From the Département de Biochimie,
Université de Montréal, Montréal, Quebec H3C 3J7,
Canada, § AstraZeneca Research and Development
Montréal, St. Laurent, Quebec H4S 1Z9, Canada, and the
¶ Department of Anatomy and Cell Biology, University of Oulu,
FIN-90014 Oulu, Finland
Received for publication, August 7, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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We have previously shown that only a fraction of
the newly synthesized human The endoplasmic reticulum
(ER)1 quality control
scrutinizes newly synthesized proteins entering the secretory pathway
and ensures that only correctly folded and, in the case of multimeric
proteins, fully assembled complexes are deployed to distal cellular
compartments (1). Those that fail to fulfill these criteria are
retained in the ER and subsequently degraded. Even minor changes in the protein primary structure can cause retention in the ER. This is
exemplified in many diseases, including cystic fibrosis and cases of
Emerging evidence indicates that degradation of aberrant ER proteins is
mediated by the cytosolic multiprotease complex, the 26 S proteasome.
Indeed, an increasing number of misfolded or unassembled yeast and
eukaryotic integral membrane and secreted proteins have been shown to
be substrates for this disposal mechanism (7-26). However,
proteasome-mediated degradation is not restricted to misfolded or
unassembled proteins, as evidenced by the observation that some
resident ER proteins are substrates for this degradation pathway in
response to cellular signals (27-29). It can thus be hypothesized that
this degradation pathway might have a role in regulating steady state
levels of normal wild type proteins traversing the secretory pathway,
as has been described for many cytosolic and nuclear regulatory
molecules (for review, see Refs. 30 and 31). Reported examples pointing
to this phenomenon are, however, scarce, exceptions being
apolipoprotein B100 (32, 33), apolipoprotein(a) (34), and tyrosinase
(35). In a recent report, Schubert and co-workers (36) showed that at
least 30% of newly synthesized proteins might be degraded by the
proteasomes. Whether these degraded proteins represent defective
ribosomal products resulting from errors in translation, as suggested,
or might also include correctly translated but incompletely folded
proteins remains to be determined.
Proteasome-mediated ubiquitin-dependent degradation of
cytosolic and nuclear proteins is a well described phenomenon (for review, see Refs. 30 and 31), but the detailed mechanisms involved in
the ER-associated proteasomal degradation remain unclear. Recent
findings have led to the emergence of models suggesting that misfolded
or incompletely folded ER proteins are retrotranslocated to the
cytosolic side of the ER membrane through the Sec61 translocon complex
and conjugated with ubiquitin before hydrolysis by the 26 S proteasomes
(for review, see Refs. 37-39).
We have previously shown that the human Materials--
EXPRE35S35S protein
labeling mix (1175 Ci/mmol) and [9-3H]bremazocine (26.6 Ci/mmol) were purchased from PerkinElmer Life Sciences. Recombinant
peptide-N-glycosidase F (PNGase F) of Flavobacterium meningosepticum, purified from Escherichia coli (EC
3.5.1.52) was from Roche Molecular Biochemicals. The proteasome
inhibitors lactacystin, Z-Leu-Leu-Leu-CHO (MG-132) and
Z-Ile-Glu(OtBu)-Ala-Leu-CHO (PSI) were obtained from Calbiochem. Cell
culture reagents were either from Life Technologies Inc. or Wisent. The
anti-FLAG M2 monoclonal antibody, the anti-FLAG M2 affinity resin, and
the FLAG peptide were products of Sigma. The anti-calnexin (SPA-860), the anti-heat shock protein 70 (Hsp70) (SPA-820), and the
anti-ubiquitin (SPA-200) antibodies were from Stressgen Biotechnologies
Corp. and Protein G-Sepharose from Amersham Pharmacia Biotech. The
anti-Sec61 Cell Culture--
Cells were cultured in either
25-cm2 or 75-cm2 culture flasks and grown to
80-90% confluence at 37 °C in a humidified atmosphere of 5%
CO2. Human embryonic kidney 293S (HEK-293S) cells stably expressing the h Metabolic Labeling with
[35S]Methionine/Cysteine--
For
[35S]methionine/cysteine labeling, cells were first
preincubated in methionine and cysteine-free DMEM for 60 min at
37 °C, and the labeling was performed in fresh medium containing 150 µCi/ml [35S]methionine/cysteine. After an incubation of
60 min at 37 °C, the pulse was terminated by washing the cells twice
with the chase medium (complete DMEM supplemented with 5 mM
methionine) and chased for different periods of time as specified in
the figures. When labeling was performed in the presence of the
proteasome inhibitors, the reagents were added 3 h before the
pulse labeling at a final concentration of 10 µM
(lactacystin) or 25 µM (MG-132 and PSI). After the
labeling, cells were washed with and harvested in phosphate-buffered saline and, unless otherwise indicated, quick-frozen in liquid nitrogen
and stored at Immunoprecipitations--
Total cellular lysates were prepared
in buffer A (0.5% n-dodecyl- Cell Fractionation--
Cells were harvested and homogenized in
buffer B (25 mM Tris-HCl, pH 7.4, 140 mM NaCl,
2 mM EDTA, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml
benzamidine, 2 µg/ml aprotinin, 0.5 mM PMSF, 2 mM 1,10-phenanthroline) with a Dounce homogenizer (Fig. 3,
A-C). Alternatively (Figs. 3 (D and
E) and 4B), cells were homogenized by freezing
and thawing the cell suspension and passing it 10 times through a
26-gauge needle. Fractionation was performed as described (42).
