(Received for publication, May 21, 1997, and in revised form, June 17, 1997)
From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
The T-cell antigen receptor (TCR) is an
hetero-oligomeric membrane complex composed of at least seven
transmembrane polypeptide chains that has served as a model for the
assembly and degradation of integral membrane proteins in the
endoplasmic reticulum (ER). Unassembled TCR chains fail to mature to
the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER
degradation" pathway that has been proposed to be autonomous to the
ER. In these studies we show that the degradation of core-glycosylated
TCR
is blocked by
N-acetyl-L-leucyl-L-leucyl-L-norleucinal
(ALLN) and lactacystin, implicating the proteasome in ER degradation.
Either acute or chronic treatment of TCR
-transfected cells with
proteasome inhibitors cause the core-glycosylated TCR
chains to
progressively shift to an ~28-kDa form that lacks
N-linked oligosaccharides and the N-terminal signal
peptide. The susceptibility of this 28-kDa species to extravesicular
protease indicates that it is not protected by the ER membrane and,
hence, cytoplasmic. These data suggest a model in which TCR
chains
that are translocated across the membrane, core-glycosylated, but fail
to assemble are dislocated back to the cytoplasm for degradation by
cytoplasmic proteasomes. Our data also suggest that covalent
modification of TCR
with ubiquitin is not required for its
degradation.
The T-cell antigen receptor
(TCR)1 is a hetero-oligomeric
complex of at least seven polypeptide chains that has served as a model
for the assembly and degradation of integral membrane proteins in the
ER (1). The clonotypic subunit (TCR
) is a type I membrane
protein containing a short (~5-amino acid) cytoplasmic domain and a
223-residue extracellular domain that has four potential sites for
N-glycosylation. Mature TCR
on the surface of the
antigen-specific T-cell hybridoma line 2B4 migrates as a broad
42-44-kDa band (2-4). However, when expressed in the absence of other
TCR subunits, TCR
is synthesized as a 38-kDa core-glycosylated
precursor that is sensitive to digestion with endoglycosidase H and is
rapidly degraded with a half-time of ~50 min (5, 6). This degradation process is not affected by inhibitors of autophagy, lysosomal proteolysis, or ER-Golgi traffic. Moreover, TCR
chains in these cells are localized to the "ER region" by immunofluorescence and electron microscopy (5). Together, these studies have led to the
conclusion that TCR
degradation occurs at a site "within or
closely associated with the ER" (5). However, efforts to identify
ER-specific proteases that participate in TCR
degradation have been
unsuccessful.
Several recent reports have suggested a role for the proteasome in the
ER degradation of some membrane or lumenal proteins (reviewed in Refs.
7 and 8). For example, misfolded cystic fibrosis transmembrane
conductance regulator (CFTR) molecules that fail to exit the ER are
rapidly degraded by a process that requires covalent modification with
ubiquitin and is blocked by lactacystin, a specific proteasome
inhibitor (9). Degradation of other ER-restricted proteins including
mutant human 1-antitrypsin (10), yeast carboxypeptidase
Y (11), and MHC class I heavy chains (12, 13) has also recently been
shown to require proteasome activity. How these proteins, which are
sequestered within the ER lumen, are recognized and delivered to the
cytoplasmic proteasome complex is unknown.
In this paper we have examined the role of the ubiquitin-proteasome
pathway in the ER degradation of newly synthesized TCR chains. Our
data suggest a model in which TCR
chains are first translocated into
the ER, cleaved by signal peptidase, and N-glycosylated with
core high mannose glycans. These chains are subsequently exported back
to the cytoplasmic face of the ER, where they are deglycosylated and
delivered to the proteasome for degradation. Moreover, our data suggest
that ubiquitination of TCR
is not required for this process.
