen activator (uPA) mRNA in LLC-PK cells. uPA mRNA became highly stable by pretreatment with either cycloheximide or pactamycin, and the stabilizing effect of cycloheximide treatment was time dependent with the full effect exerted by 60 min. Stabilization was also observed with histone H4 mRNA but only partially with c-myc mRNA. To further analyze, we developed a cell-free decay reaction system based on post-mitochondrial supernatant (PMS). In this system, uPA mRNA was completely stable when fractions were obtained from cells pretreated with cycloheximide, but very unstable in control fractions, paralleling uPA mRNA stability in intact cells. However, in contrast to uPA mRNA and the in vivo observation, histone H4 mRNA was unstable whether or not the cells were pretreated with cycloheximide. These results suggest that inhibition of protein synthesis stabilizes mRNAs in at least two different ways in LLC-PK1 cells. When PMS from cycloheximide/calcitonin-treated cells was mixed with PMS from untreated cells, uPA mRNA was not destabilized. This suggests that a putative labile factor responsible for uPA mRNA degradation is not a soluble protein.     

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.     

Mechanism of vesicular stomatitis virus mRNA decay

The chemical and functional stability of the five vesicular stomatitis virus (VSV) messenger RNAs during infection of Chinese hamster ovary (CHO) cells was studied using the temperature-sensitive mutant, tsG114. By incubating infected cells at the nonpermissive temperature (39 °C), RNA synthesis was blocked and the five VSV mRNAs decayed chemically and functionally with a half-life of 1 to 1.5 h. However, all five VSV mRNAs were stable in vivo at 39 °C when protein synthesis was blocked with either cycloheximide or emetine. In contrast, when pactamycin was used to inhibit protein synthesis, the chemical and functional decay rates of the VSV mRNAs were indistinguishable from those observed in the absence of antibiotic. On the basis of the mode of action of each of the antibiotic inhibitors, these data imply that (a) ribosome movement along VSV mRNAs plays no role in their stabilities, and (b) each VSV mRNA contains a nuclease-sensitive site, at its 5′ end at or near the initiation site, which regulates its decay in vivo.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Induction of a proteinase by heat-shock in yeast

Abstract In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCl₂, MgCl₂, or EDTA. The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.     

Effects of cycloheximide on the temperature profile of Sacharomyces cerevisiae

Tmax, the maximum temperature for growth of Saccharomyces cerevisiae, decreased linearly with increasing concentrations of cycloheximide added to the medium, to about 20 degrees C at 2.5 microgram ml-1. In this concentration range thermal death was not enhanced. The Arrhenius plot of growth was shifted to lower temperatures as a function of the cycloheximide concentration and became dissociated from the Arrhenius plot of thermal death. It was concluded that the target site of cycloheximide, the cytoplasmic ribosome, is not identical with the physiological Tmax site of S. cerevisiae and that the binding of cycloheximide to its target sites is strongly enhanced by the temperature.     

Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2.

BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

Degradation of cartilage proteoglycans by a neutral proteinase secreted by rabbit bone-marrow macrophages in culture.

When cultivated together with pieces of cartilage biosynthetically labelled with 35S in their proteoglycans, rabbit macrophages, differentiated in vitro from bone-marrow cells, cause the release of soluble 35S-labelled material into the culture medium. This process is inhibited by killing the macrophages or by cycloheximide treatment, and is due to the secretion by the cells of a metal-dependent neutral proteinase capable of degrading cartilage proteoglycan subunits into fragments of high molecular weight. Enzyme activity is optimum at about pH7, and is inhibited by EDTA, o-phenanthroline, cysteine or serum, but not by di-isopropyl phosphorofluoridate nor by 4-hydroxymercuribenzoate. The effect of EDTA is partially reversed by Co2+ or Zn2+ ions. The enzyme is eluted from Sephadex G-150 columns as a single peak of material (apparent mol.wt. 17000) that contains also most of the proteolytic activity exerted by culture media on Azocoll (denatured collagen) or on casein. The possible role of this metalloproteinase in chronic inflammatory processes is discussed, particularly in connection with joint erosions in rheumatoid arthritis.     

