©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Hepatoma Cell Mutant Defective in Cell Surface Protein Trafficking (*)

Richard J. Stockert (§) , Barry Potvin (1), Lian Tao , Pamela Stanley (1), Allan W. Wolkoff

From the (1)Marion Bessin Liver Research Center and the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To isolate a mutant liver cell defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was devised. Ovalbumin-gelonin and asialoorosomucoid (ASOR)-gelonin were incubated with mutagenized HuH-7 cells, and a rare survivor termed trafficking mutant 1 (Trf1) was isolated. Trf1 cells were stably 3-fold more resistant than the parental HuH-7 to both toxic conjugates. The anterograde steps of intracellular endocytic processing of ASOR, including internalization, endosomal acidification, and ligand degradation, were unaltered in Trf1 cells. In contrast, retrograde diacytosis of asialoglycoprotein receptor (ASGR)ASOR complex back to the cell surface was enhanced by about 250%. Selective labeling revealed an approximately 46% reduction in cell surface-associated ASGR in Trf1 cells, although their total cellular ASGR content was essentially equivalent to that in HuH-7. Similar results were obtained with the transferrin receptor. Binding of I-ASOR and I-transferrin was reduced in Trf1 cells to 49 ± 2.5% and 30 ± 2%, respectively, of HuH-7 cells. The methionine transporter was also reduced in Trf1 cells, as revealed by a 2-fold reduction in V with no change in apparent K. Pretreatment with monensin, sodium azide, or colchicine reduced surface binding of I-ASOR in HuH-7 cells by 50% but had no effect on binding to Trf1 cells. This result is predicted for a cell that expresses only State 1 ASGRs, which are resistant to modulation by metabolic and cytoskeletal inhibitors in contrast to State 2, which are responsive to these agents (Weigel, P. H., and Oka, J. A.(1984) J. Biol. Chem. 259, 1150-1154). The Trf1 mutant, having lost the ability to express State 2 receptors, provides genetic evidence for the existence of these two receptor subpopulations and an approach to identifying the biochemical mechanism by which they are generated.


INTRODUCTION

Receptor-mediated endocytosis (RME)()is a general mechanism for the uptake of macromolecules by the cell. RME is initiated by the binding of ligand to specific cell surface receptors followed by internalization of the receptor-ligand complex. Intracellular trafficking of the resulting endosomal vesicles and their ultimate destination varies with the particular receptor (for review see Ref. 1). Mutations that result in the inhibition or alteration of RME fall into two general classes. One class, exemplified by the low density lipoprotein receptor mutants(2, 3) , involves mutations of the receptor structural gene per se, limiting the effect to a single RME system. The second major class of RME mutations gives pleiotropic phenotypes arising from effects on multiple RME systems (for review see Ref. 4). The vast majority of pleiotropic endocytic mutants have been isolated from Chinese hamster ovary (CHO) cell populations. These isolates have been classified into either endocytosis (End) mutants belonging to complementation groups End 1-End 6 (4, 5) or low density lipoprotein uptake mutants belonging to complementation groups ldlB-ldlI(2, 6) . In addition, there are mutants that have not been integrated into either the End or ldl complementation groups, which also exhibit a pleiotropic phenotype(4) .

To obtain mutants affecting general steps in RME, dual selection schemes have been devised which apply selection pressure to the RME pathway(7, 8) . We have used a related strategy to isolate a defective mutant in RME from hepatocyte-derived HuH-7 cells. These cells were used partly to obtain a phenotype relevant to liver and partly to avoid reisolating the endocytosis mutants obtained with high frequency from CHO cells. In our protocol, the surface expression of two different carbohydrate-specific receptors of HuH-7 were selected against. The galactose/N-acetylgalactosamine asialoglycoprotein receptor (ASGR) and the mannose receptor, both responsible for the vesicular transport of their respective ligands to lysosomes, were utilized to deliver simultaneously to mutagenized HuH-7 cells, galactose- and mannose-terminating glycoprotein conjugates of gelonin, an inhibitor of protein synthesis(9) . Resistance to these targeted toxin conjugates would be expected to result from altered trafficking that reduces the intracellular concentration of the toxin or prevents its escape into cytosol.

In this paper we describe a mutant HuH-7 cell line termed trafficking mutant 1 (Trf1), which was isolated by the dual selection protocol. It is more resistant than parental HuH-7 cells to both of the glycoprotein conjugates, and it exhibits a defect in RME. The pleiotropic phenotype of the mutant is distinct from that of other endocytosis mutants isolated previously from CHO cell populations(4, 5, 6) . The mutation expressed by Trf1 cells reduces the surface expression of several unrelated molecules and provides genetic evidence for the existence of different subpopulations of surface receptors, in particular, the two subpopulations of ASGRs, designated State 1 and 2 by Weigel and Oka (10).


