(Received for publication, November 14, 1996, and in revised form, February 10, 1997)
From the Genetic variants of human
In eukaryotes, proteins destined for secretion are translocated as
nascent polypeptides into the lumen of the endoplasmic reticulum
(ER)1 (for a review, see Ref. 1). Folding
into the native conformation, a structure dictated by the primary amino
acid sequence (2), is facilitated through transient interaction with
one or more molecular chaperones (3). Conformational fidelity of folded structures is monitored by a poorly understood quality control system
(4) which prevents transport of incompletely folded and unassembled
proteins beyond the ER (5).
Cotranslational addition of
Glc3Man9GlcNAc2 to specific
asparagine residues and hydrolysis of attached glucose units can
accompany translocation of the nascent polypeptide (6). Reglucosylation of high mannose-type glycans has been detected in microsomal
preparations from mammals, plants, fungi, yeast, and protozoa (7, 8) and is catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (UGTR) (8-10). Importantly, only high mannose-type oligosaccharides attached to unfolded proteins function as acceptors in
the glucose transfer reaction (11-13). Results from a cell-free system
indicate that the unfolded polypeptide and asparagine-linked GlcNAc are
responsible for eliciting glucose transfer (13).
Several nascent proteins (14-18) form transient associations with
calnexin (also designated p88 or IP90), a calcium-binding protein of
the ER membrane (19). Since calnexin functions as a molecular chaperone
for glycoproteins (17, 20) and interacts with monoglucosylated
oligosaccharides (21), Hammond et al. (15) proposed that
reglucosylation by UGTR may function to initiate assembly between
unfolded glycoproteins and the molecular chaperone. In support of this
idea, Labriola et al. (22) have reported that delivery of a
nascent acid hydrolase to lysosomes of Trypanosoma cruzi is
delayed by inhibition of ER Recent evidence suggests that protein folding and quality control
machinery may participate in the molecular pathogenesis of several
human diseases caused by defective intracellular transport of an
aberrantly folded protein through the secretory pathway (23-25). Human
AAT is a member of the serine proteinase inhibitor superfamily (36).
Since elastase released by activated neutrophils is rendered inactive
by the inhibitor (37), diminished circulating levels can result in
proteolytic destruction of lung elastin, a phenomenon implicated in the
pathogenesis of chronic obstructive lung disease (38). "Loop-sheet"
polymerization is apparently responsible for accumulation of a subset
of human AAT variants in the ER of hepatocytes (31). However, impaired
secretion of the majority of variants does not include detectable
intracellular accumulation. Most "null" alleles encode a
polypeptide truncated at the carboxyl terminus (39), which is a
phenomenon predicted to preclude formation of specific secondary
structural features (31) and can prevent loop-sheet polymerization.
Variant QO Hong Kong (null(Hong Kong)) cannot attain conformational
maturation following biosynthesis, forms a physical interaction with
molecular chaperone calnexin (24), and is expected to exhibit a
persistent interaction with quality control machinery of the ER. We
demonstrate that a detectable interaction between null(Hong Kong) and
UGTR results in response to manipulations predicted to diminish ER lumenal Ca2+ stores. Interpretation of these findings with
regard to factors responsible for quality control of human AAT folding
and secretion are discussed.
Protein G-agarose was purchased from
Calbiochem. Thapsigargin was purchased from LC Laboratories. All other
detergents, buffers, and salts (including sodium fluoride and sodium
azide) were purchased from Sigma. Easy Tag
[35S]methionine was purchased from DuPont NEN. All tissue
culture media were purchased from Life Technologies, Inc. Fetal bovine serum was procured from Summit Biotechnology.
The cell line H1A/N13 was previously
generated by stable transfection of the mouse hepatoma cell line Hepa
1a with subcloned DNA encoding the human null(Hong Kong) AAT variant
(40). Cells were maintained as monolayers in Dulbecco's modified
Eagle's/Waymouth medium (3:1) containing 15% fetal bovine serum,
1 × glutamine, and 1 × penicillin/streptomycin. The cell
line remained under selective pressure with antibiotic G418 (40).
