A pool of intracellular phosphorylated asialoglycoprotein receptors which is not involved in endocytosis

One proposed function of the asialoglycoprotein receptor in hepatocytes is to mediate the endocytosis of galactose and N-acetylgalactosamine-exposing glycoproteins. Recently we defined a pool of intracellular H1 subunits of the asialoglycoprotein receptor (ASGPR) in the human hepatoma cell line HepG2 which appeared not to be involved in endocytosis (Stoorvogel, W., Geuze, H. J., Griffith, J. M., Schwartz, A. L., and Strous, G. J. (1989) J. Cell Biol. 108, 2137-2148). In addition, a pool of stably phosphorylated intracellular ASGPR has been detected (Fallon, R. J., and Schwartz, A. L. (1988) J. Biol. Chem. 263, 13159-13166). In the current study we integrate these findings and provide evidence for the existence of two types of intracellular nonexchangeable compartments containing ASGPR. A transiently phosphorylated pool of ASGPR shuttles between the plasma membrane and endosomes, via a pathway identical to that of the transferrin receptor. The second pool comprises 20% of the total intracellular ASGPR, is stably phosphorylated at a serine residue, and is located in intracellular compartments devoid of recycling transferrin receptor. We refer to this ASGPR pool as the "silent pool." We furthermore show that the two receptor pools are confined to compartments exhibiting different buoyant densities on sucrose density gradients. ASGPR in the "silent pool" is fully glycosylated, suggesting a post-Golgi sorting mechanism for trafficking to this compartment. Possible functions of the "silent" ASGPR pool are discussed.     

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.     

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.     

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 asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Vasopressin-induced changes in receptor-mediated endocytosis of asialoglycoprotein in rat hepatocytes.

The ability of second messengers to modulate receptor-mediated endocytosis was studied on isolated rat hepatocytes. A 20-min preincubation with vasopressin was used as a modulation. We observed a 20% inactivation of both surface and intracellular receptors, with no change in the affinity of those remaining active. The internalization and dissociation of a synchronous wave of ligand was not affected, but its degradation was partially inhibited. Our observations suggest that second messengers such as intracellular calcium and diacylglycerol play a complex role in the intracellular trafficking associated with endocytosis.     

Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps

The rate of endocytosis of cell surface-bound [3H]-asialo-orosomucoid was determined as a function of temperature. Freshly isolated rat hepatocytes were allowed to bind [3H]asialo-orosomucoid at 4 degrees C, washed to remove nonbound ligand, and internalization was then assessed by the resistance of cell-associated radioactivity to release by the Ca2+ chelator EDTA. At 10 degrees C or below, endocytosis is negligible. Above 10 degrees C, the rate of endocytosis is proportional to temperature but the increase of the rate of endocytosis with increasing temperature changes sharply at about 20 degrees C. From 10-20 degrees C, the apparent activation energy for endocytosis, calculated from an Arrhenius plot, is 45.9 kcal/mol and the temperature coefficient, Q10, is 15.6. However, between 20 and 41 degrees C, the calculated activation energy is 17.0 kcal/mol and the Q10 is 2.6. Although the rate of endocytosis of previously bound [3H]asialo-orosomucoid is very dependent on the temperature, the final extent of endocytosis is essentially temperature-independent between 14 and 37 degrees C. The results suggest that there are at least two steps in the overall process of endocytosis mediated by the asialoglycoprotein receptor on isolated hepatocytes which can be potentially rate-limiting, one at 10 degrees C and another at approximately 20 degrees C.     

The surface content of asialoglycoprotein receptors on isolated hepatocytes is reversibly modulated by changes in temperature

We have investigated the effect of temperature on the content of surface asialoglycoprotein receptors on isolated rat hepatocytes. Receptor was determined by measuring the specific binding of 125I- or [3H] asialo-orosomucoid at 0 degrees C. As reported previously, the receptor number/cell increases 2-3-fold within 30-60 min when freshly isolated cells are warmed from 0-37 degrees C (Weigel, P. H. (1980) J. Biol. Chem. 255, 6111-6120). This increase in receptor number is not inhibited by cycloheximide and also occurs on cells which have first been treated with EDTA to expose a population of cryptic receptors on the cell surface. The rate and extent of the receptor number increase on the cell surface are proportional to the temperature above about 17 degrees C. If cells are first equilibrated at 37 degrees C and then transferred to a lower temperature, the surface receptor number decreases at a rate and to an extent dependent on the temperature. The surface receptor number can be modulated up and down by successive temperature change cycles between 25 and 37 degrees C. In this temperature range, the number of surface receptors/cell is dependent on the final temperature but independent of the pathway to that temperature and is, therefore, a function of state with respect to temperature. The results demonstrate that temperature changes reversibly modulate the number of receptors on the hepatocyte surface. We conclude that, in the absence of ligand, surface receptors can either recycle or can be reversibly internalized or sequestered to prevent access to ligand. The results may also explain why different laboratories have reported a wide range of values for the number of receptors per hepatocyte.     

