From the Department of Molecular Pathology and
Medicine, DIBIT-San Raffaele Scientific Institute, 20132 Milan, Italy
and § Università Vita-Salute San Raffaele, 20132 Milan, Italy
Received for publication, October 20, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Quality control in the endoplasmic
reticulum must discriminate nascent proteins in their folding
process from terminally unfolded molecules, selectively degrading the
latter. Unassembled Ig-µ and J chains, two glycoproteins with five
N-linked glycans and one N-linked glycan,
respectively, are degraded by cytosolic proteasomes after a lag from
synthesis, during which glycan trimming occurs. Inhibitors of
mannosidase I (kifunensine), but not of mannosidase II (swainsonine),
prevent the degradation of µ chains. Kifunensine also inhibits J
chain dislocation and degradation, without inhibiting secretion of IgM
polymers. In contrast, glucosidase inhibitors do not significantly
affect the kinetics of µ and J degradation. These results suggest
that removal of the terminal mannose from the central branch acts as a
timer in dictating the degradation of transport-incompetent,
glycosylated Ig subunits in a calnexin-independent way. Kifunensine
does not inhibit the degradation of an unglycosylated substrate ( The endoplasmic reticulum
(ER)1 is the port of entry
and the main folding compartment for proteins destined to the exocytic pathway. A stringent quality control system is coupled to the folding
machinery, ensuring that only structurally mature proteins reach the
Golgi (1). Proteins that fail to attain their correct three-dimensional
structure are retained in the ER and eventually degraded after a lag
that varies among individual substrates (2, 3).
Cytosolic proteasomes are responsible for the degradation of many
membrane and soluble ER-synthesized proteins (4-14), implying that
proteins targeted for degradation must be delivered to the cytosol
(15). Sec61, a component of the translocon complex that mediates the
entry of proteins into the ER (16), seems to be involved also in the
retrograde translocation, or dislocation, to the cytosol (17, 18). This
observation raises intriguing questions on the mechanisms that gate
Sec61 for dislocation and determine the directionality of transport
across the ER membrane (19-22).
ER quality control prevents the deployment of potentially harmful
molecules to their final destinations and maintains homeostasis within
the ER. Somehow, the system must be capable of discriminating terminally unfolded and/or unassembled molecules from newly
translocated polypeptides, which have not had the time to complete
folding. Therefore, the processes of retention and degradation must not only be specific but also be precisely timed.
The oligosaccharide moieties present on many ER-synthesized proteins
have been shown to play a crucial role in the quality control (see Ref.
1 for a comprehensive review). A branched oligosaccharide consisting of
three glucoses, nine mannoses, and two N-acetylglucosamines
(Glc3Man9GlcNAc2) is added to the
nascent polypeptide when an appropriate sequence is recognized by
oligosaccharyl-transferase (Fig. 1).
Immediately after synthesis, the terminal glucoses are removed by two
glucosidases (I and II) localized in the ER. Another ER-resident
enzyme, UDP-glucose: glycoprotein glucosyltransferase, is
capable of adding a glucose residue to the A branch of the glycans
present on unfolded molecules, favoring their binding to calnexin or
calreticulin. These two ER chaperones bind monoglucosylated proteins
and retain them in the folding-promoting ER environment. ER
mannosidases I and II remove the terminal mannoses from the B and C
branches, respectively. It is thought that glycans lacking the terminal
mannoses are poorer substrates of glucosidase II and UDP-glucose:
glycoprotein glucosyltransferase, thereby bringing the calnexin cycle
to an end (1, 23). Hence, it is possible that the different kinetics of
the ER sugar-processing enzymes provide a molecular mechanism to time
retention and dislocation/degradation. Evidence for a role of mannose
trimming in diverting misfolded glycoproteins to proteasomal
degradation has been produced in yeast (24) and in mammalian cells
(25-29).
