From the Departments of Medicine and
Biochemistry/Molecular Biology and The Cardiovascular Institute, Mount
Sinai School of Medicine, New York, New York 10029, the
§ Department of Biochemistry and Molecular Biology, Mount
Sinai School of Medicine, New York, New York 10029, and the
¶ Department of Biological Sciences, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260
Received for publication, January 23, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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Apoprotein B (apoB) is the major protein of
liver-derived atherogenic lipoproteins. The net production of
apoB can be regulated by presecretory degradation mediated by the
ubiquitin-proteasome pathway and cytosolic hsp70. To further explore
the mechanisms of apoB degradation, we have established a cell-free
system in which degradation can be faithfully recapitulated. Human
apoB48 synthesized in vitro was translocated into
microsomes, glycosylated, and ubiquitinylated. Subsequent incubation
with rat hepatic cytosol led to proteasome-mediated degradation. To
explore whether hsp90 is required for apoB degradation, geldanamycin
(GA) was added during the degradation assay. GA increased the recovery
of microsomal apoB48 ~3-fold and disrupted the interaction between
hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system,
we also found that transfection of hsp90 cDNA into rat hepatoma
cells enhanced apoB48 degradation. Finally, apoB48 degradation was
reconstituted in vitro using cytosol prepared from wild
type yeast. Notably, degradation was attenuated when apoB48-containing
microsomes were incubated with cytosol supplemented with GA or with
cytosol prepared from yeast strains with mutations in the homologues of
mammalian hsp70 and hsp90. Overall, our data suggest that hsp90
facilitates the interaction between endoplasmic reticulum-associated
apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.
Apoprotein B (apoB)1 is
a very large (540 kDa) protein and is essential for the assembly and
secretion of hepatic lipoproteins. ApoB is the major protein component
of low density lipoprotein, the atherosclerosis-causing particle
that transports cholesterol in the blood. During the translation of
apoB in the liver, lipids are transferred to the nascent protein by the
ER-resident microsomal triglyceride transfer protein to form a
"primordial lipoprotein." In hepatocytes and
hepatocarcinoma-derived cell lines, apoB either undergoes assembly with
lipids and secretion or is subjected to ER retention and intracellular
degradation (1-3). We have previously shown that this degradation in
HepG2 cells is accomplished by the ubiquitin-proteasome pathway and
involves cytosolic hsp70 (4-6). The degree of proteasomal degradation
appeared to be regulated by the availability of the lipid
ligands for apoB (4).
There are a number of ER luminal and ER transmembrane proteins (7) that
have also been reported to undergo proteasomal degradation. This
process has been referred to as ER-associated degradation (ERAD) (8, 9)
and for a number of substrate proteins, a common scenario appears to be
that the nascent protein undergoes translocation, followed by
retrotranslocation and release into the cytosol, where it is degraded
by the proteasome. Although the route by which apoB is targeted to the
proteasome is not completely defined, certain features of its
degradation may differ from this model. In particular, apoB can assume
a bitopic topology during or shortly after translocation, resulting in
domains that can be accessed by cytosolic or ER luminal factors, with
degradation by the proteasome accomplished without the requirement of
apoB to be fully retrotranslocated (6).
As noted above, the molecular chaperone hsp70 facilitates ERAD of apoB
in HepG2 cells (4). Molecular chaperones play important roles in
assisting the folding and assembly of proteins, facilitating protein
translocation across a variety of intracellular membranes, and
targeting misfolded proteins for degradation, including secretory or
transmembrane proteins destined for ERAD (9, 10). The most abundant
chaperone in the eukaryotic cytosol is hsp90, which has been shown to
be involved in the maturation of signal transduction proteins, such as
steroid hormone receptors (11, 12). The effect of hsp90 can be
specifically inhibited by geldanamycin (GA), an ansamycin antibiotic
that competes for the ATP binding site (13). A positive role for hsp90
in the maturation of CFTR has recently been proposed (14) based on data
showing that proteasomal degradation was accelerated by the disruption
of hsp90 association with CFTR by the addition of GA to the cell
culture medium. Alternatively, other recent studies have suggested that
hsp90 can destabilize and promote the ERAD of mutant proteins
(e.g. CFTR Given this recent controversy, we were particularly interested in
investigating the role of hsp90 in the ERAD of apoB. In order to define
the mechanisms by which apoB enters the ubiquitin-proteasome pathway
and the cytosolic factors involved (including hsp90), we sought to
develop a cell-free degradation system. One particular advantage of
such a system would be the ability to examine the interactions of
chaperones with apoB without changing their steady-state levels. In
this report, we summarize the characteristics of the system and the
evidence obtained from it suggesting that hsp90 promotes apoB
degradation. Importantly, the results from the cell-free system were
consistent with those from hepatoma cells transfected with hsp90 cDNA.
Materials--
The plasmid encoding apoB48 was from V. Lingappa
(University of California, San Francisco); ubiquitin-HA protein and
proteasome inhibitor PSI were from Z. Ronai and S. Wilk, respectively
(Mount Sinai School of Medicine). Rat hepatic cytosol was purified from Harlan Sprague-Dawley rat liver (17). Yeast cytosols (from strains JN516 [SSA1], JB67 (ssa1-45), and G313N
(18-20)) were prepared as described earlier (8). The plasmid encoding
human hsp90 Cell-free Transcription/Translation and Quantification of
ApoB48--
ApoB48 was synthesized in a coupled
transcription/translation reaction (2 h, 30 °C) using the SP6-TNT
kit (Promega, Madison, WI) and 0.5 mCi/ml [35S] Protein
Labeling Mix (1000 Ci/mmol; PerkinElmer Life Sciences) in the presence
of dog pancreatic microsomes (DPM; Promega). To assess the
characteristics and the level of labeled apoB48, samples were mixed
with gel loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS,
20% glycerol, 5% Trypsin Treatment to Assess ApoB48 Topology--
The reaction
mixture at the end of the transcription/translation procedure was
treated with trypsin for 1 h at 0 °C at a final concentration
of 0.1 mg/ml. Trypsin inhibitor at final concentration of 0.2 mg/ml was
added for 10 min to stop the reaction. The concentrations of trypsin
and typsin inhibitor were optimized in pilot experiments.