Confluent monolayers
were washed with phosphate-buffered saline and then subjected to
methionine starvation for 30 min by incubation in methionine-free
Dulbecco's modified Eagle's medium. Proteins were radiolabeled during
a 15-min incubation with [35S]methionine (400 µCi) in
the same medium. Radiolabel was removed by washing monolayers with
warmed phosphate-buffered saline, and cells were "chased" for a
selected period by incubation at 37 °C in growth medium containing a
4-fold excess of unlabeled methionine. For selected dishes, chase
medium was supplemented with either sodium fluoride, sodium azide, or
thapsigargin as described in each figure legend. For cell lysis,
monolayers were washed with cold phosphate-buffered saline and scraped
with a spatula in cell lysis buffer (0.1 M Tris-HCl, pH
7.4, containing 0.5% Nonidet P-40, 0.15 M NaCl) and a
mixture of protease inhibitors as described previously (24). Insoluble
material was removed by centrifugation at 10,000 × g
for 10 min. Cellular toxicity was measured by trypan blue dye
exclusion. The hemocytometer was used to detect cells stained during a
5-min incubation with dye. Greater than 98% remained viable in
response to all reported manipulations.
Detection of proteins coprecipitating
with variant null(Hong Kong) was performed as described previously
(24). Briefly, soluble lysates were incubated for 120 min at 4 °C
with an IgG fraction of goat anti-human AAT (Organon Teknika-Cappel)
pre-immobilized to Protein G-agarose (24). Immunocomplexes were washed
with cell lysis buffer by agitation at 4 °C and then with the same buffer containing 0.5 M NaCl. Washed immunocomplexes were
heated for 10 min at 75 °C in disruption buffer containing 2%
sodium dodecyl sulfate and gel loading buffer. Eluted proteins were
separated from agarose beads by centrifugation and then fractionated by SDS-PAGE. Radiolabeled proteins were detected by fluorographic enhancement of the vacuum-dried gel.
Immunoprecipitated proteins were
fractionated by SDS-PAGE and electrophoretically transferred to Hybond
nitrocellulose (Amersham Corp.) as described previously (24) except
that 0.1% SDS (0.1%) was included in the transfer buffer. Enhanced
chemiluminescence (ECL) Western blotting (Amersham Corp.) was performed
according to the manufacturers instructions, except that SuperSignal
(Pierce) was used as the detection reagent. Calnexin was detected by
incubation of blots with a 1:1000 dilution of rabbit polyclonal
antisera raised against a synthetic polypeptide homologous to amino
acids 487-505 in the cytoplasmic tail of canine calnexin (gifts from Drs. J. J. M. Bergeron, McGill University, and A. Helenius, Yale University). Detection of UDP-glucose:glycoprotein glucosyltransferase was performed using a 1:500 dilution of rabbit polyclonal antiserum against rat UDP-glucose:glycoprotein glucosyltransferase (a generous gift from Dr. Armando Parodi, Instituto de Investigaciones Bioquimicas, Buenos Aires, Argentina).
The Howtek Scanmaster 3+
densitometric scanner was used for quantitation of band intensities
corresponding to radiolabeled proteins detected in fluorograms.
Intracellular Ca2+ distribution was examined
by scanning laser confocal microscopy using the fluorescent
Ca2+ probe Fluo-3 (Molecular Probes Inc., Eugene, OR).
Briefly, H1A/N13 cells were cultured overnight on coverslips and then
treated as described in the appropriate figure legend. Cells were then
incubated for 2 min at 37 °C with 2 µM Fluo-3.
Coverslips were transferred to an inverted glass slide to ensure
adherence and placed on an inverted Nikon Diaphot microscope (Nikon,
New York, NY). Cells were scanned for maximum fluorescence via a
section series by scanning laser confocal microscopy.
Variant null(Hong
Kong) is only 333 amino acids in length, as compared with the
full-length 394-amino acid polypeptide and results from a TC
dinucleotide deletion in the structural gene (40). Unlike the normal
M1(Val213) human AAT variant, which is efficiently secreted from stably
transfected mouse hepatoma cells, the 45-kDa variant null(Hong Kong) is
not secreted whatsoever (40) and is eventually cleared from the cell
(41). A noncovalent physical interaction between null(Hong Kong) and
molecular chaperone calnexin has been reported (24). Since ATP and
Ca2+ bind the lumenal domain of calnexin (42) and are
perceived to regulate its activity as a molecular chaperone (19, 43), we investigated the effect of intracellular ATP depletion on quality control of null(Hong Kong). For this, newly synthesized molecules were
pulse-radiolabeled with [35S]methionine, and subsequent
intracellular disposal was assessed during a 60-min chase in medium
supplemented with metabolic poisons NaF and NaN3.
