Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.     

Mechanisms of protein degradation in growing and non-growing L-cell cultures.

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20--30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

Degradation of proteins in steady-state cultures of Escherichia coli.

The degradation of proteins in Escherichia coli was investigated in cells grown under steady-state conditions in a glucose-limited chemostat. During the first 24 h, approximately 25% of pulse-labeled proteins were degraded and after 72 h up to 58% of the proteins were broken down. To examine the stability of subcellular components steady-state cultures were labeled with an initial pulse of [14C]leucine, 24 h were allowed for turnover of these proteins, and the cells were then labeled with a short pulse of [3H]leucine. By this double-label protocol, the labile proteins were preferentially labeled with [H]leucine and had high 3H/14C ratios, while the more stable proteins had lower 3//14C ratios. The 3/-labeled proteins were degraded approximately five times as rapidly as the 14C-labeled proteins in exponentially growing cells. The relative stability of subcellular fractions was determined by comparing their 3H/14C ratios to the ratio of the cells at harvest. The soluble fraction contained the most labile proteins, while the ribosomal and membrane fractions were at least as stable as the average cell protein.     

Degradation of intracellular protein in muscle. Lysosomal response to modified proteins and chloroquine

We previously showed that radioactive N-ethylmaleimide injected intramuscularly reacts with actomyosin and other muscle proteins and that a transfer of these modified proteins to lysosome-rich large granules was associated with their degradation (Gerard, K. W., and Schneider, D. L., (1979) J. Biol. Chem. 254, 11798-11805). We now show that muscle cells, when challenged by an increase in proteins modified with N-ethylmaleimide, can increase degradation by increasing the activities of enzymes involved in protein turnover. Cathepsin B activity increased 2-fold 36 h after injection of N-ethylmaleimide. In contrast, non-lysosomal proteolytic enzymes, calcium-dependent protease, and leucine aminopeptidase, did not significantly increase. Lysosomes are also involved in the degradation of normal muscle proteins labeled with [3H]leucine. Treatment with chloroquine, a known inhibitor of lysosome function, resulted in an inhibition of protein degradation, in an increase of the muscle protein content, and in the accumulation of radioactive proteins in lysosomal fractions. Chloroquine treatment for 2 days led to a 270% increase in cathepsin B and a 160% increase in lysosomal ATPase activities, but only a 30% increase in neutral proteinase activities. These results indicate a role for lysosomes in regulation of protein turnover in muscle.     

Studies on the turnover of plasma membranes in cultured mammalina cells. I. Rates of synthesis and degradation of plasma membrane proteins and carbohydrates.

The rate of turnover of membrane proteins and membrane-bound carbohydrates in exponentially growing and in confluent contact-inhibited cultures of strain MK-2 cells was investigated. Cells were labelled with [14-C]leucine and [3-H]glucosamine, incubated in isotope-free medium and, at various times thereafter, the cells were harvested and membranes isolated from them. The rate of decay of the protein and carbohydrate components was determined from specific activity dilution of the labeled components in the isolated membranes. Although the rate of membrane synthesis is different in exponential and contact-inhibited cells, the rate of degradation (turnover) of membrane proteins and carbohydrates was found to be the same (25% per generation (42 h) or 0.6%/h).     

Determination of rates of protein synthesis, gain and degradation in intact hind-limb muscle of lambs.

A model of leucine metabolism in the hind-limb muscles of the milk-fed lamb was developed which permitted simultaneous estimation of the rates of protein synthesis (Ks, days-1), degradation (Kd) and therefore gain (Kg) of muscle in vivo. The conclusions drawn from the model were: the rate of protein synthesis in muscle was related to uptake of leucine; the rate of degradation of protein was related to leucine output, as leucine, or its corresponding oxo acid, 4-methyl-2-oxopentanoic acid, or CO2. These findings support findings drawn from a wide range of studies in vitro. There was no correlation between rate of protein synthesis and rate of protein degradation, which suggests that the method can allow independent estimates of each. Estimates of protein synthesis obtained from the model (of leucine metabolism in muscle) were compared with those obtained simultaneously by constant infusion of radioisotope and analysis of incorporation into tissue. There were no significant differences between the mean values obtained for synthesis (Ks), gain (Kg) and degradation (Kd) by either method (Ks 0.051 +/- 0.002, 0.046 +/- 0.007; Kg 0.016 +/- 0.002, 0.004 +/- 0.008; Kd 0.035 +/- 0.004, 0.041 +/- 0.008 day-1, respectively, for tissue analysis and the model). However, Ks obtained from the model was significantly and positively correlated with uptake of leucine from plasma, whereas Ks obtained from tissue analysis was not.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

Studies on the turnover of plasma membranes in cultured mammalian cells.: II. Rates of synthesis and degradation of plasma membrane proteins and carbohydrates

The rate of turnover of membrane proteins and membrane-bound carbohydrates in exponentially growing and in confluent contact-inhibited cultures of strain MK-2 cells was investigated. Cells were labelled with [¹⁴C]leucine and [³H]glucosamine, incubated in isotope-free medium and, at various times thereafter, the cells were harvested and membranes isolated from them. The rate of decay of the protein and carbohydrate components was determined from specific activity dilution of the labeled components in the isolated membranes.Although the rate of membrane synthesis is different in exponential and contact-inhibited cells, the rate of degradation (turnover) of membrane proteins and carbohydrates was found to be the same (25%, per generation (42 h) or 0.6%/h).     

Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate--RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.     

Role of lysosomes in protein turnover: Catch-up proteolysis after release from NH4Cl inhibition

Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH₄Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum-free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH₄Cl. When these cells are removed from media containing NH₄Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid-soluble and acid-insoluble form. NH₄Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.     

Intracellular protein degradation in growing, in density-inhibited, and in serum-restricted fibroblast cultures.

Exponentially growing Balb/3T3 mouse fibroblasts contain protein populations with slow and fast turnover. These two stability classes were labelled selectively with 3H-leucine. The intracellular degradation of the proteins was then followed as the release into the medium of radioactive leucine. The degradation rate of both stability classes of protein is increased by about 55% in cultures whose growth is inhibited by high cell density. Serum-deprivation, which also halts cell growth, accelerates protein breakdown to a smaller extent, the increases for relatively stable and unstable proteins being 30% and 13%, respectively. The density-dependent increase in protein breakdown is also found in BHK21 cells but not in chick fibroblasts. Protein degradation in Balb/3T3 cells transformed by simian virus 40 is affected by serum-deprivation but not by cell density. The proteins which are relatively stable during growth were shown to become less stable in density-inhibited or serum-deprived cultures, and vice versa. Cycloheximide inhibits degradation to a variable extent. Dibutyryl adenosine-3',5'-cyclic monophosphate has no effect on the protein degradation under the conditions investigated here.     

Distinction between major chloroquine-inhibitable and adrenergic-responsive pathways of protein degradation and their relation to tissue ATP content in the Langendorff isolated perfused rat heart.

In the Langendorff isolated perfused rat heart, 36% of total basal protein degradation was inhibited by the lysosomal inhibitor chloroquine (30 microM), after elimination of rapid turnover proteins during a 3 h preliminary degradation period. Prior inhibition of degradation with chloroquine was additive to the 30% inhibition caused by simultaneous infusion of 50-200 nM-isoprenaline. This additivity suggests that the adrenergic-controlled process is independent of the lysosomal degradative pathway. After discontinuation of drug infusions, the isoprenaline-inhibited degradation rate returned to the previous baseline; however, the chloroquine-inhibited degradation rate transiently exceeded the previous baseline. NaN3 (0.3 mM) caused a decrease of left-ventricular myocardial ATP content of approx. 60% at 14 min and extreme impairment of contractile function; however, the total lysosomal and non-lysosomal protein degradation was not changed at this time. Conversely, left-ventricular tissue ATP content was not changed during proteolytic inhibition by 10 nM-isoprenaline or 10 microM-chloroquine at 14 min. The results indicate that depletion of myocardial energy stores in this preparation is neither necessary nor sufficient to cause inhibition of the total of lysosomal and non-lysosomal protein degradation.     

Effect of metabolic conditions on protein turnover in yeast.

1. In yeast growing on ethanol a turnover rate of up to 2%/h was measured. As much as 80% of the protein was subject to turnover, and no marked heterogeneity in the rate of degradation of protein was observed. When the yeast grew on glucose, the protein was degraded at a lower rate (0.5-1%/h). 2. Starvation for a nitrogen source increased the rate of protein degradation severalfold, whereas deprivation of phosphate had only a marginal effect (30% increase). Removal of glucose from a medium containing 50mM-phosphate did not cause marked changes in the rate of protein degradation. In contrast, when the media were low in phosphate (0.1 mM) removal of glucose increased the rate of turnover 2-4-fold. 3. Protein degradation proceeded unimpaired when the intracellular concentration of ATP decreased from 4 to 1 mM, but stopped completely when it decreased below 0.3 mM.     

Inhibition of hepatic protein degradation by synthetic analogues of chymostatin

Analogues of the microbial proteinase inhibitor chymostatin have been synthesized. The two most promising analogues were tested on protein turnover in isolated rat hepatocytes. Their effect is much similar to the effect of chymostatin, but the analogues are even more powerful inhibitors, probably due to an increased effect on lysosomal thiol proteinases. The analogues blocked most of the lysosomal (i.e. methylamine-sensitive) degradation of endogenous protein and caused a 50% inhibition of the non-lysosomal degradation; the effect occurred rapidly and was reversed upon washing the cells. One of the analogues, Z-Arg-Leu-Phe(H), is the most potent inhibitor of hepatic protein degradation so far found.     

Brief exposures of resting fibroblasts to okadaic acid stimulate DNA synthesis

To study further the factors providing for cellular quiescence, we used okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibiting type 1 and/or type 2A protein phosphatases in mammalian cell cultures. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium with 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting cells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resting state on transfer to a medium with 0.2% serum. Brief exposures of resting cells to OA did not affect the rate of protein synthesis. OA pulses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, revealed that pre-incubation of resting cells with OA for 2 h before and after fusion abrogates their ability to suppress the onset of DNA synthesis in the nuclei of proliferating cells in heterodikaryons. The results indicate that protein phosphatases of type 1 and/or 2A may be involved in the growth-arrest machinery that provides for cellular quiescence.     

Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.     

Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.     

Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).