PNGF Treatment to Assess ApoB48 Glycosylation--
At the
end of the transcription/translation procedure, the reaction mixture
was layered onto SH buffer (0.25 M sucrose, 5 mM HEPES, pH 7.4) and centrifuged at 100,000 × g for 30 min at 4 °C. The pelleted microsomal membranes
were resuspended in phosphate-buffered saline. To 10 µl of this
suspension, the following were added: 1 µl of 10% 2-mercaptoethanol,
2.5 µl of 10% Triton X-100, 1 µl of 100 mM
phenylmethylsulfonyl fluoride, 1 µl of PNGF (0.1 unit/µl), 4.7 µl
of H2O, 0.8 µl of 5% SDS. This mixture was then
incubated for 16 h at 4 °C.
Flotation Analysis of Microsome-associated ApoB48--
The
association of apoB48 with microsomes was confirmed by flotation of the
products of an in vitro transcription/translation reaction
through a sucrose gradient (21). In brief, an in vitro transcription/translation was performed as described above, after which
the reaction was diluted into 100 µl of MSB (50 mM HEPES, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol; supplemented with phenylmethylsulfonyl
fluoride, leupeptin, and pepstatin according to the manufacturers'
specifications); and then mixed with 300 µl of 2.3 M
sucrose in MSB. This mixture was overlaid onto 300 µl of 2.3 M sucrose in MSB in a centrifuge tube. Solutions of 1.5 M sucrose (600 µl) and 0.25 M sucrose (500 µl) in MSB were overlaid, and the discontinuous gradient was
centrifuged at 100,000 × g for 5 h at 4 °C,
after which 150-µl aliquots were successively removed from the top of
the gradient. Proteins in the fractions were resolved by SDS-PAGE, and
the radiolabeled apoB48 was detected by PhosphorImager analysis of a
dried gel.
ApoB48 Degradation Assay--
At the end of the
transcription/translation procedure, the reaction mixture was layered
onto SH buffer (0.25 M sucrose, 5 mM HEPES, pH
7.4) and centrifuged at 100,000 × g for 30 min at 4 °C. The pelleted microsomal membranes were resuspended in 50% (v/v) of the appropriate lysate and 50% of 2 × Ph buffer (40 mM HEPES, pH 7.4, 220 mM KCl, 10 mM
MgCl2). At the following final concentrations, 2 mM ATP, 10 mM creatine phosphate, and 100 µg/ml creatine kinase were added to the complete mixture. Aliquots of the mixture were incubated for 2 h either on ice (control) or at
37 °C. At the end of the incubation period, gel loading buffer for
SDS-PAGE was added directly to the samples. In some experiments, the
proteasome inhibitors (dissolved in Me2SO) indicated
under "Results" were added to the degradation mixture just prior to the 2-h incubation. An equivalent volume of Me2SO was added
to the control sample.
In some experiments, the following variations were made. To determine
the effect of depleting hsp90 on the recovery of apoB48 after the 2-h
degradation assay, 100-µl samples of rat hepatic lysate were
pretreated with 15 µg of hsp90 antibody and 500 µl of 5% protein
A-Sepharose (to collect the chaperone-antibody complexes). The control
lysates were treated similarly, except that the chaperone antibodies
were omitted. The control and depleted lysates were then used for the
degradation assay. To examine the dependence on the ubiquitinylation of
apoB48 for degradation in rabbit reticulocyte lysate (RRL), just prior
to its use in the degradation assay, RRL was supplemented with
ubiquitin aldehyde (Ubal), an inhibitor of deubiquitinylation (22), at
a concentration range of 0-20 µM.
Effect of GA on ApoB48 Degradation--
At the end of the
transcription/translation procedure, the reaction mixture was layered
onto SH buffer and centrifuged at 100,000 × g for 30 min at 4 °C. The pelleted microsomal membranes were resuspended in
50% (v/v) of the rat hepatic lysate and 50% of 2 × Ph buffer.
At the final concentrations below, 2 mM ATP, 10 mM creatine phosphate, and 100 µg/ml of creatine kinase
were added to the complete mixture. The mixture was halved, and GA (NCI, National Institutes of Health, Bethesda, MD) dissolved in Me2SO was added to one aliquot at a final concentration of
30 µM; an equal volume of Me2SO was added to
the other aliquot. Samples were taken every 30 min during a 2-h
incubation (37 °C) period. At the end of the incubation, gel loading
buffer for SDS-PAGE was added directly to the samples, and the apoB48
content was analyzed as above.
ApoB48 Ubiquitinylation Assay--
At the end of the
transcription/translation procedure, the reaction mixture was layered
onto SH buffer and centrifuged as above. The pelleted microsomal
membranes were resuspended in 50% (v/v) of the appropriate lysate and
50% of 2 × Ph buffer. To this mixture, ATP Cell Culture Studies--
Rat hepatoma McA-RH7777 cells
were cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum, 10% horse serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37 °C. The medium was changed
every 3 days. LipofectAMINE PLUS reagent (Life Technologies) was used to transfect McA-RH7777 cells with either the pcDNA3 vector
(control) or pcDNA3-hsp90 Immunoprecipitation and Western Blotting--
Samples for
immunoprecipitation were diluted in buffer, containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, and incubated with
appropriate antibodies at 4 °C for 2 h followed by incubation
with Protein A- or Protein G-Sepharose at 4 °C for 2 h. The
beads were washed three times with the same buffer, and
immunoprecipitated material was released by heating at 100 °C in
SDS-PAGE sample buffer. For Western blots, proteins were first resolved
by SDS-PAGE and then transferred to a polyvinylidene difluoride
membrane (PerkinElmer Life Sciences). Typically, the primary and
secondary antibodies were used at 1:1000 and 1:10,000 dilution,
respectively, with final detection of signal by the Western blot
Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).
