(Received for publication, August 16, 1994; and in revised form, November 2, 1994)
From the
HLA class II molecules are membrane proteins which are assembled
in the endoplasmic reticulum shortly after synthesis of the and
and invariant chain (Ii) monomers. DR
chains, in the absence
of DR
, are rapidly and completely degraded by the pre-Golgi
degradative pathway. Here we have examined those factors which target
DR
chains for degradation in a DR
deficient cell line,
9.22.3. The DR
monomers in 9.22.3 were initially incorporated into
a proteinaceous complex containing BiP. With time, the DR
complexes were further aggregated. In wild type cells, which can
assemble DR
-
dimers, the secondary phase of aggregation of
DR
was not seen. Additional evidence that aggregation of DR
in 9.22.3 cells was progressive was that a more mature form of DR
was found exclusively in the largest DR
complexes. Furthermore,
the most highly aggregated DR
chains were degraded more rapidly
than bulk DR
chains. These data suggest that DR
aggregates
are intermediates in the pre-Golgi pathway of DR
degradation. They
further suggest that formation of large DR
aggregates is a
proximal event to DR
degradation. We conclude that DR
chains
are targeted for degradation as a consequence of a change of state,
coincident with their aggregation into slow forming, high molecular
weight complexes.
There are two well characterized pathways for the degradation of
membrane proteins. The first involves the internalization of membrane
proteins by the invagination of plasma membrane and the removal of a
fraction of membrane proteins and their degradation in
lysosomes(1, 2) . The second involves the pre-Golgi
degradation of certain monomeric subunits or mutated proteins shortly
after their synthesis and transfer into the endoplasmic reticulum (ER) ()(3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
The first pathway is sensitive to lysomorphotrophic agents and
inhibitors of lysosomal proteases. The second is insensitive to such
agents but can be blocked by drugs or conditions which prevent the
transport of the membrane proteins beyond the
cis-Golgi(3, 5, 8, 9, 10, 11, 12) .
The events which are responsible for targeting certain membrane or
secreted proteins for pre-Golgi degradation are largely unknown. None
of the components of the proteolytic machinery have been identified.
There is good evidence that degradation occurs in the
ER(8, 10, 13) . Studies of model mutated or
truncated proteins have suggested that the pre-Golgi degradative
pathway is capable of recognizing unfolded or misfolded proteins.
Pre-Golgi degradation is frequently the fate of the subunits of
multimeric proteins which are synthesized in unequal
amounts(3, 7, 8, 12) . Degradation
of model proteins in a pre-Golgi compartment is preceded by a short lag
period after synthesis(3, 8, 12) .
Thereafter, the half-lives of membrane proteins which are degraded by
this pathway are short, usually approximately 1 h. We have studied the
degradation of HLA-DR
monomers and have found that they are
degraded by the pre-Golgi pathway(12) . Degradation of DR
monomers is comparable to that of several other subunits of multimeric
membrane proteins, and occurs rapidly in a pre-medial Golgi
compartment. The pre-Golgi pathway is insensitive to lysomorphotropic
agents, but degradation can be inhibited by conditions or drugs which
deplete energy stores in the ER(7) .
Recognition of misfolded or incompletely folded proteins must occur shortly after synthesis. Studies of protein folding in the ER indicate that folding of the nascent polypeptide chain begins before translation is completed(14, 15, 16) . A class of proteins, termed chaperones, associate transiently with newly synthesized proteins and are believed to assist in the folding process. The chaperones BiP-GRP78 (hereafter referred to as BiP), calnexin, protein disulfide isomerase, and GRP94 bind a broad but select set of newly synthesized polypeptides and likely assist in their folding(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . Folding is not completely efficient, however, and a fraction of proteins may be misfolded. Misfolded proteins can be detected shortly after synthesis and, in general, are retained in the ER as aggregates(15, 29, 30) . Severe misfolding of proteins can result in formation of large aggregates; precipitates may also form which can exist in the ER for several hours (15, 20, 22, 29, 30, 31) . It is not clear how these model proteins are degraded and there is no indication that such aggregated proteins are substrates of the pre-Golgi degradative pathway. In contrast, subunits of multimeric proteins which remain unassembled appear to be handled differently(29, 31) . The unassembled subunits, though retained in the ER, are usually not found in highly aggregated states and they retain the capability of assembling into multimers for long periods of time (29, 31, 32, 33) . In general, unassembled subunits appear to interact with BiP, but these interactions are less avid and more reversible than BiP interactions with highly aggregated proteins(28) .
