By
mura,
From the * Laboratory of Immune Cell Biology, Division of Basic Sciences, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892-1152; The Kitasato Institute, Tokyo,
Japan; and the § Cell Biology and Metabolism Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892-5430
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T
cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane.
TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal
processes that have been referred to as "ER degradation". The molecular basis for the loss of
the TCR CD3- and TCR-
subunits from the ER was investigated in lymphocytes. For
CD3-
, we describe a process leading to its degradation that includes trimming of mannose
residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of
CD3-
was markedly curtailed and CD3-
remained membrane bound in a complex with
CD3-
. TCR-
was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-
,
and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target
proteins from the ER to the cytosol for degradation.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The multi-subunit TCR is comprised of six distinct
type I transmembrane polypeptides that assemble in
the endoplasmic reticulum (ER)1 as an octameric complex.
On most T cells, these receptor subunits consist of clonotypic TCR-/
heterodimers in association with a set of
invariant subunits including heterodimers of CD3-
and -
,
and CD3-
and -
, and a TCR-
homodimer (1). In T
cells, cell surface expression of TCRs is dependent on the
proper assembly of complete TCRs in the ER, which then
traverse the secretory pathway to arrive at the plasma membrane (1). Partial receptors lacking only
homodimers also
assemble and leave the ER. However, these are largely degraded in lysosomes (2). Other partial receptors and unassembled subunits, except for
, are retained in the ER from
where they are degraded with varying efficiencies by
poorly understood mechanisms that have been referred to
as "ER degradation"(3, 4). ER degradation is believed to
play a particularly prominent role in immature (CD4+
CD8+) thymocytes undergoing selection in the thymus.
These cells express only 10% of the number of cell surface
TCRs as mature thymocytes despite adequate synthesis of
all TCR subunits. This low TCR expression occurs as a
consequence of as yet undefined posttranslational mechanisms that include a relative block in egress of TCRs from the ER and increased degradation of TCR components
from this organelle (5, 6).
Among the TCR subunits, TCR- and CD3-
are particularly susceptible to degradation from the ER, whereas
CD3-
and CD3-
generally exhibit considerably longer
half-lives (3, 4, 7). The molecular basis for the selectivity of
targeting for degradation among receptor subunits is largely
unknown; however, it has been shown that TCR-
is
uniquely unstable as a transmembrane protein due to the
presence of two basic amino acids within its transmembrane domain (8, 9).
The modification of proteins with chains of ubiquitin is
well-established as an important and regulated means of
disposing of cytosolic and nuclear proteins by targeting for
degradation in 26S proteasomes (10, 11). Recently, however, there have been several reports implicating proteasomes in the degradation of transmembrane and soluble
yeast (12) and mammalian (15) proteins that were
initially cotranslationally inserted into the ER. A well-studied example is that of MHC class I heavy chains, which,
like TCR subunits, are type 1 transmembrane proteins
with a single transmembrane domain. Newly synthesized
MHC class I heavy chains undergo rapid proteasomal degradation in cells that express certain human cytomegalovirus gene products, are defective in peptide transport into
the ER, or lack expression of 2 microglobulin (19).
These MHC molecules are dislocated from the ER membrane to the cytosol with the concomitant total removal of
N-linked oligosaccharides by a cytosolic N-glycanase activity. Notably, this removal from the ER membrane occurs
independently of the catalytic activity of proteasomes.
These dislocated proteins are then degraded in the cytosol
in a proteasome-dependent manner, without evidence of
ubiquitinated intermediates. Based on these observations, as
well as others involving a mutant form of a soluble luminal
yeast protein (13), models have been proposed for the degradation of ER proteins in which the proteins are first dislocated into the cytosol from whence they are degraded by
proteasomes (20, 22).
ER forms of the multi-membrane spanning cystic fibrosis conductance regulator (CFTR) are also degraded by proteasomes, in this case with the generation of ubiquitinated intermediates (15, 16). It has been speculated that misfolded forms of this complex protein are recognized as such by enzymes of the ubiquitin-conjugating system and are therefore targeted for destruction. However, no information is available as to the subcellular location of ubiquitinated CFTRs, nor is it known whether CFTRs are dislocated from ER membranes before ubiquitination.
In this study, we have evaluated the degradation of murine CD3- and TCR-
from the ER in T lymphocytes,
which continually synthesize and degrade these subunits (3,
4, 7). CD3-
has a core molecular weight of 16 kD; the
addition of three N-linked oligosaccharide chains results in
its migration at ~26 kD by SDS-PAGE (25). This protein
has a luminal domain of ~79 amino acids and an intracytoplasmic domain of 46 amino acids that includes three lysines. The TCR-
subunit has four sites of N-glycosylation and, in most T cells, forms disulfide-linked heterodimers with the TCR-
subunit after its cotranslational
membrane insertion. TCR-
has a small cytoplasmic domain of five amino acids, none of which are lysines. As previously mentioned, the transmembrane domain of TCR-
includes two basic amino acids (Arg and Lys) that may destabilize this molecule in the ER, perhaps functioning as an inefficient stop-transfer signal (8, 9). We now report that both CD3-
and TCR-
are ubiquitinated and are degraded from the ER in a proteasome-dependent manner and
that for CD3-
, removal from the ER requires the catalytic
activity of proteasomes and the activity of mannosidases.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells and Reagents.
