Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [¹²⁵I]bovine serum albumin (BSA) was unaffected by chloroquine, NH₄Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [¹²⁵I]lactate dehydrogenase (LDH) and [¹²⁵I]pyruvate kinase (PK) was inhibited one-third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of ¹²⁵I-PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [¹⁴C]sucrose-labeled BSA-polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia. Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected ¹²⁵I-BSA and the modest inhibition of degradation of injected ¹²⁵I-LDH or ¹²⁵I-PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.     

Hepatic processing of insulin. Characterization of differential inhibition by weak bases

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied. 1. Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed. 2. Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products. 3. None of the weak bases changed the species of the degradation products found within the endocytic vesicles. 4. Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the rate-limiting process in degradation. 5. Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition of insulin degradation in isolated endocytic vesicles. 6. The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting by switching the receptor into a tight-binding conformation thereby slowing dissociation.     

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.     

Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods.

1. The method of estimating the intralysosomal pH by measuring the distribution of [14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. In intact lysosomes, the binding of [14C]methylamine is only partly inhibited by monovalent cations at 25 degrees C. 4. THe accumulation of [14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [14C]methylamine accumulation is obtained with NH4Cl. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. The breakdown of endocytosed 123I-labelled bovine serum albumin by intact isolated lysosomes is much less sensitive to the pH of the medium than the breakdown of added protein by lysed lysosomes. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 125I-labelled albumin in intact lysosomes as a function of medium pH with that of added 125I-labelled albumin by lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [14C]methylamine. 8. Methylamine and NH4Cl inhibit the breakdown of 125I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. It is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within the lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anions (or of OH- in exchange for anions).     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

Role of a low-pH environment in adenovirus enhancement of the toxicity of a Pseudomonas exotoxin-epidermal growth factor conjugate

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.     

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.     

Effects of weak bases on the degradation of endogenous and exogenous proteins by rat yolk sacs.

In rat yolk sacs incubated in vitro, the rates of degradation of endogenous [3H]leucine-labelled proteins and of pinocytically ingested 125I-labelled bovine serum albumin were both decreased in the presence of either ammonium, methylammonium or ethylammonium ions (0-20 mM) or much lower concentrations of chloroquine (0-500 microM). These effects were also accompanied by an inhibition of pinocytosis, as measured by the rate of uptake of 125I-labelled polyvinylpyrrolidone, and by a fall in the [ATP]/[ADP] ratio within the tissue. Re-incubation in inhibitor-free medium of yolk sacs previously exposed to a weak base restored pinocytic and proteolytic capacities, except for tissues exposed to chloroquine at concentrations above 0.1 mM (these appeared to be cytotoxic); an attendent rise in [ATP]/[ADP] ratios to near normal values was also observed. Weak bases, at concentrations that fully arrested the breakdown of 125I-labelled albumin, failed to inhibit by more than 45% the degradation of [3H]leucine-labelled endogenous proteins. Since 125I-labelled bovine serum albumin has been shown to be degraded entirely intralysosomally by yolk sacs, this suggests either that the hydrolysis of endogenous proteins is shared between lysosomes and some other site or that, unlike 125I-labelled albumin, some endogenous proteins can be degraded within lysosomes at abnormally high pH.     

Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein-labelled control immunoglobulin G (control IgG) a marker for non-specific adsorptive endocytosis and of anti-(plasma membrane) IgG (specific IgG), a specific ligand of cell-surface antigens. At 4 degrees C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture medium. They do not affect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return of the cell surface of membrane antigens and of membrane fragments bearing 5'-nucleotidase or binding sites for control IgG. We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.     

Swainsonine inhibits glycoprotein degradation by isolated rat liver lysosomes

Rat liver lysosomes, isolated from metrizamide gradients by the method of Wattiaux et al. (Wattiaux, R., Wattiaux-de Coninck, S., Ronveaux-Dupol, M.F., and Dubois, F. (1978) J. Cell Biol. 78, 349-368) took up from the medium and degraded several marker protein preparations, viz. 125I-asialofetuin, [35S]methionine-labeled hemoglobin, and [3H]leucine-labeled rat liver cytosol proteins. Rates were indistinguishable for all the markers, indicating that uptake was by a nonspecific process analogous to fluid pinocytosis. No effect of added MgATP or K+ was observed. Lysosomal degradation of all the markers was inhibited by 10(-4) M chloroquine. Swainsonine, on the other hand, at 10(-5) M, inhibited the breakdown only of the glycoprotein, 125I-asialofetuin. In the presence of the inhibitors, there was an accumulation of markers in the lysosomes in amount corresponding to the decreased breakdown, indicating that uptake was unaffected. Degradation and inhibition were measured at pH 7.0, 6.0, and 5.0 with both intact lysosomes and with lysosomes disrupted by the addition of 0.2% Triton X-100. Degradation with intact lysosomes was relatively independent of pH. On the other hand, activity with disrupted lysosomes was negligible at pH 7.0 and rose rapidly with decreasing pH. Inhibition by 10(-4) M chloroquine and 10(-5) M swainsonine with intact lysosomes decreased sharply with decreasing pH and did not occur with disrupted lysosomes.     