Briefly, homogenates were first centrifuged at 1000 × g for 10 min to pellet unbroken cells and nuclei. The supernatant was then centrifuged at 10,000 × g for 30 min to pellet the crude membranes, and the supernatant was further
centrifuged at 100,000 × g for 60 min to clarify the
cytosolic fraction. DDM was added to the soluble fraction to a final
concentration of 0.5% (w/v) to decrease nonspecific binding of
proteins to either the anti-FLAG M2 affinity resin or Protein
G-Sepharose. Pellets were washed with buffer B, solubilized in buffer
A, and insoluble material removed by centrifugation at 100,000 × g for 60 min. Immunoprecipitations from the soluble fraction
and the solubilized membranes were carried out as described above.
Deglycosylation of the Immunoprecipitated H SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)--
SDS-PAGE
was performed as described (43), using 4% stacking gels and 10%
separating gels. Samples were heated at 95 °C for 2 min in the
presence of 50 mM dithiothreitol and run on a Bio-Rad Mini-Protein II apparatus. Molecular weight markers (Bio-Rad) detected
by staining with Coomassie Brilliant Blue (Bio-Rad) were used to
calibrate the gels. For the detection of radioactivity, the gels were
treated with EN3HANCE® (PerkinElmer Life
Sciences) according to the manufacturer's instructions, dried, and
exposed at Miscellaneous Procedures--
Total protein was measured using
the Bio-Rad DC assay kit and bovine serum albumin as a standard.
Binding assays were carried out using [3H]bremazocine,
essentially as described (40).
Degradation of ER-retained Newly Synthesized h The Mr 42,000 and 39,000 h The Mr 39,000 h
The fact that inhibition of proteasomal degradation significantly
increased the amount of the Mr 39,000 h Inhibition of Proteasomal Degradation Leads to Accumulation of
Polyubiquitinated h
To assess whether the ubiquitinated h
The observation that a significant proportion of the soluble receptors
migrates as a nonubiquitinated Mr 39,000 species
(Fig. 4B, lane 4) is consistent with
previous observations (8, 17, 35, 48). Such nonubiquitinated species
probably results from constant deubiquitination by ubiquitin
isopeptidases (50). It is very unlikely that this form could result
from a lactacystin-mediated interference in cotranslational protein
translocation into the ER membrane since this species accumulates in
the cytosol during the chase period of the metabolic labeling
experiment shown in Fig. 3E.
The ER-retained Newly Synthesized h The results of the present study indicate that core-glycosylated
h Several lines of evidence demonstrate the role of cytoplasmic
proteasomes in the high degradation rate of newly synthesized h Association of the h The finding that the Mr 39,000 h Inhibition of proteasomal degradation resulted in accumulation of
polyubiquitinated forms of the h Proteasomal degradation of the h Whether the degradation pathway uncovered for the h Results of the present study support the notion that the cytosolic
proteasomal disposal mechanism is involved in the elimination of newly
synthesized h opioid receptors is able to leave the
endoplasmic reticulum (ER) and reach the cell surface
(Petäjä-Repo, U. E, Hogue, M., Laperrière, A.,
Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727-13736). In the present study, we investigated the fate of those
receptors that are retained intracellularly. Pulse-chase experiments
revealed that the disappearance of the receptor precursor form
(Mr 45,000) and of two smaller species (Mr 42,000 and 39,000) is inhibited by the
proteasome blocker, lactacystin. The treatment also promoted
accumulation of the mature receptor form (Mr
55,000), indicating that the ER quality control actively routes a
significant proportion of rescuable receptors for proteasome
degradation. In addition, degradation intermediates that included
full-length deglycosylated (Mr 39,000) and
ubiquitinated forms of the receptor were found to accumulate in the
cytosol upon inhibition of proteasome function. Finally,
coimmunoprecipitation experiments with the
-subunit of the Sec61
translocon complex revealed that the receptor precursor and its
deglycosylated degradation intermediates interact with the translocon.
Taken together, these results support a model in which misfolded or
incompletely folded receptors are transported to the cytoplasmic side
of the ER membrane via the Sec61 translocon, deglycosylated and
conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin deficiency and familial
hypercholesterolemia, which are characterized by an "ER storage
phenotype" (2). Although less thoroughly characterized, numerous
naturally occurring mutations leading to ER retention of G
protein-coupled receptors (GPCRs) have also been evoked as the cause of
inherited diseases. For example, ER-retained mutants of rhodopsin, the
luteinizing hormone receptor, and the V2-vasopressin receptor have been
implicated in some forms of retinitis pigmentosa, male
pseudohermaphroditism, and nephrogenic diabetes insipidus, respectively
(3-6).
opioid receptor (h
OR) is
an example of integral membrane proteins that are partially retained in
the ER, possibly due to difficulties in the folding process (40).