HEK293 cells were grown and
transiently transfected by calcium phosphate precipitation as described
previously (14). In some experiments,
N-acetyl-L-leucinal-L-leucinal-L-norleucinal (ALLN, calpain inhibitor I, Calbiochem) or lactacystin (a kind gift
from S. Omura, Kitasato Institute, Tokyo) at indicated concentrations was added to the fresh media. The cDNA corresponding to 2B4 TCR (15) in pCDM8 was a kind gift from J. Bonifacino (National
Institutes of Health).
The 11 lysine residues in TCR
were mutated to arginine by 8 sequential rounds of polymerase chain
reaction-based "megaprimer" mutagenesis (16). The mutant construct
(K
R) was verified by sequence analysis, which revealed the presence
of an additional mutation of Phe154 to Tyr. Functional
comparison with a K
R lacking this additional mutation showed that
this conservative change does not influence either the kinetics of
degradation or its sensitivity to proteasome inhibitors.
HEK293 cells transiently transfected with
TCR were processed for immunoblot analysis as described previously
(9). Samples were resolved in 11% SDS-polyacrylamide gels and
electroblotted to nitrocellulose. Blots were probed with the
appropriate antibody, and immunoreactive bands were detected by
enhanced chemiluminescence. Metabolic labeling and immunoprecipitation
was carried out as described (14) with the following modifications.
Cells were pulse-labeled with 500 µCi/ml [35S]Met/Cys
(>1,000 Ci/mmol, NEN Life Science Products). Immunoprecipitation was
performed using A2B4 (17) and/or H28-710 (18) and GammaBind Plus
Sepharose (Pharmacia Biotech Inc.). The samples were fractionated in
11% SDS-polyacrylamide gel and analyzed by fluorography. For steady
state labeling, cells were incubated with 125 µCi/ml
[35S]Met/Cys in 90% Met/Cys-free and 10% complete media
for 14 h. PNGase F (New England Biolabs) digestion was carried out
on TCR
immunoprecipitated by H28-710 and bound to GammaBind Plus
Sepharose according to the manufacturer's directions.
HEK293 cells were treated with 50 µg/ml ALLN for 12 h prior to homogenization in buffer H (150 mM NaCl, 10 mM Hepes, pH 7.4, 1 mM EGTA) supplemented with protease inhibitors using a glass/Teflon homogenizer. Unbroken cells and nuclei were removed by centrifugation at 800 × g for 5 min. Total cell membranes from the resulting post-nuclear supernatant were then sedimented in a TLA100.2 rotor (Beckman) at 100,000 × g for 45 min. The membrane pellet was resuspended in buffer H containing 10 µM CaCl2. Samples were treated with Proteinase K (Life Technologies, Inc.) in the presence or absence of 1% Triton X-100 for 1 h at 0 °C. Protease digestion was terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 5 mM. Samples were analyzed by SDS-PAGE and immunoblotting.
Cell-free TranslationRadiolabeled TCR was synthesized
in a coupled transcription and translation reaction using a
T7-TNT kit (Promega) and 0.5 mCi/ml [35S]Met
(1000 Ci/mmol, Amersham). Canine pancreatic microsomes were prepared
according to Walter and Blobel (19). TCR
translated in the presence
or absence of microsomes in 50-µl reactions were immunoprecipitated
with mAb H28-710 and 50 µl of GammaBind Sepharose beads.
To determine if proteasomes play a role in the
degradation of incompletely assembled TCR, we examined the effect of
proteasome inhibitors on the steady state levels of TCR
in HEK cells
transfected with cDNA encoding the 2B4
clonotype. Immunoblot
analysis (Fig. 1A) reveals
that these cells contain a predominant immunoreactive species with a
mobility of 38 kDa, corresponding to the size of core-glycosylated
TCR
, as previously observed in transfected fibroblasts (5). A minor
band with ~2-kDa faster mobility, probably corresponding to a
partially glycosylated form of the protein (see below), was also
detected occasionally. In the absence of proteasome inhibitors, the
38-kDa species was completely soluble in nonionic detergent. However,
overnight treatment with the proteasome inhibitors ALLN or lactacystin
increased the steady-state level of the 38-kDa band in the
detergent-soluble fraction and also led to its appearance in the
detergent-insoluble fraction. Strikingly, these proteasome inhibitors
also induced the accumulation of a novel, detergent-insoluble 28-kDa
band. Because the mobility of this band corresponds to the predicted
mobility of core, unglycosylated TCR
, we examined the effects of
PNGase on the TCR
species which accumulated in proteasome-inhibited
cells.