Thymi from C57Bl/6 ×DBA/2 mice that had been interbred for 20 generations were provided by Astrid Eder (National Cancer Institute, Bethesda, MD). 21.2.2 cells (26) and BW5147 cells (27) were maintained in complete medium as previously described (26). COS-7 cells were maintained and transfected as previously described (28). Anti-CD3-
|
Experimental Techniques.
For "pulse-chase" metabolic labeling experiments, cells were incubated at 37°C for 20 min in methionine-free medium before labeling with Tran35S-label (ICN Radiochemicals, Costa Mesa, CA) at 1 mCi/ml (thymocytes) or 300 µCi/ml (cell lines). After labeling, cells were washed and resuspended in complete medium with the indicated additions. Cells were lysed and immunoprecipitates were washed in buffers containing Triton X-100 as previously described (33), except that 0.1% SDS was added to the wash buffer in all immunoprecipitates except those with 2C11, and 0.1% SDS was added to the lysis buffer in experiments in which samples were immunoprecipitated with anti-TCR-
|
|
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine if proteasomes play a role in degradation of
CD3-, unfractionated thymocytes were subject to pulse-chase metabolic labeling followed by immunoprecipitation
with an anti-peptide rabbit polyclonal antibody that recognizes the cytoplasmic domain of CD3-
(anti-CD3-
; reference 29). Newly synthesized CD3-
was found to be
rapidly degraded (Fig. 1 A); however, a peptide-aldehyde that inhibits proteasome function, LLnL (35), blocked its
degradation, whereas an inhibitor of lysosome function,
NH4Cl, was significantly less effective. A second potent
peptide-aldehyde proteasome inhibitor, MG132 (36), as
well as LCN, a chemically unrelated and highly specific
proteasome inhibitor (37), both also markedly inhibited loss of CD3-
(Fig. 1 B).
To establish whether CD3- undergoes ubiquitination
in thymocytes, anti-CD3-
immunoprecipitates were immunoblotted with anti-Ub antibodies capable of detecting
low levels of multi-ubiquitinated proteins (reference 30,
Fig. 1 C). Consistent with CD3-
ubiquitination, in cells
treated with the proteasome inhibitor LLnL discrete species as well as a higher molecular weight "smear" was easily detected in anti-CD3-
immunoprecipitates but not in immunoprecipitates with control antiserum (CA). These were
not detected in cells incubated without LLnL, indicating
that these species are normally efficiently degraded by proteasomes.
To further assess proteasomal degradation of CD3- we
turned to 21.2.2 cells (26). This cell line is a variant of the
T cell hybridoma 2B4 (38) distinguished by its failure to
express any form of TCR-
. This cell line is also deficient
in expression of the 2B4-specific TCR-
chain, but it does
express TCR-
chains derived from BW5147, the fusion
partner used in the generation of 2B4 (27). Because 21.2.2 cells lack TCR-
, TCRs do not assemble, and CD3-
remains in the ER from whence it is degraded (33). When
pulse-chase metabolic labeling was carried out on these
cells, LLnL and MG132 each inhibited degradation of
newly synthesized CD3-
, while a closely related peptide-
aldehyde that is ineffective in blocking proteasome function, LLM (35), did not substantively affect CD3-
loss
(Fig. 2 A). As expected for this cell line, NH4Cl failed to
block CD3-
loss (Fig. 2 A), and LCN was as effective as
LLnL and MG132 (data not shown).
As with thymocytes, inhibition of proteasome function
also resulted in an accumulation of specifically immunoprecipitated ubiquitinated species in 21.2.2 (Fig. 2 B). In Fig. 2
B immunoprecipitates were washed under conditions that
minimize coimmunoprecipitation of other proteins with
CD3-. To verify that CD3-
itself was being ubiquitinated, anti-CD3-
immunoprecipitates from LLnL-treated cells were denatured in SDS-PAGE sample buffer and then
reimmunoprecipitated (Fig. 2 C). Only the sample specifically reimmunoprecipitated with anti-CD3-
recovered
ubiquitinated material.
Accompanying the proteasome-dependent loss of CD3- (Figs. 1
and 2) is a drop in molecular weight suggestive of the posttranslational action of mannosidases, which are present
both in the ER and cis-Golgi. Mannosidases are responsible
for the trimming of N-linked oligosaccharides after the
rapid cleavage of the three glucose residues from the cotranslationally added Glc3Man9GlcNAc2 structure (Glc, glucose; Man, mannose; GlcNAc, N-acetyl glucosamine; reference
39). The resultant Man5GlcNAc2 may be further processed
to generate complex carbohydrates in the medial- and trans-
Golgi. To ascertain whether the change in migration is due
to mannose trimming, cells were treated with dMNJ, a
specific inhibitor of ER and Golgi mannosidases. As is evident, the change in migration of CD3-
during the chase was largely abrogated by this treatment (Fig. 3 A). Strikingly, however, dMNJ also markedly inhibited the loss of
CD3-
during the chase. This effect was specific for inhibition of mannosidases, as a closely related compound, dNJ,
which inhibits glucosidases but not mannosidases, affected
the migration of CD3-
but was ineffective in altering its
fate (Fig. 3, B and C). Results similar to those obtained
with dNJ were obtained with another glucosidase inhibitor, castanospermine (data not shown). As is evident (Fig. 3, B and C), the ability of dMNJ to inhibit loss of CD3-
was similar in magnitude to effects seen with LLnL; and exposure of cells to the two together was not additive (data
not shown). This suggests that the proteasome inhibitor
and mannosidase inhibitor are functioning on the same
population of CD3-
molecules. Moreover, the half-life
CD3-
was prolonged by either LLnL or dMNJ when this
TCR subunit was expressed transiently in COS-7 cells
(data not shown), indicating that the effects seen do not require other T cell-specific proteins.
