Polysomal Populations Research Articles

Deciphering the effect of Endoplasmic Reticulum (ER) stress on near‐mitochondrial localized translation

Local synthesis of proteins near their active site is a critical cellular phenomenon. Recent studies have demonstrated that a subpopulation of cytoplasmic ribosomes is localized to organelles such as the endoplasmic reticulum and mitochondria. Due to the presence of different membranous compartments within eukaryotic cells, proper mRNA localization or targeted translation is required to focus the expensive process of translation to where it is needed most. During stress while global translation has been downregulated translation of stress responsive proteins as well other essential proteins remains active. During ER stress, substantial amounts of remodeling happen near ER localized translation populations. Due to its physical and functional relation with mitochondria it is also essential to regulate the translation process of mitochondrial destined proteins during the ER stress condition.In the current study, a digitonin based extraction method has been successfully established to separate the free cytoplasmic ribosome and mitochondria bound ribosome from HEK293 cells and to perform ribosome profiling in order to capture the actively translating polysome populations of free and mitochondria bound translating ribosome populations. This method can successfully separate the two ribosomal populations and it is enriched with known near‐mitochondria localized ribosome. Previous studies identified a population of mRNAs which are translating near mitochondrial surface in yeast and most of them has been found to be conserved in higher organisms. Interestingly, a set of known near mitochondria localized RNA has been found to involve in active translation in our global riboseq data collected from Hela upon ER stress. It indicates a correlation between the near mitochondrial localized translation and ER stress regulation. Specifically, higher expression of different sub units of oxidative phosphorylation complexes has been found to be enriched upon ER stress. This motivates further detailed study to understand organeller cross talk at translation level during stress. Understanding the near mitochondria localized populations in presence and absence of ER stress in higher organisms would be helpful to understand the complex ER‐mitochondrial cross talk events.Support or Funding InformationNIHR01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Formation and stability of polysomes and polysomal populations in roots of germinating seeds of soybean (Glycine max L.) under cold, osmotic and combined cold and osmotic stress conditions

Abiotic stress factors such as extreme temperatures or osmotic stress are among the major causes of inferior crop yields. In response to a stress, plants have evolved various defense mechanisms. In our study, we have demonstrated how cold stress, osmotic stress and a combination of both stresses retard the growth of roots and inhibit the process of ribosomes binding into polysomes. The tested stresses also limited the ability of root tissues to synthesize proteins. At the same time, most of the analyzed samples were found to contain elevated shares of the fractions of cytoskeleton-bound polysomes (CBP, CMBP) in the total population of polysomes. Using a polysome-based degradation system, it was shown that polysomes formed under stress conditions were much more resistant to the effect of exogenous ribonuclease than the control ones. The highest tolerance to digestion was demonstrated by the cytoskeleton-bound (CBP) and cytoskeleton-membrane bound polysomes (CMBP). The increasing share of CBP and their stability in roots of seeds germinating under stress conditions can be a target for physiological regulation. It seems that modifications in the stability and percentages of particular polysomal populations play an important role in the adaptation of plants to stress conditions, which may indicate that these forms of polysomes, i.e., cytoskeleton-bound ones, are involved (via selective translation) in the synthesis of stress proteins in soybean roots.

RNA complexity during seed formation and germination.

ABSTRACT The sequence composition and quantity of cotton cotyledon mRNAs present after 24 h of germination were evaluated by physical techniques, in vitro translation in wheat germ extracts, and hybridization with complementary DNA. The results indicate that most of the 25,000 individual mRNAs exist in both the poly(A)plus mRNA and the poly(A)minus mRNA populations in similar concentrations. Polysomal and nonpolysomal mRNA populations also appear to be virtually identical. The cotyledon mRNAs present during cotyledon development were compared by hybridization of poly(A)plus mRNA from each of three stages with DNA complementary to RNAs present in the other stages. Dramatic changes in the concentration of most of the abundant mRNA sequences and many of the less abundant mRNA sequences occur during cotyledon development. Most of the about 25,000 rare mRNA sequences appear to be common to all three stage mRNAs. A summary is presented of the mRNA sequences in other plant issues, as reported from other laborato...