Another example is the cystic fibrosis transmembrane conductance
regulator (CFTR) (for review, see Ref. 41). Only about 40% and 25% of
the newly synthesized wild type h
OR and CFTR molecules,
respectively, are able to leave the ER and reach the cell surface. We
thus set out to determine whether the ER-retained h
ORs are targeted
for degradation by the 26 S proteasomes and, if so, to delineate the
mechanisms involved in this process. The results presented here
indicate that these receptors are transported back to the cytosol, in a
process that involves the Sec61 translocon, and deglycosylated before
being degraded by the proteasomes. Furthermore, inhibition of the
proteasomal pathway led to the accumulation of ubiquitinated receptor
forms, pointing to the fact that polyubiquitination may be a targeting
signal for disposal of misfolded or incompletely folded h
OR molecules.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antibody was a generous gift from Dr. Tom A. Rapoport
(Harvard Medical School, Boston, MA). All the other reagents were of
analytical grade and obtained from various commercial suppliers.
OR tagged at the C terminus with the FLAG epitope (DYKDDDDK) (40) were maintained in Dulbecco's modified Eagle's medium
(DMEM) containing 10% (v/v) fetal calf serum, 1000 units/ml penicillin, 1 mg/ml streptomycin, 1.5 µg/ml fungizone (complete DMEM)
and 400 µg/ml Geneticin. A clone expressing 10 pmol of receptor/mg of
membrane protein was chosen for this study.
80 °C.
-D-maltoside
(DDM) (w/v), 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM EDTA, 5 µg/ml leupeptin, 5 µg/ml soybean
trypsin inhibitor (STI), 10 µg/ml benzamidine, 2 µg/ml aprotinin,
0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM 1,10-phenanthroline). Insoluble material was removed by
centrifugation at 5000 × g for 30 min, and bovine serum albumin was added to a final concentration of 0.1% (w/v). The
receptor was immunoprecipitated using the anti-FLAG M2 affinity resin
as described (40). For the Sec61
immunoprecipitation, DDM was
replaced by digitonin while NaCl, EDTA, and 1,10-phenanthroline were
omitted from the buffer. For the ubiquitin immunoprecipitation, N-ethylmaleimide (NEM) was included in all buffers to
distinguish the high molecular weight ubiquitinated receptor forms from
putative aggregated receptors. Before immunoprecipitation of calnexin, Hsp70, Sec61
, and ubiquitin, samples were precleared for 60 min with
15 µl of Protein G-Sepharose. The appropriate antibody (dilutions: 1:200, 10 µg/ml, 1:100, and 1:100, respectively) and 15 µl of Protein G-Sepharose were then added and incubated overnight at 4 °C
with gentle agitation. Following washing of the resin as described
(40), the bound antigens were eluted in SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromphenol blue) at 95 °C for 5 min. If two or more consecutive immunoprecipitation steps were performed, the antigens were
eluted with 1% (w/v) SDS, 25 mM Tris-HCl, pH 7.4, at
95 °C for 5 min and the eluates diluted 10-fold with buffer A prior to the next immunoprecipitation.
ORs--
The
immunoprecipitated receptors were deglycosylated following elution from
the anti-FLAG M2 affinity resin with 1% (w/v) SDS, 50 mM
sodium phosphate, pH 7.5. Before the enzyme reaction, the eluates were
diluted 10-fold with 0.5% (w/v) DDM, 50 mM sodium phosphate, pH 7.5, 50 mM EDTA, 0.5 mM PMSF, 2 mM 1,10-phenanthroline, 5 µg/ml leupeptin, 5 µg/ml STI,
10 µg/ml benzamidine, and 1% 2-mercaptoethanol. PNGase F was added
at a final concentration of 0.01-10 units/ml, samples incubated at
37 °C for 16 h, and the reaction terminated by the addition of
SDS-sample buffer.
80 °C for 1-15 days using Biomax MR film and
intensifying screens (Eastman Kodak Co.). The relative intensities of
the labeled bands on the fluorographs were analyzed by densitometric scanning with an Agfa Arcus II laser scanner, and the data were quantified using the NIH image program, version 1.61, subtracting the
local background from each lane.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ORs Is Mediated by
the Proteasomal Pathway--
We have previously shown that a large
fraction of newly synthesized h
ORs in HEK-293S cells remains in a
pre-Golgi compartment as core-glycosylated Mr
45,000 precursors that are not transported to the cell surface (40).
Now we set out to investigate the fate of these intracellularly
retained receptors. To determine whether proteasomes are involved in
the degradation of these receptor forms, cells were incubated with
lactacystin, a specific inhibitor of the 26 S proteasome (44, 45).
HEK-293S cells stably expressing the receptor carrying a C-terminal
FLAG epitope (HEK-293S-h
OR-FLAG cells) were metabolically labeled
with [35S]methionine/cysteine and after a chase in a
medium containing an excess of unlabeled methionine, cell lysates were
prepared and the receptors isolated by immunoprecipitation (Fig.
1). A major band of
Mr 45,000 and two minor ones of
Mr 42,000 and 39,000 were apparent in untreated
cells at the end of the pulse (Fig. 1A). During the
subsequent chase, label incorporated into these bands decreased and a
slower, more diffusely migrating band of Mr
55,000 appeared, representing the fully mature receptor (40). When the
cells were treated with lactacystin, an increase in labeling of all
receptor species was detected (Fig. 1B). Furthermore,
proteasome inhibition significantly retarded the disappearance of the
Mr 45,000, 42,000, and 39,000 receptor species,
the half-life of the last one being the most dramatically affected
(Table I). Similar findings were obtained
using two other proteasome inhibitors, the peptide aldehydes MG-132 and
PSI (45) (data not shown). Taken together, these results suggest an
active role of proteasomes in the elimination of newly synthesized
h
ORs. The fact that lactacystin promoted increase in receptor
labeling, even within a shorter pulse period of 15 min, suggests that
targeting to degradation begins very early, either cotranslationally or
immediately after the receptor synthesis is complete. Inhibition of
this rapid degradation is most likely responsible for the increase in
the amount of mature receptors observed in pulse-chase labeling
experiments (compare lanes 4 and 5 in
Fig. 1, A and B). This increase was accompanied by a 38% increase in the number of
[3H]bremazocine-binding sites detected. Our findings are
not due to the presence of a FLAG-epitope tag at the C terminus of the receptor since the same results were also obtained using a receptor harboring the tag at its N terminus (data not shown). Unfortunately, the lack of a high affinity and selective antibody for the h
OR makes
it impossible to carry out these experiments on the native untagged
receptor.