EXPERIMENTAL PROCEDURES

Materials

Human asialoorosomucid receptor (ASOR) was prepared by acid hydrolysis as described previously(11) . ASOR, protein A (Sigma), and ovalbumin (Sigma) were iodinated by a chloramine-T method (12) or by a coupled lactoperoxidase/glucose oxidase method following the manufacturer's instructions (Bio-Rad). The specificity of the polyclonal antibody to ASGR has been described previously(13) . Antibody to the transferrin receptor was kindly provided by Dr. C. Enns, Oregon Health Science University. Gelonin and N-succinimidyl-3-(2 pyridyldithio) propionate (SPDP) were obtained from Pierce Chemical Co. Methyl-thiazolyl-tetrazolium (MTT) was obtained from Sigma. The following toxins were also obtained: ricin (EY Laboratories, San Mateo, CA); wheat germ agglutinin (Sigma); abrin (Vector, Burlingame, CA); Pseudomonas toxin and modeccin (kindly provided by Dr. April Robbins, NIH); and diphtheria toxin (Biological Laboratories, Campbell, CA).

Preparation of Glycoprotein-Gelonin Conjugates

ASOR or ovalbumin (1 mg/ml) was dissolved in phosphate-buffered saline (PBS), pH 7.2, containing 0.5 mM EDTA. Gelonin (1 mg/ml) was dissolved in PBS, pH 8.0. A 10-fold molar excess of SPDP, dissolved just prior to use at a concentration of 20 mM in absolute ethanol, was added to each protein solution. The reaction was allowed to proceed for 1 h at room temperature and was terminated by gel filtration through a column of Sephadex G-25 equilibrated with the protein dissolution buffer. The number of covalently linked dithiopyridyl groups was determined by the increase in absorbance measured at 314 nm following reduction with increasing amounts of dithiothreitol(14) . The SPDP-activated glycoproteins were dialyzed against PBS, pH 6.0, for 4 h at 4 °C. After dialysis, the modified glycoproteins were incubated for 15 min at 22 °C with dithiothreitol in a 50-fold molar excess to the number of linked dithiopyridyl groups. The reaction was terminated by filtration through a Sephadex G-25 column equilibrated with PBS, pH 7.4. The protein-containing fractions were immediately added to an equal quantity of SPDP-activated gelonin, and the coupling reaction was allowed to proceed for 18 h at 4 °C. The reaction was terminated by the addition of iodoacetamide to a final concentration of 20 mM and incubation for 30 min at 22 °C. Following centrifugation (10,000 g, 10 min) to remove the small amount of insoluble material, the reaction mixture was concentrated in an Amicon P-30 and the conjugate was purified by filtration on Sephadex G-200 equilibrated with PBS, pH 7.4. Aliquots of protein-containing fractions were resolved by reducing and nonreducing 10% SDS-PAGE. The presence of the desired conjugate was confirmed by the detection of hetero-oligomers and dimers which, upon reduction, were resolved into the appropriately sized monomers.

Properties of Glycoprotein Conjugates

Nonreducing SDS-PAGE analysis of the glycoprotein-conjugates resolved the conjugates into major bands of approximately 70 and 100 kDa for ASOR-gelonin and 70 kDa for ovalbumin-gelonin, respectively. The addition of dithiothreitol to the sample buffer resulted in the reduction of each conjugate into its constituent proteins. Densitometric analysis of the resolved proteins suggested that the recovered conjugate of ASOR-gelonin varied between a 1:1 and 2:1 molar ratio of gelonin to glycoprotein, whereas ovalbumin-gelonin was mainly a 1:1 molar conjugate.

Cell Culture

HuH-7 cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were plated in 60-mm plastic dishes (Falcon) and were grown near or at confluence before each experiment unless otherwise indicated. The number of viable cells was estimated by an MTT assay(15) .

Cell Surface Binding ofI-ASOR and I-Transferrin

Cells were preincubated for 1 h in serum-free MEM made 2.5 mM in CaCl (MEM-CA) at 37 °C. To assay surface binding, cells were chilled to 4 °C and incubated for 1 h with I-ASOR (2 µg) or I-transferrin (1 µg) added to 1 ml of MEM-CA. Nonspecific ligand binding was estimated from culture dishes that also contained 100 µg of unlabeled ligand. Unbound I-ASOR or I-transferrin was removed by two washes with 1.5 ml of MEM-CA. In the case of I-ASOR, a final wash with MEM-CA made 5 mM in N-acetylgalactosamine (GalNAc) was performed(16) . Surface-bound I-ASOR was released with 50 mM GalNAc(15, 16) . In a typical binding assay, 5-7 ng of I-ASOR (5 10 cpm/ng) was specifically bound to 1 10 HuH-7 cells. Surface-bound I-transferrin was determined from the radioactivity associated with cells harvested at 4 °C(10) . In a typical transferrin assay, 1 10 HuH-7 cells bound 4 ng of I-transferrin (4 10 cpm/ng).

