The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent

In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.     

Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.     

Loss of surface galactosyl receptor activity on isolated rat hepatocytes induced by monensin or chloroquine requires receptor internalization via a clathrin coated pit pathway

We studied the effect of hyperosmotic inhibition of the clathrin coated pit cycle on the monensin- and chloroquine-dependent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes. Cells treated for 60 min without ligand at 37 degrees C with 25 microM monensin or 300 microM chloroquine in normal medium (osmolality congruent to 275 mmol/kg) bound 40-60% less 125I-asialo-orosomucoid (ASOR) at 4 degrees C than untreated cells. Cells exposed to monensin or chloroquine retained progressively more surface Gal receptor activity, however, when the osmolality of the medium was increased above 400 mmol/kg (using sucrose as osmolite) 10 min prior to and during drug treatment. Cells pretreated for 10 min with hyperosmolal media (600 mmol/kg) alone internalized less than or equal to 10% of surface-bound 125I-ASOR. Thus, the ligand-independent loss of surface Gal receptor activity on monensin- and chloroquine-treated hepatocytes requires internalization of constitutively recycling receptors via a coated pit pathway.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

Recycling of the hepatic asialoglycoprotein receptor in isolated rat hepatocytes. Receptor-ligand complexes in an intracellular slowly dissociating pool return to the cell surface prior to dissociation

We recently reported that the dissociation of internalized receptor-125I-asialo-orosomucoid (ASOR) complexes by isolated hepatocytes is a biphasic process; most complexes dissociate rapidly but 25-50% dissociate slowly (Oka, J. A., and Weigel, P. H. J. Biol. Chem. 258, 10253-10262). Cells were allowed to endocytose a pulse of surface-bound 125I-ASOR, and were washed and then incubated at 37 degrees C in the presence or absence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Without EGTA, very little intact ASOR appeared in the medium. With EGTA present, a large amount of intracellular ligand appeared undegraded in the medium in a time-dependent manner. N-Acetylgalactosamine, but not ASOR, in the medium also caused release of intact 125I-ASOR. Within 15 min, more than 50% and by completion at least 80% of the internalized ligand in the slow dissociation compartment was released into the medium. If cells containing internalized ligand were incubated at 37 degrees C for increasing times before the addition of EGTA, then progressively less ligand accumulated in the medium. Experiments at 18 degrees C, a temperature at which neither degradation nor slow dissociation occurred, demonstrated that in the presence of EGTA the intracellular free 125I-ASOR pool did not change. The amount of receptor-bound ligand in the slowly dissociating pool decreased and the amount of intact ligand in the medium increased by essentially equal amounts. The temperature dependence for the return of internal 125I-ASOR to the cell surface was similar to that for endocytosis, with a cut-off temperature of about 12 degrees C. We conclude that a normal part of the endocytic process involves the return of receptor-ligand complexes to the cell surface from an internal slowly dissociating pool. This might reflect either an obligatory step or a reversible statistically random step in the endocytic/recycling pathway.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

Fluid endocytosis in isolated rat parenchymal and non-parenchymal liver cells

Fluid endocytosis in rat liver parenchymal (hepatocytes) and non-parenchymal cells was studied by measuring uptake of [¹²⁵I]polyvinylpyrrolidone (PVP). Radioactive sucrose preparations were also tested but turned out to be unsuitable because of impurities of radioactive glucose and fructose. Fluid endocytosis was temperature dependent without any transition temperature. The rate of endocytosis was inhibited by inhibitors of the glycolytic and the respiratory pathway. Colchicine, but not cytochalasin B, inhibited the uptake of [¹²⁵I]PVP in hepatocytes. Therefore, intact microtubuli, but not microfilaments may be required for normal rate of fluid endocytosis in hepatocytes. Colchicine reduced the rate of fluid endocytosis in the non-parenchymal liver cells. Subcellular fractionation by isopycnic centrifugation in sucrose gradients indicated that [¹²⁵I]PVP taken up by the hepatocytes accumulated in the lysosomes. The rate of uptake expressed as volume of fluid internalized per unit time (endocytic index) was calculated to 0.08 μl/h/10⁶ cells for hepatocytes and 0.07 μl/h/10⁶ cells for non-parenchymal liver cells.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.     

Internalization of lucifer yellow in Candida albicans by fluid phase endocytosis

Lucifer yellow (LY), an impermeable fluorescent dye used as a marker for fluid phase endocytosis, was internalized by Candida albicans. As observed by fluorescence microscopy, incubation of C. albicans with LY in potassium phosphate buffer (pH 6.0) and glucose (2%, w/v) resulted in localization of the dye inside vacuoles. Sodium azide and carbonyl cyanide m-chlorophenylhydrazone, which are inhibitors of energy metabolism, decreased the uptake of the dye. The optimum temperature for uptake was 30 degrees C; no internalization was observed at 0 degrees C. Quantification of cell-associated LY by fluorescence spectrometry showed an uptake linear with time and not saturable over a 400-fold range of concentration. Thus, C. albicans internalized LY into vacuoles by a nonsaturable and time-, temperature- and energy-dependent process consistent with fluid phase endocytosis. Both the yeast and mould phase of this dimorphic fungus endocytosed LY. Growth in complex medium appeared to be required to enable the cells to internalize LY. However, addition of peptone or yeast extract to the phosphate buffer/glucose assay medium interfered with LY uptake by causing an apparent increase of exocytosis. These studies provide the first evidence of fluid phase endocytosis in C. albicans and may explain how some large molecules, such as toxins and cationic proteins, enter C. albicans.     

Copper and zinc ions differentially block asialoglycoprotein receptor-mediated endocytosis in isolated rat hepatocytes.

Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 degrees C for 2 h with ZnCl2 (0-220 microM) or CuCl2 (0-225 microM) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t1/2 = 62 min) and 99% (t1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost approximately 50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 degrees C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by approximately 33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

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 cellular internalization of recombinant gamma interferon differs from that of natural interferon gamma

Purified natural and recombinant murine gamma interferons (MuIFN-gamma) bind at 4 degrees C to cultured L929 mouse fibroblasts with comparable receptor-binding affinity (Kd = 9 x 10(-10) M). Both 125I-labeled MuIFNs are rapidly internalized by cells at 37 degrees C, although recombinant IFN is internalized somewhat more slowly than natural IFN (t1/2 = 90 sec and 45 sec, respectively). Immunoelectronmicroscopy showed that the majority of bound recombinant MuIFN-gamma was located on the plasma membrane outside of coated areas, whereas natural interferon was found mainly in coated pits. At 37 degrees C most of the recombinant molecules entered the cytoplasm in pinocytotic vesicles, while natural interferon was internalized by the specific mechanism of receptor-mediated endocytosis [1]. However, nearly equal amounts of immunocytochemically detectable molecules of both IFNs were found in the cell nucleus within 2-3 min incubation at 37 degrees C. Thus, the process of translocation of the recombinant IFN-gamma appears to differ from that of the natural product.