Ig
light chains) or of chimeric µ chains extended with the
transmembrane region of the
T cell receptor chain, implying the existence of additional pathways for extracting
proteins from the endoplasmic reticulum lumen for proteasomal degradation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Processing of N-glycans
in vivo and in vitro. A branched
glycan consisting of two N-acetylglucosamines
(squares), nine mannoses (open circles), and
three glucoses (striped or dotted circles) can be
added to the nascent polypeptide when an asparagine (Asn) is followed
by the sequence Aaa-(Ser/Thr), where Aaa is any amino acid but
proline (59). Soon after translation, glucosidases (Glu) I
and II remove the glucoses, whereas UDP-glucose: glycoprotein
glucosyltransferase (UGT) can add a single glucose
(striped circle) to the terminal mannose of the A branch,
yielding a monoglucosylated glycan. ER mannosidases (ER Man)
I and II remove the terminal mannoses from the B and C branches,
respectively. Castanospermine and dN can be used to inhibit
glucosidases, whereas kifunensine and swainsonine block the activity of
ER mannosidase I and ER mannosidase II, respectively. dM inhibits both
ER mannosidases (1, 43 and references therein). Jack bean
-mannosidase (
-Man) can be used in vitro to
discriminate between glycans carrying or not carrying a glucose on the
A branch (46). Only in the absence of the glucose (G0),
Man cleaves the mannoses of the A branch.
To investigate how ER-synthesized proteins are diverted from retention
into the degradative pathway, we compared the fate of Ig µ, J, and
chains in assembly-deficient myeloma transfectants. These Ig
subunits fold primarily under the assistance of BiP (30-34). Whereas µ and J carry five N-linked glycans and one
N-linked glycan, respectively,
are not glycosylated.
Evidence is presented indicating that the trimming of the B branch
terminal mannose by ER mannosidase I times the dislocation of µ and J
chains from the ER lumen to the cytosol for proteasomal degradation,
without the involvement of calnexin and/or calreticulin.
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EXPERIMENTAL PROCEDURES |
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Cell Lines--
NSO, a myeloma cell line that produces
endogenous J chains but no heavy or light chains, and the transfectants
expressing wild type secretory (µs) or membrane (µm) µ chains
(N[µ1] and N[µm], respectively) or a mutant chain
(N[
Gly-214]), were described previously (34-36).
N[µ-TCR
TM], N[
2b-TCR
TM] were generated by
stably transfecting NSO with chimeric µ or
2b heavy chains extended at their C termini with the transmembrane region of the T cell
receptor
chain (37).
Antibodies and Reagents--
A rabbit anti-mouse J chain
antiserum (38) was kindly provided by Dr. R. M. E. Parkhouse
(Pirbright Laboratory, Surrey, UK). Horseradish peroxidase-conjugated
goat anti-mouse µ, goat anti-mouse Ig, and goat anti-rabbit Ig were
from Southern Biotechnology Associates Inc. (Birmingham, AL). Purified
rabbit anti-µ and anti- antibodies were obtained from
Zymed Laboratories Inc. (San Francisco, CA).
Dithiothreitol, leupeptin, N-ethylmaleimide, thapsigargin, and Triton X-100 were purchased from Sigma; the protease
inhibitors Complete and endoglycosidase H were from Roche Molecular
Biochemicals. The proteasome inhibitor
carboxybenzyl-leucyl-leucyl-leucinal (ZL3H) (39, 40), was a kind gift
of Drs. M. Bogyo and H. Ploegh (Harvard University, Cambridge, MA).
Phenylarsine oxide (Sigma) was used at 20-50 µM
(26).