Internalization and Degradation ofI-ASOR

Internalization and degradation of I-ASOR were quantitated after uptake at 37 °C. At various times, cells were cooled to 4 °C, and an aliquot of medium was added to an equal volume of 20% trichloroacetic acid, 4% phosphotungstic acid to assess ligand degradation(16, 17) . The cells were then washed twice and incubated in 50 mM GalNAc or 20 mM EGTA for 10 min at 4 °C to remove residual surface radioactivity. Cells were washed again and harvested in 1 ml of PBS, and the amount of residual, cell-associated I-ASOR was quantified.

Diacytosis

Cells were incubated with I-ASOR (2 µg/ml) for 10 min at 37 °C to allow ligand internalization. Cells were then washed at 4 °C, and residual surface ligand was removed by a 10-min incubation in 50 mM GalNAc. Pregassed and warmed MEM containing 50 mM GalNAc was added, and the cells were incubated at 37 °C. Over time, aliquots of medium were withdrawn, and radioactivity was determined. Degradation products were quantitated by trichloroacetic acid precipitation as described above. The cells were then washed with MEM and harvested. Intact, I-ASOR released into the medium (diacytosed) was measured as acid-precipitable radioactivity. There was no cellular contamination of the medium as centrifugation (5 min in Eppendorf centrifuge 5412) to remove intact cells before acid precipitation did not alter the results.

Immunoprecipitation of ASGR

[S]Cysteine/methionine metabolically labeled (13) or I-surface labeled (11) cells were lysed in 1.0 ml of lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 1% Nonidet P-40, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride containing 100 units of aprotinin, 1 µg of leupeptin, 1 µg of pepstatin). Aliquots of cell lysates containing an equal amount of protein were precleaned by the addition of 20 µl of Gammabond G (50% suspension) prebound with 10 µl of preimmune serum and were incubated at 4 °C with constant mixing for 30 min. The lysates were centrifuged, and 10 µl of polyclonal antiserum and 50 µl of Gammabond G were added to the supernatant. Following 4-6 h of incubation at 4 °C with constant mixing, the lysates were centrifuged, and the pellets were washed three times with a solution containing 10 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS, pH 7.4, and a final wash with 50 mM Tris, 150 mM NaCl, pH 7.4. An equal volume of 2 SDS-PAGE sample buffer was added, and the pellets were heated at 90 °C for 10 min prior to electrophoresis on a 10% gel. The fixed gel was prepared for either fluorography with Enhance (DuPont NEN) for S or autoradiographic analysis for I. To estimate the amount of S-amino-acid incorporated into protein, a 50-µl aliquot of the lysate was added to 0.5 ml of 10 mM Tris, pH 7.4, 150 mM NaCl containing 0.1% BSA to which an equal volume of 20% trichloroacetic acid was added. The precipitated protein was collected on a Whatman GF/A glass fiber filter under reduced pressure and was washed with 10 ml of methanol. The air-dried filter was dispersed in 10 ml of Hydrofluor (National Diagnostics), and the amount of S was determined.

Acidification of Endosomes

The conversion of Semliki Forest virus (SFV) to the acid conformation was utilized as an indicator of endosomal acidification as described by Phalen and Kielian (18). Metabolically radiolabeled SFV (kindly provided by Dr. M. Kielian, Albert Einstein College of Medicine, New York) was prebound to cells on ice with continuous shaking for 2 h. Following removal of unbound SFV, cells were warmed to 37 °C to initiate endocytosis. At timed intervals, cells were shifted to 4 °C and washed two times with ice-cold PBS. Cells were lysed, and duplicate aliquots were processed for immunoprecipitation (see ``Immunoprecipitation of ASGR''). The recovered precipitates were analyzed by nonreducing 10% SDS-PAGE. Acid-converted E1 protein was selectively bound by the monoclonal antibody E1a-1, which is specific for the acid conformation, and surface-bound SFV was precipitated with polyclonal antibody (provided by M. Kielian)(19) . Acid-converted E1 immune complexes were precipitated with rabbit anti-mouse antibody prebound to fixed Staphylococcus aureus (Zysorbin; Zymed Laboratories, San Francisco) and were analyzed by fluorography following SDS-PAGE.