Approximately 30% of the radiolabeled molecules were degraded in
untreated cells (Fig. 1A, compare lanes
1 and 2), and disposal was slightly enhanced in
response to incubation with metabolic poisons (Fig. 1A,
compare lanes 2 and 3). A modest increase in
electrophoretic mobility was observed (Fig. 1A, compare lanes 2 and 3), and results of endoglycosidase H
digestion indicated that the phenomenon had resulted from
post-translational trimming of asparagine-linked oligosaccharides (not
shown).
In addition to these slight alterations, a radiolabeled 150-kDa protein
(p150) was detected in the null(Hong Kong) immunoprecipitate from
treated cells (Fig. 1A, lane 3) but was absent
from untreated cells (Fig. 1A, lane 2).
Furthermore, p150 was not detected in a mock immunoprecipitate from
nontransfected Hepa 1a cells treated with metabolic poisons (Fig.
1B, lane 3). These findings indicated that an
authentic physical interaction between p150 null(Hong Kong) was
detectable in response to incubation of cells with metabolic poisons.
To rule
out the possibility that intracellular association with p150 was merely
an artifact of cellular toxicity, pulse-radiolabeled cells were
incubated for 60 min in medium containing NaF and NaN3 and
then incubated for an additional 60-min period in regular growth
medium. Coimmunoprecipitation of p150 was not detectable following the
second incubation (Fig. 2A, compare
lanes 2 and 3), suggesting that the interaction
was reversible. In a parallel experiment, trypan blue was excluded by
>98% of cells (not shown), confirming that treatment with metabolic
poisons had not resulted in cell death.
Intensity of coimmunoprecipitating p150 increased approximately 5-fold
in cells incubated for 120 min with medium containing NaF and NaN3.
(Fig. 2A, lane 4) as compared with those
incubated for only 60 min (Fig. 2A, lane 2).
Because intracellular disposal was unaffected by the longer incubation
period increased coimmunoprecipitation of p150 likely reflected gradual
intracellular accumulation of the null(Hong Kong)-p150 complex.
As combinations of metabolic poisons are required for efficient
reduction of intracellular ATP levels (44), we tested the ability of
NaF and NaN3 individually to facilitate coprecipitation of
p150. Incubation with either compound alone did not result in
detectable coimmunoprecipitation of p150, and there was no apparent
increase in electrophoretic mobility of radiolabeled molecules (Fig.
2B, lanes 3 and 4). Importantly,
coimmunoprecipitation of p150 required incubation with both compounds
(Fig. 2B, lane 2). These data are consistent with
the idea that detectable interaction between null(Hong Kong) and p150
had resulted from reduction of intracellular ATP levels.
Since coimmunoprecipitation of p150 resulted from
incubation of cells with metabolic poisons we asked whether the complex would dissociate in the presence of nucleotide triphosphates. In
separate experiments cell lysates containing the accumulated complex
were incubated for 10 min at 37 °C with exogenous ATP or GTP prior
to immunoprecipitation of null(Hong Kong). Coimmunoprecipitation of
p150 was unaffected (not shown). Reduction of intracellular ATP levels
can inactivate microsomal Ca2+ATPase, the enzyme
responsible for maintaining the high ER lumenal Ca2+
concentration (45, 46), so we considered the possibility that the
formation of the complex might have resulted from mobilization of
intracellular Ca2+ stores. To test this hypothesis cell
lysates containing the accumulated complex were incubated for 10 min at
37 °C in the presence of 1 mM CaCl2. As
shown in Fig. 3, coimmunoprecipitation of p150 was
diminished to 36% of that detected in the absence of the divalent cation. In three separate experiments incubation with CaCl2
diminished co-immunoprecipitation of p150 to a mean value of 40.1 ± 5.3% (not shown).