|
Subcellular fractionation
was carried out to determine whether the CD3- that accumulates when proteasome function is inhibited is membrane-bound or cytosolic. After a 20 min labeling with
[35S]methionine and a chase in medium without [35S]methionine (Fig. 4 A, lanes 1-4), cells were broken by mechanical shearing without detergent and fractionated into
residual unbroken cells and nuclei (U), membranes (M), and
cytosol (C) (Fig. 4 A, lanes 5-10), and immunoprecipitated
with anti-CD3-
. CD3-
was not found in the cytosolic
fractions of cells. Instead, CD3-
localized to the membrane fraction, with LLnL-treated cells exhibiting greater
levels of membrane-associated CD3-
, commensurate with the increased amount of CD3-
present at the end of the
chase (Fig. 4 A, lanes 1-4). To verify that CD3-
, if
present, could be immunoprecipitated from the cytosolic
fraction by anti-CD3-
, CD3-
that had been translated in
vitro in rabbit reticulocyte lysate without microsomes was
mixed with the cytosolic fraction. As shown (Fig. 4 B), this
exogenously added material could be specifically immunoprecipitated from the cytosolic fraction with anti-CD3-
.
CD3- is the only nonglycoprotein of the three CD3
subunits (1). At an early stage in the assembly of the octomeric TCR complex, CD3-
and CD3-
each dimerize
with CD3-
through their luminal domains. Dimerization
with CD3-
or CD3-
allows heterodimers containing
CD3-
to be immunoprecipitated by 2C11, a monoclonal antibody that recognizes a conformation-dependent luminal
epitope on CD3-
(7, 31). The association of CD3-
with
CD3-
and their coimmunoprecipitation with 2C11 was
used to further assess the localization of CD3-
. As is evident in both LLnL-treated and untreated samples (Fig. 4
C), the large majority of membrane-bound CD3-
is immunoprecipitated with 2C11. This demonstrates that CD3-
salvaged from degradation is largely bound to CD3-
.
The results presented in Fig. 4, A and C strongly suggest
that, when its degradation is inhibited, CD3- accumulates
in ER membranes in a native configuration. To establish
the topological orientation of CD3-
with certainty, protease-protection studies were carried out. In these experiments 21.2.2 cells were labeled with [35S]methionine, and
after a 3-h chase cells were broken by mechanical shearing
and nuclei and residual unbroken cells were removed. The
resultant postnuclear supernatant, consisting of cytosol and membrane-bound organelles, was subject to proteinase K
digestion. Such treatment will degrade portions of proteins
that are exposed to the cytosol, but leave protected transmembrane and luminal domains intact. Since the epitope
recognized by anti-CD3-
is cytosolic and, as expected,
lost on treatment with proteinase K (data not shown), we
took advantage of the luminal epitope recognized by 2C11
to immunoprecipitate residual CD3-
after proteinase K digestion (Fig. 4, D and E). As predicted for CD3-
in its
native topological membrane orientation, its ~4-kD cytosolic domain was degraded by proteinase K, while the luminal and transmembrane domains of CD3-
were largely
protected. As expected, solubilization of membranes with
Triton X-100 before exposure to proteinase K abrogated
this protection. Importantly, increased levels of CD3-
were protected in cells in which CD3-
degradation was
prevented by inhibition of either proteasome function (Fig.
4 D) or mannosidase activity (Fig. 4 E). Also, the amount
of material remaining after protease treatment (as compared
with nondigested samples) was similar in cells in which
proteasome or mannosidase activity was inhibited and in
untreated samples. This establishes that when either proteasome function or mannosidase activity is inhibited, the
CD3-
that remains is still membrane-embedded in its native topological orientation.
To determine the location of ubiquitinated CD3-, anti-Ub immunoblotting of immunoprecipitates from fractionated cells was carried out (Fig. 4 F). Although some ubiquitinated CD3-
was found in the cytosolic (C) fraction
from LLnL-treated cells, the majority was membrane-bound (M; Fig. 4 F). Thus, it is apparent that ubiquitination of CD3-
takes place while still in the ER membrane.
TCR- chains are products of genes that
undergo somatic rearrangement. In most cells TCR-
forms disulfide-linked dimers with TCR-
, also a product
of a rearranged gene. The membrane proximal portion of
the intraluminal domain (extracellular domain) and the entire transmembrane and cytoplasmic segments of mouse
TCR-
are derived from a constant region that is invariant
among TCR-
's in different cells (40). The TCR-
transmembrane segment is remarkable in having two charged
amino acids (Lys and Arg), and its short cytoplasmic domain has no Lys residues (41). Recent studies in non-T
cells have provided evidence for proteasome-independent dislocation of TCR-
from the ER to the cytosol accompanied by removal of its N-linked sugars (42, 43), similar to
that observed for MHC class I (19), without evidence
of accompanying ubiquitination. To assess the degradation
of TCR-
we evaluated BW5147, the fusion partner used
in the generation of T cell hybridomas such as 2B4 (38).