Biogenesis of ApoB48 in Vitro--
For our studies, we have used
the apoB48 species, which is expressed in the intestine of all mammals
and in the rodent liver. Although shorter than apoB100 (expressed only
in mammalian liver), apoB48 still displays in cell culture studies many
similar properties with respect to translocation, membrane integration,
and degradation (23). ApoB48 (~240 kDa) was synthesized from its
cDNA by in vitro transcription/translation in RRL (Fig.
1). Translation performed in the presence
of DPM resulted in the appearance of two products (Fig. 1,
lane 2), implying translocation and
post-translational processing. Insertion of apoB48 into the microsomal
lumen was confirmed by a protease protection assay. At the end of the
transcription/translation procedure, the reaction mixture was incubated
with trypsin in the presence or absence of Triton X-100 (Fig.
2A). Note that the upper band
was protected from trypsin digestion in the absence of Triton X-100
(Fig. 2A, lane 2), indicating that
this product translocated across the microsomal membrane, with the
lower band representing untranslocated apoB48. This interpretation is
consistent with the data reported by Rusinol et al. (24). In
the presence of Triton X-100 (used to destroy membrane integrity), the
protein represented by the upper band was digested by trypsin (Fig.
2A, lane 3). Thus, the protease
resistance in the absence of Triton X-100 resulted from proper
integration of apoB48 and not from protein aggregation or inadequate
protease activity.
To investigate whether the upper band in Fig. 1 represented apoB48 that
was modified by glycosylation, microsome-associated apoB48 was
separated from the reaction mixture by centrifugation and treated with
PNGF. The effect of PNGF treatment is shown in Fig. 2B. Note
that PNGF reduced the apparent molecular weight of the upper band to
that of the lower band (lane 2 versus
lane 3). For comparison, the total reaction
(lane 1) and supernatant after pelleting the
microsomes (lane 4) are also displayed. This reduction of apparent molecular weight of the upper band by PNGF treatment not only demonstrates modification of apoB48 by
glycosylation, an event also known to occur with apoB in intact cells
(25), but also confirms that in the presence of DPM, there was
translocation of apoB48. Treatment with sodium carbonate at pH 11.5 (26), however, released less than 5% of apoB48 from the microsomal
membrane (data not shown).
To confirm that apoB48 was associated with the microsomal membrane, an
in vitro transcription/translation reaction in the presence
of DPM was performed, after which the microsomes were floated through a
sucrose gradient as previously described (21). As shown in Fig.
3, we found that the majority of apoB48
had migrated into fractions of lower density than that at which it was
loaded onto the gradient (denoted by the arrow), indicative
of it being membrane-associated.
Together, the sodium carbonate and sucrose gradient data imply that
translocated apoB48 strongly interacts with membrane lipid or protein
components, consistent with previous results using isolated microsomes
or intact cells (5, 6, 27).
ApoB48 Is Ubiquitinylated and Degraded in the Cell-free
System--
Recent studies have shown that degradation of apoB in
HepG2 cells is mediated by the ubiquitin-proteasome pathway
(e.g. Refs. 4, 28, and 29). To determine whether nascent
apoB48 was ubiquitinylated in vitro, HA-tagged ubiquitin was
added at the end of the transcription/translation procedure, and the
incubation was continued at 37 °C for 1 h in the presence of
ATP and an ATP regenerating system (Fig.
4). Ubiquitinylated apoB48 was
immunoprecipitated with anti-HA-tag antibody (Fig. 4, lane
4). That the high molecular weight material represents
bona fide modification of apoB by ubiquitinylation was
supported by three results: 1) if the antibody to HA tag was omitted,
no labeled material was detected (Fig. 4, lane
5); 2) when the reaction was depleted of ATP by the
ATP-hydrolyzing enzyme apyrase, ubiquitinylation was not observed (Fig.
4, lane 3); and 3) in the absence of the apoB48
translation product, no labeled material was recovered by the anti-HA
antibody (Fig. 4, lane 6). For comparison, the
contents of the transcription/translation reactions (with or without
apoB48 cDNA) prior to immunoprecipitation are displayed (in
lanes 1 and 2, respectively). Thus,
apoB48 become ubiquitinylated in the cell-free system, and this
process, as in intact cells, is ATP-dependent.
Overall, the data in Figs. 1-4 demonstrate that the early events
during apoB biogenesis are faithfully reproduced in the cell-free system and suggest that this represents a valid model for the study of
the mechanisms leading to apoB degradation. Toward this goal,
microsome-associated apoB48 was studied in the next series of
experiments, because we have previously shown in HepG2 cells that apoB
destined for proteasomal degradation remains stably associated with
microsomes (4-6). Thus, at the end of transcription/translation, microsomes were pelleted and resuspended in RRL or rat hepatic cytosol.
The latter cytosol was utilized to study apoB48 degradation because it
derives from the tissue in which it is normally expressed.
Aliquots of each reaction mixture were incubated on ice (control
condition) and at 37 °C for 2 h in the presence of ATP and an
ATP regeneration system. Independent of the source of the cytosol, after 2 h, the majority (~75%) of apoB48 was degraded compared with the control (Fig. 5, A
and B, lane 2 versus
lane 1). To determine whether this degradation
was mediated by the proteasome, the experiments were repeated in the
presence or absence of the proteasome inhibitor lactacystin,
benzyloxycarbonyl-Leu-Leu-leucinal (MG132), or
benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) (30). Note that proteasome inhibitors did not significantly reduce degradation in RRL (Fig. 5, A and C),
consistent with the data for CFTR reported by Xiong et al.
(31), who showed that lactacystin is ineffective and that a high
concentration of MG132 (200 µM) is only partially
effective at blocking degradation of ubiquitinylated substrates in RRL.
Based on their results, they hypothesized that RRL contains a factor or
factors that interfere with the actions of some proteasome inhibitors.