Changes in intracellular
Ca2+ distribution was monitored by scanning laser confocal
microscopy after incubation with the fluorescent Ca2+ probe
Fluo-3 (see "Materials and Methods"). Intense staining of
intracellular Ca2+ stores became very diffuse in response
to treatment of cells with metabolic poisons (Fig. 4,
compare panels A and B), indicating that
coimmunoprecipitation of p150 did coincide with mobilization of
intracellular Ca2+ stores. Toxicity of Ca2+
ionophores A23187 and ionomycin (not shown) prevented their use in our
analyses. To ask whether mobilization of ER Ca2+ stores was
important pulse-radiolabeled cells were incubated for 60 min with
medium containing 10 µM thapsigargin, a specific inhibitor of microsomal Ca2+ATPase (45, 47, 48).
Coimmunoprecipitating p150 was detected in thapsigargin-treated cells
(Fig. 5, lane 2) which was consistent with
the notion that reduction of ER lumenal Ca2+ stores did
coincide with intracellular accumulation of the null(Hong Kong)-p150
complex.
Since
variant null(Hong Kong) normally exhibits a Ca2+-sensitive
interaction with molecular chaperone calnexin (24), in the next set of
experiments ECL Western blotting was used as a method to quantitate
changes in coprecipitation of the molecular chaperone at conditions
that favored detectable formation of null(Hong Kong)-p150. Incubation
for 60 min with 10 µM thapsigargin diminished
coprecipitation of calnexin to only 5.3% of that detected in untreated
cells (Fig. 6, compare lanes 3 and
4). Association with calnexin was reduced to 35.4% of
normal in response to a 60-min incubation with metabolic poisons NaF
and NaN3 (Fig. 6, lane 5), and this was restored
to normal values following an additional 60-min incubation with regular growth medium (Fig. 6, lane 6). The data indicated that
detectable binding of p150 to null(Hong Kong) coincided with reduced
levels of associated calnexin.
ECL Western blotting was employed as a method
to confirm the identity of coprecipitating p150. p150 did not
cross-react with antiserum against human AAT or calnexin, indicating
that it did not consist of an SDS-resistant aggregate of null(Hong
Kong) or contain the molecular chaperone, respectively (not shown). In the course of our analyses we tested the hypothesis that p150 might
actually be UGTR, which is composed of two identical 150-kDa subunits
(49) and has been implicated as a component of glycoprotein quality
control (15, 17). The prediction was verified in that coprecipitated
p150 exhibited cross-immunoreactivity with a polyclonal antiserum
raised against rat UGTR (Fig. 7, lane 3).
Furthermore, mobility of p150 in SDS-PAGE was identical to that of
immunoreactive murine UGTR detected in the cell lysate (Fig. 7,
lane 1), and the signal was absent from a null(Hong Kong)
immunoprecipitate generated from untreated cells (Fig. 7, lane
4). It should be noted that the intensity of the band migrating
slightly faster than authentic UGTR (Fig. 7, lane 1) is
over-represented because of the longer length of exposure needed to
detect coprecipitating UGTR.
In the present study, a physical association between
immunoreactive UGTR and secretion-incompetent variant null(Hong Kong) was detected in response to incubation of cells with metabolic poisons.
The interaction was cumulative and required incubation of cells with
both metabolic poisons, sodium fluoride and sodium azide. The exclusion
of trypan blue from treated cells and the absence of coprecipitating
UGTR following removal of metabolic poisons suggest that formation of
the complex did not represent an artifact of cell death. Detection of
radiolabeled UGTR in the context of our radiolabeling procedure
suggests that it may contain a significant number of methionine
residues or exhibit rapid intracellular turnover. Intracellular
association between null(Hong Kong) and calnexin was diminished at
conditions that induced coprecipitation of UGTR. Because ATP is
required to stabilize calnexin-glycoprotein interactions in the ER
(43), and reduction of nucleotide levels would be expected to
dissociate the null(Hong Kong)-calnexin complex.
Exogenous ATP had no influence on stability of the accumulated
null(Hong Kong)-UGTR interaction, suggesting that lowered intracellular availability of the nucleotide was not solely responsible for intracellular accumulation of the complex. Ability of thapsigargin treatment to result in detectable coprecipitation of p150, plus destabilization of the interaction during a 37 °C incubation with exogenous Ca2+, supported the hypothesis that depletion of
ATP by metabolic poisons might have resulted in inactivation of the
microsomal Ca2+ATPase pump, leading to reduction of ER
lumenal Ca2+ stores. In support of this concept, depletion
of ATP in rat liver microsomes induced calcium release and pump
inhibition, and these effects were mimicked by thapsigargin treatment.