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.     

Vanadate modulates the activity of a subpopulation of asialoglycoprotein receptors on isolated rat hepatocytes: active surface receptors are internalized and replaced by inactive receptors

In the absence of ligand, sodium vanadate causes a time- and dose-dependent loss of up to approximately 50% of the surface galactosyl receptor (GalR) activity in rat hepatocytes at 37 degrees C. The effect on total (surface plus intracellular) GalR activity is also dependent on exposure time and vanadate concentration. At less than 1 mM, vanadate induces a transient decrease and then partial recovery of cell surface GalR activity. At greater than 3 mM vanadate, surface GalR activity decreases rapidly (t1/2 approximately 2 min). Lost surface activity is initially recovered in digitonin-permeabilized cells, indicating that active surface GalRs redistribute to the cell interior. However, an antibody assay for GalR protein showed that although surface activity decreased, there was no decrease in surface receptor protein. The active intracellular GalRs then slowly inactivate over 30-60 min. With 8 mM vanadate, the loss of both surface and total cellular GalR activity is more rapid and coincident; no lag is observed. Maximal activity loss, however, was still only approximately 50%. Again, no net change was seen in the distribution of GalR protein between the cell surface and the interior. These results indicate that vanadate causes active GalRs to move from the surface to the inside and be replaced by inactive receptors moving from the inside to the cell surface. The Gal receptor system is comprised of two functionally different receptor subpopulations that operate via two distinct intracellular pathways. Only the State 2 GalRs, which recycle constitutively, are sensitive to modulation by vanadate. Consistent with this, vanadate inhibits the endocytosis of 125I-asialoorosomucoid (ASOR) only partially. The rate of uptake and the steady state level of ASOR intracellular accumulation were maximally inhibited by 50 and 70%, respectively, at 0.2 mM vanadate. The rate and extent of degradation of 125I-ASOR were also inhibited by 50-70%. Residual ASOR uptake and degradation is accounted for by the minor vanadate-resistant State 1 Gal receptor pathway.     

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.     

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.     

Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes.

We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.     

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.     

A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes.

Following in vivo administration of cycloheximide (20 mg/kg body weight i.p.) protein synthesis was completely inhibited (99%) in rat liver. No newly synthesized asialoglycoprotein receptor (ASGP-R) could be detected by metabolic labeling. Fluorescence immunocytochemistry of several secretory proteins and plasma membrane proteins, including the receptors for polymeric IgA (IgA-R), demonstrated a rapid loss from the Golgi complex following cycloheximide administration. On the other hand, two membrane proteins, the receptors for ASGP-R and mannose 6-phosphate (MP-R), were not altered in their cellular localization including the Golgi. Using quantitative immunoelectron microscopy with colloidal gold, we found that 2 h and 4 h after cycloheximide administration, the densities of ASGP-R and MP-R in the membranes of the Golgi complex were unaltered compared with control liver. Similarly, there was no significant effect of cycloheximide on the receptor labeling in coated vesicles and compartment of uncoupling receptors and ligands (CURL). These observations are consistent with an involvement of the Golgi and CURL pools of the receptors in intracellular trafficking, endocytosis and receptor recycling.     

The intracellular distribution of high density lipoproteins taken up by isolated rat hepatocytes

The subcellular distribution of 125I-labelled HDL taken up by rat hepatocytes in vivo and in vitro has been studied with subcellular fractionation techniques: differential centrifugation and isopycnic centrifugation in sucrose gradients. 125I-labelled HDL bind to plasma membranes both in vivo and in vitro and part of the membrane-bound 125I-labelled HDL can be dissociated by the addition of unlabelled HDL. The hepatocytes also internalize 125I-labelled HDL. The 125I-labelled HDL accumulate, however, at different intracellular sites in the in vivo and in vitro situation. The subcellular distribution pattern of 125I-labelled HDL taken up by the cells in vivo is similar to that of the lysosomal marker enzyme acid phosphatase. Peak activity was found at a density of 1.20 g/ml. In vitro 125I-labelled HDL accumulate in an organelle with a medium density of about 1.13 g/ml. This distribution was similar to that of the plasma membrane marker 5'-nucleotidase. The subcellular distribution of radioactivity taken up in vivo was changed to lower density by incubating the cells with chloroquine, a drug known to render the lysosomes more boyant. Chloroquine had no effect on the distribution of 125I-labelled HDL taken up by hepatocytes in vitro.