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Fig. 1.
H OR synthesis and
maturation in untreated and lactacystin-treated HEK-293S cells.
HEK-293S-h
OR-FLAG cells were untreated (A) or treated
with 10 µM lactacystin (B) for 3 h,
pulse-labeled for 60 min with [35S]methionine/cysteine,
and then chased for the indicated times in the absence (A)
or continued presence of the proteasome inhibitor (B).
Cellular lysates were prepared and the solubilized receptors isolated
by a two-step immunoprecipitation using the anti-FLAG M2 antibody as
described under "Experimental Procedures." The samples were
analyzed by SDS-PAGE and fluorography and the dried gels exposed to
x-ray films. The fluorographs shown are representative of four
independent experiments. Arrows indicate the molecular
weights of the different receptor forms. Markers used to calibrate the
gels are indicated (from the top: myosin,
-galactosidase,
phosphorylase b, bovine serum albumin, ovalbumin, and
carbonic anhydrase).
Half-lives of the hOR species in the absence and presence of
lactacystin
OR Species Represent
Receptors Carrying One or No N-Linked Oligosaccharides,
Respectively--
It has been reported previously that
N-linked oligosaccharides of newly synthesized glycoproteins
are removed before proteasomal degradation (13-17, 26, 35, 42, 46). We
therefore assessed whether the smaller molecular weight receptor
species of Mr 42,000 and 39,000 represent
deglycosylated receptors rather than proteolytic fragments. For this
purpose, immunoprecipitated [35S]methionine/cysteine
pulse-labeled samples were subjected to enzymatic deglycosylation using
PNGase F to remove the two N-linked glycans from the
receptor (40). As can be seen in Fig. 2
(lane 5), complete deglycosylation of the
Mr 45,000 receptor precursor led to the
appearance of a single species migrating at Mr
39,000. The fact that the Mr 42,000 species was
found to be an intermediate in the stepwise deglycosylation of the
receptor precursor suggests that this species represents a receptor
carrying one N-linked glycan. The mobility of the
Mr 39,000 species that was stabilized in
lactacystin-treated cells was indistinguishable from that of the PNGase
F-digested core-glycosylated Mr 45,000 receptor
form (Fig. 2, compare lanes 5-7), confirming the fact that
this species represents an intact receptor polypeptide with no
N-linked glycans. Protein translocation into the ER membrane
and core glycosylation are believed to occur cotranslationally in
mammalian cells (47). Thus, it is very likely that the
Mr 39,000 receptor species represents a
previously core-glycosylated receptor that has been deglycosylated and
is an intermediate in the degradation pathway.
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Fig. 2.
Deglycosylation of the metabolically labeled
h OR with PNGase F. HEK-293S-h
OR-FLAG
cells were pulse-labeled for 60 min with
[35S]methionine/cysteine in the absence or presence of
lactacystin as indicated. Receptors were then isolated from cellular
lysates by immunoprecipitation as described in Fig. 1. The
immunoprecipitates were incubated for 16 h at 37 °C with
increasing concentrations (0.01-1 unit/ml) of PNGase F as described
under "Experimental Procedures." The enzyme-free controls
(lanes 1 and 6) contained buffer only.
Reactions were stopped by adding SDS-sample buffer followed by SDS-PAGE
and fluorography.
OR Species Dislocates from the ER
Membrane and Accumulates in the Cytosol--
To further verify the
nature and the subcellular localization of the
Mr 39,000 h
OR species, fractionation of
cellular homogenates was performed after metabolic labeling (Fig.
3). Receptors were immunoprecipitated
from four fractions: 1000 × g pellet containing cellular debris, nuclei and trapped soluble proteins, 10,000 × g pellet containing crude membranes, 100,000 × g pellet containing residual microsomal membranes, and
100,000 × g supernatant containing cytosolic proteins.
As a control, the ER membrane protein, calnexin, and the cytosolic
protein Hsp70 were immunoprecipitated from aliquots of the same four
fractions. Calnexin was detected in the membrane fraction (Fig.
3B, lanes 3 and 4) but not
in the cytosolic one (lanes 7 and 8),
whereas the opposite was true for Hsp70 (Fig. 3C). As
expected, both proteins were also detected in the 1000 × g pellet (Fig. 3, B and C,
lanes 1 and 2). As can be seen in Fig.
