Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Localization of proteins in the inner and outer membranes of Caulobacter crescentus

Cytoplasmic and outer membranes of Caulobacter crescentus were separated by isopycnic sucrose gradient centrifugation into two peaks with buoyant densities 1.22 and 1.14 g/cm³. These peaks were identified as outer and cytoplasmic membranes by the enrichment of malate dehydrogenase and NADH oxidase in the lower density peak and the presence of flagellin, a cell surface protein, in the heavier peak. The identity of the heavier peak as outer membrane was confirmed by labeling of cells with diazotized [³⁵S]sulfanilic acid, a reagent that does not penetrate intact cells. Under these conditions only outer membrane proteins were substituted by the sulfanilic acid. The distribution of proteins between the cytoplasmic and outer membranes were examined by the analysis of [³⁵S]methionine-labeled membranes by SDS-polyacrylamide and two-dimensional gel electrophoresis. These results showed that the inner and outer membranes contain approximately equal numbers of proteins, and that the distribution of these proteins between the two layers is highly asymmetric. Although many of the proteins could be assigned to one or the other membrane fraction, a number of the outer membrane proteins in the 32 000–100 000 molecular weight range frequently contaminate the inner membrane fractions. The implications of these results for membrane isolation and separation in C. crescentus are discussed.     

The inhibition of protein synthesis by IgG containing anti-ribosome P autoantibodies from systemic lupus erythematosus patients

Autoantibodies found in approximately 15% of patients with systemic lupus erythematosus recognize three 60 S ribosomal phosphoproteins P0, P1, and P2. Fab fragments obtained from sera of these patients inhibited globin mRNA translation in an in vitro protein synthesizing system which was reversed by the addition of excess ribosomes. Further studies suggested that these antibodies bind to ribosomes in the intact cell. Thus, when IgG fractions from these sera were microinjected into cultured human fibroblasts [35S]methionine incorporation into cellular proteins was inhibited.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Preferential release of new outer membrane fragments by exponentially growing Escherichia coli

We have examined whether the outer membrane fragments released by normally growing Escherichia coli contain relatively old or new outer membrane.Double-label experiments show that after incorporation of radioactive leucine into E. coli protein, there is a preferential release of outer membrane material which contains a high percentage of newly labeled protein. This implies that outer membrane fragments are preferentially released from those regions where newly synthesized proteins are inserted into the outer membrane. We estimate that these insertion regions cover no more than 13% of the total outer membrane, and that newly inserted proteins diffuse in the plane of the outer membrane with a diffusion constant ? 5 · 10^?13 cm²/s.     

The presence and synthesis of the NADPH-protochlorophyllide oxidoreductase in barley leaves with a high temperature-induced deficiency of plastid ribosomes

High-temperature-induced deficiency of plastid ribosomes in barley plants (Hordeum vulgare L.) was used as a system for studying the role of the cytoplasm in the synthesis of the NADPH-protochlorophyllide oxidoreductase. The enzyme is present in 33° C-grown plants. The failure of high-temperature-grown plants to accumulate chlorophyll during illumination is not caused by the absence of the protochlorophyllide-reducing enzyme. The synthesis of the NADPH-protochlorophyllide oxidoreductase was studied by feeding [³⁵S]methionine to the seedling and by following the incorporation of the radioactively labeled amino acid into plastid proteins. The NADPH-protochlorophyllide oxidoreductase was labeled in high-temperature-grown barley plants to the same extent as in control plants grown at 25° C. It is concluded that the 36,000-M_r polypeptide of the NADPH-protochlorophyllide oxidoreductase is synthesized outside the plastid on cytoplasmic 80S ribosomes.     

Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [³⁵S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, ³⁵S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.

Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [³⁵S]methionine or ¹⁴C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.

Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.

     

Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [³⁵S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.     

Erythrocyte membrane proteins. Sequential accumulation in the membrane during reticulocyte maturation

Reticulocytes of increasing maturity were separated by dextran gradient centrifugation. The accumulation in the membrane of the anion transport protein and other erythrocyte membrane proteins was studied during reticulocyte maturation by separating reticulocytes after incubation with [³⁵S]methionine. The incorporation of the reticulocyte membrane proteins was shown to be sequential, the anion transport protein being inserted at a very early stage in the cells' maturation.     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Effects of Carboxylemethylation and Polyamine Synthesis Inhibitors on Methylation of Trypanosoma brucei Cellular Proteins and Lipids

ABSTRACT. The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-α-difluoromethylomithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5′-{[(Z)-4-amino-2-butenyl]-methylamino}-5′-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [¹⁵S] or [U-¹⁴C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fraction. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 μM) and sinefugin (2 nM) on [³⁵S] and [U-¹⁴C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U¹⁴C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-¹⁴C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-¹⁴C]methionine as AdoMet. Exogenous AdoMet had no effect on [³⁵S]methionine incorporation. The agents studied are curative for African trypnosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.     