The pre-Golgi
degradative pathway is thought to degrade proteins which are misfolded,
but it is unclear how unfolded proteins are recognized and once
recognized, selectively sorted for disposal. It has been suggested that
the ER chaperones such as BiP may play a role in the determining a
protein's fate toward native conformation(16, 17, 32, 34, 35, 36) or
toward misfolding and ultimately,
degradation(19, 25, 31) . Although much
progress has been made in understanding the functions of the ER
resident chaperones, the potential role of ER resident chaperones in
either identifying or targeting misfolded proteins for degradation has
not been fully explored. Here we have examined the possible role of one
ER stress protein, BiP, in determining the fate of DR chains
destined for degradation. We find that BiP initially interacts
transiently with DR
chains shortly after their synthesis but at a
time when no DR
degradation takes place, suggesting that binding
of BiP to DR
chains per se does not target them for degradation.
However, at later times, multiple copies of BiP bind to DR
monomers, resulting in the formation of tightly associated high
molecular weight oligomers. The aggregation of DR
chains into high
molecular weight BiP
DR
complexes suggests a means by which
DR
chains are segregated within the ER and targeted for
degradation.
Figure 1:
BiP associates
strongly with DR monomers in a DR
-deficient cell line. 9.22.3
cells (20
10
) (lanes 1-7) or 8.1.6
cells (4
10
) (lane 8) were labeled for 3 h
with [
S]methionine, and extracts were reacted
with anti-BiP (lanes 2 and 4), anti-DR
(lanes 1 and 3), anti-class I (lanes 5 and 6), normal mouse IgG (lane 7), or VI.15 (anti-DR
dimer) (lane 8). Immunoprecipitates were washed six times,
including twice with buffers including either 0.65 M NaCl (lanes 1, 2, 5, 7, and 8)
or 0.5 M LiCl (lanes 3, 4, and 6).
Note that anti-BiP preferentially coprecipitated the p27 form of
DR
.
Figure 2:
BiP
binding to DR is biphasic. 9.22.3 cells were pulsed with
[
S]methionine for short periods (A) or
longer periods (B) and chased for the times indicated. Cells
were pulsed for 8 or 10 min in (A) or for 20 min in (B). Samples were immunoprecipitated with HB10.A and the BiP
and DR
regions of the gel were quantitated by PhosphorImager-based
analysis. The BiP/DR
ratios, uncorrected for methionine content,
were normalized to the BiP/DR
ratios found at 0 chase time. The
initial decline in BiP/DR
ratio was seen in five of five
experiments and the extent of decline appeared to relate to the length
of the pulse. It is likely that with the longer pulse time shown in B, the secondary increase of BiP binding tends to mask the
initial decline in BiP/DR
ratio.
Figure 5:
Size distribution of DR complexes in
wild type cells. 8.1.6 cells (8
10
) were pulsed for
15 min (Panel A) or pulsed and chased for 2 h (Panel
B) and analyzed in sucrose gradients by immunoprecipitation with
HB10.A as in Fig. 3. In A, note that not much assembly
of DR dimers has occurred during the pulse. In A, Ii is the
most prominent band, followed by DR
, with small amounts of DR
seen only in fractions 8-11. Quantitation of DR
is
shown in C. Note that the average size DR
complex in wild
type cells becomes smaller with time.
Figure 3:
Aggregation of DR chains is
progressive in DR
deficient cells. Sucrose gradient and
immunoprecipitation analysis of 9.22.3 cells which were pulsed with
[
S]methionine for 20 min and then chased for 0 (A), 1 h (B), or 2 h (C) or pulsed and
cross-linked with 500 µg/ml dithiobis(succinimidyl propionate) for
30 min at 25 °C (D). Panels A-D were
immunoprecipitated with HB10.A, whereas Panel E was
immunoprecipitated with a mixture of W6/32 (anti-class I) and HB10.A (E). For Panels A-D, fractions 2, 12, 14, 16,
and 17 were analyzed on a separate gel and the PhosphorImaging
quantitation of DR
is shown in Fig. 5. Because of the brief
exposure time (4 h) in Panel E, only MHC class I is visible.
The above experiment was performed three times with essentially
identical results.