This well-characterized thymoma expresses TCR-
and
TCR-
but not CD3-
(44). Using a monoclonal anti-
TCR-
antibody that recognizes a constant region intraluminal epitope, newly synthesized TCR-
was found to be
rapidly degraded (Fig. 5, A and B). LLnL markedly inhibited TCR-
loss, and this was accompanied by increased
density both above and below TCR-
(Fig. 5 A), perhaps representing ubiquitination and partial degradation, respectively (similar results were obtained using LCN; data not
shown). However, dMNJ had minimal effects on loss of
this receptor subunit (Fig. 5, A and B). To ascertain the extent to which TCR-
is disulfide-linked to TCR-
, samples
were resolved by two-dimensional nonreducing/reducing SDS-PAGE (Fig. 5 C). The large majority of TCR-
, including most that was salvaged by LLnL, was found not to
be disulfide-linked to TCR-
. The findings obtained in
BW5147 cells were verified in 21.2.2 cells (Fig. 6 A), where
dMNJ inhibited degradation of CD3-
but not TCR-
. In
21.2.2 and BW5147 a minor band above the predominant
form of TCR-
is often distinguished, which may reflect expression from a second BW-specific TCR-
allele in these
cells (45). Inhibition of TCR-
degradation was accompanied
by the appearance of lower molecular species of 14-30 kD
(Fig. 6, A and B), which unlike full-length TCR-
, were
unaffected by treatment with N-glycanase (Fig. 6 B) and
which were immunoreactive with anti-TCR-
by immunoblotting (data not shown). These therefore likely represent incompletely proteolyzed deglycosylated TCR-
.
|
|
The location of TCR- in T cells was assessed by separation of membrane and cytosolic fractions. In BW5147 as
well as 21.2.2 cells, the large majority of TCR-
that accumulated was found in the membrane fraction (Fig. 7, A and
B). In some samples treated with LLnL, more rapidly migrating forms, consistent with deglycosylation, are disproportionately represented in the cytosolic fraction. The topological orientation of TCR-
was assessed by proteinase K digestion of cells that had been broken open by mechanical shearing (Fig. 8). In contrast to CD3-
, where a similar
proportion of material was protected in samples treated
with or without proteasome inhibitors, a substantially
smaller percentage of the TCR-
was protected in samples
in which proteasome function was inhibited as compared
with samples not exposed to proteasome inhibitors (Fig.
8 A). Strikingly, loss of full length TCR-
was accompanied by the appearance of a discrete lower molecular
weight immunoreactive band of ~23 kD (Fig. 8 A). That
this is a fragment of TCR-
and not an associated protein
was established by heating of immunoprecipitates to 95°C
in SDS sample buffer under reducing conditions and reimmunoprecipitation with anti-TCR-
(Fig. 8 C). As is evident, this 23-kD band was recovered with the same efficiency as full length TCR-
. A similar fragment was
visualized when broken cells were digested with trypsin instead of proteinase K (data not shown). Since the 23-kD
fragment drops from the predicted molecular weight of
glycosylated TCR-
, it is likely to also be glycosylated. In
fact, treatment with N-glycanase resulted in a drop in molecular weight consistent with at least one N-linked oligosaccharide (data not shown).
To assess whether this partially protected form of TCR-
represents material that never fully traversed the ER membrane, or if this reflects ongoing removal of TCR-
that
had been initially fully translocated into the ER, TCR-
was labeled in the absence of LLnL and chased in its presence or absence. As can be seen in Fig. 8 D, a small amount
of this partially protected TCR-
was present at the end of
the pulse (lane 2); however, the amount detected clearly
increased when LLnL was present during the chase. This
indicates that TCR-
that had fully translocated across the ER membrane undergoes partial retrotranslocation, resulting in the retention of a discrete form of TCR-
within
the ER lumen/membrane. This partial retrotranslocation
occurs independent of the activity of proteasomes. However, to complete the removal/degradation of TCR-
from the ER membrane, as is the case with CD3-
, proteasome activity is required.
The smearing seen above
TCR- in lanes from LLnL-treated cells in Figs. 5 and 6 is
suggestive of ubiquitination. However, recently published
studies in which TCR-
was expressed in non-T cells
failed to reveal evidence for this modification (42, 43). To
assess whether ubiquitinated forms of TCR-
were being
generated in T cells, anti-Ub immunoblotting was carried
out on 21.2.2 cells. Similar to CD3-
, specifically immunoprecipitated ubiquitinated species were, in fact, detected
in the presence of the proteasome inhibitor LLnL (Fig. 9
A). These species were detected both in the cytosolic and
membrane compartments. Confirmation that these represent ubiquitinated TCR-
was obtained by dissociation of samples in SDS and reimmunoprecipitation with anti-
TCR-
(data not shown). Since TCR-
, unlike CD3-
,
has no lysine residues that are normally exposed to the cytosol (25, 41), ubiquitination of this subunit is either occurring on residues that have become exposed to the cytosol
after the initiation of retrograde movement or is taking
place on luminal residues. However, as there is no evidence
for luminal ubiquitin-conjugating enzymes, the latter possibility seems unlikely. To evaluate this, anti-Ub immunoblotting was carried out on samples that had been digested
with proteinase K. As is evident in Fig. 9 B, ubiquitin attached to TCR-
was largely removed with proteinase K,
indicating that the lysines to which ubiquitin has been attached on TCR-
are in the cytosol. It is therefore apparent that, for TCR-
, initiation of retrograde movement
out of the ER precedes ubiquitination.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent findings have led to the emergence of models for degradation of transmembrane and soluble luminal proteins from the ER where fates are largely decided by proteasome-independent retrotranslocation from the ER to the cytosol through protein conducting channels followed by proteasomal degradation (20, 22). For MHC class I heavy chains (20) and for a mutant form of a yeast luminal protein, carboxypeptidase y (46), it has been proposed that newly synthesized proteins associate with the Sec61 translocon complex which then facilitates their movement back into the cytosol. In the case of retrotranslocation of MHC class I heavy chains, this is accompanied by complete removal of N-linked oligosaccharides, without evidence of ubiquitinated intermediates (19).