TCR was immunoprecipitated from detergent-soluble and insoluble
fractions of transfected HEK cells that had been metabolically labeled
to steady state with [35S]Met/Cys in the presence of ALLN
(Fig. 1B). In this 15% acrylamide gel the 28-kDa species
was resolved into a doublet of closely spaced bands, both of which were
resistant to PNGase treatment, and thus, not glycosylated. The upper
band of this doublet comigrates with TCR
translated in a cell-free
protein synthesis extract in the absence of microsomes, indicating that
it has an intact signal sequence. Together, these results suggest that
translocation of TCR
is not completely efficient and that HEK and
possibly other cells possess a proteasome-mediated degradation pathway that normally masks inefficiency in the process of ER
translocation.
The lower band of the 28-kDa doublet comigrates with the limit product
of PNGase-deglycosylated 38-kDa TCR, corresponding to the
signal-cleaved form of
TCR
.2 This suggests that
some TCR
chains either fail to become glycosylated following
translocation and signal peptide cleavage in the ER or that a fraction
of glycosylated, signal-cleaved TCR
chains are deglycosylated
in vivo.
To distinguish between these two possibilities, we examined the effect
of proteasome inhibitors on the fate of newly synthesized TCR (Fig.
2). Following a 10-min pulse with
[35S]Met/Cys, TCR
migrated primarily as a
detergent-soluble 38-kDa core-glycosylated protein that was rapidly
degraded with a half-time of 65 min (Fig. 2A). In contrast,
core-glycosylated TCR
in cells treated with the proteasome
inhibitors ALLN (Fig. 2B) or lactacystin (Fig.
2C) was markedly stabilized. Both proteasome inhibitors induced the formation of bands corresponding to partially glycosylated TCR
intermediates and a 28-kDa species which accumulated over time
in the detergent-insoluble fraction. Together, these data suggest that
degradation of newly synthesized TCR
by the proteasome is preceded
by progressive deglycosylation of the core-glycosylated protein.
TCR
We used cell
fractionation and protease protection to test the possibility that
TCR degradation by proteasomes is associated with its dislocation
from the ER to the cytoplasm. Cells were lysed by mechanical
disruption,and the post-nuclear supernatant was centrifuged at
100,000 × g. A small amount (<5%) of TCR
(both the 38-kDa and the 28-kDa forms) was recovered in the supernatant, even
after a second round of 100,000 × g centrifugation,
suggesting that some TCR
had been released to the cytosolic fraction
(data not shown). However, the majority of TCR
chains sedimented
with the microsomal pellet fraction, suggesting that they are
associated with ER membranes or are present as high molecular weight
aggregates. To determine the orientation of these TCR
chains with
respect to the ER membrane, the microsomal pellet fraction was
subjected to digestion with proteinase K (Fig.
3). The endogenous lumenal proteins BiP
and GRP94 were resistant to digestion by proteinase K in the absence,
but not the presence, of detergent. By contrast, the ~10-kDa
cytoplasmic tail of calnexin was readily cleaved by the protease
indicating that this fraction contained ER vesicles that were sealed
and of uniform membrane orientation. Core-glycosylated TCR
in the
100,000 × g pellet was completely protected from
protease digestion, confirming that it had been correctly translocated. Strikingly, both bands of the 28-kDa unglycosylated doublet were highly
susceptible to proteinase K digestion, indicating that they must be
present on the exterior, i.e. cytoplasmic side of the
vesicles. These data strongly suggest that reverse translocation of
TCR
from the ER accompanies its degradation by the proteasome.