Sensitivity of Cells to Lectins and Other Toxins

The sensitivity of cells to lectins and various toxins was determined essentially as described(20) . Briefly, the cells were removed from nearly confluent T-75 flasks by treatment with PBS containing 5 mM EDTA, pelleted by centrifugation, resuspended in -MEM + 10% FBS and counted. Each cell line was then diluted in the same medium to 2 10 cells/ml. A range of toxin concentrations was prepared in -MEM + 10% FBS, and 0.1 ml of each toxin was loaded into a well of a 96-well microtiter plate. Cells (2 10) were then added to the wells, and the plates were incubated at 37 °C until the control wells (without toxin) reached confluence. The concentration of each toxin required to kill 90% of the cells (D value) was determined by microscopic examination of surviving cells and after the plates were stained and fixed using a solution of 0.2% methylene blue in 50% methanol.

Immunoblotting

Aliquots of cell lysate in SDS-PAGE sample buffer were heated at 90 °C for 10 min before resolution of the proteins on a 10% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose and detected with antibody and iodinated protein A as described previously (21) or by chemiluminescence as per the manufacturer's instructions (ECL kit, Amersham Corp.).

Methionine Transport

Cells (1 10/dish) were preincubated in methionine minus RPMI supplemented with 10% dialyzed FBS for 1 h at 37 °C. Medium was replaced with prewarmed (37 °C) methionine minus RPMI supplemented with dialyzed FBS containing increasing concentrations of methionine and a constant amount of [S]methionine (1 10 cpm). Following the incubation period, uptake was terminated by the addition of 1.0 ml of ice-cold PBS containing 0.1% BSA(22) , and the cells were transferred immediately to an ice bath. Cells were washed three times with ice- cold PBS/BSA and harvested for counting. The extent of nonspecifically associated [S]methionine was estimated by an incubation at 4 °C, and this value was subtracted as an uptake blank.


RESULTS

Toxicity of Glycoprotein Conjugates

Prior to mutant selection, the toxicity of the glycoprotein-gelonin conjugates for HuH-7 cells was determined. Cells were exposed to various concentrations of gelonin conjugates for 1 h in serum-free Dulbecco's modified Eagle's medium (DMEM) containing 2.5 mM Ca. The medium was replaced with DMEM supplemented with 10% FBS, and cell viability was determined at 18 h by an MTT assay (Fig. 1). Neither unconjugated glycoproteins nor gelonin alone had any significant effect on cell viability at concentrations up to 20 µg/ml. However, the addition of ASOR-gelonin or ovalbumin-gelonin reduced cell viability in a concentration-dependent fashion, resulting in 80-90% cell death at 20 µg/ml of either conjugate. Although 5 µg of either conjugate reduced cell viability by only 20%, when cells were incubated with 5 µg/ml of both conjugates, cell death was increased to 80% ± 5%, similar to results upon incubation with 10 µg/ml of either conjugate alone (Fig. 1).


Figure 1: Toxicity of glycoprotein-gelonin conjugates. Near confluent HuH-7 cells were incubated for 1 h in serum-free DMEM supplemented with 1.8 mM CaCl containing increasing concentrations of ASOR-gelonin (ASOR-G) or ovalbumin-gelonin (Ova-G). The medium was replaced with DMEM supplemented with 10% FBS, and incubation was continued for 18 h. The number of viable cells was estimated by an MTT assay (see ``Experimental Procedures'') and direct cell counts. In a typical cell viability determination by an MTT assay, 100% equals 2-4 OD units before dilution, measured at a wavelength of 560 nm. Values reported are the means for triplicate determinations from three independent experiments expressed as a percent of the viable cell number obtained in the absence of glycoprotein-gelonin conjugates.



Mutant Selection

With the expectation that any profound alteration in the endocytic pathway might result in cell death, the selection protocol was designed to obtain conditional mutants. Cells (about 2 10 previously mutagenized with 0.04 µg/ml N-methyl N-nitrosoguanidine for 2 h and allowed to grow at 34 °C for 12 days), were incubated at 39 °C overnight before exposure to a combination of 8 µg/ml of each gelonin conjugate for 18-22 h. Cells were then incubated at 34 °C in fresh medium without toxin. At the end of 14 days at 34 °C, two colonies were recovered. The isolate Trf1 did not express a temperature-sensitive growth phenotype, and therefore all subsequent experiments were performed at 37 °C. In the course of assessing the selection protocol, it was found that ASOR binding activity in control HuH-7 cells, as well as Trf1, was reduced by 50% as a result of the 39 °C preincubation. This may have contributed to the survival of Trf1.