Because of this limitation, to support the interpretation that in RRL,
apoB48 degradation is accomplished by the ubiquitin-proteasomal pathway and not by another protease, we performed degradation assays in which
RRL was supplemented with Ubal, an inhibitor of deubiquitinating enzymes. Ubal has previously been reported to decrease apoB degradation in semipermeabilized HepG2 cells (32) and exerts a negative effect on
proteasomal degradation by interfering with the removal of ubiquitin
chains from substrates prior to their entry into the proteasome
(e.g. see Ref. 22). The addition of Ubal to RRL resulted in
a dose-dependent increase in the recovery of apoB48 at the
end of the 2-h degradation assay, from 50% to 80 and 93% at 0, 5, and
20 µM of Ubal, respectively. Overall, the results from
these and the prior studies (31) imply that the degradation of apoB48
in RRL is accomplished by the ubiquitin-proteasome pathway, but the
response of proteasomes in RRL to some inhibitors may be anomalous.
In contrast, the proteasome inhibitors effectively inhibited
degradation in rat hepatic cytosol (Fig. 5, B and
D). In addition to the increased recovery of apoB48 in the
presence of these inhibitors, further evidence for a role of the
ubiquitin-proteasome pathway was suggested by a reduction in apoB48
degradation when the cytosol was pretreated with the ATP-depleting
agent apyrase (data not shown). Consistent with the rat hepatic lysate
studies, similar results were obtained using human hepatic cytosol
(data not shown).
Hsp90 Is Required for ApoB Degradation--
As reviewed in the
Introduction, hsp90 has been shown to either promote or inhibit the
interaction of substrate proteins with components of the
ubiquitin-proteasome pathway. In light of this controversy and our
finding that in the cell-free system apoB48 can be ubiquitinylated and
degraded by proteasomes (Figs. 4 and 5), we next explored whether hsp90
plays a role in apoB degradation.
Cytosolic hsp90 has been shown to be the specific target for the
ansamycin benzoquinone antibiotics (33, 34), particularly GA.
Therefore, GA, the most potent drug from this class antibiotics (35),
was added to the degradation mixture, and apoB recovery was monitored
every 30 min during the subsequent 2-h incubation. The results shown in
Fig. 6 indicate that the recovery of
apoB48 was increased over 3-fold in the presence of GA. It has been
previously shown with purified proteasomes that GA did not reduce the
degradation of the model substrate SLLVT-AMC (36), suggesting that GA
did not function simply as a proteasome inhibitor in the cell-free degradation assay but directly interfered with a prodegradative effect
of hsp90 on apoB48.
The homologue of hsp90 in the ER, GRP94, which also binds the same
class of antibiotics (37), has been shown to interact with apoB100 in
HepG2 cells (38). In that study, GRP94 was found to interact with
apoB100 upon a reduction of the ATP level by apyrase treatment. Note
that under our conditions, ATP and an ATP regeneration system are added
(see "Experimental Procedures"), making it unlikely that GA's
effect was mediated by disrupting an apoB48-GRP94 interaction.
Nonetheless, to show that the GA effect was specific for cytosolic
hsp90, we determined whether either chaperone interacts with apoB48.
Using antibodies to either hsp90 or GRP94, we attempted to
co-immunoprecipitate apoB48, and the results are presented in Fig.
7. As shown, apoB48 could be co-immunoprecipitated with hsp90, but not with GRP94 (Fig.
7A, lane 3 versus
lane 5). Notably, GA added to the incubation
mixture disrupted the interaction of apoB48 with hsp90 (lane
4). The interaction between apoB48 and hsp90 and its
disruption by GA was independent of the antibody (anti-hsp90 or
anti-apoB) used for the immunoprecipitation (Fig. 7, compare
A with B). These results could not be explained by a perturbation of the general pool of hsp90, because Western blotting (Fig. 7C) indicated comparable signal intensities
for hsp90 in samples untreated and treated with GA. Taken together, the
results shown in Figs. 6 and 7 imply that hsp90 interacts with apoB48
and promotes apoB48 degradation.
Effect of GA on Ubiquitinylation of ApoB48--
Because ubiquitin
marks proteins for proteasome-mediated degradation, we wished to
determine whether GA treatment mediated the increased recovery of
apoB48 by a decrease in its ubiquitinylation. Microsome-associated
apoB48 was incubated at 37 °C for 1 h in rat hepatic lysate
with HA-ubiquitin, in the presence or absence of GA. Because GA
inhibits apoB48 degradation, apoB48 recovery increases (Fig. 6).
Therefore, to control for this in the analysis of the amount of
ubiquitinylated apoB48, the content of apoB48 in each sample was first
determined by SDS-PAGE/densitometry. Then aliquots containing
equivalent amounts of apoB48 were immunoprecipitated with an antibody
against the HA epitope. As shown in Fig.
8, ubiquitinylated apo48
(ub-apoB48) appeared as a smear reaching to the top of the gel; as confirmed by densitometry, there is no significant decrease in
the ubiquitinylated apoB48 signal in the sample treated with GA (Fig.
8, lane 3 versus lane
2).
Transfection with hsp90 cDNA Increases ApoB Degradation in Rat
Hepatoma Cells--
To establish in intact hepatic cells the relevance
of our results implicating hsp90 in apoB48 degradation, rat hepatoma
McArdle-RH7777 cells were transfected with hsp90 cDNA. These cells
express primarily the apoB100 form of native apoB, the recovery of
which we have shown to be increased by lactacystin treatment (39). We
took this transfection approach rather than treating the cells with GA,
because treatment of cells with ansamycin antibiotics not only directly
affects protein-chaperone interactions but increases the level of
multiple heat shock proteins by promoting the activation of heat shock
transcription factor (HSF1) (40, 41). For example, treatment of yeast
with ansamycin antibiotics inhibits ERAD due to a secondary increase in
BiP concentration.2
After transfection either with pcDNA3 vector (control condition) or
pcDNA3-hsp90
As shown in Fig. 9B,
transfection with the hsp90 cDNA did not affect albumin synthesis
or secretion. In contrast, the total recovery of apoB100 from lysate
and medium was lower at the 30 min (21% reduction) and 90 min (48%
reduction) chase time points in the cells transfected with the hsp90
cDNA (Fig. 9A). These results are consistent with the
data from the cell-free system and support a prodegradative role for
hsp90 in apoB degradation in the liver.
In addition to studying the effects of increasing hsp90 on apoB
degradation in hepatic cells, it would have been desirable to also
determine the consequences of decreasing the level of a chaperone.