Ribosome Fractionation in Yeast

This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1).

Effect of osmotic stress on in vitro translational capacity of polysomes and on the composition of polysome-associated proteins in germinating seeds of pea (Pisum sativum L.)

Plant growth throughout the world is often limited by unfavourable environmental conditions. This paper reports results of a study on long- and short-term osmotic stress (−0.5 MPa) followed by a recovery on in vitro translational capacity of polysomes and on the composition of polysome-associated proteins in germinating pea (<em>Pisum sativum </em>L.) seeds. Here we show that, under osmotic stress, cytoskeleton-bound polysomes were charaterized by the highest translation activity, which may be indicative of an important role that this population of polysomes plays in the synthesis of the so-called “stress proteins”. We also find out that in response to osmotic stress, new proteins (22.01, 96.47 and 105.3 kDa), absent in the unstressed sample, associated with the total pool of polysomes, whereas the protein of 22.95 kDa, which was present in the embryonic tissue of seeds germinating under unstressed conditions, disappeared. These changes may have affected both the stability and the translational capacity of polysomes.

Effect of osmotic stress on the formation of a population of polysomes and their stability in pea (Pisum sativum L.) seeds

Plants growing under natural conditions are constantly exposed to various stress factors, which can restrain their productivity and limit yields. This paper deals with the effect of long- and short-term osmotic stress followed by recovery on the formation of polysomes and their stability during germination of pea (Pisum sativum L.) seeds. By isolating polysomes, it is possible to obtain an index which evidences the ability of tissues to synthesize proteins. Changes in the distribution of polysomes often precede measurable changes in amounts of proteins. Under osmotic stress, the dominant population of polysomes was the population of free polysomes (FP). The share of membrane-bound polysomes (MBP) and cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP) in the total fraction of ribosomes increased under intensive (−1.0 and −1.5 MPa) osmotic stress. These results can suggest that the bound forms of polysomes play an important role in the synthesis of stress proteins. In addition, the stability of polysomes isolated from pea early seedlings growing under unstressed control and osmotic stress conditions was tested. It turned out that polysomes formed under osmotic stress conditions (especially the CMBP) were more resistant to the activity of exogenous ribonucleases than the polysomes in the control samples. Under stress conditions it is highly likely that ribosomes become more densely packed on mRNA thus making it more resistant to ribonuclease. This is just one of the many mechanisms regulating stability of mRNA.

Polysome trafficking of transcripts and microRNAs in regenerating liver after partial hepatectomy

Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysomal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs.

Application of a Translational Profiling Approach for the Comparative Analysis of CNS Cell Types

Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.

Exogenous abscisic acid increases stability of polysomes in embryos of triticale caryopses during germination

Some posttranslational processes that occur in embryos of germinating triticale caryopses treated with different concentrations of abscisic acid (ABA) were examined. ABA increased the ratio of cytoskeleton-bound polysomes in the total population of polysomes and depressed the share of free and membrane-bound polysomes. Using exogenous RNase, stability of the total polysomal population as well as each polysomal fraction was investigated. The total extractable polysomes isolated from embryonic tissues of germinating triticale caryopses treated with ABA were more stable than the polysomes isolated from the control sample caryopses. The contribution of the polysomes that were not digested by RNase was increased by higher concentrations of ABA applied during germination. At high concentrations of ABA (50, 100 µM), the quantitative contribution of polysomes in the total ribosomal fraction was almost 100% of the amount of polysomes before digestion and the modifications observed consisted mainly of the shift of the so-called heavy polysomes towards light polysomes, containing a few ribosomes. Within each polysomal population, cytoskeleton-bound polysomes (CBP and CMBP) were the most stable, which may imply that the bonds between polysomes and these protein filaments, created in all eukaryotic cells increased their stability. It is assumed that mRNAs are stabilised or destabilised by interaction of proteins with their various sequences. A plant hormone may depress or elevate the quantities of these proteins, thus regulating the stability of different mRNAs. The results confirm the multi-faceted mechanism of ABA-induced response, where one of the constituents is the effect of ABA on the stability of mRNAs molecules. The co-ordinated regulation of mRNAs synthesis and their stability provide plants with improved adaptability.