Toxin-Conjugate Resistance

A comparison of the toxicity of the conjugates to parental HuH-7 and mutant cells indicated a pleiotropic effect of the mutation (). Trf1 cells were approximately 3-fold more resistant than HuH-7 cells to both of the conjugates (10 µg/ml). Specificity of the conjugates was indicated by inhibition of their uptake in the presence of a 10-fold excess of unconjugated glycoprotein (). The toxicity of 10 µg/ml conjugate was reduced from 72 ± 5% for ASOR-gelonin and from 70 ± 3% for ovalbumin-gelonin to 8 ± 5% and 14 ± 2%, respectively. In contrast, the addition of 100 µg/ml ovalbumin to 10 µg/ml ASOR-gelonin or 100 µg/ml ASOR to 10 µg/ml ovalbumin-gelonin did not inhibit the toxic effect of the gelonin conjugates. That there was no significant difference in the sensitivity of parental and mutant cells in the presence of a 10-fold excess of the relevant unconjugated glycoprotein indicated that conjugate toxicity was exerted following binding to ASGRs and mannose receptors, respectively. These results were consistent with the notion that a common element affecting both ASGR and mannose receptor endocytosis was altered in Trf1.

Assessment of ASGR RME

Binding of I-ASOR was used to estimate the number of bioactive ASGRs and showed that cell surface binding activity was reduced by 49 ± 2.5% in Trf1 cells (Fig. 2). Consistent with this finding, the total amount of I-ASOR internalized by mutant cells was reduced by 50% when compared with internalization by HuH-7 cells (Fig. 2). Once internalized, ASOR enters a number of functionally distinct intracellular compartments(22, 23, 24) . A large proportion of internalized ASOR has been shown to recycle intact to the cell surface in association with the ASGR(23, 24, 25) , a phenomenon termed diacytosis. When the extent of diacytosis by Trf1 and HuH-7 cells was determined after normalizing to the amount of internalized ASOR, the return of ligand-receptor complex to the cell surface was found to be increased by approximately 2.5-fold in the mutant (Fig. 2). The final destination of internalized ASOR is the lysosome, the commonly accepted site of ligand degradation(23, 24, 25) . Endosomal trafficking to this subcellular compartment appears unaffected by the trf1 mutation, since the levels of acid-soluble I detected in the medium 1 h after internalization were 50% of the I-ASOR internalized for both HuH-7 and Trf1 cells (Fig. 2).


Figure 2: Determination of RME in Trf1 cells. Cells (2 10/dish) were grown to near confluence prior to assay to assure maximum binding activity. Following saturation of cell surface ASGRs at 4 °C, unbound I-ASOR was removed and the culture transferred to 37 °C to determine the extent of ligand internalization, diacytosis at 30 min, and degradation at 60 min, as described under ``Experimental Procedures.'' The values presented for internalization, diacytosis, and degradation are the means ± S.D., normalized to surface-bound I-ASOR for each cell line; data are presented as a ratio of these values. In a typical binding assay, 10-14 ng of I-ASOR (5 10 cpm/ng) was specifically bound to 2 10 HuH-7 cells.



Endosomal Acidification

The unaltered rate of ASOR degradation by Trf1 cells suggested that acidification within the lysosomes was not significantly affected by the mutation. However, it was important to test endosome acidification directly, since a failure to acidify this organelle appears to be the underlying defect in a number of endocytic mutants(4) . To assess endosomal pH, we took advantage of the acid-dependent, irreversible conformational change in the E1 spike protein of SFV(18) . Monoclonal antibody Ela-1, specific for the acid form of E1, gave rise to immunoprecipitates with the same kinetics in infected Trf1 and HuH-7 cells, providing evidence that endosomal acidification was unaltered by the trf1 mutation (Fig. 3).


Figure 3: Endosome acidification in Trf1 cells. [S]Methionine-labeled SFV was prebound for 2 h at 4 °C to HuH-7 or Trf1 cells. The cells were subsequently warmed to 37 °C for the indicated time, lysed, and duplicate aliquots analyzed by immunoprecipitation and nonreducing 10% SDS-PAGE. The first zero time point samples were precipitated with a polyclonal antibody against the SFV spike protein. The second zero and all subsequent time point samples were precipitated with the monoclonal antibody E1a-1, which recognizes the acid conformation of E1.



Additional evidence that the endosomal compartment in Trf1 was acidic was obtained from the sensitivity of the cells to a panel of toxins requiring endosomal acidification for their action (). Trf1 cells were not resistant to any of several toxins. In fact, Trf1 cells were significantly more sensitive to each toxin and to the lectin WGA. This contrasts with the CHO endocytosis mutants End 1-End 5, which are all resistant to diphtheria toxin (4) and to two ricin internalization mutants that are resistant to ricin and to Pseudomonas toxin(26, 27) . In addition, the sensitivity of Trf1 cells to lectins such as wheat germ agglutinin, ricin, abrin, and modeccin, which bind to cell surface carbohydrates, provides evidence that Trf1 cells are not defective in cell surface glycosylation.