Perhaps because of the abundance and relatively long half-lives of many
chaperones, it has been difficult to achieve major decreases in intact
cells. For example, a ribozyme approach undertaken independently by two
laboratories resulted in no more than a 25-30% reduction in cellular
GRP94 levels (42, 43). Thus, we tried to immunodeplete hsp90 from rat
hepatic lysate (see "Experimental Procedures") and to use the
depleted lysate in the cell-free degradation assay. Compared with
control rat hepatic lysate, there was a 4-fold increase in apoB
recovery from the reaction mixtures containing lysate that had been
depleted of ~75% of its hsp90. This increase in apoB recovery is
again consistent with the transfection results and the other data from the cell-free system indicating that hsp90 is a prodegradative factor
for apoB.
Degradation of Microsomal ApoB48 in Yeast Cytosols--
The
availability of specific hsp70 and hsp90 chaperone mutants in yeast
(18-20, 44) prompted us to explore whether yeast cytosol would support
apoB48 degradation. Cytosols from wild type yeast or from isogenic
strains with either a temperature-sensitive mutation in hsp82
(the yeast homologue of mammalian hsp90) and deleted for the gene
for the constitutive hsc90 homologue, hsc82, or a
temperature-sensitive mutation in the hsp70 homologue
ssa1 (ssa1-45) were prepared from cells
shifted for 1 h to the non-permissive temperature. These cytosols
were then used in the degradation assay (see "Experimental
Procedures").
As shown in Fig. 10 (panel
A, lane 2 versus lane
1; panel E), after the 2-h incubation,
68% of apoB48 was degraded in the presence of wild type yeast cytosol.
Note that GA (lane 3) blocked this degradation.
The degradation of apo48 in yeast cytosols was also reversed by
proteasome inhibitors (data not shown). Thus, the characteristics of
apoB48 degradation in the presence of yeast cytosols were quite similar
to those in the presence of rat hepatic cytosol (Figs. 5 and 6).
The experiments were then repeated using the cytosols from the mutant
strains. The results clearly show that apoB48 is almost completely
protected from degradation when the hsp82 or
ssa1 mutant cytosols were used (Fig. 10,
B, C, and E), similar to the effect of
GA on wild type cytosol.
It is known that hsp90 acts in association with several different
co-chaperones, which bind to hsp90 and organize it into discrete
subcomplexes (12). In particular, hsp70 has been found in such a
complex, associated with hsp90 through the Hop protein (45). We
previously found that hsp70 promotes apoB degradation (4). Thus, the
protection of apoB48 from degradation when only one of these chaperones
is mutated suggested that hsp70 and hsp90 may have cooperative effects
on apoB degradation. To test this hypothesis, cytosols from the two
mutant yeast strains were mixed at a 1:1 ratio and used in the
degradation assay. The degradation of apoB48 was restored to wild type
levels, and under these conditions, apoB48 was protected when GA was
added to the reaction (Fig. 10, D and E).
The majority of apoB is subjected to proteasome-mediated ERAD in
cells of hepatic origin that are either deprived of exogenous fatty
acids (which stimulate lipid synthesis) or deficient for microsomal
triglyceride transfer activity (4, 28, 29). It is assumed that the
association of apoB with lipid ligands is required for the achievement
of its native conformation, and when the concentration or transfer of
lipids is reduced, apoB is detected by quality control mechanisms that
prevent the exit of malfolded proteins from the ER (46).
That chaperones may have important functions in apoB degradation stems
from the growing recognition that these molecules influence the ERAD
process of other proteins (for a review, see Ref. 9) and from our
demonstration that increasing the expression of hsp70 in fatty
acid-deprived human liver-derived HepG2 cells promoted the proteasomal
degradation of apoB (4, 47). Further hints that hsp70 and other
chaperones may be involved in the quality control of apoB come from
Linnik and Herscovitz (38), who showed in HepG2 cells that BiP/GRP78,
calreticulin, Erp72 and GRP94 co-immunoprecipitate with apoB, and from
Olofsson and colleagues (48), who have found recently that BiP, PDI,
calreticulin, and GRP94 are associated with apoB-containing lipoprotein
particles in rat hepatoma cells.
Because of the limited ability in intact hepatic cells to modulate the
interactions of chaperones without affecting their steady state levels,
we developed a cell-free degradation assay system in order to best
explore the roles of these proteins in apoB ERAD. The previous report
that apoB48, synthesized in a coupled transcription/translation system,
was translocated into dog pancreatic microsomes (24) suggested the
feasibility of this goal. Under such conditions, there is neither
microsomal triglyceride transfer protein activity nor active lipid
synthesis, thereby mimicking the lipid-ligand-deficient state in
liver-derived cells deprived of fatty acids or lipid transfer
activity. In the present studies, we have extended the previous results
by showing that the nascent protein was modified by glycosylation and
ATP-dependent ubiquitinylation, both known to occur under
native conditions (4, 25, 28, 49). By isolating the
microsome-associated apoB48 and demonstrating its proteasome-mediated
degradation after resuspension in hepatic cytosol, we have established
a cell-free system in which ERAD of apoB can be reconstituted. As with
other mammalian and yeast ERAD substrates studied in similar systems
(e.g. Refs. 8, 50, and 51), this now allows us to decipher
the molecular requirements for the degradation of apoB by the
ubiquitin-proteasome pathway.
As noted in the Introduction, we were interested in studying the
potential influence of hsp90 on apoB degradation, given its abundance
in mammalian cells and its controversial role as a protective or
susceptibility factor in the proteasomal degradation of other proteins
(14-16). Multiple lines of evidence implicate hsp90 as a
prodegradative factor for apoB. First, there was increased recovery of
apoB in the cell-free degradation assay when GA was added to the rat
hepatic cytosol (Fig. 6). GA is widely considered to be a specific
inhibitor of protein interactions with members of the hsp90 family
(e.g. Refs. 34 and 37) and in fact was used in two of
studies cited above (14, 15).