TCR
Substrates destined for degradation by the 26 S
proteasome are commonly "tagged" by the covalent attachment of
multiubiquitin chains (20). Inhibition of proteasome function in
vitro or in vivo usually induces the accumulation of a
significant fraction of highly ubiquitinated proteins, including ER
degradation substrates like CFTR (9). As TCR is a small protein,
attachment of even a single ubiquitin moiety (~7 kDa) would result in
a readily detectable decrease in gel mobility. In the present study, no
such mobility shift was observed in proteasome-inhibited cells (Figs. 1
and 2). However, ubiquitinated TCR
could have been missed if the ubiquitin linkages were labile to cleavage by cellular isopeptidases. To directly test whether TCR
ubiquitination is required for its degradation by the proteasome, we constructed a TCR
mutant (K
R) in which all 11 lysine residues were substituted by arginine. Attachment of ubiquitin to substrates occurs via an isopeptide linkage
between a lysine
-amino group on the substrate and the C-terminal
glycine of ubiquitin (21). Cells transfected with K
R were
pulse-labeled with [35S]Met/Cys for 10 min, and the K
R
protein was immunoprecipitated from both the detergent-soluble and
insoluble fractions with anti-TCR
antibody (Fig.
4A). Like wild-type TCR
,
K
R was core-glycosylated and rapidly degraded. Remarkably, this
degradation was efficiently inhibited by the proteasome inhibitor ALLN,
giving rise to the appearance of partially and completely
deglycosylated forms in both detergent-soluble and insoluble fractions.
K
R degradation was similarly inhibited by 5 µM
clasto-lactacystin
-lactone, the active form of lactacystin (22).
These data suggest that either ubiquitination of TCR
is not required
for its degradation by the proteasome or ubiquitin moieties can be
attached to TCR
at alternate non-lysine residue(s). Future studies
will be required to distinguish between these two possibilities.
Selective proteolysis is the final step in the elaborate network
of proofreading and editing processes that have evolved to protect
eukaryotic cells against the potentially deleterious consequences of
errors that can accrue between genes and proteins. These include alterations in primary sequence due to mutation or to transcriptional and translational errors, as well as the effects of inappropriate spatial and temporal expression. Selective degradation is also required
to eliminate unassembled or misassembled subunits of hetero-oligomeric
plasma membrane complexes such as the heptameric TCR (4). Eukaryotic
cells contain two major proteolytic systems: proteasomes, which are
present in the cytoplasm and the nucleus, and lysosomes. In contrast to
the lysosome-mediated disposal of mature or partially assembled TCR
oligomers, the rapid, nonlysosomal degradation of unassembled TCR
subunits had suggested the existence of a unique degradation system
associated with the endoplasmic reticulum (23). However, several recent
studies have demonstrated that some misfolded proteins in the ER can be
degraded by cytoplasmic proteasomes following their reverse
translocation from the ER (7, 8). The data in this paper demonstrate
that unassembled TCR
subunits that have been biosynthetically
translocated into the ER and core-glycosylated are exported or
"dislocated" into the cytoplasm, where they are deglycosylated and
degraded by the proteasome.
TCR was synthesized in HEK cells as a 38-kDa core-glycosylated
precursor that was rapidly degraded. Our data show that lactacystin and
ALLN stabilize this core-glycosylated form of TCR
, implicating the
proteasome in its degradation. Although the effect of ALLN in
stabilizing TCR
has been previously reported (24), neither its
activity against the proteasome nor its ability to induce the
accumulation of dislocated and deglycosylated forms were recognized at
that time. Our data show that either acute or chronic treatment of
TCR
-transfected cells with proteasome inhibitors cause the core-glycosylated 38-kDa TCR
chains to progressively shift to an
~28-kDa form that also lacks both N-linked
oligosaccharides and an N-terminal signal peptide. As signal peptidase
has its active site at the lumenal face of the ER, these data establish that some TCR
chains must have been at least partially translocated across the ER membrane. The susceptibility of the 28-kDa species to
extravesicular protease indicates that it is not protected by the ER
membrane and, hence, is cytoplasmic.
