From the
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)
Receptor-mediated endocytosis (RME)
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).
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
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
Confluent Trf1 and HuH-7 cultures (
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.
We thank Subha Sundaram and Hsiao-Nan Hao for
excellent technical assistance and Anna Caponigro for the preparation
of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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) .
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 of
Cells were preincubated
for 1 h in serum-free MEM made 2.5 mM in CaClI-ASOR
and
I-Transferrin
(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 of
Internalization and
degradation of I-ASOR
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
En
hance (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.
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.
for methionine uptake in the absence of any
significant alteration in apparent K
extends 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.
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
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.