Castanospermine and deoxynojirimycin (dN) were purchased from Sigma and used at the final concentration of 1 mM to inhibit glucosidases I and II. Kifunensine and swainsonine were purchased from Toronto Research Chemicals (Toronto, Canada), dissolved in water, and used at a final concentration of 2 µg/ml and 100 µM, respectively, to selectively block mannosidase I and mannosidase II, respectively (41-44). 1-deoxymannojirimicin (dM, Sigma) was used at 1 mM as an inhibitor of ER mannosidases I and II. The effects of kifunensine and swainsonine on the degradation and electrophoretic mobility of µ and J chains were similar when the drugs were added either before or immediately after the pulse.2 Therefore, to simplify the experimental procedures and to reduce variability in incorporation, cells were pulse-labeled in bulk and then aliquoted and chased with the different drugs.
Pulse-Chase Assays, Immunoprecipitation, and SDS-PAGE-- Pulse-chase assays were performed as previously described (21). In the experiment shown in Fig. 7, cells were chased in Krebs-Ringer Hepes medium (125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5.5 mM glucose, 25 mM Hepes, 0.5 mM MgCl2, 2% fetal calf serum, pH 7) supplemented with different concentrations of CaCl2 (3 µM-1 mM). Ionomycin was added at the final concentration of 1 µM. Films were scanned, and relevant bands were quantitated by ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Glucosylation Assay--
Following extensive washings,
immunoprecipitates on protein A beads were incubated overnight at
37 °C with jack bean -mannosidase (Sigma) in 20 µl of 50 µM sodium citrate, pH 4.4 before boiling in sample buffer
and electrophoresis.
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RESULTS |
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Glycan Trimming Determines Diversion of µ and J Chains into
the Proteasomal Degradation Pathway--
In the absence of chains,
µs and J chains cannot assemble into transport-competent IgM polymers
and are degraded by cytosolic proteasomes (21). When cells were
pulse-labeled for a short period (5 min), a lag was evident before
degradation ensued (Fig. 2, A
and C). During this period, both subunits undergo some
folding, as determined by the formation of intrachain disulfide
bonds3 and, in the case of
µ, by acquisition of partial protease resistance (21). Changes in the
electrophoretic mobility of µ chains were evident during the lag
period (Fig. 2A). These mobility shifts probably
corresponded to glycan processing, because they were no longer
detectable after treatment with endoglycosidase H (data not shown). In
contrast, if N[µ1] cells were pulse-labeled for a long period (20 h) so as to label the whole intracellular pool, µ and J chain
degradation could be observed immediately (Fig. 2, B and
C), consistent with the fact that a fraction of the
molecules had reached a degradation-competent state.
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Inhibiting Binding to Calnexin and Calreticulin Does Not Alter
Significantly the Kinetics of µ or J Chain
Degradation--
Because some newly synthesized µ chains can
be coprecipitated with calnexin and calreticulin (21), the effects
described above could reflect a role of the two chaperones in µ chain
quality control, as described for certain 1-antitrypsin mutants
(23). To verify this, the glucosidase inhibitor castanospermine was added before the pulse, so as to inhibit the rapid removal of glucose
residues from newly made proteins. Consistent with the presence of
additional glucoses, µ and J chains synthesized in the presence of
castanospermine migrated more slowly (Fig.
3A). Under these conditions, µ chains were not coimmunoprecipitated by anti-calnexin antibodies
(data not shown). Preventing the binding to calnexin and calreticulin,
however, did not affect the kinetics of µ chain degradation;
after the characteristic lag, clearance was efficient (Fig.
3B, lanes 1-4). Castanospermine also had little, if any, effect on the degradation of J chains (Fig. 3C,
lanes 2-5). Pre-treatment with castanospermine did not
affect the dislocation of J chains, which can be monitored by the
appearance of the two faster migrating bands (Fig. 3C,
>
and
) when cells are chased in the
presence of proteasome inhibitors (21). Similar amounts of cytosolic J
chains accumulated in the presence or absence of castanospermine (Fig.
3C, compare lanes 1 and 9 and the
histograms below).