Subcellular Distribution of ASGR and Transferrin Receptor

Estimation of total ASGR expression by Western blot analysis using a polyclonal antibody to both receptor subunits indicated that the resistance of Trf1 cells to ASOR-gelonin is not the result of a reduction in the amount of their receptor protein. However, selective labeling of ASGRs at the cell surface, followed by subsequent immunoprecipitation, indicated that Trf1 mutant cells had a 46 ± 5% (n = 3) reduction in the expression of cell surface receptors compared with HuH-7 cells. This suggested an altered distribution of ASGRs in Trf1 cells (Fig. 4).


Figure 4: Steady-state distribution of ASGRs in HuH-7 and Trf1 cells. For the estimation of total receptor, lysates (20 µg of protein/lane) of HuH-7 (lane A) and Trf1 (lane B) were resolved by 10% SDS-PAGE and transferred to nitrocellulose. The membrane was probed with polyclonal antibody against affinity-purified receptor; the signal was detected using I-protein A and autoradiography. The surface content of receptor in HuH-7 (lane C) and Trf1 (lane D) was determined following the selective iodination of cell surface proteins, immunoprecipitation, and resolution on 10% SDS-PAGE. This figure is representative of three studies that were quantitated by densitometry.



The subcellular distribution of transferrin receptor was also found to be altered. Western blot analysis indicated that the Trf1 mutation had no effect on the amount of transferrin receptor in Trf1 cells (Fig. 5). However, the cell surface expression of transferrin receptor was reduced by 44%. The reduction in cell surface transferrin receptor was confirmed by a direct binding assay of I-transferrin at 4 °C. When compared with the parental HuH-7, the Trf1 cells exhibited a 30 ± 2% (n = 3) reduction in I-transferrin binding. Although the effect of the mutation on transferrin receptors was not as dramatic as that for ASGRs, it was highly significant and supports receptor redistribution as one of the major phenotypes of Trf1 cells. The resistance of Trf1 cells to ovalbumin-gelonin is consistent with a redistribution of mannose receptors; however, this could not be tested directly, as an antibody specific for mannose receptors was not available, and binding experiments with I-ovalbumin-BSA gave a high background of nonspecific binding.


Figure 5: Steady-state distribution of the transferrin receptors in HuH-7 and Trf1 cells. For the estimation of total receptor, cell lysates (20 µg of protein/lane) of HuH-7 (lane A) and Trf1 (lane B) were resolved by 10% SDS-PAGE and transferred to nitrocellulose. The membrane was probed with a polyclonal antibody against the purified receptor, and the signal was detected by chemiluminescence. The surface content of receptor in HuH-7 (lane C) and Trf1 (lane D) was determined following the selective iodination of the cell surface, immunoprecipitation, and SDS-PAGE. This figure is representative of two experiments quantitated by densitometry.



Biosynthetic Processing of ASGR

Metabolic labeling with [S]cysteine/methionine indicated that the reduced expression of ASGRs at the cell surface of Trf1 cells was not due to an altered rate of intracellular processing of its subunit polypeptides (Fig. 6). Although the amount of S-labeled receptor was markedly reduced in Trf1, the intracellular ASGRs were processed in a fashion identical to that in control cells, which was consistent with the discrete processing steps described previously (21, 28).


Figure 6: Biosynthetic processing of ASGR by HUH-7 and Trf1 cells. Near confluent HuH-7 (upper panel) and Trf1 (lower panel) cells (1 10/dish) were metabolically labeled with a mixture of [S]cysteine/methionine for 15 min. At various times of chase, the cells were harvested, and ASGRs were immunoprecipitated from aliquots of cell lysates (see ``Experimental Procedures'') containing equal amounts of protein. The recovered immunoprecipitates were resolved on 10% SDS-PAGE and autoradiographed.



Determination of trichloroacetic acid-precipitable radioactivity suggested that the Trf1 mutation results in either a 47 ± 2% reduction in total protein synthesis or in transport of the radiolabeled amino acid. As total cellular protein was the same in HuH-7 and Trf1 (0.5 mg/10 cells), we suspected that methionine transport was reduced. To investigate this possibility, the kinetic characteristics of [S]methionine uptake were determined (Fig. 7). In preliminary experiments, it was established that the uptake of both 100 and 2500 µM methionine was linear over 4 min (data not shown). A 0.5-min uptake period was used to avoid confounding the data by potential differences in the rates of protein synthesis between Trf1 and HuH-7. The reduced uptake of methionine by Trf1 cells reflected a 2.5-fold reduction in V (3.05 ± 0.3 versus 1.55 ± 0.1 nmol/min/mg of protein, n = 3) with no appreciable change in the apparent K of transport (0.69 ± 0.1 versus 0.70 ± 0.09 µM, n = 3), suggesting a probable reduction in the number of Phe transporters in the plasma membrane.