This study reveals that, in T cells, components of the
TCR are also degraded from the ER as a consequence of
pathways that terminate in proteasomes. Our evaluation of
CD3- and TCR-
illuminates novel aspects of these degradative pathways and underscores that, although cytosolic
proteasomes are likely a final destination for a large number
of proteins, the mechanisms involved in targeting for proteasomal degradation vary significantly. Strikingly, even between two normal components of the TCR, the route and
the determining factors leading to degradation differ. TCR-
undergoes partial retrograde movement regardless of proteasome activity, with a 23-kD glycosylated fragment remaining sequestered within the ER when proteasome function is inhibited. The partial retrotranslocation of TCR-
in the absence of proteasome function should be compared with results obtained with MHC class I and more recent
studies in which TCR-
was overexpressed ectopically in
nonlymphoid cells (42, 43). In these instances complete removal from the ER membrane occurred even when proteasome function was inhibited. Further distinguishing our
findings from the aforementioned examples is the existence
of ubiquitinated membrane-associated TCR-
. In contrast
to TCR-
, the large majority of CD3-
retains its native transmembrane location when proteasome function is inhibited. Thus, proteasome function is required both for detectable retrograde movement and destruction of this TCR
subunit. Moreover, it is evident that there is a requirement
for cellular mannosidase activity in proteasome-dependent
CD3-
degradation. As with TCR-
, CD3-
also undergoes ubiquitination while still associated with the ER
membrane.
When assessing differences between TCR- and CD3-
that might account for their relative susceptibilities to proteasome-independent retrotranslocation, the nature of their
transmembrane segments must be considered. CD3-
has a
single acidic residue within its transmembrane domain, a
feature common to all of the invariant TCR components
(1). TCR-
, on the other hand, has two bulky basic amino
acids that have been postulated to contribute to its inherent
instability as a transmembrane protein (8, 9). Because of
this, it is reasonable to speculate that the relatively hydrophilic nature of TCR-
's transmembrane domain has the
potential to facilitate movement into and through protein-conducting channels that mediate retrograde movement.
Whether or not this is a major factor in determining susceptibility to retrograde movement will require further
evaluation.
Branch chain N-linked oligosaccharides are cotranslationally added to luminal asparagine residues of proteins as
Glc3Man9GlcNAc2 (39). The three terminal Glc residues
are generally rapidly cleaved in the ER, and ER and cis-Golgi mannosidases carry out trimming of these "high
mannose" chains. This trimming involves removal of four
of the mannose residues generating Man5GlcNAc2 before
further processing in the Golgi complex to "hybrid" and "complex" chains (39). For a number of proteins, oligosaccharide processing plays important roles in ER "quality
control". This has been analyzed predominantly with regard to removal of glucose residues in calnexin- and calreticulin-mediated folding in the ER (47). Previous evidence
of a role for mannosidase activity in determining the fate of
proteins in the ER was limited to a study in which yeast
pre-pro- factor was expressed ectopically in mammalian
cells (48), and more recent work on mutant
1-antitrypsin (49). Both pre-pro-
factor and
1-antitrypsin are luminal
proteins without membrane anchors. Our results establish a
previously unappreciated role for mannosidase activity in
targeting a normal transmembrane protein, CD3-
, for
proteasomal degradation. They also demonstrate that mannosidases and proteasomes function along the same pathway leading to destruction of this receptor subunit. When
21.2.2 cells were treated with tunicamycin, which blocks
the addition of N-linked oligosaccharides to proteins, nonglycosylated CD3-
was rapidly degraded in a proteasome-dependent manner, and, in COS-7 cells, CD3-
in which
the three sites of N-glycosylation were mutated was degraded rapidly (our unpublished observations). This suggests that N-linked oligosaccharides in which mannose residues have not been trimmed stabilize CD3-
. This could
occur by enhancing the association of CD3-
with the
more stable CD3-
. Arguing against this possibility are the
findings that trimmed and untrimmed CD3-
both coimmunoprecipitate with anti-CD3-
, and that degradation of
CD3-
in COS-7 cells is also inhibited by dMNJ.
Man9GlcNAc2 species undergo cycles of reversible monoglucosylation (47). Since the carbohydrate-binding luminal
chaperones calnexin and calreticulin preferentially associate
with monoglucosylated oligosaccharides, interactions with
these proteins could stabilize CD3- in which mannose residues have not been trimmed. However, CD3-
does not
associate with calreticulin (50) and we and others have
found that the glycosylation state of CD3-
does not substantially affect its association with calnexin (51 and our unpublished observations). Although we can not exclude with
certainty that differences in the nature of calnexin interactions explain the protective effects of mannosidase inhibitors, we are left to consider that other, as yet to be characterized, carbohydrate-binding "chaperones" play a role in
stabilization of untrimmed CD3-
.