Macrophage endosomes contain proteases which degrade endocytosed protein ligands

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.     

Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts

Pseudomonas exotoxin (PE) was used as a probe to study the mechanism by which protein ligands are internalized by mammalian cells. Both biochemical and electron microscopic methods were used to look at the internalization of PE by mouse LM cell fibroblasts. Our data suggest that PE enters cells by receptor-mediated endocytosis, a process previously thought to be restricted to the entry of biologically significant molecules such as lysosomal enzymes and peptide hormones. Biochemical studies showed that methylamine (20 mM) and chloroquine (10 microM) protected LM cells from the action of PE. Full protection was observed if methylamine or chloroquine was added to the monolayers simultaneously with toxin or if they were added up to 10 min after toxin binding. Later addition of amine or chloroquine afforded partial protection to the monolayers. With immunoelectron microscopy we observed that in the cold toxin bound diffusely to the cell surface but was rapidly internalized when cells were warmed to 37 degrees C. In the presence of methylamine, chloroquine or ammonium chloride, internalization did not occur. We propose that PE enters mouse fibroblasts by receptor-mediated endocytosis and that chloroquine and methylamine, agents which are known to block this process, prevent expression of toxicity.     

PROTEIN DEGRADATION IN CULTURED CELLS : II. The Uptake of Chloroquine by Rat Fibroblasts and the Inhibition of Cellular Protein Degradation and Cathepsin B1

The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B₁ but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B₁.     

Structural and physical changes in lysosomes from isolated rat hepatocytes treated with methylamine

Incubation of isolated rat hepatocytes with 10 mM methylamine resulted in an inhibition of endogenous protein degradation and a microscopically visible enlargement of the lysosomes. Lysosomes from methylamine-treated cells exhibited increased buoyancy in metrizamide gradients and increased fragility as measured by the release of acid phosphatase activity in vitro, despite the fact that no methylamine remained in the gradient-isolated organelles. When methylamine was extracted from intact cells, the inhibition of protein degradation was immediately relieved, whereas the lysosomal enlargement (and to a certain extent also the increased fragility) persisted for some time. The methylamine-induced osmotic swelling of the lysosomes would thus seem to involve not merely a passive stretching of the lysosomal membrane, but rather some structural change (e.g., an increased amount of membrane material) which is relatively slowly reversible, but without effect on lysosomal function.     

Insulin degradation in 3T3-L1 adipocytes: Role of the endocytic lysosomal pathway

Insulin bound to 3T3-L1 adipocytes at 12 °C rapidly becomes processed to higher and lower molecular weight components at 37 °C. A part of this insulin processing (degradation) appears to have no role in the expression of its biological effects on hexose and amino acid transport. Degradation is strongly (~70%) inhibited by bacitracin, and very weakly inhibited (~5%) by methylamine, monodansylcadavarine, and bromophenacylbromide. All the other compounds tested for inhibition—azide, dinitrophenol (inhibitors of energy-dependent endocytosis), chloroquine (a lysosomotropic agent), chlorpromazine, phenylglyoxal (reported inhibitors of macromolecular internalization)—inhibited degradation partially to about the same extent (~20%), suggesting that the endocytic lysosomal pathway accounts for only a fifth of insulin degradation in 3T3-L1 adipocytes.     

Weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes

Effects of weak amines on the proteolytic conversion of proalbumin to serum albumin were studied in primary culture of rat hepatocytes. In control culture proalbumin was converted to serum albumin before discharge into the medium. However, in the presence of chloroquine the conversion to serum albumin was inhibited and proalbumin per se was released into medium. A similar inhibition of the processing was also observed in the presence of other amines such as methylamine and NH4Cl. Thus weak amines mimic the carboxylic ionophore monensin with regard to the effect on proalbumin conversion [Oda & Ikehara (1982) Biochem. Biophys. Res. Commun. 105, 766-772]. Since proteolytic conversion of proalbumin is believed to occur at the Golgi complex, these results suggest that weakly basic amines perturb the Golgi complex in addition to lysosomes and endosomes.     

Inhibition of myoblast fusion by lysosomotropic amines

Fusion of cultured chick embryo myoblasts was inhibited by treatment with several lysosomotropic amines. The concentrations required for half-maximal inhibition of fusion were approximately 2 μM for chloroquine, 30 μM for tributylamine, 3.2 mM for ammonium chloride, and 3.3 mM for methylamine. All the amines inhibited fusion appreciably at concentrations lower than those that reduced cell density. Both the rate and extent of fusion were affected by the amines, which had to be present for about 20 hr before the usual onset of fusion. Inhibition of fusion was reversible by transfer of inhibited cells to fresh medium. The amines did not cause accumulation of a nondialyzable inhibitor in the culture medium. Levels of creatine kinase increased by eight-fold or more between 18 and 65 hr in cultures treated with tributylamine or chloroquine, although this increase was not as pronounced as in control cultures. The increased creatine kinase activity in amine-treated cultures was due mainly to the BB and MB isozymes, with relatively little increase in the MM isozyme.     

Internalization of transforming growth factor-beta and its receptor in BALB/c 3T3 fibroblasts

The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.