Postribosomal Supernatant Fraction Research Articles

Azetidine-induced Accumulation of Class I Small Heat Shock Proteins in the Soluble Fraction Provides Thermotolerance in Soybean Seedlings

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

Actin—an inhibitor of eukaryotic elongation factor activities

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger. Some component(s) in the postribosomal supernatant (S-130) fraction and also DNase I prevent actin-promoted inhibition of polyPhe synthesis.

Overexpression and Purification of Elongation Factor 3 fromSaccharomyces cerevisiae

The translational elongation factor 3 (EF-3) fromSaccharomyces cerevisiaewas overexpressed and purified to near homogeneity from the post-ribosomal supernatant fraction (S-100). A detailed protocol for the isolation of overexpressed EF-3 is presented. The procedure involves ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose and affinity chromatography on ATP-agarose. A protein purity of ≥96% was established by quantitating the silver-stained SDS/polyacrylamide gel. The present method facilitates isolation of EF-3 in large amounts in high yield.

A Reevaluation of the Cap-binding Protein, eIF4E, as a Rate-limiting Factor for Initiation of Translation in Reticulocyte Lysate

The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (approximately 75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported. The association of a small proportion of eIF4E with the ribosome fraction appears to be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate.

Soluble Factor Requirements for the Tetrahymena Peptide Elongation System and the Ribosomal ATPase as a Counterpart of Yeast Elongation Factor 3 (EF-3)1

Peptide elongation factor 3 (EF-3), which is widely present in yeasts and fungi (Eumycota), does not occur in another lower eukaryote, the unicellular protozoan Tetrahymena pyriformis, as was shown by the following findings: (a) there is no activity to satisfy the EF-3 requirement of yeast ribosomes in the post-ribosomal supernatant fraction from Tetrahymena, and (b) the Tetrahymena ribosomes displayed their full capacity for polyphenylalanine synthesis with purified EF-1 alpha and EF-2 alone from either Tetrahymena or yeast, and their activity on the Tetrahymena ribosomes was not further enhanced by the addition of yeast EF-3, in contrast to the case of the yeast ribosomes. However, as a substitute for the ribosome-activated nucleotidase activity of EF-3, Tetrahymena ribosomes were shown to harbor strong, firmly bound ATPase and GTPase activities, which probably involve the same active site. The ribosome-bound ATPase activity was inhibited by a polyclonal antibody raised against yeast EF-3 with the same inactivation profile as that of polyphenylalanine synthesis on Tetrahymena ribosomes, indicating that the ribosomal ATPase plays an essential role in the elongation process on Tetrahymena ribosomes as previously revealed in the yeast system. It was also shown that the ribosomal nucleotidase plays a pivotal role in the elongation cycle in other eukaryotes.

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria.

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.

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

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

Mitochondrial and cytoplasmic fumarases in Saccharomyces cerevisiae are encoded by a single nuclear gene FUM1.

Respiratory defective pet mutants of Saccharomyces cerevisiae assigned to complementation group G5 are deficient in fumarase. A representative mutant from this complementation group was used to clone a nuclear gene (FUM1) whose sequence encodes a protein homologous to bacterial fumarase. Based on the primary structure homology and the elevated levels of fumarase in transformants harboring FUM1 on a multicopy plasmid, this gene is concluded to code for yeast fumarase. In wild type yeast, fumarase is detected in both mitochondria and the soluble postribosomal protein fraction. Several lines of evidence indicate that the two compartmentally distinct fumarases are isoenzyme products of FUM1. Mutations in FUM1 simultaneously abolish both activities. Transformation of a fumarase mutant with a plasmid containing FUM1 leads to increased fumarase activity in mitochondria and in the postribosomal supernatant fraction. Transformation of the same mutant with a plasmid construct in which the region of FUM1 coding for the amino-terminal 17 amino acids of fumarase is deleted results in a preferential increase of nonmitochondrial fumarase. Northern and S1 nuclease analysis of fumarase transcripts in wild type yeast and in a mutant transformed with FUM1 on an episomal plasmid indicate that the gene is transcribed from multiple start sites, some of which are located inside the coding sequence. The major transcript presumed to code for mitochondrial fumarase has a 5'-untranslated leader of 185 nucleotides. The most abundant shorter transcripts have 5' termini from 57 to 68 nucleotides downstream of the first ATG; their translation products lacking the amino-terminal mitochondrial import signal are proposed to target fumarase to the cytoplasm.