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These results indicated that calnexin and calreticulin do not play a
major role in timing the degradation of the two Ig subunits. We
therefore investigated other glycan processing steps known to occur in
the ER lumen, in particular mannose trimming, which has been implicated
in the quality control of mutated 1-antitrypsin (23, 25-27) and
thyroglobulin (29).
Inhibitors of ER Mannosidase I Prevent µ Chain Degradation-- Inhibiting the activity of ER mannosidase I with kifunensine prevented the degradation of µ chains synthesized in the presence of castanospermine as efficiently as did proteasome inhibitors (Fig. 3B, compare lanes 6 and 9). The simultaneous presence of kifunensine and castanospermine did not have synergistic effects (Fig. 3B, lane 8). As shown in Fig. 3C, kifunensine partially inhibited J chain degradation as well (see below). These results suggested that the trimming of the B branch mannose is important in timing the degradation of unassembled Ig subunits.
To further confirm this, specific inhibitors of the main ER
glycosidases were added after a 5-min pulse without castanospermine preincubation (Fig. 4). As observed above
in castanospermine pre-treated cells, kifunensine was as effective as
ZL3H in preventing the degradation of µ chains (Fig. 4A,
compare lanes 2, 3, 6, and
9). dM, an inhibitor of both ER mannosidase I and ER
mannosidase II, also inhibited µ chain degradation (Fig.
4A, lane 17). In contrast, the ER mannosidase II
inhibitor swainsonine was not effective (Fig. 4A, lane
12). Consistent with the presence of additional mannoses, µ chains accumulating in the presence of dM, kifunensine, or swainsonine
migrated more slowly than those present in untreated cells (Fig.
4A, lane 2) or in cells incubated with proteasome inhibitors (lane 3). When both swainsonine and kifunensine
were present during the chase, the effects of the ER mannosidase I inhibitor prevailed, and abundant µ chains accumulated
intracellularly (Fig. 4A, lane 22). The mobility
shift was more pronounced than that induced by kifunensine or
swainsonine alone. Densitometric quantitations (Fig. 4A, see
bars below each lane) confirmed that the removal of the
terminal mannose from the central branch is crucial for the
degradation of µ chains.
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Because the removal of mannoses may alter the substrate recognition by glucosidases and UDP-glucose: glycoprotein glucosyltransferase, it was possible that the drugs influenced the binding of µ chains to calnexin. To exclude this possibility, castanospermine or dN was added after the pulse so as to block glycans in a monoglucosylated state. The effects of castanospermine and dN were confirmed by the mobility shift of µ chains accumulating in treated cells (Fig. 4A, compare lanes 18-20) and by mannosidase sensitivity assays (see Fig. 6 below). Under these experimental conditions, which should favor the binding of unfolded glycoproteins to calnexin and/or calreticulin (1), castanospermine and dN had minor effects on µ chain degradation.
Analysis of the anti-J immunoprecipitates confirmed the role of mannose trimming in targeting unassembled Ig subunits to degradation. Castanospermine and dN had minor effects (Fig. 4A, lanes 10, 18, 19). Kifunensine and dM inhibited the degradation of J chains, although to a lesser extent than µ chains. A single J chain band was precipitated from treated cells, with mobility slightly slower than the upper band accumulating in the presence of proteasome inhibitors (Fig. 4B, compare lanes 3 and 4). Inhibitors of ER mannosidase I prevented only in part the dislocation of J chains, as indicated by the appearance of the two bands with faster mobility, corresponding to deglycosylated, cytosolic J chains (21), when proteasome inhibitors were added during the chase (Fig. 4B, compare lanes 4-9). Therefore, glycan trimming is not essential for the dislocation of J chains. The differences between µ and J chains may reflect the number of N-glycans present on the two substrates and may correlate with our previous observation that some J but no µ chains can dislocate to the cytosol when proteasomal degradation is inhibited (21).
