Regulation of the breakdown rates of biotin-containing proteins in Swiss 3T3-L1 cells.

1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases.

1. A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabelled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. 2. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. 3. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mm-inhibitor. 4. The inhibition of proteolysis and of cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. 5. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those Z-Phe-Ala-CHN2, and were also largely reversible. 6. It is concluded that thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.     

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

Ionophores and weak bases inhibit phagolysosomal proteolysis in Paramecium

In a recent report we showed that ionophores and weak bases inhibit digestive vacuoles (DV) acidification primarily and lysosome-DV fusion secondarily but have no effect on lysosome-DV fusion when acidification is normal. In this study we attempted 1) to show that fluorescein isothiocyanate (FITC)-albumin taken up by phagocytosis could be used for a sensitive proteolytic assay, 2) to use this assay to determine the effect of ionophores and weak bases on proteolysis and 3) to learn how an inhibition of acidification and/or lysosome-DV fusion would affect proteolysis. When cells were pulsed with FITC-albumin and latex beads for 3 min and chased, the amount of albumin degraded increased linearly from 9 to 27 min, reaching a plateau by 30 min, and was inhibited by leupeptin and pepstatin A by 47 to 89%. These results showed that the degradation of FITC-albumin occurred in the phagolysosomes. When added before acidification had commenced, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), monensin and NH4Cl partially inhibited lysosome-DV fusion (25-50%) and strongly inhibited proteolysis by 64 to 79%. Added between acidification and lysosome-DV fusion, fusion was unaffected while proteolysis was reduced by 40 to 50%. Added after lysosome-DV fusion was completed, proteolysis was still reduced by the same amount. Chloroquine at 0.25 mM had no effect on proteolysis except when added before acidification, it inhibited fusion by 22% and proteolysis by 16%. These data, together with those published recently, showed that 1) ionophores and weak bases inhibited acidification first, lysosome-DV fusion second and proteolysis third, but they also inhibited proteolysis directly and independent of the prior steps and 2) the proteolysis inhibitory effects were additive.     

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.     

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.     

Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors

Most previous studies on inhibitors of lysosomal protein breakdown have been performed on isolated or cultured cells or on perfused organs. We have tested various inhibitors of proteolysis on lysosomes isolated from livers of rats injected with [14C]leucine 15 min (short labeling time) and 16 h (long labeling time) before killing. Intact lysosomes were incubated with different inhibitors (leupeptin, propylamine, E-64, pepstatin, and chloroquine) in increasing concentrations. None of these caused more than a 40-75% inhibition of proteolysis irrespective of labeling protocol. Chloroquine was the most effective inhibitor, followed by leupeptin, propylamine, E-64, and pepstatin. When lysosomes were incubated with various combinations of inhibitors, including a weak base and an enzyme inhibitor, a somewhat higher inhibition (86%) was obtained. To assess if lysosomes are active in the degradation of both short and long lived proteins, lysosomes were isolated from livers of rats labeled with [14C]leucine for various time intervals. The highest fractional proteolytic rates were seen for short lived proteins. If the recovery of the isolated lysosomes is taken into consideration, about 80% (short labeling time) and 90% (long labeling time) of the total proteolysis in the homogenate could be accounted for by lysosomes. Isolated Golgi, mitochondrial, and microsomal fractions displayed negligible proteolytic activities. The cytosol contributed one-fifth of the total protein breakdown of short lived proteins, whereas insignificant proteolysis was recovered in the cytosolic fraction following long time labeling. Accordingly, we propose that 1) lysosomal inhibitors do not completely suppress proteolysis in isolated lysosomes and that 2) both short and long lived proteins are degraded in lysosomes.     

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.     

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.     

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.     

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.     

Effects of microbial proteinase inhibitors on the degradation of endogenous and internalized proteins by rat yolk sacs.