3A, in addition to be found in the membrane fraction, the Mr 39,000 receptor species was recovered from
the cytosolic fraction of lactacystin-treated (lane
8) but not of untreated cells (lane 7). This is in agreement with the notion that it represents
an intermediate in the degradation pathway that would normally be broken down by the cytosolic proteasomes. Higher molecular weight bands
forming a regularly spaced ladder, of which the smallest had an
apparent molecular weight of 45,000, were also found in the cytosolic
fraction of lactacystin-treated cells (Fig. 3A, lane 8). This Mr 45,000 species does not represent the core-glycosylated receptor precursor
seen in Fig. 3A (lanes 1-4), because
PNGase F was not able to decrease its molecular weight (Fig.
3D, lanes 3 and 4). It is
more likely that the ladder and the accompanying high molecular weight
smear comprise polyubiquitinated forms of the Mr
39,000 receptor (see below).
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Fig. 3.
Subcellular fractionation of metabolically
labeled HEK-293S-h OR-FLAG cells.
HEK-293S-h
OR-FLAG cells were pulse-labeled for 60 min with
[35S]methionine/cysteine in the presence or absence of
lactacystin as indicated and either harvested immediately
(A-D) or chased for the indicated times in the continued
presence of the proteasome inhibitor (E). The total cellular
homogenates were fractionated by differential centrifugation as
described under "Experimental Procedures." H
OR (A,
D, and E), calnexin (B), and Hsp70
(C) were immunoprecipitated from the fractions obtained
using the appropriate antibodies. The immunoprecipitates were then
either analyzed directly by SDS-PAGE and fluorography (A-C
and E) or deglycosylated with PNGase F prior to SDS-PAGE
(D). Arrows indicate migration of the different
receptor species, calnexin and Hsp70. Results shown are representative
of four (A-C) or two (D and E)
independent experiments.
OR
species in the cytosolic fraction (Fig. 3A, lanes
7 and 8), suggests that this species may
dislocate from the ER membrane and accumulate in the cytosol prior to
proteasomal degradation. To test this hypothesis, receptors were
immunoprecipitated from the soluble fraction after pulse-chase labeling
of lactacystin-treated cells. As seen in Fig. 3E, a small
amount of the Mr 39,000 receptor species was present in the cytosol at the end of the pulse. However, the amount detected clearly increased during the chase, indicating that the Mr 39,000 species originates from a pool of
newly synthesized receptors that are retained in the ER and not from a
failure or inefficiency of the translocation machinery. Thus, this
species most likely results from deglycosylation of the
Mr 45,000 receptor precursor that is destined
for degradation and eventually dislocates from the ER membrane. Removal
of the N-linked glycans most likely occurs prior to
retrotranslocation to the cytosol since the deglycosylated h
OR
species was found both in the cytosolic and the membrane fractions
(Fig. 3A, lanes 8 and 4, respectively).
ORs--
To directly test the hypothesis that
the h
OR is modified by ubiquitination, cellular lysates from
[35S]methionine/cysteine pulse-labeled cells were
subjected to sequential immunoprecipitation. First, the h
ORs were
immunoprecipitated with the anti-FLAG M2 antibody and the purified
material was denatured by SDS. Proteins were then reimmunoprecipitated
using an anti-ubiquitin antibody. All these immunoprecipitation steps
(Fig. 4, A and B) were carried out in the presence of NEM to prevent inappropriate aggregation of proteins. In lactacystin-treated cells, the
immunoprecipitated material migrated as a high molecular weight smear
that extended upwards toward the top of the gel (Fig. 4A,
lane 4), a feature that is characteristic of
ubiquitinated proteins (8, 10, 17, 20, 22, 33, 35, 48, 49). This smear
is probably due to the variable number of ubiquitin molecules that are
added to individual receptor molecules during the process of
polyubiquitination. Ubiquitinated proteins were barely detectable in
the absence of lactacystin (Fig. 4A, lane
3), indicating that they normally are efficiently disposed
by the proteasomes.
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Fig. 4.
Immunoprecipitation of the ubiquitinated
h OR species from metabolically labeled
HEK-293S-h
OR-FLAG cells.
HEK-293S-h
OR-FLAG cells were pulse-labeled for 60 min with
[35S]methionine/cysteine in the absence or presence of
lactacystin as indicated, and cellular lysates were prepared
(A) or membrane (B, lanes
1-3, 7, and 8) and soluble fractions
(B, lanes 4-6) were isolated. The
total pool of receptors (A (lanes 1 and 2) and B (lanes 1 and
4)) and the ubiquitinated receptor forms (A
(lanes 3 and 4) and B
(lanes 2, 3, and 5-8))
were isolated by sequential immunoprecipitation using the anti-FLAG M2
and the anti-ubiquitin antibodies as indicated. The immunoprecipitates
were then subjected to SDS-PAGE and fluorography. Lanes
7 and 8 in B represent longer
exposures of lanes 2 and 3,
respectively. Results shown are representative of three independent
experiments.