Neither Kifunensine nor Castanospermine Inhibits the Degradation of
Unglycosylated Chains--
To exclude the possibility that the
different drugs acted on general cellular functions, we investigated
their effects on
, a nonglycosylated Ig subunit. Neither kifunensine
nor castanospermine had any effect on the degradation of
G213, a
mutant that is also degraded by proteasomes (Fig.
5), albeit with slower kinetics than µ or J chains (36, 45).4
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µ Chains Accumulating in the Presence of Kifunensine Are Not
Glucosylated--
The presence of a terminal glucose on the A branch
of an N-glycan can be monitored by a mannosidase sensitivity
assay (46). When the glucose is present (Fig. 1, G1),
the mannosidase cannot remove the underlying mannoses, yielding a small
mobility shift. In contrast, glycans lacking the glucose (see Fig. 1,
G0) are digested to a greater extent. As expected, µ chains present in castanospermine-treated cells were only
marginally affected by mannosidase (Fig.
6A, compare lanes 9 and 10). In contrast, ZL3H (Fig. 6A, lanes
5 and 6) and kifunensine (lanes 7 and
8) caused the accumulation of µ chains that were sensitive
to the enzyme. Similarly, J chains appeared to be sensitive to
mannosidase except after chase in the presence of castanospermine (Fig.
6B). µ and J chains accumulating in the presence of
swainsonine (Fig. 6, A, lanes 11 and
12 and B, lane 6) were largely
resistant to mannosidase, suggesting that the presence of a terminal
mannose on the C branch inhibits glucosidase activity on the two Ig
subunits.
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Calcium Is Required for the Degradation of Unassembled µ Chains--
The recent solution of the structure of yeast class I
1,2-mannosidase I revealed a crucial role for calcium ions in
enzymatic activity (28). Therefore, we investigated whether perturbing calcium homeostasis in the ER lumen affected µ chain degradation. As
shown in Fig. 7, the ER
Ca2+-ATPase inhibitor thapsigargin caused the intracellular
accumulation of µ chains, which migrated with a mobility slower than
that of those present in untreated cells (Fig. 7, compare lanes
2 and 3). Similar to that reported above for
kifunensine, thapsigargin also partially inhibited J chain degradation
(data not shown).
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The requirements for calcium were further confirmed by chasing pulse-labeled N[µ1] cells with ionomycin and different Ca2+ concentrations (Fig. 7, lanes 4-8). In the presence of the ionophore, the Ca2+ levels in the ER are expected to be similar to those in the extracellular medium. Based on this assumption, it would seem that 100 µM Ca2+ is required to sustain the processes underlying µ chain degradation. The reduced degradation of µs in this experiment (Fig. 7, lane 2) could be due to the low extracellular Ca2+ concentration.
Mannose Trimming Is Important for the Degradation of µm Chains as
Well--
Both substrates analyzed so far, µs and J chains, are
soluble proteins retained in the ER lumen. It was of interest to
analyze the role of glycan processing on an integral membrane protein. Alternate RNA processing generates two forms of µ chains, µs and µm, which differ only in their C-terminal ends (47) and are degraded
with similar kinetics (35). Clearly, kifunensine prevented the
degradation of membrane bound µ chains (Fig.
8). Cytosolic proteasomes are responsible
for the degradation of µm, because ZL3H caused their accumulation
(Fig. 8, lane 11). Castanospermine had little, if any,
effect on the clearance of membrane-bound µm chains (Fig. 8,
lane 10).
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Kifunensine Does Not Inhibit IgM Secretion--
In the presence of
chains, most µ chains synthesized by plasma cells are assembled
into IgM polymers and secreted (34). Only part of them, corresponding
to molecules that fail to assemble with
or to polymerize, are
degraded (37). We therefore tested the effects of kifunensine and ZL3H
on J[µ1] cells, which express µ,
, and J chains. As shown in
Fig. 9, kifunensine did not inhibit IgM
secretion (compare lanes 5 and 6). The proteasome
inhibitor ZL3H had only a marginal effect (Fig. 9, lane 7).