Ubiquitinated CD3- and TCR-
are easily detected when proteasome function is inhibited. Ubiquitinated CD3-
is primarily membrane-associated, whereas
ubiquitinated TCR-
is found to a similar extent in both
membranes and cytosol. A model integrating ubiquitination with retrograde movement is one in which ubiquitination is a requisite event preceding the initiation of retrograde movement, with this covalent modification serving
to facilitate association with proteasomes and subsequent
entry into protein conducting channels. However, there
are observations with both TCR-
and CD3-
that make
such a simple model untenable. First, TCR-
has no
lysines within its short cytoplasmic tail (41), necessitating cytoplasmic exposure of transmembrane and luminal lysines
to serve as sites of ubiquitination. Second, when all of the
cytoplasmic lysines of CD3-
were mutated and this protein was transiently expressed in non-T cells, TCR-
was
still subject to ubiquitination while associated with the ER
(Yang, Y., J.S. Bonifacino, and A.M. Weissman, unpublished observations). These findings strongly suggest that
for both TCR-
and for CD3-
initiation of retrograde
transport can precede ubiquitination.
Since the steady state level of ubiquitinated forms are
low relative to the total number of molecules subject to
degradation, and initiation of retrograde movement can
precede ubiquitination, what then is the significance of
ubiquitination in degradation from the ER? First, we point
out that in most instances, when proteasome function is inhibited, proteins that are known to be ubiquitinated accumulate primarily in non-ubiquitinated forms. This is due,
at least in part, to the ongoing activity of cellular deubiquitinating enzymes (11). As there are no cell permeant selective inhibitors of deubiquitinating enzymes, the percent of TCR subunits that exist transiently in a ubiquitinated
form can not be determined. Since multiubiquitin chains
are potent proteasome targeting signals (11), ubiquitination
is required for the destruction of some ER membrane proteins (12), and proteasomes are known to associate with the
ER (18, 52), we favor a model in which transmembrane proteins are predisposed for degradation from the ER
by either having never left, or reversibly entering, protein-conducting channels, such as the translocon. In this hydrophilic milieu they have the opportunity to undergo variable
degrees of movement across the membrane, enhancing
their susceptibility to ubiqutination. Ubiquitination leads to
association with 26S proteasomes, which facilitates complete retrograde movement and degradation. Among proteins, the relative requirements for ubiquitination and proteasomes in completing retrograde movement likely varies,
with human cytomegalovirus-enhanced loss of MHC class
I heavy chains at one end of the spectrum, and CD3- at
the other. An alternative model that warrants consideration
is that proteasomes are drawn to translocons by ubiquitinated chaperone-like translocon-associated proteins. This
facilitates the removal/degradation of target proteins that
are susceptible to retrotranslocation. For TCR subunits
there is no need to postulate such an indirect mechanism,
since TCR-
and CD3-
are both ubiquitinated. However, there is at least one in vitro situation where ubiquitination of calnexin has been suggested to facilitate the degradation of a mutant form of a secreted protein (
1-antitrypsin; reference 17).
When considering the enzymes responsible for the ubiquitination of ER membrane proteins it is of note that two yeast ubiquitin conjugating enzymes (E2s), UBC6 and UBC7, have been implicated in the ubiquitination/degradation of proteins from the ER (12, 55). One of these, UBC6, has its catalytic domain localized to the cytoplasmic face of the ER membrane through a hydrophobic COOH-terminal anchor (28, 55). Interestingly, both of these E2s are implicated in the ubiquitin-mediated degradation of mutant forms of the Sec61 translocon complex (12), and we now have evidence for the existence of an enzymatically active mammalian UBC6 homolog (Tiwari, S., and A.M. Weissman, unpublished observations).
Conclusion.Eukaryotic cells have evolved sophisticated
mechanisms to degrade both transmembrane and soluble
proteins of the secretory pathway that are either made in
excess or that are abnormally folded. It is apparent that
even among subunits of the TCR, multiple pathways exist
that ultimately lead to their breakdown by cytosolic proteasomes. Degradation of CD3- from the ER entails trimming of mannose residues from N-linked oligosaccharides,
and recognition and proteolysis by proteasomes with concomitant removal from ER membranes. For TCR-
there
is no evidence that mannosidase activity plays a role, and a
significant degree of exposure of this protein to the cytosol
occurs regardless of proteasome function. Although light is
now being cast on what has been, until recently, a mysterious process, much remains to be learned regarding the molecular details of the multiple pathways leading to degradation from the ER.
![]() |
Footnotes |
---|
Address correspondence to Allan M. Weissman, Bldg. 10, Rm. 1B34, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1152. Phone: 301-496-3557; Fax: 301-402-4844; E-mail: amw{at}nih.gov
Received for publication 17 June 1997 and in revised form 24 November 1997.
1Abbreviations used in this paper: anti-Ub, anti-ubiquitin; CA, control antiserum; CFTR, cystic fibrosis conductance regulator; CHX, cycloheximide; dMNJ, deoxymannojirimycin; dMJ, deoxynojirimycin; ER, endoplasmic reticulum; Glc, glucose; GlcNAc, N-acetyl glucosamine; LCN, lactacystin; LLM, LLnL, N-acetyl-Leu-Leu-methioninal; N-acetyl-Leu-Leu-norleucinal; Man, mannose; Mg132, Z-Leu-Leu-Leu CHO.We thank Kelly Kearse, Jennifer Lippincott-Schwartz, and Jocelyn Weissman for their comments on this manuscript and Jonathan Ashwell and Richard Klausner for helpful discussions. We thank Proscript for MG132, and Astrid Eder for mouse thymi.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Weissman, A.M.. 1994. The T-cell antigen receptor: a multisubunit signaling complex. Chem. Immunol. 59: 1-18 [Medline]. |
2. |
Sussman, J.J.,
J.S. Bonifacino,
J. Lippincott-Schwartz,
A.M. Weissman,
T. Saito,
R.D. Klausner, and
J.D. Ashwell.