POLYSOMES OF THE SEA URCHIN EMBRYO: AN IMPROVED METHOD FOR EXTRACTION OF INTEGRATED POLYSOMES

Suitable conditions for extracting integrated polysomes from embryos of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus were investigated.
Integrated polysomes could not be extracted under the conditions reported by other investigators. It was found, however, that use of 5 m m MgCl₂, 0.30 m KCl, 0.5 m m EDTA and 2 m m cycloheximide was effective for maintaining the integrity of polysomes. At higher concentrations of Mg²⁺, and even at higher concentrations of K⁺, monosomes and polysomes aggregated to form polysome-like particles which had sedimentation patterns with a small amount of nascent peptide. Thus, a medium consisting of 0.05 m Tris-HCl buffer, pH 7.5, 0.30 m KCl. 5 m m MgCl₂, 0.5 m m EDTA-2K, 2 m m cycloheximide, 5 m m mercaptoethanol and 0.5% (v/v) Nonidet P-40 is concluded to be the most suitable for extraction of sea urchin polysomes. Under the conditions used EDTA did not suppress polysome degradation completely and their degradation was linear with time.     

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.     

Characteristics of the macrophage uptake of proteinase-alpha-macroglobulin complexes.

Complexes formed between labelled proteolytic enzymes (trypsin, subtilopeptidase A) and the alpha-macroglobulins of plasma are rapidly and selectively taken up by rabbit alveolar macrophages. The uptake occurs over a narrow zone of pH. Kinetics of the uptake is affected by temperature; in particular, incubation of macrophages at 37 degrees C before the addition of the labelled complex reduces the capacity to take up complexes. EDTA prevents the association of labelled complexes with macrophages, and can dissociate previously bound label. The effect of EDTA is reversed by the addition of calcium or magnesium or both. Iodoacetamide does not prevent the uptake of complexes but causes them to remain available for dissociation from the cells by EDTA. Incubation of complexes with macrophages at 37 degrees C with no iodoacetamide results in the appearance of trichloroacetic acid soluble products of the enzyme in the supernatant fluid. These observations indicate that the selective uptake of proteinase-alpha-macroglubin complexes by rabbit alveolar macrophages can be resolved into three phases: (1) membrane binding which depends upon divalent cations and is pH sensitive, (2) endocytosis inhibitable by iodoacetamide and (3) temperature-dependent hydrolysis of the contained labelled enzyme.     

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.     

Protein synthesis by synaptosomes from rat brain. Contribution by the intraterminal mitochondria

(1) The characteristics of protein synthesis in microsomal and synaptosomal fractions from rat brain were examined. A high sensitivity to ribonuclease and to cycloheximide, and the need for the presence of pH5 enzymes distinguished protein synthesis in microsomal fractions from protein synthesis in synaptosomes. (2) Under various conditions of incubation synaptosomal fractions prepared in sucrose showed limited protein synthesis compared with synaptosomal fractions prepared by using Ficoll. Such discrepancies could not be attributed to: (i) animal age, (ii) the metabolic state of the synaptosomal fraction, (iii) the absence of bivalent cations in the incubation medium or (iv) the temperature. (3) Protein synthesis in synaptosomal fractions was inhibited 50-65% by cycloheximide, 38-50% by chloramphenicol, 95% by puromycin, 70% by azide and 40% by deoxyglucose; ribonuclease had only a negligible inhibitory effect. (4) As a first approximation to the localization of the protein-synthetic machinery present in the synaptosomal fraction, the distribution of enzymes and radioactivity in subfractions of prelabelled synaptosomes was determined after osmotic shock with water. Approximately 60% of the total protein synthesis in the synaptosomal fraction occurred in the intraterminal mitochondria. (5) Protein synthesis in the intraterminal mitochondria did not show any fundamental difference from synthesis in somatic mitochondria, with respect to inhibition by cycloheximide and chloramphenicol. (6) It was concluded that if extramitochondrial protein synthesis occurs in synaptosomes, it must be very low.