Considering these data, it is reasonable to conclude that reduction of
ER lumenal Ca2+, occurring as an indirect effect of
depleting intracellular ATP, likely played a significant role in
causing detectable accumulation of the null(Hong Kong)-UGTR complex and
is also supported by coprecipitation of UGTR in response to
mobilization of the thapsigargin-sensitive Ca2+ pool.
The physical interaction between null(Hong Kong) and molecular
chaperone calnexin (24, 50) is disrupted by incubation with
Ca2+ chelators (24). Consistent with this finding was the
observation that thapsigargin treatment diminished interaction between
calnexin and null(Hong Kong). Furthermore, the treatment diminished
interaction with calnexin to a greater extent than did metabolic
poisons. Despite this fact, coprecipitation of UGTR was not greater in thapsigargin-treated cells, and longer incubations did not result in
gradual accumulation of the null(Hong Kong)-UGTR complex (not shown).
An explanation for this observation is that thapsigargin treatment may
have caused gross structural alterations in the ER in response to rapid
mobilization of ER Ca2+ stores which could impair
accumulation of the null(Hong Kong)-UGTR complex. In this regard,
gradual mobilization of the divalent cation in response to incubation
with metabolic poisons might have provided more favorable conditions
for intracellular accumulation.
A recently proposed model (15, 17) suggests that asparagine-linked
oligosaccharides of unfolded glycoproteins participate in a cycle of
reglucosylation/deglucosylation in which these events are catalyzed by
UGTR and Asparagine-linked oligosaccharides of unfolded glycoproteins with the
structure of Man7-9GlcNAc2 function as
acceptors in the glucose transfer reaction (11). Since catalysis of
glucose transfer by UGTR is a Ca2+-dependent
process (49) it has been suggested that hydrolysis of UDP-glucose,
which is coupled to glucose transfer, may provide the energy necessary
for dissociating bound glycoproteins from UGTR (8). We have been unable
to demonstrate transfer of glucose to oligosaccharides of null(Hong
Kong) during Ca2+-dependent dissociation of
UGTR, but this may reflect poor detection limits associated with our
analysis. Also, Ca2+-dependent dissociation of
UGTR does not occur following coprecipitation of the complex, even in
the presence of UDP-glucose.3 If
dissociation is coupled to glucose transfer then binding of the
antibody to null(Hong Kong) may somehow interfere with recognition of
oligosaccharides by UGTR.
The concept that UGTR might participate in glycoprotein folding and
quality control originally emerged from experiments performed in
cell-free systems. However, in a recent report, Fernandez et al. (8) detected a 2-9-fold induction of UGTR mRNA in
Schizosaccharomyces pombe at conditions known to perturb
glycoprotein folding and induce synthesis of stress proteins. The
structural gene was cloned and compared with that in Drosophila
melanogaster. A conserved yeast heat shock promoter sequence was
detected in the 5
Departments of Pathology and Cell Biology,
1-antitrypsin unable to fold into the native
structural conformation are poorly secreted from hepatocytes. The
molecular chaperone calnexin coimmunoprecipitates with
secretion-incompetent variant null(Hong Kong) retained in stably
transfected mouse hepatoma cells (Le, A., Steiner, J. L., Ferrell, G. A., Shaker, J. F., and Sifers, R. N. (1994) J. Biol.
Chem. 269, 7514-7519). Mobilization of intracellular
Ca2+ stores with metabolic poisons diminished interaction
with calnexin and coincided with coimmuoprecipitation of a 150-kDa
protein (p150). Mobilization of endoplasmic reticulum lumenal
Ca2+ with thapsigargin, an inhibitor of the microsomal
Ca2+ATPase, gave a similar result. Coimmunoprecipitation of
p150 was specifically disrupted in response to incubation of the cell
lysate with exogenous CaCl2. Finally, in ECL Western
blotting, p150 was recognized by polyclonal antiserum against
UDP-glucose:glycoprotein glucosyltransferase that likely functions in
glycoprotein folding and quality control (Sousa, M. C., Ferrero-Garcia,
M. A., and Parodi, A. J. (1992) Biochemistry 31, 97-105).
The data are consistent with a model in which perturbation of
endoplasmic reticulum Ca2+ results in a stable physical
association between unfolded human
1-antitrypsin and
UDP-glucose:glycoprotein glucosyltransferase.