1988.
Failure to synthesize the T cell CD3-![]() |
3. | Klausner, R.D., J. Lippincott-Schwartz, and J.S. Bonifacino. 1990. The T cell antigen receptor: insights into organelle biology. Annu. Rev. Cell. Biol. 6: 403-431 . |
4. | Bonifacino, J.S., and J. Lippincott-Schwartz. 1991. Degradation of proteins within the endoplasmic reticulum. Curr. Opin. Cell. Biol. 3: 592-600 [Medline]. |
5. | Bonifacino, J.S., S.A. McCarthy, J.E. Maguire, T. Nakayama, D.S. Singer, R.D. Klausner, and A. Singer. 1990. Novel post-translational regulation of TCR expression in CD4+ CD8+ thymocytes influenced by CD4. Nature 344: 247-251 [Medline]. |
6. |
Kearse, K.P.,
J.L. Roberts,
T.I. Munitz,
D.L. Wiest,
T. Nakayama, and
A. Singer.
1994.
Developmental regulation of
![]() ![]() ![]() |
7. | Bonifacino, J.S., C.K. Suzuki, J. Lippincott-Schwartz, A.M. Weissman, and R.D. Klausner. 1989. Pre-Golgi degradation of newly synthesized T cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly. J. Cell Biol 109: 73-83 [Abstract]. |
8. | Bonifacino, J.S., C.K. Suzuki, and R.D. Klausner. 1990. A peptide sequence confers retention and degradation in the endoplasmic reticulum. Science 247: 79-82 [Medline]. |
9. |
Shin, J.,
S. Lee, and
J.L. Strominger.
1993.
Translocation of
TCR![]() |
10. | Coux, O., K. Tanaka, and A.L. Goldberg. 1996. Structure and function of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65: 801-847 [Medline]. |
11. | Weissman, A.M.. 1997. Regulating protein degradation by ubiquitination. Immunol. Today 18: 189-198 [Medline]. |
12. | Biederer, T., C. Volkwein, and T. Sommer. 1996. Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. EMBO (Eur. Mol. Biol. Organ.) J. 15: 2069-2076 [Abstract]. |
13. |
Hiller, M.M.,
A. Finger,
M. Schweiger, and
D.H. Wolf.
1996.
ER degradation of a misfolded luminal protein by the
cytosolic ubiquitin-proteasome pathway.
Science
273:
1725-1728
|
14. |
Werner, E.D.,
J.L. Brodsky, and
A.A. McCracken.
1996.
Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.
Proc. Natl. Acad. Sci. USA.
93:
13797-13801
|
15. |
Ward, C.L.,
S. ![]() |
16. | Jensen, T.J., M.A. Loo, S. Pind, D.B. Williams, A.L. Goldberg, and J.R. Riordan. 1995. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129-135 [Medline]. |
17. |
Qu, D.,
J.H. Teckman,
S. ![]() ![]() |
18. |
McGee, T.P.,
H.H. Cheng,
H. Kumagai,
S. ![]() |
19. | Wiertz, E.J., T.R. Jones, L. Sun, M. Bogyo, H.J. Geuze, and H.L. Ploegh. 1996. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84: 769-779 [Medline]. |
20. | Wiertz, E.J., D. Tortorella, M. Bogyo, J. Yu, W. Mothes, T.R. Jones, T.A. Rapoport, and H.L. Ploegh. 1996. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384: 432-438 [Medline]. |
21. |
Hughes, E.A.,
C. Hammond, and
P. Cresswell.
1997.
Misfolded major histocompatibility complex class I heavy chains
are translocated into the cytoplasm and degraded by the proteasome.
Proc. Natl. Acad. Sci. USA.
94:
1896-1901
|
22. | Lord, M.J.. 1996. Protein degradation: go outside and see the proteasome. Curr. Biol 6: 1067-1069 [Medline]. |
23. | Bonifacino, J.S.. 1996. Reversal of fortune for nascent proteins. Nature 384: 405-406 [Medline]. |
24. | Kopito, R.H.. 1997. ER quality control: the cytoplasmic connection. Cell 88: 427-430 [Medline]. |
25. | van den Elsen, P., B.-A. Shepley, M. Cho, and C. Terhorst. 1985. Isolation and characterization of a cDNA clone encoding the murine homolgue of the human 20K T3/T-cell receptor glycoprotein. Nature 314: 542-544 [Medline]. |
26. |
Sussman, J.J.,
T. Saito,
E.M. Shevach,
R.N. Germain, and
J.D. Ashwell.
1988.
Thy-1- and Ly-6-mediated lymphokine
production and growth inhibition of a T cell hybridoma require co-expression of the T cell antigen receptor complex.
J.
Immunol.
140:
2520-2526
|
27. | Hyman, R., and V. Stallings. 1974. Complementary patterns of Thy-1 variants and evidence that antigen loss variants "pre-exist" in the parental population. J. Natl. Cancer Inst. 52: 429-437 [Medline]. |
28. |
Yang, M.,
J. Ellenberg,
J.S. Bonifacino, and
A.M. Weissman.
1997.
The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic
reticulum.