Figure 7: Methionine uptake by HuH-7 and Trf1 cells. In this representative experiment, HuH-7 () and Trf1 () cells (10/dish) were preincubated for 1 h in methionine minus RPMI supplemented with 10% dialyzed FBS. Methionine uptake was measured for 30 s at 37 °C as described under ``Experimental Procedures'' at increasing concentrations of amino acid. The values expressed are means ± S.D. of triplicate determinations.



ASGR Subpopulations

Recently the concept of two subpopulations of ASGRs, designated States 1 and 2 by Weigel and Oka (10), and normally present in equal amounts at the surface of hepatocytes, was proposed (for review see Ref. 29). Levels of State 2 receptors, but not State 1 receptors, are modulated by various metabolic inhibitors(30) . To investigate the nature of State 1 and State 2 receptors in HuH-7 and Trf1, cells were subjected to a 1-h preincubation with either 10 mM sodium azide, 40 µg/ml colchicine, or 50 µM monensin prior to determining surface ASGR expression by the I-ASOR binding assay. In agreement with previous observations with isolated hepatocytes(29, 30, 31, 32) , the amount of I-ASOR bound to the cell surface of the parental HuH-7 line was reduced by approximately 50% with each treatment (Fig. 8). In contrast, there was no significant change in the cell surface binding of I-ASOR by Trf1 cells under any condition, suggesting that the reduced number of cell surface receptors expressed by the mutant is solely in the State 1 configuration.


Figure 8: Effect of sodium azide, colchicine, and monensin on cell surface expression of ASGRs. Cells (2 10/dish) were preincubated for 1 h with 10 mM sodium azide, 40 µg/ml colchicine, or 50 µM monensin prior to determination of cell surface I-ASOR binding as described under ``Experimental Procedures.'' In a typical binding assay, 100% equals 10-14 ng of I-ASOR (50-70 10 cpm/dish) of HuH-7 cells. The values presented are the means ± S.D. of three independent experiments.




DISCUSSION

The dual ligand-toxin selection protocol described in this paper was designed to exclude the isolation of simple receptor mutants, and as expected, the mutant Trf1 was equally resistant to both toxin conjugates (). As there was no significant reciprocal inhibition between the mannose- and galactose-terminated glycoproteins and their respective toxin conjugates (), the mutation harbored by Trf1 cells arose as a consequence of an alteration in a common step in their endocytic pathways.

Trf1 cells exhibit a pleiotropic phenotype that alters not only ASGR and mannose receptor expression (both objects of the dual selection protocol) but also transferrin receptor expression. The Trf1 mutation results in an approximately 50% reduction in cell surface expression of ASGR and transferrin receptor at the plasma membrane. The 2-fold reduction in V for methionine uptake in the absence of any significant alteration in apparent Kextends the effect of the mutation to yet another type of cell surface protein, the Phe transporter (Fig. 5). Although direct evidence for the redistribution of the Phe amino acid transporter and the mannose receptor could not be obtained, the reduction of both ASGRs and transferrin receptors specifically at the cell surface makes it reasonable to assume that the trf1 mutation also causes redistribution of these proteins.

Early studies established the functional (33) and metabolic (34) separation of the cell surface ASGR population from that of the larger intracellular receptor pool. More recently, the concept of two subsets of surface receptors, designated States 1 and 2 by Weigel and Oka(10) , has gained support (for review see Ref. 29). Originally defined on the basis of the biphasic kinetics of ligand-receptor complex dissociation (35), numerous characteristics have since been described which differentiate these two receptor states and the proposed endocytic pathways they mediate(29) . Although the biochemical basis for these two types of receptor has not been established, only State 2 receptors are proposed to be susceptible to modulation by a variety of cell perturbants, including reduced temperature, colchicine, cytokines, phorbol esters, monensin and chloroquine, azide, vanadate, or ethanol (for review see Refs. 29 and 36). Each of these agents causes an approximately 50% reduction in cell surface ASGR activity, and in some cases, this loss is accompanied by the loss or redistribution of cell surface receptor protein.

State 2 receptors, once they are internalized, undergo a reversible inactivation and reactivation before they return to the cell surface (37). It has been proposed that this inactivation may be an important factor in ligand-receptor dissociation and segregation(35) . The selective reduction of cell surface ASGR in Trf1 cells (Fig. 4) appears to be due to the loss of State 2 receptors. A failure to respond to treatment with sodium azide, monensin, or colchicine (Fig. 8) further supports the notion that the mutation limits the expression of cell surface ASGRs to State 1. The differential response of State 1 and State 2 receptors to these perturbants has been utilized to demonstrate alternative intracellular trafficking pathways for their receptor-ligand complexes. For example, State 1 receptors have been proposed to be responsible for the observed diacytosis of internalized ligand(10, 36) , and this is consistent with the increased rate of diacytosis observed in Trf1 cells ().