Both kifunensine and ZL3H caused a small accumulation of intracellular µ chains (Fig. 9, lanes 2 and 3), suggesting
that secretory µ chains that failed to polymerize were degraded by
proteasomes. These results imply that the molecular mechanisms
that recognize µ chains lacking the central mannose(s) and that
divert them to proteasomal degradation are specific for structurally
immature molecules (either unassembled or unpolymerized).
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The Degradation of µ-TCR TM Chains Does Not Require
Mannose Trimming--
We have previously shown that appending the
transmembrane region of the TCR
chain to the C terminus of Ig µ and
2b chains induces brefeldin A-insensitive degradation of
chimeric immunoglobulins (37). When expressed in the absence of light
chains in NSO, degradation of µ-TCR
TM was rapid. Unlike
in the case of µs and µm chains, however, kifunensine did not alter
the kinetics of µ-TCR
TM degradation (Fig.
10A). Thapsigargin failed to
inhibit the proteasomal disposal of µ-TCR
TM as well (Fig.
10B), in agreement with the notion that Ca2+ is
not required for the degradation of T cell receptor subunits (48). In
addition, the proteasomal degradation of
2b-TCR
TM chains, which
occurs with kinetics slower than µ-TCR
TM (37), was insensitive to
glycosidase inhibitors and thapsigargin (Fig. 10C),
suggesting that the extraction of these chimeric molecules follows a
different pathway, independent from the activity of ER mannosidase
I.
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It has been recently reported that inhibitors of tyrosine phosphatases,
such as phenylarsine oxide prevent the degradation of certain
1-antitrypsin mutants (26). Phenylarsine oxide clearly inhibited the
degradation of both µs and µ-TCR
TM chains (Fig. 10D). Unlike kifunensine, phenylarsine oxide inhibited the
dislocation of J chains, because the simultaneous presence of
phenylarsine oxide and proteasome inhibitors during the chase did not
yield the characteristic cytosolic J isoforms (Fig. 10D, see
arrows) in either N[µ-TCR
TM] or N[µ1] cells
(lanes 5 and 9). Pervanadate (data not shown) and
the oxidant diamide (21) gave similar results.
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DISCUSSION |
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To mediate folding efficiency and homeostasis, the processes of retention and degradation that underlie quality control within the ER must be precisely coordinated. Sufficient time must be allocated to allow the folding of newly made polypeptides in a milieu that provides optimal assistance by specialized chaperones and enzymes. On the other hand, molecules that fail to attain the correct three-dimensional structure after some time should be degraded to prevent their accumulation or aggregation in the ER lumen (49). The data presented in this paper demonstrate that the trimming of N-linked glycans provides a molecular mechanism for coordinating the quality control of two glycosylated unassembled Ig subunits. This timer function seems to be based on the sequential activity of the ER sugar-processing enzymes. Whereas glucosidases act rapidly as substrates emerge from the translocon, ER mannosidases I and II come into the scene later (1). The absence of the terminal mannose on the B branch of asparagine-linked glycans enables the targeting of terminally unfolded glycoproteins for dislocation to the cytosol and proteasomal destruction.
The distinct patterns of µ and J chain electrophoretic mobility and
in vitro sensitivity to jack bean -mannosidase confirmed the specificity of the pharmacological inhibitors. Interestingly, µ chains isolated from cells chased in the presence of swainsonine are
largely resistant to mannosidase, whereas kifunensine leads to the
accumulation of sensitive molecules. This suggests that, at least in
myeloma cells, removal of the B branch mannose does not impair
glucosidase II to a greater extent than UDP-glucose: glycoprotein
glucosyltransferase. On the other hand, on glycans lacking a C branch
terminal mannose, the activity of UDP-glucose: glycoprotein
glucosyltransferase prevails, and glucosylated molecules accumulate,
which are nonetheless dislocated and degraded by proteasomes. Thus, it
appears that calnexin has a minor role in the clearance of µ chains.