1. The effects of leupeptin and other microbial proteinase inhibitors were measured in rat yolk sacs on the uptake and degradation of formaldehyde-denatured 125I-labelled bovine serum albumin as well as on the degradation of 3H-labelled endogenous protein. 2. Leupeptin, at concentrations between 1 and 100 micrograms/ml, inhibits the degradation of added albumin without affecting pinocytic uptake. Accordingly large amounts of undegraded albumin accumulate within the tissue. 3. Removal of leupeptin produces a rapid recovery of the capacity to degrade albumin. 4. Endogenous protein degradation is rapidly inhibited by leupeptin, but to a far lesser extent than the breakdown of albumin. However, the inhibition is only slightly reversed on removal of leupeptin. 5. Degradation of both albumin and endogenous protein in intact yolk sacs is inhibited by the microbial proteinase inhibitors in the order: leupeptin greater than antipain greater than chymostatin; elastatinal, pepstatin and bestatin are ineffective. 6. Similar results are found when albumin is incubated in yolk-sac homogenates at pH 4 with the inhibitors. 7. The marked inhibitory effects of leupeptin, antipain and chymostatin suggest that cathepsin B and possibly cathepsin L participate in the degradation of 125I-labelled albumin in yolk sacs. By comparison, the smaller inhibitory effects of the proteinase inhibitors on endogenous protein breakdown imply a minor role of lysosomal cathepsins in this process.     

Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

Binding, internalization, and degradation of 125I-labeled-rat atrial natriuretic peptide (rANP) were studied in cultured rat aortic vascular smooth muscle cells (VSMC). At 37 degrees C, 125I-labeled-rANP rapidly bound to VSMCs, but the cell-bound radioactivity rapidly decreased upon subsequent incubation, while the binding was slow at 4 degrees C, reaching to an apparent equilibrium after 6 hrs. The cell-bound 125I-labeled-rANP at 37 degrees C is rapidly dissociated from VSMC (t 1/2: approximately 40 min) with the appearance of degradaded product(s) of radioligand in the medium, whereas the degradation was minimal at 4 degrees C. This degradative process was blocked by inhibitors of metabolic energy production (azide, dinitrophenol), inhibitors of lysosomal cathepsins (leupeptin, pepstatin), and lysosomotropic agents (NH4Cl, chloroquine, lidocaine, methylamine, dansylcadaverine), but not by inhibitors of serine or thiol proteases. 125I-labeled-rANP initially bound to the cell-surface was rapidly internalized, and delivered to lysosomal structures, which was confirmed by autoradiographic studies. These data indicate that rANP, after binding to the cell-surface receptors, is rapidly internalized into the cells through receptor-mediated endocytosis, and subsequently degradaded by lysosomal hydrolases.     

Regulation of adipocyte lipolysis by degradation of the perilipin protein: nelfinavir enhances lysosome-mediated perilipin proteolysis

A decrease in the lipid droplet-associated protein perilipin may constitute a mechanism for enhanced adipocyte lipolysis under nonstimulated (basal) conditions, and increased basal lipolysis has been linked to whole body metabolic dysregulation. Here we investigated whether the lipolytic actions of the human immunodeficiency virus protease inhibitor, nelfinavir, are mediated by decreased perilipin protein content and studied the mechanisms by which it occurs. Time course analysis revealed that the decrease in perilipin protein content preceded the increase in lipolysis. A causative relationship was suggested by demonstrating that nelfinavir potently increased lipolysis in adipocytes derived from mouse embryonal fibroblasts expressing perilipin but not in mouse embryonal fibroblast adipocytes devoid of perilipin and that adenoviral mediated overexpression of perilipin in 3T3-L1 adipocytes blocked the lipolytic actions of nelfinavir. Nelfinavir did not alter mRNA content of perilipin but rather decreased perilipin proteins t((1/2)) from >70 to 12 h. Protein degradation of perilipin in both control and nelfinavir-treated adipocytes could be prevented by inhibiting lysosomal proteolysis using leupeptin or NH(4)Cl but not by the proteasome inhibitor MG-132. We propose that proteolysis of perilipin involving the lysosomal protein degradation machinery may constitute a novel mechanism for enhancing adipocyte lipolysis.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells

Biologically active, iodine-labeled derivatives of human choriogonadotropin in which all the iodine is localized either in the alpha or beta subunits have been prepared. It is found that upon binding to Leydig tumor cells these derivatives are ultimately degraded to 3'-monoiodotyrosine. A comparison of the rates of degradation of the derivatives labeled exclusively in the alpha or beta subunits show that the alpha subunit is degraded somewhat faster than the beta subunit. It was also found that NH4Cl, chloroquine and leupeptin inhibited the degradation of both subunits to the same extent. These results show that the Leydig tumor cells degrade both subunits of the receptor-bound human choriogonadotropin, and suggest that the two subunits are degraded by the same mechanism(s).     

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).     

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.