Further support that GA was operating through a specific hsp90-mediated
mechanism was our finding that it disrupted the interaction between
hsp90 and apoB48 (Fig. 7) without perturbing the total pool of hsp90.
Importantly, despite the interaction of apoB and GRP94 in HepG2 cells
(38), there was no evidence for this in the cell-free system,
consistent with the suggestion that GRP94 plays a role in the
maturation of lipoproteins containing apoB escaping ERAD (48).
The prodegradative effect of hsp90 on apoB in the cell-free assay was
also found in rat hepatoma cells, McArdle-RH7777, transfected with
hsp90 cDNA (Fig. 9). This cell line is a standard model of hepatic
mammalian lipoprotein metabolism and also exhibits ERAD of apoB (39,
52). Although the cells produced predominately apoB100, the small
amount of apoB48 in the control cells was also reduced in the hsp90
cDNA-transfected cells (data not shown). These effects on cellular
apoB were not attributable to a nonspecific effect of hsp90 or the
transfection procedure, since albumin recoveries from the cell and
medium were not significantly changed and the control cells were
subjected to the same transfection protocol. Overall, these results
imply that the cell-free system is an accurate reflection of the role
of hsp90 in intact cells of hepatic origin.
The availability of yeast strains with mutations in the homologues of
mammalian hsp90 and hsp70, hsp82 and ssa1,
respectively, has allowed us to confirm and to examine further the
roles of these chaperones in apoB degradation (Fig. 10). The
suitability of this system was first evidenced by the fact that the
degradation of microsome-associated apoB48 in the presence of the wild
type yeast cytosol had characteristics resembling those observed with rat hepatic cytosol; i.e. >60% of apoB48 was degraded,
which was reversed by the proteasome inhibitor (data not shown) or GA.
Second, consistent with the results summarized here and our previous
studies implicating a prodegradative effect of hsp70 (4, 47), cytosols from the strains with either chaperone mutated did not support significant degradation. This implied that both chaperones are required
for the proteasomal degradation of apoB. This requirement was specific,
as supported by our finding that degradation was reconstituted in the
complementation (mixing) experiment. Other studies have shown that
hsp90 and hsp70 cooperate. For example, both chaperones participate in
the activation of hormone binding by glucocorticoid receptor (53).
Multichaperone complexes containing hsp90 and hsp70 have also been
shown to mediate refolding of denatured luciferase and
How do we envision hsp90 and hsp70 to function in the ERAD pathway for
apoB? Based on our recent studies (5, 6, 47, 56), we find no evidence
for a complete retrotranslocation into the cytosol of apoB destined for
proteasomal degradation, as for major histocompatibility complex class
I molecules in cytomegalovirus-infected cells (57) or mutant prepro- Increased expression of hsp70 enhances apoB ubiquitinylation (4, 47).
In contrast, GA did not change apoB ubiquitinylation (Fig. 8), implying
that the hsp90 acted after ubiquitin tagging of apoB48. Because hsp90
has been shown to be associated with the proteasome, it has been
proposed recently that it may facilitate the unfolding of substrates
into the relatively narrow mouth of the 19 S cap of the proteasome (59,
60). In this scenario, we envision hsp70 and hsp90 working
sequentially, with hsp70 being an early participant as an
"extraction" and, perhaps, a ubiquitinylation factor and hsp90 as a
factor acting later to facilitate the association with and entry into
the proteasome of the ubiquitinylated apoB. Thus, for maximal
degradation, both chaperones should be expected to be required for apoB
degradation. Because of the length of apoB (2152 and 4536 amino acids
for apoB48 and apoB100, respectively) and the nature of an extraction
process, however, it is likely that at steady state in the hepatic
cell, both chaperones are bound to apoB targeted for ERAD, as we have
recently observed in HepG2 cells
(6).3 The participation of
both chaperones in apoB degradation is consistent also with the recent
finding that hsp70 and hsp90 have roles in the folding and
ubiquitinylation of wild type and In summary, we have established a powerful and convenient cell-free
system to study the ERAD pathway for apoB. With this system and
confirmation in transfection studies, we have now obtained clear
evidence that both hsp90 and hsp70 promote apoB ERAD. The effects of
these two chaperones and other cytosolic factors on this process can
now be studied by using purified factors or cytosols from yeast strains
with relevant mutations, singly or in combination, to allow the
biochemical characterization and the kinetic ordering of events related
to the targeting of apoB to the proteasome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
508 (15) and insulin receptor
Ex13-IR
(16)).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was provided by W. Sessa (Yale University). Mouse
anti-HA monoclonal antibody (HA11) was purchased from Babco (Berkeley,
CA). Anti-hsp90 (SPA-835 and SPA-845) and anti-GRP94 (SPA-850)
antibodies were obtained from StressGen (Victoria, Canada). Protein
A-Sepharose and
-Bind-plus-Sepharose were purchased from Amersham
Pharmacia Biotech. Ubiquitin aldehyde, lactacystin, and MG132 were
purchased from Calbiochem. Peptide:N-glycosidase F (PNGF)
was obtained from Roche Molecular Biochemicals. Cell culture media and
related supplies were purchased from Life Technologies, Inc. Other
reagents were purchased from Sigma unless otherwise stated.
-mercaptoethanol, 2.5% bromphenol blue), the
proteins were resolved by SDS-PAGE, and a fluorogram was made from the
resulting gel. The intensities of the apoB48 band signals were measured
by densitometry of the fluorograms.
S and HA-tagged
ubiquitin protein were added to final concentrations of 5 mM and 0.1 mg/ml, respectively. The mixture was incubated
at 37 °C for 1 h. Ubiquitinylated apoB48 was immunoprecipitated with anti-HA antibody.
plasmid according the protocol of the
manufacturer. After 48 h, cells were pulse-labeled with
[35S] Protein Labeling Mix (PerkinElmer Life Sciences)
(100 µCi/ml) and chased in label-free Dulbecco's modified Eagle's
medium, containing 0.5% fetal bovine serum, 0.5% horse serum, 2 mM L-glutamine, 10 mM
L-methionine, 3 mM L-cysteine. Cell
lysates and conditioned media were analyzed by
immunoprecipitation with anti-apoB antibody or anti-rat albumin
antibody (Bethyl laboratories, Montgomery, TX). The amount of
labeled immunoprecipitated proteins was measured by
SDS-PAGE/fluorography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
In vitro synthesis of apoB48 in
the cell-free system. ApoB48 was synthesized in the absence ( )
or presence (+) of dog pancreatic microsomes in a coupled
transcription/translation reaction in which rabbit reticulocyte lysate
(containing [35S]methionine/cysteine) was programmed with
apoB48 mRNA. The reaction mixture was then separated by SDS-PAGE,
and the resulting fluorogram is displayed. The migration of the 207-kDa
marker is indicated.