In contrast, kifunensine and dM were as effective as proteasome
inhibitors in preventing µ chain degradation. Under both conditions, µ chain dislocation across the ER membrane was blocked, and
glycosylated heavy chains accumulated. Kifunensine (Figs. 3 and 4) and
proteasome inhibitors (21) only partially blocked the dislocation of J
chains, perhaps reflecting the different number of glycans or folding
state of the two subunits.
The involvement of mannose trimming in targeting glycoproteins for
proteasomal destruction has been demonstrated in several experimental
models. In yeast, biochemical and genetic evidence indicates that the
degradation of CPY* requires the activity of a Class I
1,2-mannosidase (50, 51). Trimming of the central mannose is
important also for the degradation of yeast pre-pro-
-factor ectopically expressed in mammalian cells (52) and of certain
1-antitrypsin mutants associated with familial emphysema (23, 25-27). The degradation of a truncated
1-antitrypsin mutant was shown to depend on a prolonged interaction with calnexin (23). In this
respect the degradation of µ and J chains seems to be mechanistically
different, because it is only marginally affected by castanospermine.
This indicates that more than one pathway can coordinate the
dislocation/degradation of glycoproteins, a notion that is not
surprising because at least two parallel systems exist in the ER to
assist glycoprotein folding. Whether a glycoprotein selects
calnexin/calreticulin or BiP seems to depend on the location of the
glycan(s). The closer the oligosaccharide moieties are to the N
terminus, the higher are the chances that the protein engages with
calnexin (53). This may explain why µ chains, in which the first of
the five N-linked glycans is located at position 171 (see
Ref. 54 and references therein), fold primarily on BiP. However, a
fraction of newly made µ chains can be coprecipitated with calnexin
(21). It is not known whether these have detached from a first round of
interaction with BiP or represent direct binding to the lectin,
possibly facilitated by the numerous glycan moieties present on µ chains. Although the single glycan of J chains is bound to asparagine
48, we failed to detect interactions of this small Ig subunit with
calnexin or calreticulin (21). In contrast, J chains can be easily
coprecipitated with BiP, especially in tunicamycin-treated
cells.2 Because unassembled µ and J chains preferentially
associate with BiP, it is not surprising that pre-incubation with
castanospermine (which prevents binding to calnexin/calreticulin) fails
to affect their degradation. The minor effects observed when the
glucosidase inhibitor is administered after the pulse could be due to a
reduced activity or accessibility of ER mannosidase I on
monoglucosylated substrates. Therefore, our results suggest the
involvement of glycan trimming in timing glycoprotein degradation
independently from interactions with calnexin or calreticulin.
Many ER resident proteins bind Ca2+, and perturbing the homeostasis of this ion is bound to have pleiotropic effects. However, because ER mannosidase I contains Ca2+ (28), thapsigargin could inhibit µ chain degradation by interfering with the function of this enzyme, as also suggested by the similar electrophoretic mobility of µ chains accumulating in thapsigargin- or kifunensine-treated cells.
How can removal of the B branch terminal mannose activate dislocation? Terminally unfolded substrates, in particular soluble molecules like µs and J chains, must be recruited in the vicinities of a functional dislocon, which must eventually be opened and activated. Dislocation across the ER membrane, which in the case of µ chains requires active proteasomes (21), is probably facilitated by unfolding and reduction of the substrate. It is tempting to speculate that lectin molecules endowed with specificity for Man8 glycans play an important role in this chain of events (24). In principle, if such lectins had a binding specificity similar to ER mannosidase I, kifunensine, thapsigargin, and other drugs may compete with or inhibit the lectin itself. The observation that kifunensine does not affect IgM secretion indicates that, if a lectin plays a role in timing dislocation, it must be specific for unfolded molecules. It also suggests that the inhibition of degradation caused by kifunensine is not due to prolonged retention in the ER lumen. Taken together, these results are consistent with an active role of Man8-binding lectin molecules in targeting misfolded or unassembled glycoproteins to dislocation/degradation.
