Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex

The effects of chloroquine, verapamil and monensin on secretion of very-low-density lipoproteins (VLDLs) were studied in cultured rat hepatocytes. Maximum inhibition of VLDL-triacylglycerol secretion by 50–90% of control was reached at 200 μM chloroquine, 200 μM verapamil and 5 μM monensin, whereas no effect on cellular triacylglycerol synthesis was observed. The inhibition could be seen within 15 min and was reversible after washout of the drugs. Chloroquine and verapamil inhibited both cellular protein synthesis and protein secretion, whereas monesin reduced protein secretion without any effect on protein synthesis. Control experiments with cycloheximide revealed that intact protein synthesis was not necessary for secretion of VLDL-triacylglycerol during 2 h. Electron micrographs of cells treated with chloroquine, verapamil or monensin showed swollen Golgi cisternae containing VLDL-like particles. By morphometry, a more than 2-fold increase in volume fractions and size indices of Golgi complexes and secondary lysosomes was observed, except that monensin had no significant effect on these parameters of secondary lysosomes. These results suggest that the inhibition of VLDL secretion by chloroquine, verapamil and monensin which takes place in the Golgi complex might be due to disruption of trans-membrane proton gradients. An increase in pH of acidic Golgi vesicles may cause swelling and disturb sorting and membrane flow through this organelle.     

Effect of monensin on the neuronal ultrastructure and endocytic pathway of macromolecules in cultured brain neurons

1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the 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.     

Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.     

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.     

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Morphological localization of a major lysosomal membrane glycoprotein in the endocytic membrane system

We have raised specific polyclonal immunoglobulin G (IgG) against a major lysosomal membrane sialoglycoprotein (LGP107) taken from rat liver and have prepared a conjugate of its Fab' fragment with horseradish peroxidase (HRP-anti LGP107 Fab') as a probe for the subcellular antigen. Electron immunocytochemistry in primary cultured rat hepatocytes showed that LGP107 resided primarily within lysosomes and was associated with luminal amorphous materials as well as limiting membranes. In addition, LGP107 was shown to be substantially distributed throughout the endocytic vacuolar system. The glycoprotein was found clustered in coated pits at the cell surface and localized along the surrounding membranes in endocytic vesicles. When cultured cells were exposed to HRP-anti LGP107 Fab', the antibody which was bound to its antigen within the coated pits was internalized via a system of endocytic vesicles and transported to lysosomes. During 20 min of incubation at 37 degrees C, the HRP tracer appeared at an early stage in small vesicles and moved progressively to larger vesicles, including multivesicular bodies. After 1 h, the tracer could be clearly seen in lysosomes heterogeneous in shape and size. The existence of LGP107 in endocytic compartments and the uptake of anti LGP107 antibody by hepatocytes were not blocked by prior treatment of the cells with cycloheximide and excess amounts of anti LGP107 IgG. These data suggest that LGP107 circulates between the cell surface and lysosomes through the endocytic membrane traffic in hepatocytes.     

The role of pH in the intracellular acetylcholine receptor ligand processing

Previous studies have shown that the internalized AChRs are transported through many vesicular compartments: Golgi associated vesicles, coated vesicles, smooth vesicles, endosome-like structures and lysosomes. These compartments have an acidic pH ranging from 4.5 to 6.5. The pH differences between organelles suggests that these differences may influence the sorting and final expression of AChRs. To test this hypothesis, we measured the number of counts of 125I-alpha BTX or 125I-Mab35 dissociated from myotube membranes containing AChRs as a function of pH. Neither the 125I-alpha BTX nor 125I-Mab35 showed an enhanced dissociation in the pH range 4.0-7.0, whereas lowering the pH to 6.0 or below enhanced the dissociation of 125I-alpha 2-macroglobulin from myotubes. In other experiments using Torpedo membrane we showed that neither 125I-alpha BTX nor 125I-Mab35 appreciably dissociated from the AChR unless the pH was less than 4 or above 11. Double-label studies using a novel membrane permeable acidotropic molecule DAMP (3-(2,4 nitroanilino) 3'amino-N-methyl-dipropylamine), facilitated mapping the pH of the intracellular compartments containing internalized AChRs. This molecule accumulates inside acidic compartments in the cell and has a dinitrophenol (DNP) group recognized by DNP specific antibodies. Cells were treated with 30 micrograms DAMP for 30 min and allowed to internalize Mab35-gold (15 nm) for various periods (0-15 h). At each time point we fixed and washed the cells, and incubated with anti-DNP monoclonal antibodies followed by incubation with anti-mouse IgG and protein A colloidal gold (5 nm). Different sized gold particles allowed us to simultaneously identify the AChR compartments and estimate their pH. Sister cultures were exposed to acidotropic drugs to destroy pH gradients. Under those conditions, AChR delivery to lysosomes was blocked. Our studies show that AChRs are transported through acidic compartments ranging from pH 4.5 to 6.5 and in contrast to other ligands they do not dissociate from the intracellular membranes at low pH.     