Site of synthesis of the proteins of mammalian mitochondrial ribosomes. Evidence from cultured bovine cells

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeling in the absence of cytoplasmic protein synthesis produced a "blank" fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [35S]methionine into the enumerated mitoribosomal proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.     

The transport of proteins into yeast mitochondria. Kinetics and pools

By double isotope pulse-labeling of yeast cells, we determined the kinetics of labeling at 9 degrees C of total mitochondrial membrane, mitochondrial matrix, and cytosolic proteins, the alpha, beta, and gamma subunits of F1 ATPase, and glyceraldehyde-3-phosphate dehydrogenase. We find that none of the mitochondrial proteins show a lag in the incorporation of label compared to cytosolic proteins. These results argue against the existence in the cytosol of large pools of mitochondrial proteins awaiting transport into the organelle. Cycloheximide addition during the pulse stops [35S]methionine incorporation into mitochondrial membrane and cytosolic proteins rapidly (approximately 1 min) and with identical kinetics. Compared to cytosolic protein, however, there is a persistent incorporation of label into mitochondria after a chase with cold methionine (t1/2 approximately 1.5 min at 9 degrees C) which cannot be accounted for solely by chain completion. We conclude that this continued incorporation reflects some transport process in addition to a completion of a round of translation. When cells are labeled during a synchronous "restart" of protein synthesis, where ribosome run-off from mRNA was first induced either by incubating cells for 4 h at 0 degrees C or by treatment with 5 mM aurintricarboxylic acid, the initial rate of incorporation of label into mitochondrial protein now lags behind that of cytosolic proteins. From these results and those in the accompanying report (Ades, I.Z., and Butow, R.A. (1980) J. Biol. Chem. 255, 9918-9924) we propose that the translation of mRNA specific for mitochondrial proteins takes place in the cytoplasm and that at least a portion of the polysomes are then transported and bind to the outer mitochondrial membrane, followed by completion of translation and transfer of the newly synthesized polypeptides into the mitochondria. From a consideration of all of the available data on protein transport into mitochondria in yeast, we conclude that cytoplasmic polysomes bound to the outer mitochondrial membrane function in the transport of proteins into mitochondria by a process not necessarily mutually exclusive of post-translational transport.     

Nerve-dependent changes in content of ribosomes, polysomes, and nascent peptides in newt limb regenerates.

Regeneration of a newt limb requires a constant supply of adequate amounts of a neuronal contribution at the amputation site. Denervation during the early stages of regeneration precludes its growth and morphogenesis. It has been reported that denervation of a regenerating limb lowers the efficiency of incorporation of radioactive amino acids to 60% of contralateral control levels. To gain more insight into the mechanism responsible for this decrease, we examined the effects of denervation on the size distribution and quantity of regenerate polysomes. We characterized the [³⁵S]methionine-labeled nascent peptidyl-tRNA from polysomes by hydroxyapatite chromatography. Moreover, we show that the labeled nascent peptides on polysomes can serve as a measure to quantitate the relative amounts of ribosomes on polysomes and the relative size of the translational machinery. Thus, we report that [³⁵S]methionine-labeled nascent polypeptides on polysomes from denervated regenerates contain about 48% less radioactivity than those from controls. Despite decreased incorporation of [³⁵S]methionine into nascent peptides, the relative distribution of radioactivity across linear sucrose gradients is not significantly altered by denervation. Studies of polysomes labeled with [³H]uridine prior to denervation indicate that ribosome content is depressed by denervation. Our results suggest that the nerve-dependent decrease in protein synthesis is mediated by decreasing the number of ribosomes active in protein synthesis. In addition, similarities in the ratios of free monosomes to polysomes and the relative size distribution of polypeptides between denervated and innervated regenerates indicate that in denervated regenerates the number of translatable mRNA molecules decreases in a coordinate manner with the number of ribosomes active in protein synthesis.     

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Summary Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mitochondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [¹⁴]leucine and [³⁵]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Some effects of aluminium on rat brain protein synthesis

1. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, 10 or 20 mg Al/kg body weight) every third day from one to four weeks of age.2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [¹⁴C] leucine, [¹⁴C] phenylalanine, or [³⁵S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits.3. The incorporation of [³⁵S]methionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction.4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.     

Cell-free translation of human pheochromocytoma messenger RNA yields protein(s) containing methionine-enkephalin

Cell-free translation of messenger RNA extracted from a human pheochromocytoma yields protein(s) of apparent molecular weight >70,000 daltons which contain the pentapeptide methionine-enkephalin. It is estimated that 0.8–1.0% of the total pheochromocytoma mRNA codes for the methionine-enkephalin-containing protein, based on percent incorporation of [³⁵S]methionine into methionine-enkephalin during cell-free translation. These results demonstrate that human pheochromocytoma mRNA contains the message for a high-molecular weight methionine-enkephalin-containing protein or proteins, presumably the methionine-enkephalin precursor molecule(s).