Our data indicate that the majority of dislocated TCR sediments at
relatively low speed and is insoluble in nonionic detergent. This
change in detergent solubility is probably the result of the formation
of high molecular weight aggregates. TCR
contains an unconventional
transmembrane domain that is interrupted by four polar or potentially
charged amino acids. In the absence of oligomeric partners that could
shield these side chains from the hydrophobic core of the lipid
bilayer, these polar residues have a dominant destabilizing influence
over the rest of the molecule (6, 25, 26). It is unlikely, therefore,
that nascent TCR
chains are able to effectively partition from the
hydrophilic environment of the translocon into the lipid bilayer. At
the same time the remaining 16 hydrophobic residues that constitute the TCR
transmembrane domain are unlikely to be able to effectively partition into the cytosol and may facilitate aggregation of the undegraded dislocated chains. It is possible that the inability of this
heterodox transmembrane to effectively partition into the lipid bilayer
may facilitate its dislocation without ever fully dissociating from the
translocon.
The data presented in this paper suggest that ubiquitination of TCR
chains is not required for their degradation by cytoplasmic proteasomes. Although the attachment of high molecular weight ubiquitin
polymers has been demonstrated to increase the susceptibility of
substrate for degradation by 26 S proteasome, modification by ubiquitin
is neither a necessary (27, 28) nor a sufficient signal (29) for
degradation. The requirement for ubiquitination of membrane and
secretory proteins degraded by the proteasome is also variable. For
example, inhibition of proteasome-mediated degradation of
1-antitrypsin (10) or MHC class I heavy chain (13, 30)
does not appear to lead to the accumulation of ubiquitinated forms,
although the lack of an evident ubiquitin "ladder" is not sufficient evidence upon which to exclude a role for ubiquitin. By
contrast, there is evidence supporting a requirement for substrate ubiquitination in the degradation of other membrane and secretory proteins including connexin 43 (31) and CFTR in mammalian cells (9) and
Sec61p (32) and carboxypeptidase Y (11) in yeast.
In the absence of ubiquitination, what signals are used to target ER
degradation substrates to the proteasome? In cytomegalovirus-infected cells, two gene products appear to possess the capacity to induce the
dislocation of MHC class I heavy chains from the ER and accompany them
to the proteasome. We speculate that in non-virus-infected cells such
targeting could be accomplished by direct coupling of proteasomes to
the dislocation apparatus. Possibly, the presence of a misfolded
protein in association with the dislocation apparatus could provide a
signal that would recruit the docking of proteasome. Such a signal
could be transmitted via a transmembrane chaperone like calnexin, as
has been suggested recently (10). For this model to be true,
dislocation of substrate would be predicted to depend on proteasome
activity. In our studies <30% TCR was dislocated (as measured by
the appearance of deglycosylated chains) after 3 h in the presence
of proteasome inhibitor, even though >75% TCR
would have been
degraded during the same interval in the absence of proteasome
inhibitors. Although preliminary, these data suggest that dislocation
of TCR
from the ER may be coupled to the activity of the
proteasome.
Taken together, the data presented above support the conclusion that
TCR chains are dislocated from the ER for degradation by cytoplasmic
proteasomes. Thus, TCR
joins a growing number of membrane and
secretory proteins which appear to be disposed of by a process
involving dislocation from the ER and subsequent degradation by
cytoplasmic proteasomes. Since TCR
has served as a prototype that
has largely defined the process of ER degradation, we propose that the
cytosolic degradation pathway may be the major pathway for degradation
of misfolded or unassembled proteins in the ER.
We are indebted to Juan Bonifacino for
providing us with the 2B4 cDNA and the 2B4 mAb used in these
studies. We also thank John Moorhead for generously providing us with
the mAb anti- H28-710. We are grateful to Cristina Ward for help in
the early stages of this study and for critical discussion of the data
and manuscript.