Water-deficit-induced changes in cytoskeleton-bound and other polysomal populations in embryonic tissue during triticale caryopsis germination

We studied the influence of water-deficit stress on the process of formation of different polysomal populations, their abundance and stability in embryonic tissue during triticale caryopsis germination. Osmotic stress retarded the ability of seeds to germinate and decreased the content of the total ribosomal fraction in embryos. In control samples, the fraction of free polysomes was the most abundant and this population of polysomes decreased sharply in osmotic stress conditions. Water-deficit stress applied during germination profoundly changed the proportions between different polysome populations in the total ribosomal fraction of embryonic tissue. The predominant population in these conditions was the cytoskeleton-bound fraction. This may indicate an important role for cytoskeleton-bound polysomes in the synthesis of stress-induced proteins. We hypothesize that there must be an active mechanism of translational control that permits specific proteins to be synthesized despite a reduction in total protein synthesis.

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 µM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.

Plant polysome binding to the actin cytoskeleton as a target for physiological regulation

One of the principal functions of the actin cytoskeleton is to serve as a support for diverse physiological and biochemical processes, protein synthesis in particular. About 25 years ago, in experiments with cultured animal cells, it was discovered that most mRNAs, ribosomes, and protein synthesis factors were attached to the actin microfilaments (Lenk et al., 1977). Later, a similar phenomenon was demonstrated for plant tissues (Davis et al., 1991; Zak et al., 1995). The physiological basis for such an attachment is evident in some cases but obscure in others. Attachment of polysomes to the microfilaments might provide for the correct spatial arrangement of the translational machinery and protein synthesis activation. If the binding of polysomes to the cytoskeleton is considered an additional level of protein synthesis regulation, its physiological control is expected but is not shown, at least for plant cells. To evaluate the proportion of the cytoskeleton-bound polysomes (CBP) in the total polysome population, we used the differential sensitivity of membranes and cytoskeleton to nonionic detergents that destroy membranes but leave microfilaments intact. A difference in the polysome peak areas in sucrose density gradients of plant tissue homogenates treated and nontreated with Triton X-100 was a rough measure for the amount of CBP (stretched areas in Fig. 2). * Corresponding author. Tel.: +7-95-432-0233; fax: +7-95-977-8018.

Annexin II is associated with mRNAs which may constitute a distinct subpopulation

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of 32P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)+-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.

Annexin II is associated with mRNAs which may constitute a distinct subpopulation

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.

Significant role for polysomes associated with the cytoskeleton in the control of protein synthesis during germination of triticale caryopses in the presence of abscisic acid

The influence of abscisic acid (ABA) on the process of polysome formation and synthesis of newly-formed proteins by different polysome populations was studied. Triticale caryopses were germinated in water or various ABA concentrations for 48 hrs, and afterwards they were transferred to a solution of 14C-amino acids and germinated for an additional 30 min. Embryos were separated from caryopses, and four polysome populations were isolated: the FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). ABA retarded both the process of polysome formation and their activity in forming new proteins in vivo in all studied fractions. Participation of polysomes in total ribosomal materials (sub-units, monosomes and polysomes) of each polysome population in the control sample was as follows: FP — 77; MBP — 72; CBP — 70 and CMBP — 66 %, whereas in sample treated by ABA (100 µM) it was accordingly: 17; 23; 27 and 28%. The largest population made up FP (in control sample 69%), participation of MBP was always lower and ranged from about 19 to 30 %. Participation of polysome populations bound with the cytoskeleton CBP and CMBP, both in control sample as well as in samples treated with 1 and 10 µM ABA solution, was only a few per cent. It should be noted that when the ABA concentration was higher (100 µM) (process of germination was strongly inhibited), participation of those two populations (CBP and CMBP) was much increased in embryos, respectively to about 18 and 20 %. In both the control group and in embryonal tissue treated with ABA increasing incorporation of radioactive precursors to newly-formed proteins in vivo in fractions of polysomes isolated by following buffers: C (FP), C + PTE (MBP), C + Tris (CBP) and buf. U (CMBP) was observed. It should be noted, that the biggest incorporation of 14C-amino acids into nascent polypeptide chains was found in the last polysome population (CMBP). In the sample treated with ABA (100 µM) the activity of this fraction (CMBP) in forming new proteins is several times, and in the case of FP dozens of times, more intense. Increased participation of CBP and CMBP in embryos of triticale caryopses treated with ABA (100 µM) and the largest incorporation of 14C-amino acids into nascent polypeptide chains synthesised by CMBP, may indicate the important role of proteins formed by polysomes associated with cytoskeleton in inhibition of germination and seedling growth by ABA.

