Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Free ribosomes and growth stimulation in human peripheral lymphocytes: Activation of free ribosomes as an essential event in growth induction

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.     

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.     

Stored and polysomal ribosomes of mouse ova

RNP particles of ovulated mouse ova, labeled by exposure of growing oocytes to [³H]uridine, were displayed on sucrose gradients. Under standard salt conditions, radioactivity was observed coinciding with liver ribosomal subunits, monomers, and polysomes. The RNA from each region of the gradient was isolated and was found to contain the expected species of labeled 18S and/or 28S ribosomal RNA. Heterogeneous RNP particles were widely distributed in the gradient. From data on RNase sensitivity and resistance to dissociation in high salt, it was estimated that 20–25% of the total ribosomes were in polysomes. No difference in the distribution was observed when ribosomes were labeled in the early or late growth phase of the oocyte. The evidence suggested that the nonpolysomal subunits and monomers were unable to form a high salt-stable complex in the presence of poly(U) and factors for protein synthesis. Thus, the bulk of the ribosomes are inactive in protein synthesis in ovulated ova and are apparently stored for use in embryonic development.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

Isolation of stable ribosomal subunits from spores of Bacillus cereus.

Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction.     

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.     

Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.     

Starvation-induced alterations of ribosomal protein phosphorylation in Bombyx mori L. Evidence for different phosphorylation kinetics in free and membrane-bound ribosomes

In the posterior silk gland of Bombyx mori, ribosomal protein S1, homologous to S6 in mammals, is partially phosphorylated in a normally fed animal. Before the first meal of the fifth larval instar, S1 is completely dephosphorylated. Likewise, starvation induces rapid dephosphorylation of the protein in both free and membrane-bound ribosomes. Upon refeeding after 48 h of starvation, S1 becomes phosphorylated again, first on membrane-bound ribosomes, then on free ribosomes, with a lag time of about 3 h. Following 48 h of refeeding, the most highly phosphorylated form of S1 predominates in both populations of ribosomes. These variations in phosphorylation are correlated with the level of protein synthesis in the posterior silk gland, 70% of the ribosomes occurring in polysomes upon feeding and only 30% upon starvation [Prudhomme, J.-C. & Couble, P. (1979) Biochimie (Paris) 61, 215-227]. After in vivo 32P labelling, the phosphopeptides of S1 from free and membrane-bound ribosomes were found to be identical and phosphoserine (only) was found in each S1. These results suggest the involvement of S1 phosphorylation in the regulation of protein synthesis at the translational level and the existence of at least two different pathways controlling this phosphorylation: one for the free ribosomes, the other for the membrane-bound ribosomes.     

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [³⁵S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [³⁵S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.     

Mechanism of protein synthesis inhibition during stationary phase in Bacillus stearothermophil US

Summary The appearance of a protein (association factor I) in ribosomes from Bacillus stearothermophilus at stationary phase of growth is described. Association factor I is present on 30S subunits and 30S–50S ribosomal couples, but not on 50S subunits. This protein is responsible for the low levels of polyphenylalanine synthesis shown by stationary phase ribosomes. Association factor I is able to bind to free 30S–50S ribosomal couples but not to polysomes, and exerts its effect by inhibiting the initiation step of protein synthesis. Ribosomes preincubated with association factor I have a decreased ability for polypeptide snythesis directed phage mRNA or poly(U).     

Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

Expression and functional assembly into bacterial ribosomes of a nuclear-encoded chloroplast ribosomal protein with a long NH2-terminal extension

Chloroplast ribosomal protein L13 is encoded in the plant nucleus and is considerably larger than its eubacterial homologue by having NH2- and COOH-terminal extensions with no homology to any known sequences (Phua et al., J Biol. Chem. 264, 1968-1971, 1989). We made two gene constructs of L13 cDNA using the polymerase chain reaction (PCR) and expressed them in Escherichia coli. Analysis of the ribosomes and polysomes from these cells, using an antiserum specific to chloroplast L13, shows that the expressed proteins are incorporated, in the presence of the homologous E. coli L13, into functional ribosomes which participate in protein synthesis (i.e. polysomes). Evidence is obtained that the large NH2-terminal extension probably lies on the surface of these 'mosaic ribosomes.' This first report of the assembly into E. coli ribosomes of nuclear-coded chloroplast ribosomal protein with terminal extensions thus suggest an extraordinary conservation in the function of eubacterial type ribosomal proteins, despite the many changes in protein structure during their evolution inside a eukaryotic system.     

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.