-glucosidase activity and is a
predictable response if attached monoglucosylated oligosaccharides are
interacting with calnexin.
1-antitrypsin (AAT) is a 394-amino acid protein (26, 27)
glycosylated at three specific asparagine residues (28). It is folded
into a highly ordered tertiary structure containing three
-sheets,
nine
helices, and three internal salt bridges (29). The human AAT
structural gene is highly polymorphic (30), and several alleles exhibit
a distinct mutation predicted to preclude conformational maturation of
the encoded polypeptide following biosynthesis (31). Secretion of AAT
from hepatocytes (32, 33) is impaired in response to incomplete folding
of the polypeptide (34, 35).
Chemicals and Reagents
Coimmunoprecipitation of a 150-kDa Protein
Fig. 1.
Coprecipitation of a 150-kDa protein in
response to treatment of cells with metabolic poisons. A,
H1A/N13 cell monolayers were pulse-radiolabeled for 15 min with
[35S]methionine (lane 1) and then chased for
60 min in regular growth medium (lane 2) or in medium
supplemented with 10 mM NaF and 40 mM
NaN3 (lane 3). B, non-transfected
Hepa 1a cell monolayers were treated identical to that described in
panel A. AAT was immunoprecipitated from cell lysates, and
radiolabeled proteins were fractionated by SDS-PAGE prior to detection
by fluorography. The molecular mass of radiolabeled proteins is shown
in kilodaltons (kDa).
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
Association with p150 is reversible and
cumulative. A, H1A/N13 cells were pulse-radiolabeled for 15 min with [35S]methionine. Cells were chased for 60 min
with regular growth medium (lane 1) or medium supplemented
with 10 mM NaF and 40 mM NaN3
(lane 2). In lane 3, cells were treated as in
lane 2 followed by an additional 60-min incubation with
regular growth medium. In lane 4, cells were incubated for
120 min in medium supplemented with 10 mM NaF and 40 mM NaN3. B, cells were
pulse-radiolabeled as described above and then chased for 120 min with
regular growth medium (lane 1), medium supplemented with 10 mM NaF and 40 mM NaN3 (lane
2), 10 mM NaF (lane 3), or 40 mM NaN3 (lane 4). Variant null(Hong
Kong) (NHK) was immunoprecipitated from cell lysates, and
radiolabeled proteins were detected by fluorography after being
fractionated by SDS-PAGE. The molecular mass of radiolabeled proteins
is shown in kilodaltons (kDa).
[View Larger Version of this Image (36K GIF file)]
Fig. 3.
Exogenous Ca2+ facilitates
dissociation of bound p150. H1A/N13 cells were pulse-radiolabeled
for 15 min with [35S]methionine and chased for 2 h
in medium supplemented with 10 mM NaF and 40 mM
NaN3. A, cell lysates were incubated for 10 min at 37 °C in the absence (lane 1) or presence of 1 mM CaCl2 (lane 2). Variant null(Hong
Kong) (NHK) was immunoprecipitated, and radiolabeled
proteins were fractionated by SDS-PAGE and detected by fluorography.
The molecular mass of radiolabeled proteins is shown in kilodaltons
(kDa). B, quantitation of coimmunoprecipitated p150 described from the experiment shown in panel A
following incubation with 1 mM CaCl2
(Ca2+) or with no additions
(Control). The amount of associated p150 (% UGTR
association) was calculated as a percent of the control value,
which was 100%.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Changes in the intracellular distribution of
Ca2+ stores following incubation with metabolic
poisons. H1A/N13 cells were cultured on glass coverslips and
incubated with regular medium (A) or medium supplemented
with 10 mM NaF and 40 mM NaN3
(B). After treatment, cells were stained with the
fluorescent Ca2+ probe Fluo-3 and then scanned via a
section series via scanning laser confocal microscopy (see "Materials
and Methods").
[View Larger Version of this Image (70K GIF file)]
Fig. 5.
p150 coprecipitates in response to
thapsigargin treatment. H1A/N13 cell monolayers were
pulse-radiolabeled for 15 min with [35S]methionine and
chased for 60 min with medium either containing no additions
(lane 1) or supplemented with 10 µM
thapsigargin (lane 2). Null(Hong Kong) (NHK) was
immunoprecipitated from cell lysates, and radiolabeled proteins were
fractionated by SDS-PAGE and detected by fluorography. The molecular
mass of radiolabeled proteins is shown in kilodaltons
(kDa).