OR species are associated with
the ER membranes or represent dislocated cytosolic receptors, [35S]methionine/cysteine-labeled receptors were subjected
to sequential immunoprecipitation following fractionation of
lactacystin-treated cells. As seen in Fig. 4B, the presence
of ubiquitinated receptors in the cytosolic fraction could be easily
detected following re-immunoprecipitation of the purified h
ORs with
the anti-ubiquitin antibody (lane 5). Although
hardly any ubiquitinated material could be seen in the membrane
fraction (lane 2), longer exposure revealed that
some ubiquitinated receptors were also associated with this fraction (lane 7). These results suggest that
ubiquitinated receptors accumulate in the cytosol after the receptor
dislocates from the ER membrane but that addition of ubiquitin
molecules to the receptor begins prior to dislocation. To further
verify that the receptor itself was being ubiquitinated and did not
coimmunoprecipitate with other ubiquitinated proteins, the
anti-ubiquitin antibody immunoprecipitates were purified a second time
with the anti-FLAG M2 antibody following SDS denaturation. As expected
for an ubiquitinated receptor, the diffusely migrating material was
recovered again (Fig. 4B, lane 6). The
distinct higher molecular weight species detected in Fig. 4
(panels A (lane 2) and
B (lane 4) but not in
panels A (lane 4) and
B (lane 5)) most likely represent
labeled proteins that interact with the newly synthesized receptor in a
SDS-resistant manner or receptor oligomers that are resistant to NEM.
Polyubiquitination of the receptor did not result from the presence of
a FLAG-epitope tag at its C terminus since a N-terminally tagged
receptor form was also found to be ubiquitinated (data not shown).
ORs Are Retrotranslocated to
the Cytosol via the Sec61 Complex--
The cellular fractionation
experiments clearly showed that the ER-retained newly synthesized
h
ORs dislocate from the ER membrane into the cytosol (Figs. 3
(A and E) and 4B). To assess whether this dislocation involves retrotranslocation through the Sec61 translocon, as has been reported for other membrane proteins that are
degraded by the proteasomes (13, 17, 21), coimmunoprecipitation experiments with the
-subunit of the Sec61 complex were performed. Cells treated or not with lactacystin were pulse-labeled with [35S]methionine/cysteine and chased for 60 min. As shown
in Fig. 5A (lanes
5 and 6), re-immunoprecipitation of the Sec61
immunoprecipitates with the anti-FLAG M2 antibody recovered the
Mr 45,000, 42,000, and 39,000 receptor species,
suggesting that they interact with the translocon. This interaction was
shown to be specific because these species were absent when the
anti-Sec61
antibody was replaced with a preimmune serum in the first
immunoprecipitation step (Fig. 5A, lanes
3 and 4) or when the anti-FLAG M2 affinity
resin was replaced with the anti-mouse IgG agarose in the second step
(data not shown). Although the three receptor species were found to coimmunoprecipitate with the
-subunit of the Sec61 complex in both
untreated and lactacystin-treated cells, treatment with the proteasome
inhibitor increased their amount and the Mr
39,000 species became prevalent (Fig. 5A, compare
lanes 5 and 6). The sensitivity of the
Mr 45,000 and the Mr
42,000 receptors to PNGase F (Fig. 5B, lanes
3 and 4) confirmed their identity as the
precursor and the deglycosylation intermediate, respectively. Because
the cells were chased for 60 min before the lysates were prepared, the
receptor species that interact with the translocon are more likely to
be in transit to exit the ER rather than being translocated into the
membrane. The fact that the fully deglycosylated
Mr 39,000 receptor as well as the glycosylated
Mr 42,000 and 45,000 species all associate with
the Sec61 complex confirms that deglycosylation occurs before complete
retrotranslocation of the receptor to the cytosol.
View larger version (34K):
[in a new window]
Fig. 5.
Immunoprecipitation of the Sec61
complex-associated h OR species from
metabolically labeled HEK-293S-h
OR-FLAG
cells. HEK-293S-h
OR-FLAG cells were pulse-labeled for 60 min
with [35S]methionine/cysteine and chased for 60 min in
the presence or absence of lactacystin as indicated. The total pool of
receptors (A (lanes 1 and
2) and B (lanes 1 and
3)) and those interacting with the Sec61 complex
(A (lanes 5 and 6) and
B (lanes 2 and 4)) were
isolated from total lysates by sequential coimmunoprecipitation using
the anti-FLAG M2 and the anti-Sec61
antibodies as indicated, and the
immunoprecipitates were subjected to SDS-PAGE and fluorography either
directly (A) or after PNGase F digestion (B). The
results shown are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
OR precursors that are not competent to enter the maturation pathway are transported to the cytosol in a process that involves the
Sec61 translocon, removal of N-linked oligosaccharides, and addition of ubiquitin molecules. This process leads to targeting of a
significant proportion of newly synthesized receptors for degradation
by the 26 S proteasomes. The h
OR thus appears to be a protein that,
due to inherent difficulties in acquiring its native conformation, is
very efficiently disposed of by the ER quality control. This
elimination could result either from a high level of irreversible
misfolding or from limitations in the folding kinetics. Because
proteasomal blockade was found to enhance ER export and maturation of
the receptor, kinetic limitations leading to the premature degradation
of folding intermediates most likely contribute to the observed
phenomenon. Irrespective of the nature of the receptor forms that are
targeted for degradation (irreversibly misfolded or folding
intermediates), the present results show that the ER quality control
can eliminate a high proportion of wild type proteins. This does not
seem to be unique to the h
OR since Schubert and colleagues (36)
recently suggested that at least 30% of newly synthesized proteins are
degraded by the proteasomes (36). It was hypothesized that degradation
of what was referred to as defective ribosomal products could serve as
an important adaptation for early antigen presentation during viral
infection (36, 51). Whether the high rate of degradation of the newly synthesized h
ORs also serves a biological purpose remains to be
determined. Alternatively, this process could merely reflect the
exceedingly stringent control system monitoring the rapid but
error-prone protein synthesis machinery. Because expression to a
relatively high level (~10 pmol/mg of protein for the clone used) is
needed to perform the type of studies presented here, one cannot
entirely exclude that overexpression is in part responsible for the
proteasomal-mediated degradation observed. However, we feel that this
is unlikely since identical data were obtained using a clone expressing
a smaller number of receptors (4 pmol/mg of protein; data not shown).