J. Biol. Chem.
272:
1970-1975
|
29. |
Samelson, L.E.,
A.M. Weissman,
F.A. Robey,
I. Berkower, and
R.D. Klausner.
1986.
Characterization of an anti-peptide
antibody that recognizes the murine analogue of the human
T cell antigen receptor-T3 ![]() |
30. |
Cenciarelli, C.,
K.J. Wilhelm Jr.,
A. Guo, and
A.M. Weissman.
1996.
T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation.
J. Biol. Chem.
271:
8709-8713
|
31. | Leo, O., M. Foo, D.H. Sachs, L.E. Samelson, and J.A. Bluestone. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA. 84: 1374-1378 [Abstract]. |
32. |
Kubo, R.T.,
W. Born,
J.W. Kappler,
P. Marrack, and
M. Pigeon.
1989.
Characterization of a monoclonal antibody
which detects all murine ![]() ![]() |
33. | Chen, C., J.S. Bonifacino, L.C. Yuan, and R.D. Klausner. 1988. Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J. Cell Biol 107: 2149-2161 [Abstract]. |
34. | Cenciarelli, C., D. Hou, K.-C. Hsu, B.L. Rellahan, D.L. Wiest, H.T. Smith, V.A. Fried, and A.M. Weissman. 1992. Activation-induced ubiquitination of the T cell antigen receptor. Science 257: 795-797 [Medline]. |
35. | Rock, K.L., C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, and A.L. Goldberg. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761-771 [Medline]. |
36. |
Chen, Z.,
J. Hagler,
V.J. Palombella,
F. Melandri,
D. Scherer,
D. Ballard, and
T. Maniatis.
1995.
Signal-induced site-specific phosphorylation targets I![]() ![]() |
37. |
Fenteany, G.,
R.F. Standaert,
G.A. Reichard,
E.J. Corey, and
S.L. Schreiber.
1994.
A ![]() |
38. | Hedrick, S.M., L.A. Matis, T.T. Hecht, L.E. Samelson, D.L. Longo, E. Heber, Katz, and R.H. Schwartz. 1982. The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c. Cell 30: 141-152 [Medline]. |
39. |
Elbein, A.D..
1991.
Glycosidase inhibitors: inhibitors of N-linked
oligosaccharide processing.
FASEB (Fed. Am. Soc. Exp. Biol.)
J.
5:
3055-3063
|
40. | Ashwell, J.D., and A.M. Weissman. 1995. T Cell Antigen Receptor Genes, Gene Products and Co-receptors. R.R. Rich, T.A. Fleisher, B.D. Schwartz, W.T. Shearer, and W. Strober, editors. Mosby-Year Book, Inc., St. Louis, MO. 69-93. |
41. | Chien, Y., D.M. Becker, T. Lindsten, M. Okamura, D.I. Cohen, and M.M. Davis. 1984. A third type of murine T-cell receptor gene. Nature 312: 31-35 [Medline]. |
42. |
Huppa, J.B., and
H.L. Ploegh.
1997.
The ![]() |
43. |
Yu, H.,
G. Kaung,
S. Kobayashi, and
R.R. Kopito.
1997.
Cytosolic degradation of T-cell receptor ![]() |
44. | Lippincott-Schwartz, J., J.S. Bonifacino, L.C. Yuan, and R.D. Klausner. 1988. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell 54: 209-220 [Medline]. |
45. |
Kearse, K.P.,
D.B. Williams, and
A. Singer.
1994.
Persistence of glucose residues on core oligosaccharides prevents association of TCR-![]() ![]() ![]() |
46. | Plemper, R.K., S. Bohmler, J. Bordallo, T. Sommer, and D.H. Wolf. 1997. Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature 388: 891-895 [Medline]. |
47. | Hammond, C., and A. Helenius. 1995. Quality control in the secretory pathway. Curr. Opin. Cell. Biol. 7: 523-529 [Medline]. |
48. |
Su, K.,
T. Stoller,
J. Rocco,
J. Zemsky, and
R. Green.
1993.
Pre-Golgi degradation of yeast prepro-![]() |
49. |
Liu, Y.,
P. Choudhury,
C.M. Cabral, and
R.N. Sifers.
1997.
Intracellular disposal of incompletely folded human ![]() |
50. |
van Leeuwen, J.E.M., and
K.P. Kearse.
1996.
The related
molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum.
J. Biol. Chem.
271:
25345-25349
|
51. |
van Leeuwen, J.E.M., and
K.P. Kearse.
1996.
Calnexin associates exclusively with individual CD3 delta and T cell antigen receptor (TCR) alpha proteins containing incompletely
trimmed glycans that are not assembled into multisubunit
TCR complexes.
J. Biol. Chem.
271:
9660-9665
|
52. | Palmer, A., A.J. Rivett, S. Thomson, K.B. Hendil, G.W. Butcher, G. Fuertes, and E. Knecht. 1996. Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol. Biochem. J 316: 401-407 [Medline]. |
53. |
Rivett, A.J.,
A. Palmer, and
E. Knecht.
1992.
Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.
J. Histochem. Cytochem
40:
1165-1172
|
54. |
Yang, Y.,
K. Fruh,
K. Ahn, and
P.A. Peterson.
1995.
In vivo
assembly of the proteasomal complexes, implications for antigen processing.
J. Biol. Chem.
270:
27687-27694
|
55. | Sommer, T., and S. Jentsch. 1993. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365: 176-179 [Medline]. |