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Effect of metabolic poisons and thapsigargin
on coprecipitation of calnexin. H1A/N13 cells were incubated with
medium containing compounds described below. Null(Hong Kong) was
immunoprecipitated from cell lysates and immunocomplexes were
fractionated by SDS-PAGE. Proteins were electrophoretically transferred
to nitrocellulose membranes and coimmunoprecipitating calnexin
(CXN) was detected by ECL Western blotting using
calnexin-specific antiserum (see "Materials and Methods"). Shown
are a crude H1A/N13 cell extract (lane 1), a blank lane
(lane 2), cells incubated for 60 min with regular growth
medium (lane 3), and after the same incubation in medium
supplemented with 10 µM thapsigargin (lane 4).
Cells were incubated for 60 min with 10 mM NaF and 40 mM NaN3 (lane 5) or incubated as in
lane 5 followed by an additional 60-min incubation with
regular growth medium (lane 6). The molecular mass of
immunoreactive proteins is shown in kilodaltons (kDa).
[View Larger Version of this Image (26K GIF file)]
Fig. 7.
Coprecipitating p150 is recognized by
antiserum against rat UDP-glucose:glycoprotein
glucosyltransferase. H1A/N13 cell monolayers were incubated for
120 min as described below prior to immunoprecipitation of null(Hong
Kong) from cell lystates. Immunoprecipitated proteins were fractionated
by SDS-PAGE and electrophoretically transferred to nitrocellulose
membranes. Membranes were incubated with antiserum against rat
UDP-glucose:glycoprotein glucosyltransferase (UGTR) to
detect immunoreactivity by ECL Western blotting (see "Materials and
Methods"). Shown are a crude H1A/N13 cell extract (lane
1), a blank lane (lane 2), cells incubated in a medium
supplemented with 10 mM NaF and 40 mM
NaN3 (lane 3), or regular growth medium
(lane 4). The molecular mass of immunoreactive proteins is
shown in kilodaltons (kDa).
[View Larger Version of this Image (24K GIF file)]
-glucosidase II, respectively. Cycles of transient
interaction with UGTR, each resulting in reglucosylation of attached
oligosaccharides, is believed to facilitate interaction between
unfolded glycoproteins and calnexin and ensure the retention of
improperly folded glycoproteins in the ER. We have observed post-translational incorporation of mannosidase-resistant radiolabel into asparagine-linked oligosaccharides of null(Hong Kong) in H1A/N13
cells incubated with
[3H]galactose,2 suggesting
that they likely participate as substrates for UGTR. Conceivably, the
steady-state concentration of the transiently-formed complex is beyond
the detection limits of ECL Western blotting. Conceivably,
intracellular accumulation of the null(Hong Kong)-UGTR complex would
result if the two components normally interact with one another in a
transient fashion but require Ca2+ for dissociation. Data
generated in the present study lend support to the proposed
precursor-product relationship between UGTR association and subsequent
assembly with calnexin. However, we cannot exclude the possibility that
formation of these null(Hong Kong)-containing complexes, in the context
of this study, occurred as mutually exclusive events.
terminus, providing an explanation of how its
expression is induced similar to that of several stress proteins. Our
observations demonstrating that intracellular accumulation of the
null(Hong Kong)-UGTR complex lend additional support to this
hypothesis. Co-immunoprecipitation of intracellular complexes
containing AAT furnishes an opportunity to subject the quality control
model to experimental analysis, which will provide further insight into
the molecular pathogenesis of pulmonary emphysema resulting from severe
plasma AAT deficiency.
*
This work was supported in part by an American Heart
Association grant-in-aid and an American Lung Association research
career investigator award (to R. N. S.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Section of Molecular Pathobiology, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-3169; Fax: 713-798-5838; E-mail: rsifers{at}bcm.tmc.edu.
1
The abbreviations used are: ER, endoplasmic
reticulum; AAT, 1-antitrypsin; PAGE, polyacrylamide gel
electrophoresis; UGTR, UDP-glucose:glycoprotein
glucosyltransferase.
2
R. Sifers, unpublished observations.
3
P. Choudhury, unpublished observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.