Additionally, the fact that several endogenously expressed ER proteins
have been shown to be targeted for proteasomal degradation in a
regulated manner (27-29) reinforces the idea that this proteolytic
pathway is a general mechanism used to regulate protein biosynthesis.
ORs.
First, the turnover rate of the core-glycosylated
Mr 45,000 precursor as well as that of the two
smaller receptor species of Mr 42,000 and 39,000 was reduced considerably in cells treated with proteasomal inhibitors,
lactacystin, MG-132, and PSI. Second, the amount of the fully mature
receptor was increased upon proteasomal inhibition. Finally, the
Mr 39,000 receptor and its polyubiquitinated forms accumulated in the cytoplasm following proteasomal blockade. Given that the Mr 39,000 receptor species
contains the full-length receptor polypeptide, these data support the
hypothesis that integral membrane proteins are released completely from
the ER membrane prior to proteasomal degradation rather than cleaved
partially within the membrane (13-15, 17, 42).
OR precursor and that of the two full-length
deglycosylated intermediates with the Sec61 translocon supports the
recently proposed model, suggesting that retrotranslocation of
misfolded or incompletely folded ER proteins to the cytoplasm is
mediated by the Sec61 complex (13, 17, 21, 46, 52, 53), the multimeric
protein complex that is also responsible for the cotranslational
translocation of proteins into the ER (54). Like the h
OR, the major
histocompatibility class I molecule and the
F508 mutant of the CFTR
were found to interact with the Sec61 translocon during their transit
out of the ER, which leads to their degradation by the proteasomes (13,
17). It can be hypothesized that dislocation of these integral membrane
proteins may involve a reverse process of membrane integration
involving lateral opening of the translocon toward the lipid bilayer
(39, 54). However, how these misfolded or incompletely folded proteins are recognized and delivered to the dislocation machinery is unknown. Similarly, the precise mechanism by which these proteasomal substrates are extracted from the lipid bilayer as well as the origin of the
driving force for their retrotranslocation are still unresolved issues.
OR
degradation intermediate accumulates in the cytosol following
lactacystin treatment suggests that proteasomal function is not
obligatory for dislocation of membrane proteins from the ER membrane.
However, this does not rule out the possibility that, in normal
conditions (i.e. when proteasomes are not impaired),
dislocation and degradation of misfolded or incompletely folded
proteins may be tightly coupled events. In fact, three observations in
the present study suggest coupling between proteasome function and
dislocation of proteins from the ER membrane. Inhibition of the
proteasome was found to: 1) increase the amount and reduce the
turnover-rate of the membrane bound Mr 42,000 and 45,000 forms of the receptor, 2) increase the total amount of
receptor coimmunoprecipitated with the
-subunit of the Sec61
complex, and 3) increase the relative proportion of the
translocon-associated Mr 39,000 degradation
intermediate that ultimately dislocates from the ER membrane into the
cytosol. Thus, although proteasomal function is not obligatory for
dislocation, its impairment appears to lead to attenuation of the
process and prolongation of association with the translocon. These
results are consistent with findings showing that mutant (21) and
chimeric (55) transmembrane proteins were stabilized within the ER
membrane of yeast mutants expressing functionally impaired proteasomes. Coupling of dislocation and degradation could be achieved by direct transfer of protein substrates to the proteasomes. Indeed, although proteasomes are mainly detected in the cytosol and the nucleus, some
have been found to be associated with ER membranes (56). Coupling of
membrane dissociation and proteolysis would clearly increase the
efficiency of proteolysis, because direct transfer of the unfolded
retrotranslocated proteins to the proteasome would eliminate the need
for additional unfolding of the substrate that is a prerequisite for
proteasomal degradation (31). At the same time, the proteasome itself,
via its regulatory subcomplex, PA700, could provide the driving force
for the retrotranslocation (31). Such coupling would also be a very
effective way to prevent cytoplasmic aggregation of dislocated membrane
proteins. In this context, it is noteworthy that proteasomal blockade
has been shown to promote the formation of high molecular weight
insoluble aggregates (8, 16). For example, both wild type and
F508
mutant forms of the CFTR were shown to accumulate, together with
cytosolic chaperones, in a specific pericentriolar structure, called
the aggresome (57, 58).
OR in the cytosol, suggesting that
this modification may target the misfolded or incompletely folded
receptors to the proteasome. Based on the finding that a small amount
of ubiquitinated receptor forms was detected in the membrane fraction,
it can also be envisioned that polyubiquitination could provide a
direct signal for proteasome recruitment to the ER membrane. However,
it is unclear whether ubiquitination is a prerequisite for the rapid
breakdown of the receptor, as has been shown for many other integral
membrane and secretory proteins that are degraded by the proteasomes.