The existence of two subpopulations of receptors is not limited to the ASGR and has been suggested for a wide variety of receptors on many different cell types(29, 36) . It is reasonable to hypothesize that a common biochemical basis may underlie differentiation into the two states. Interestingly, total cell ASGR and transferrin receptor contents in Trf1 do not differ from those in HuH-7 ( Fig. 4and Fig. 5). This suggests that State 2 receptors are ``trapped'' within Trf1. Although no particular post-translational modification of the ASGR has been described to account for its segregation into States 1 and 2, changes in phosphorylation (38) and acylation (39) have recently been suggested to affect the transition from State 1 to State 2. These post-translational modifications are common to most, if not all, cell surface receptors, and a change in one of them might define the receptor state.

Mutant CHO cells defective in various stages of the endocytic process have been described by several laboratories(4, 5, 6, 7) . Somatic cell genetic analysis has been used to assign these mutants into complementation groups, End 1-End 6 (4, 5) or ldlA-I(6, 7) . In all cases, the gene defect harbored by the endocytosis mutants results in a pleiotropic phenotype, suggesting multiple functions for the gene product. Fluid phase endocytosis and lysosomal biogenesis are affected in the End 1, End 3, End 4, and End 6 complementation groups(40, 41, 42, 43) . Golgi trafficking is affected by the respective gene product defining End 2 and End 4 mutants(44, 45) . The End 6 gene product appears to play a role in receptor recycling from endosomes back to the cell surface(46) . The unaltered pH-dependent conversion of SFV E1 polypeptide (Fig. 3) and enhanced susceptibility of Trf1 cells to pH-dependent toxins would appear to preclude the most common explanation for the pleiotropic effect of End 1-End 4 mutants(4) , namely a defect in endosomal acidification. Whereas Trf1 cells are not resistant to any toxins or lectins tested, End 1-End 5 mutants are resistant to diphtheria toxin, and some endocytosis mutants, as well as two ricin internalization mutants, are resistant to Pseudomonas toxin (26, 27, 46) or to modeccin(47) . When compared with endocytosis complementation groups, the Trf1 mutation most closely resembles End 6, in which the fast receptor recycling process is inhibited(5) . However, End 6 cells have a conditional lethal phenotype. Within the ldl complementation groups, ldlB-G mutants were found to carry general defects affecting synthesis of N-linked, O-linked, and lipid-linked oligosaccharides(48) , resulting in lectin resistance and altered processing of the low density lipoprotein receptor. The normal size and processing of ASGR in Trf1 cells and their relative sensitivity to plant lectins () distinguish them from each of the ldl mutants (Fig. 6). The ldlA locus is the structural gene for the low density lipoprotein receptor(49) , and ldlH is a conditional lethal (6). ldlI mutants have a constitutive phenotype like Trf1 but, in contrast to Trf1, these mutants are 80-fold hypersensitive to ricin (6). The Trf1 phenotype is therefore unique, and definition of the trf1 mutation should provide new insight into the mechanisms regulating receptor expression and the endocytic pathway.

  
Table: Toxicity and specificity of glycoprotein-toxin conjugates

Confluent Trf1 and HuH-7 cultures (1 10 cell/60-mm dish) were exposed to ASOR-gelonin (ASOR-G) or ovalbumin-gelonin (Ova-G) in the absence or presence of unconjugated glycoprotein at the indicated concentration. Following a 1-h incubation in serum-free medium, the cells were washed free of glycoprotein(s) and incubated overnight (18 h) in medium with serum. Values shown are the means ± S.D. from three independent experiments, expressed as a percent of vaiable cells in treated versus untreated cultures and determined by an MTT assay or direct cell counting (see ``Experimental Procedures'').


  
Table: Toxin and lectin toxicity

Cells were incubated in increasing concentrations of toxic agent until control wells were confluent, as described under ``Experimental Procedures.'' The concentration that killed 90% of the cells in a representative experiment is given. The relative sensitivity of Trf1 is given as the range of fold differences compared with HuH-7 cells in two or more experiments. A dash indicates no increase in the sensitivity of the Trf1 cells.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK41918 and DK41296. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Ullmann 517, Bronx, NY 10461. Tel.: 718-430-2098; Fax: 718-918-0857.

The abbreviations used are: RME, receptor-mediated endocytosis; CHO, Chinese hamster ovary; End, endocytosis; ldl, low density lipoprotein; ASGR, asialoglycoprotein receptor; Trf1, trafficking mutant 1; ASOR, asialoorosomucoid; SPDP, N-succinimidyl 3-(2-pyridyldithio) propionate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; MEM, minimal essential medium; FBS, fetal bovine serum; BSA, bovine serum albumin; SFV, Semliki Forest virus; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We thank Subha Sundaram and Hsiao-Nan Hao for excellent technical assistance and Anna Caponigro for the preparation of this manuscript.


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