Pathway and kinetics of vitellogenin-gold internalization in the Xenopus oocyte

After in vitro incubation of Xenopus oocytes with vitellogenin (VTG)-gold conjugate, the gold particles are distributed on the whole plasma membrane. Their concentration in coated pits still occurs at 0 degrees C. At +20 degrees C the label quickly (30 sec) appears in multi-vesicular endosomes (MVE) which segregate together with primary endocytic vesicles into distinct clusters below the plasma membrane. From this step up to crystallization of the yolk platelets, the gold particles stay in the same compartment. During 5.5 h the label progressively increases along the MVE membrane, first (1.5 h) by fusion of primary endocytic vesicles with consecutively enlarging endosomes, then (4 h) by decreasing of the MVE membrane. As concerns the yolk platelet formation, concentration of primordial yolk platelets (PYP) occurs at 5.5 h from the incubation onset, the labeling of preexisting yolk platelets starts at 7 h, while crystallization of PYP begins only after 12-13 h. Our results indicate that VTG receptors are not preclustered in coated pits and their lateral translation is not inhibited at 0 degrees C. The yolk protein processing takes place within one compartment only. The VTG condensation begins with a long concentration phase of receptor-VTG complexes still integrated in the endosome membrane. It occurs in MVE by: i) a repeated fusion of primary endocytic vesicles; ii) removing part of the endosome membrane by internal vesiculation. Fusion between endosomes occurs only after VTG has dissociated from its receptors and VTG dissociates only when when the density of the VTG-receptor complexes in the endosome membrane is sufficient. Crystallization begins after a 7-8 h delay. The endosome migration into the oocyte is also controlled by the binding of VTG to its receptors. Our results also demonstrate that binding of VTG colloidal gold modifies neither the vitellogenic pathway nor the duration of the vitellogenin internalization. However when vitellogenin is bound to colloidal gold, dissociation of ligand-receptor complexes is delayed because the amount of ligand in the incubation medium is necessarily low.     

Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts

It has been shown that endocytic vesicles in BALB/c 3T3 cells have a pH of 5.0 (Tycko and Maxfield, Cell, 28:643-651). In this paper, a method for measuring the effect of various agents, including weak bases and ionophores, on the pH of endocytic vesicles is presented. The method is based on the increase in fluorescein fluorescence with 490-nm excitation as the pH is raised above 5.0. Intensities of cells were measured using a microscope spectrofluorometer after internalization of fluorescein-labeled alpha 2-macroglobulin by receptor-mediated endocytosis. The increase in endocytic vesicle pH was determined from the increase in fluorescence after addition of various concentrations of the test agents. The following agents increased endocytic vesicle pH above 6.0 at the indicated concentrations: monensin (6 microM), FCCP (10 microM), chloroquine (140 microM), ammonia (5 mM), methylamine (10 mM). The ability of many of these agents to raise endocytic vesicle pH may account for many of their effects on receptor-mediated endocytosis. Dansylcadaverine caused no effect on vesicle pH at 1 mM. The observed increases in vesicle pH were rapid (1-2 min) and could be reversed by removal of the perturbant. This reversibility indicates that the vesicles themselves contain a mechanism for acidification. The increase in vesicle pH due to these treatments can be observed visually using an SIT video camera. Using this method, it is shown that endocytic vesicles become acidic at very early times (i.e., within 5-7 min of continuous uptake at 37 degrees C).     

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.     

Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.     

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.