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.

Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor.

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.

Characterization of a translational inhibitor isolated from rabbit brain following intravenous administration of d-lysergic acid diethylamide.

Intravenous administration of d-lysergic acid diethylamide (LSD) to rabbits results in a transient inhibition of brain protein synthesis in vivo and in vitro. A translational inhibitor that appears in the postribosomal supernatant fraction of cerebral hemispheres following LSD administration was partially purified by gel filtration on Sephadex G-150 and precipitation with 60% ammonium sulfate. This inhibitor, which was proteinaceous, reduced the translational capacity of an initiating cell-free protein synthesis system derived from brain. It also inhibited a messenger RNA-dependent reticulocyte lysate programmed with brain polysomes and a globin-synthesizing reticulocyte lysate system. Addition of the partially purified inhibitor to a brain cell-free protein synthesis system resulted in the decreased formation of ternary complexes as well as 40 and 80S initiation complexes, suggesting that the inhibitor affects an early step in the initiation of protein synthesis in brain.

Immunological detection of the messenger RNA cap-binding protein.

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.

Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000).

A ribosomal subunit antiassociation activity has been purified from both the postribosomal supernatant and ribosomal salt-wash protein fractions of rabbit reticulocyte lysates. A majority (greater than 90%) of the activity is associated with a low molecular weight protein of Mr of approximately 25,000. A small but significant level of antiassociation activity (less than 10%) was found to be associated with higher molecular weight protein fractions. The purified 25,000-dalton antiassociation factor interacts with 60 S ribosomal subunits to prevent them from reassociating with 40 S ribosomal subunits. The factor does not seem to interact directly with 40 S subunits nor does it dissociate 80 S monosomes. The properties of this factor are thus similar to the eukaryotic initiation factor 6 isolated from both wheat germ and calf liver extracts.

Purification to homogeneity and partial characterization of a 56,000-dalton protein phosphatase from rabbit reticulocytes.

A 56,000-Da peptide with inherent protein phosphatase activity was isolated from the postribosomal supernatant fraction of rabbit reticulocytes. The peptide appears to form complexes with other proteins that are present in crude fractions. It exhibits atypical retention on steric exclusion columns during high performance liquid chromatography, an unusual characteristic that facilitated its isolation. The protein phosphatase activity of the 56,000-Da peptide is dependent on Mn2+ ions, but is not activated by either the FA, ATP/Mg2+ protein phosphatase activator system or by proteolysis. The protein phosphatase activity of the peptide is increased 3-fold or more by the antigen peptides described in the accompanying paper (Fullilove, S., Wollny, E., Stearns, G., Chen, S.C., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2493-2500).

Purification of the messenger RNA cap-binding protein using a new affinity medium.

The p-aminophenyl gamma-ester of 7-methylguanosine 5'-triphosphate (m7GTP) was synthesized and coupled to Sepharose 4B. A 0.5 M salt extract of rabbit reticulocyte ribosomes was passed over a column containing the affinity medium. After extensive washing, a solution of m7GTP was passed through the column, and a single polypeptide species of 24 kilodaltons (kDa) was eluted. This had an electrophoretic mobility identical with that of the mRNA cap-binding protein. This assignment was confirmed by the fact that the eluted material was enriched nearly 200-fold in the ability to specifically bind 32P-labeled capped oligonucleotides. A control affinity medium consisting of GTP similarly coupled to Sepharose failed to retain the 24-kDa species. The postribosomal supernatant fraction yielded slightly more of the 24-kDa species than the ribosomal wash fraction when passed over this affinity medium.

Effect of temperature on ppp(A2′ p) nA binding protein activities in rabbit reticulocyte lysates and other mammalian extracts

Lysates of rabbit reticulocytes and other mammalian cells are known to contain an activity which binds with high specificity ppp(A2′p) 3A,3′-[ 32P]pCp. The binding activity shows a marked dependence on preincubation of lysates at different termperatures (4°C – 45°C). For example, binding was increased 50% by preincubation of rabbit reticulocyte lysates at 37°C for 60 minutes. An identical preincubation of mouse brain extracts results in a greater than 90% loss of binding activity. Fractionation of rabbit reticulocyte lysates into the postribosomal supernatant (PRS) and the ribosomal salt wash (RSW), followed by heparin-agarose column chromatoraphy, showed that with the PRS fraction, most of the binding activity is eluted with 600 mM KCl. With the RSW fraction, more than 50% of the binding activity is eluted with 250 mM KCl. These data suggest that multiple ppp(A2′p) nA binding protein activities exist in mammalian cells.