View larger version (21K):
[in a new window]
Fig. 2.
Topology and glycosylation of in
vitro synthesized apoB48. A, trypsin
digestion of apoB48. ApoB48, synthesized in a coupled
transcription/translation reaction in which rabbit reticulocyte lysate
(containing [35S]methionine/cysteine) was programmed with
apoB48 mRNA in the presence of dog pancreatic microsomes
(lane 1), was treated with trypsin (0.1 mg/ml,
1 h, on ice) in the absence (lane 2) or
presence of Triton X-100 (lane 3). B,
glycosylation of apoB48. ApoB48 was synthesized as in A in
the presence of dog pancreatic microsomes (lane
1). Microsome-associated apoB48 was collected by
centrifugation and incubated (4 °C, 16 h) with (lane
2) or without (lane 3) the glycosidase
PNGF (lane 2). Lane 4 contains the
untreated supernatant after centrifugation (i.e.
nonmicrosome-associated apoB48). Samples were resolved by SDS-PAGE, and
representative fluorograms are displayed in both panels. The
migration of the 207-kDa marker is indicated.
View larger version (27K):
[in a new window]
Fig. 3.
In vitro synthesized apoB48 is
microsome-associated. ApoB48, synthesized in a coupled
transcription/translation reaction in which rabbit reticulocyte lysate
(containing [35S]methionine/cysteine) was programmed with
apoB48 mRNA in the presence of dog pancreatic microsomes, was
analyzed by floatation through a discontinuous sucrose gradient as
described under "Experimental Procedures." T, ~1% of
the diluted reaction prior to the gradient; the numbered
fractions represent SDS-PAGE analysis of portions of
150-µl aliquots taken from the top (fraction 1) to the bottom
(fraction 13) of the gradient after centrifugation. The
arrow denotes the position at which the sample was
loaded.
View larger version (27K):
[in a new window]
Fig. 4.
Ubiquitinylation of apoB48 in the cell-free
system. ApoB48 was synthesized in the presence of dog pancreatic
microsomes in a coupled transcription/translation reaction in which
rabbit reticulocyte lysate (containing
[35S]methionine/cysteine) was programmed with apoB48
mRNA (lane 1). In a control reaction, the
apoB48 cDNA was omitted (lane 2). Unlabeled
methionine and cysteine were added at the end of the reaction in a
100-fold excess relative to the corresponding labeled amino acids
present at the beginning of the reaction. To aliquots of this mixture
were added 0.1 mg/ml of HA-tagged ubiquitin and either 10 units/ml of
apyrase (lane 3) or 2 mM ATP, 10 mM creatine phosphate, and 100 µg/ml of creatine kinase
(lanes 4-6). The mixtures were then incubated at
37 °C for 1 h and immunoprecipitated with (lanes
3, 4, and 6) or without
(lane 5) anti-HA antibody. Samples were resolved
by SDS-PAGE, and a representative fluorogram is displayed.
View larger version (30K):
[in a new window]
Fig. 5.
Degradation of microsome-associated apoB48 in
rabbit reticulocyte lysate and rat hepatic cytosol. ApoB48 was
synthesized in the presence of dog pancreatic microsomes in a coupled
transcription/translation reaction in which rabbit reticulocyte lysate
(containing [35S]methionine/cysteine) was programmed with
apoB48 mRNA. The reaction mixture was layered onto a sucrose
cushion and centrifuged at 100,000 × g for 30 min (see
"Experimental Procedures"). The pelleted microsomal membranes were
resuspended in 50% (v/v) of rabbit reticulocyte lysate (A
and C) or rat hepatic cytosol (B and
D) containing ATP and an ATP regenerating system. Aliquots
of each mixture were then incubated on ice (lane
1) or at 37 °C for 2 h (lane
2, without proteasome inhibitor; lanes
3-5, with the indicated proteasome inhibitors, each at 50 µM). Gel loading buffer was then directly added, and
apoB48 recovery was assessed by SDS-PAGE/fluorography (A and
B). The apoB48 bands from repeated experiments were scanned
with a Molecular Dynamics densitometer and plotted as the mean signal
intensity ± S.D. (n = 3) for each lane
(C and D).
View larger version (27K):
[in a new window]
Fig. 6.
GA, an inhibitor of hsp90, blocks the
degradation of microsome-associated apoB48. A and B,
apoB48 was synthesized in the presence of dog pancreatic microsomes in
a coupled transcription/translation reaction in which rabbit
reticulocyte lysate (containing [35S]methionine/cysteine)
was programmed with apoB48 mRNA. Microsomes were isolated and
resuspended in rat hepatic cytosol as in Fig. 5, except that the
mixture was divided, and to each half either GA (30 µM,
in Me2SO) or Me2SO alone was added. Samples
were taken every 30 min during a 2-h incubation at 37 °C. SDS-PAGE
and densitometric analyses were performed as described in the legend to
Fig. 5. Representative fluorograms are shown in A and B. The
apoB48 signal intensity at each time point was measured in three
separate experiments and is plotted as the mean ± S.D. in
C.
View larger version (24K):
[in a new window]
Fig. 7.
Co-immunoprecipitation analysis of
microsome-associated apoB48 and the chaperones hsp90 and GRP94.
A, apoB48 was synthesized and microsomes were isolated and
resuspended in rat hepatic cytosol with or without GA, as in Fig. 5,
except that the final mixtures were incubated at 37 °C for 1 h.