The degradation of the PiZ and null variants of
1-antitrypsin seems to follow different pathways (23, 26, 27). The results obtained with µ-TCR
TM indicate that indeed more than one
pathway can be utilized to dislocate a protein from the ER to the
cytosol. In µ-TCR
TM, the last residues of µs, including the
C-terminal N-glycan (54) and Cys-575, had been replaced with
the transmembrane and cytosolic domains of the
-TCR chain, known to
contain a dominant ER-associated degradation (ERAD) targeting signal (11, 14, 55, 56). Like µm, µ-TCR
TM contains four of the
five glycans present in wild type µs and is an integral membrane
protein with a short cytosolic tail. Therefore, neither the number of
sugars nor the different topology can explain the distinct requirements
of the three ERAD substrates for dislocation and degradation.
In addition,
2b-TCR
TM, whose slower kinetics of degradation
allow glycan processing by ER mannosidase I, is efficiently degraded in
the presence of kifunensine. These observations suggest that
information contained in the transmembrane and cytosolic tail of the
-TCR chain can induce mannosidase I-independent degradation of a
polypeptide that normally requires removal of the B terminal mannoses.
In addition, this pathway seems to utilize cytosolic proteasomes,
because it can be inhibited by ZL3H (Fig. 10), and in this respect
differs from the pathway described for the PiZ
1-antitrypsin variant (26). In view of the importance of maintaining ER homeostasis and fidelity of quality control, it is not surprising that multiple pathways are operative in mammalian cells to eliminate misfolded proteins from the ER.
Although differing in sensitivity to kifunensine and thapsigargin, µs
and µ-TCR TM share the property of being stabilized by inhibitors
of tyrosine phosphatases, such as phenylarsine oxide (Fig.
10D) and pervanadate (data not shown). In addition, the
degradation of endogenous J chains is blocked by phenylarsine oxide.
The absence of deglycosylated J isoforms suggests that this drug blocks
dislocation from the ER lumen to the cytosol, a molecular phenotype
similar to that induced by the reversible oxidant diamide (21). The involvement of tyrosine phosphatases could be important for integrating ERAD with other cellular signaling pathways, including the
responses to unfolded proteins and ER overload (57, 58 and references
therein). However, because many steps in the complex process of
ERAD are redox-sensitive, the precise targets of
phenylarsine oxide, pervanadate, and diamide remain to be identified.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Aebi, M. Alessio, A. Cabibbo, A. Fassio, A. M. Fra, A. Helenius, C. Jakob, R. Mancini, A. Mezghrani, and T. Simmen for helpful discussions and suggestions; Drs. M. Bogyo, H. Ploegh, and R. M. E. Parkhouse for generously providing reagents; and S. Trinca for impeccable secretarial assistance.
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FOOTNOTES |
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* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche (Target Project on Biotechnology, 97.01296.PF49 and 5% 99.0089.PF31), and Ministero della Sanità (Special AIDS Project and Ricerca Finalizzata RF 98.53).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Via Olgettina 58, 20132 Milan, Italy. Tel.: 39 02 2643 4763; Fax: 39 02 2643 4723; E-mail: r.sitia@hsr.it.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M009603200
2 C. Fagioli and R. Sitia, unpublished observations.
3 A. Mezghrani, C. Fagioli, and R. Sitia, unpublished results.
4 A. Sparvoli and R. Sitia, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; µs, secretory µ; µm, membrane µ; TM, tunicamycin; ZL3H, carboxybenzyl-leucyl-leucyl-leucinal; dN, deoxynojirimycin; dM, deoxymannojirimicin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TCR, T cell receptor; ERAD, ER-associated degradation.
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