Inhibition of translation in lysates of mouse L cells infected with vesicular stomatitis virus: presence of a defective ribosome-associated factor

Lysates of L cells infected for 4 h with vesicular stomatitis virus were inhibited in their in vitro translational activity to about the same extent as protein synthesis was inhibited in vivo in infected L cells. Inhibition of translation occurred at the level of the ribosome as determined by reciprocal cross-reconstitution studies with polyribosomes and postribosomal supernatant fractions isolated from virus-infected and mock-infected cells. Inhibition of protein synthesis in reconstituted lysates of virus-infected cells was found to be at the level of initiation of translation as evidenced by reduction in incorporation into acid-precipitable proteins of formylatable [35S]methionine. Ribosomes from virus-infected and mock-infected cells were exposed to 0.5 M KCl and fractionated by centrifugation into salt-washed polyribosomes and supernatant fractions containing ribosome-associated proteins; reciprocal reconstitution of translational activity by a mixing of salt-washed polyribosomes and ribosome-associated proteins revealed that the defect in initiation of translation was in the ribosome-associated proteins released by salt wash from the infected-cell ribosomes. Differential ammonium sulfate precipitation of the supernatant ribosome-associated proteins from virus-infected and mock-infected cells indicated by reciprocal reconstitution studies that the defective ribosomal initiation factor(s) was (were) present primarily in the 0-40% ammonium sulfate fraction that is considered to contain primarily eIF-3 and eIF-4B. These results are similar to those found in earlier studies of defective initiation factors responsible for impaired protein synthesis in cells infected with plus-strand viruses quite different from the rhabdovirus studied in these experiments.

Evidence for non-coordinated synthesis of 5 S RNA in the thermophilic fungus,Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.

Occurrence of aminoacyl-tRNA synthetase complexes in calf brain

Eighteen aminoacyl-tRNA synthetases of the postribosomal supernatant fraction of brain cortex were characterized by glycerol density gradient centrifugation and gel filtration analysis. On the basis of sedimentation properties and gel elution profiles, four groups of enzyme activities were determined in the postribosomal supernatant fraction; the first group sedimenting at about 6 S contained 18 individual synthetase activities, the next successive groups of greater molecular sizes contained synthetase complexes, and the last group possessed activities of 15 synthetases. Each aminoacyl-tRNA synthetase appeared at least in two forms: free and bound in complexes of varying sizes and different enzyme compositions. Conventional purification methods of lysyl-tRNA synthetase from the post-ribosomal supernatant fraction of brain cortex gave a preparation containing four groups of aminoacylation activities. The obtained preparation contained a large complex, reduced number of intermediate complexes and some individual synthetases.

Effect of intravenous administration of d-lysergic acid diethylamide on subsequent protein synthesis in a cell-free system derived from brain.

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of d-lysergic acid diethylamide (LSD) to rabbits induced a transient inhibition of translation following a brief stimulatory period. Subfractionation of the brain cell-free system into postribosomal supernatant (PRS) and microsome fractions demonstrated that LSD in vivo induced alterations in both of these fractions. In addition to the overall inhibition of translation in the cell-free system, differential effects were noted, i.e., greater than average relative decreases in in vitro labeling of certain brain proteins and relative increases in others. The brain proteins of molecular weights 75K and 95K, which were increased in relative labeling under conditions of LSD-induced hyperthermia, are similar in molecular weight to two of the major "heat shock" proteins reported in tissue culture systems. Injection of LSD to rabbits at 4 degrees C prevented LSD-induced hyperthermia but behavioral effects of the drug were still apparent. The overall decrease in cell-free translation was still observed but the differential labeling effects were not. LSD appeared to influence cell-free translation in the brain at two dissociable levels: (a) an overall decrease in translation that was observed even in the absence of LSD-induced hyperthermia and (b) differential labeling effects on particular proteins that were dependent on LSD-induced hyperthermia.