Then equal aliquots were taken for immunoprecipitation with antibodies
(Ab) to apoB (lanes 1 and
2), hsp90 (lanes 3 and 4),
or GRP94 (lanes 5 and 6). The
immunoprecipitates were resolved by SDS-PAGE, and the resulting
fluorogram is displayed. B, Western blot of the material in
lanes 1 and 2 of A using
the hsp90 antibody. C, Western blot of an aliquot of the
sample analyzed in lanes 3 and 4 of
A using the hsp90 antibody.
View larger version (41K):
[in a new window]
Fig. 8.
Effect of GA on the ubiquitinylation of
microsome-associated apoB48 in rat hepatic cytosol. ApoB48 was
synthesized in the presence of dog pancreatic microsomes in a coupled
transcription/translation reaction in which rabbit reticulocyte lysate
(containing [35S]methionine/cysteine) was programmed with
apoB48 mRNA. Microsomes were isolated as in Fig. 5 and were
resuspended in a 1:1 mixture of rat hepatic cytosol/2 × Ph
buffer (see "Experimental Procedures"). This mixture was
supplemented with 0.1 mg/ml of HA-tagged ubiquitin and either 10 units/ml of apyrase (lane 1) or 5 mM
ATP S (lanes 2 and 3). Either
Me2SO (lanes 1 and 2) or
GA (lane 3; 30 µM in
Me2SO) was then added, and the samples were incubated for
1 h at 37 °C. Samples containing equivalent amounts of apoB48
were then analyzed by immunoprecipitation with the anti-HA antibody,
followed by SDS-PAGE and fluorography. The migration of the 207-kDa
marker is indicated.
plasmid, cells were pulse-labeled with
[35S]methionine/cysteine for 15 min and chased in
isotope-free medium for 30 and 90 min. Cell lysates and conditioned
media were then analyzed by immunoprecipitation with antibodies to apoB
and rat albumin (as a control). Successful transfection of the cells
and expression of the plasmids were confirmed by reverse
transcription-polymerase chain reaction detection of the plasmid region
encoding neomycin antibiotic resistance (data not shown).
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[in a new window]
Fig. 9.
Transfection with hsp90 cDNA increases
apoB100 degradation in rat hepatoma cells. A and B, rat
hepatoma (McArdle-RH7777) cells were transfected either with pcDNA3
(control; lanes 1 and 3) or
pcDNA3-hsp90 plasmid (lanes 2 and
4). Forty-eight hours after transfection, cells were
pulse-labeled with [35S]methionine/cysteine for 15 min
and chased in isotope-free medium for 30 min (lanes
1 and 2) or 90 min (lanes 3 and 4). Cell lysates and conditioned medium samples
containing an equal amount of trichloroacetic acid-insoluble
radioactivity (cpm) per mg of total protein were immunoprecipitated
with antibodies to apoB (A) or albumin (B), and
the immunoprecipitates were analyzed by SDS-PAGE and fluorography. The
lack of apoB100 in the conditioned medium samples at the 30-min time
point was expected based on previous data indicating that it takes
~40 min for newly synthesized apoB100 to be secreted by hepatic cells
(e.g. Ref. 2). Data were reproducible in three separate
experiments, and typical fluorograms are shown.
View larger version (30K):
[in a new window]
Fig. 10.
Degradation of microsome-associated apoB48
in cytosols from wild type and mutant yeast strains.
A-D, apoB48 was synthesized, and the microsomes were
isolated as in Fig. 5. The pelleted microsomal membranes were
resuspended in yeast cytosols (wild type (WT), mutant
hsp82 (yeast homologue of mammalian hsp90), mutant
ssa1 (homologue of hsp70), or an equal mixture of cytosols
containing mutant hsp82 and ssa1) and then mixed
1:1 with 2 × Ph buffer containing ATP and an ATP regenerating
system (see "Experimental Procedures"). Aliquots of each mixture
were incubated at either 4 °C (control condition) or at 37 °C for
2 h. As indicated, some samples also contained GA (30 µM). At the end of the incubation, gel loading buffer was
added, and the samples were analyzed by SDS-PAGE and fluorography.
Representative fluorograms are shown in A-D. The
densitometric signal intensities from repeated experiments are plotted
in E and are expressed as means ± S.D.
(n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase in a cooperative manner (54, 55).
factor (8); rather, there appears to be an extraction process in which
factors interact with apoB domains as they appear on the cytosolic
surface of the ER membrane. It has been proposed that hsp70 (58) can
facilitate such an extraction, perhaps by binding to a domain and
serving as a molecular motor or a ratchet. This would be consistent
with our finding that increased expression of hsp70 decreased the
secretion of apoB-containing lipoproteins even when lipid
synthesis was stimulated in HepG2 cells (4), suggesting a
competition between the cytosolic factors targeting apoB to ERAD and
the ER luminal factors promoting lipoprotein assembly and exit from the
ER.
F508 CFTR in mammalian cells (15)
and that hsp70 facilitates the degradation of CFTR in yeast (21).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Chris Cardozo for helpful comments.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL58541 (to E. A. F.), National Science Foundation Grant MCB-9722889 (to J. L. B.), a grant from the Irma T. Hirschl Trust (to A. J. C.), and a grant from the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine (to E. A. F.).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: Box 1269, Mount
Sinai School of Medicine, 1 Gustave Levy Pl., New York, NY 10029. Tel.:
212-241-7152; Fax: 212-828-4178; E-mail: edward.fisher@mssm.edu.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M100633200
2 J. L. Brodsky, unpublished data.
3 R. Pariyarath and E. Fisher, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
apoB, apoprotein B;
DPM, dog pancreatic microsomes;
GA, geldanamycin;
ER, endoplasmic
reticulum;
ERAD, ER-associated degradation;
hsp, heat shock protein;
HA, hemagglutinin;
PNGF, peptide:N-glycosidase F;
PAGE, polyacrylamide gel electrophoresis;
Ubal, ubiquitin aldehyde;
ATPS, adenosine 5'-O-(thiotriphosphate);
RRL, rabbit reticulocyte
lysate;
CFTR, cystic fibrosis transmembrane conductance
regulator.
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