For example, many misfolded or unassembled ER proteins were shown to be
stabilized by mutant ubiquitin-activating (8, 46, 48) or
ubiquitin-conjugating enzymes (9, 10, 18, 20, 21, 59) or by
dominant-negative ubiquitin mutants that prevent polyubiquitination of
substrate proteins (8-10, 18, 21, 48, 60). When considering the enzymes that could be responsible for the ubiquitination of the h
OR
while it is still membrane-bound, it is noteworthy that two yeast
ubiquitin-conjugating enzymes, the Ubc6p and the Ubc7p, have been
implicated in the ubiquitination and degradation of ER proteins (9, 10,
18, 20, 21, 28, 59). Both of these enzymes are localized to the ER,
thus possibly enabling ubiquitination of misfolded or incompletely
folded proteins prior to their dislocation from the membrane. The Ubc6p
is an integral membrane protein, which is anchored to the membrane via
a C-terminal hydrophobic sequence (61), whereas the Ubc7p is docked
onto the membrane protein Cue1p (59). In a recent report, several mammalian homologs of the Ubc6p were identified (62). Whether these
enzymes are actually involved in ubiquitination and degradation of ER
proteins remains to be determined.
OR is preceded by removal of the two
N-linked oligosaccharides from the Mr
45,000 receptor precursor. This is in agreement with previous reports
on glycoproteins for which proteasomal degradation has been described
(13-17, 26, 35, 42, 46). Although PNGase-type activity has been
detected in both cytosolic and microsomal membrane fractions of
mammalian tissues (63, 64), the enzymes mediating the deglycosylation of misfolded or incompletely folded proteins as well as their precise
location have remained elusive. However, recent cloning of a cytosolic
yeast PNGase (65) may be pointing to the first candidate involved in
deglycosylation of proteasomal substrates. In any case, our data
suggest that deglycosylation of the h
OR occurs prior to dislocation
from the ER membrane and the translocon. Indeed, neither the
Mr 45,000 receptor precursor nor the partially deglycosylated Mr 42,000 form were found in the
cytosolic fraction. Furthermore, the fully deglycosylated
Mr 39,000 degradation intermediate was found to
coimmunoprecipitate with the
-subunit of the Sec61 complex.
OR in the present
study is a unique characteristic of this receptor or represents a
general mechanism regulating the maturation and cell surface expression
of most GPCRs remains an open question. Indeed, very little is known
about the processes involved in the folding/maturation/degradation of
other GPCRs. Inhibition of proteasome function was found to lead to an
increase in the
2-adrenergic receptor expression in HEK-293 cells (66) and only three GPCRs, rhodopsin (67) and the yeast
- and a-mating factor receptors (68, 69), have previously been shown
to be ubiquitinated. For rhodopsin, no specific role has yet been
attributed to the modification whereas ubiquitination has been proposed
to be an internalization signal for the activated
-mating factor
receptor, leading to its lysosomal/vacuolar degradation (68, 70-72).
Interestingly, monoubiquitination rather than polyubiquitination normally associated with the proteasome degradation pathway appears to
be sufficient to promote such internalization (70, 72). It will be
interesting to determine whether ubiquitination of the h
OR and other
mammalian GPCRs can, in addition to its role in the proteasome-mediated
degradation of ER-retained receptors, also be implicated in their
internalization and lysosomal degradation. Such a dual role for
ubiquitination has been proposed for a number of yeast and mammalian
membrane proteins (38) but has not been documented yet for mammalian GPCRs.
ORs, resulting ultimately in degradation of even
salvageable folding intermediates. This raises several important
questions. For example, it becomes important to determine the
mechanisms by which the stringent ER monitoring system discriminates conformational variants of the receptor and sorts them either for
degradation or allows their transport downstream within the secretory
pathway. Future studies are also needed to identify the pathways and
components that facilitate folding of the receptor and to determine
whether these are under the control of dynamic regulatory processes.
Although inhibiting proteasomal function favored ER export and
maturation of the receptor, a large proportion of the newly synthesized
receptors still remained entrapped within the ER. Whether approaches
acting at earlier off-pathway steps could be more effective in
enhancing receptor folding still remains to be investigated.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Tom A. Rapoport
(Harvard Medical School, Boston) for providing us the anti-Sec61
antibody. We also thank Dr. Thierry Groblewski for critical reading of
the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council of Canada (to M. B.) and from the Ella and Georg Ehrnrooth Foundation, the Helsinki University Pharmacy, and the Oulu University Scholarship Foundation (to U. P.-R.).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 may be addressed: Dept. of Anatomy and
Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. E-mail: ulla.petaja-repo@ oulu.fi.
** Scientist of the Medical Research Council of Canada. To whom correspondence may be addressed: Dépt. de Biochimie, Faculté de Médicine, Université de Montréal, P.O. Box 6128, Succursale Centre-Ville, Montréal, Quebec H3C 3J7, Canada. E-mail: bouvier@bcm.umontreal.ca.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M007151200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic
reticulum;
CFTR, cystic fibrosis transmembrane conductance regulator;
DDM, n-dodecyl--D-maltoside;
DMEM, Dulbecco's modified Eagle's medium;
GPCR, G protein-coupled receptor;
h
OR, human
opioid receptor;
HEK-293S, human embryonic kidney
293S;
Hsp70, heat shock protein 70;
MG-132, Z-Leu-Leu-Leu-CHO;
NEM, N-ethylmaleimide;
PAGE, polyacrylamide gel electrophoresis;
PMSF, phenylmethylsulfonyl fluoride;
PNGase F, peptide-N-glycosidase F;
PSI, Z-Ile-Glu(OtBu)-Ala-Leu-CHO;
STI, soybean trypsin inhibitor.
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