Apomixis in plants: structural and functional aspects of diplospory inPoa nemoralis andP. palustris

Data on structural and functional aspects of mitotic diplospory and later stages of apomictic seed formation are reported forPoa palustris andP. nemoralis. In this study, the plant material of two Russian populations ofP. nemoralis andP. palustris were used for transmission electron microscope observations. Seed formation was investigated by phase contrast microscopy in two populations ofP. nemoralis collected in The Netherlands. The processes of transformation of the megasporocytes to the megaspore mother cells of diplosporous embryo sacs, and thereafter to one- and two-nucleate diplosporous embryo sacs (Antennaria type) were characterized by an increase of cell size, structural and functional reorganization of the nucleus, nucleolus, and cytoplasm, and cell isolation as a result of thickening of the cell wall. These were accompanied by an increase in the cell metabolic activity inferred from visual evidence of the activation of nucleus, nucleolus, endoplasmic reticulum, dictyosomes, mitochondria, and from the appearance of a dense population of ribosomes and polysomes. The diplosporous embryo sac of the Antennaria type was characteristic for bothP. nemoralis andP. palustris. No signs of the presence of synaptonemal complexes were observed during the process of diplosporous-embryo-sac megaspore mother cell differentiation and division. About 90–95% of the diploid egg cells of diplosporous embryo sacs were able to produce apomictic embryos. These embryos developed before anthesis. However, many of them degenerated at the globular stage because of lack of endosperm. The ultrastructural events occurring during the process of diplospory of apomictic species, and meiosis and megagametogenesis of sexually reproduced plants are discussed.

Changes in Protein and Amino Acid Levels during Growth and Senescence of Nicotiana rusticaCallus

Summary The degradation of protein, changes in the polysome population and amino acid levels were investigated during the growth and senescence of tobacco ( Nicotiana rustica L. cv. Gansu Yellow Flower) callus. During the growth period, the synthetic rate of protein increased concommitantly with the elevation of proteinase activity. After the onset of callus senescence, a dramatic decline in the polysome population was observed after day 25. The activity of proteinase was markedly enhanced between day 20 and day 25, and then decreased. The activity of aminopeptidase was low throughout the growth period and the early stage of senescence, and eventually decreased during the late stage of senescence. Therefore, it is suggested that the loss of proteins is not mainly due to the role of aminopeptidase but that of proteinase. Free amino acids accumulated rapidly during senescence. The levels of 14 free amino acids, except for alanine and cysteine, increased significantly after the onset of senescence.

The effect of insulin on proteins associated with free, cytoskeletal-bound and membrane-bound polysome populations.

It is known that insulin treatment increases the rate of protein synthesis in many cells and tissues and that it causes changes in the distribution of ribosomes between free (FP), cytoskeletal-bound (CBP) and membrane-bound polysome (MBP) populations. This paper concerns an analysis of the pattern of proteins in high-salt extracts of FP, CBP and MBP isolated from Krebs II ascites and MPC-11 cells. A combined detergent/salt extraction procedure was used to isolate the three fractions of polysomes from control cells and from cells following short-term stimulation with insulin. There were differences in the protein patterns in the individual fractions and changes occurred after insulin stimulation.