The coprecipitation of BiP by
anti-DR antibody does not appear to be simply the gratuitous
interaction with an abundant ER protein. Evidence that the interaction
was not gratuitous was that binding exhibited both a high degree of
specificity and was strong. No BiP was immunoprecipitated with normal
mouse IgG (lane 7), indicating that coprecipitation of BiP
with anti-DR antibody was not due to the well known ability of BiP to
bind to free or partially denatured immunoglobulin heavy
chains(16) . Moreover, BiP was not detected in
immunoprecipitates using antibodies to fully assembled DR dimers from
wild type cells (lane 8). Trace amounts of BiP, constituting
some 4% of that seen in anti-DR
immunoprecipitates, was
coprecipitated from 9.22.3 cells with antibodies to MHC class I
molecules (lanes 5 and 6). The coprecipitation of BiP
by anti-DR
antibodies was quite strong; it did not require the use
of chemical cross-linkers, and BiP
DR
complexes were
resistant to stringent wash conditions of either 0.5 M LiCl or
0.65 M NaCl (Fig. 1, compare lanes 1 and 2 with lanes 3 and 4).
DR was a prominent
band in the anti-BiP immunoprecipitate, comprising
6% of the total
non-BiP material. This value seems unexpectedly high, given that
DR
chains make up only 0.1% of the total mRNA and protein in wild
type B cell lines(41) . Thus DR
chains, when they exist as
monomers, are more likely to bind to or interact more stably with BiP
than the average membrane protein. The corollary of this is that some
membrane proteins would be expected either not to interact with BiP or
to interact only very transiently. In support of this, neither proteins
the size of Ii or MHC class I were evident in anti-BiP
immunoprecipitates (Fig. 1, lanes 2 and 4). An
estimate by another method, quantitative Western blotting, of the BiP
found in anti-DR
immunoprecipitates compared to the BiP found in
the starting cellular extract indicated that, under steady state
conditions,
3% of the total BiP was complexed with DR
monomers in DR
-deficient cells (data not shown).
The detection of a secondary
increase in DR-BiP complexes after 10-20 min of DR
synthesis suggested that the ratio of BiP to DR
might be
increasing during this time. Therefore we measured the ratio of DR
to BiP as a function of time following DR
synthesis in
DR
-deficient 9.22.3 cells. The BiP to DR
ratio was measured
at three different times: during a 10-min pulse, at 3 h when DR
but not BiP is labeled to equilibrium, and at 48 h, a time when the
labeling of BiP approached equilibrium. (
)The amounts of
DR
and BiP coprecipitated by anti-BiP and anti-DR
,
respectively, were quantitated by densitometry and corrected for the
relative methionine content of the two proteins as previously
described(15) . The BiP to DR
ratio went from 0.2 after a
10-min pulse, to 0.9 after 3 h and increased to an average (in three
experiments) of 2.2 at 48 h. These data indicate that, at equilibrium
in the absence of DR
chains, the average DR
molecule is in a
complex with about 2 BiP molecules. The intensity of the BiP band
coprecipitated by anti-DR
antibody probably significantly
underestimates the stoichiometry of the DR
BiP complex for
short labeling periods because it does not account for the large amount
of unlabeled BiP present in the anti-DR
immunoprecipitate. The
kinetics of BiP binding to DR
in DR
deficient cells and a
BiP/qDR
ratio of about 2 at steady state suggested that, in the
absence of DR
chains, DR
monomers increasingly enter into a
complex with BiP.
Figure 4:
Quantitation of the DR complex size
in sucrose gradients after chemical cross-linking or as a function of
time. The amount of DR
in each sucrose gradient fraction of
pulse-chased DR
immunoprecipitates of 9.22.3 in Fig. 3, A-D, was analyzed by PhosphorImaging. The DR
in
each fraction is plotted and was calculated as the percent of the total
DR
in the entire gradient.
The
observation that DR chains are found in increasingly aggregated
states in DR
deficient cells contrasted sharply with the kinetics
of DR
complex formation in wild type cells. Pulse-chase and
sucrose gradient analysis of DR
complex formation in wild type
8.1.6 cells revealed that DR
was initially found as a 6 S complex (Fig. 5, fractions 9-10). After 2 h of chase, the
peak size of DR
complexes decreased (Fig. 5, B and C, fractions 12-13). Thus in both wild type and
DR
-deficient cells newly synthesized DR
chains are
incorporated into a 5-6 S complex, probably composed of one or
more members of the ER stress response family. Thereafter the fate of
DR
chains in mutant and wild type cells diverges.
The large
size (7-12 S) of late forming DR complexes in
DR
-deficient cells cannot be explained by the contribution of
bound detergent because other membrane proteins of comparable size
sediment much more slowly in sucrose gradients. Mature MHC class I
molecules (M
57,000) for example, sedimented at
5.3 S, just ahead of the albumin marker (Fig. 3E).
Moreover, 2 h after synthesis the size of mature DR heterodimers from
wild type cells sedimented at 4.5-5 S (Fig. 5B).
These data indicate that the size of DR
complexes in sucrose
gradients must be due to the binding of other proteins to DR
chains.
BiP appears to be a major component of the large, late
forming DR complexes found in DR
-deficient cells. A 78-kDa
band which co-migrated with BiP was found in fractions 1-9
(6.5-20 S) after 1 h of chase (Fig. 2B). In the
fastest sedimenting fractions, the intensity of the BiP band approached
that of DR
, in spite of the fact that only a fraction (<10%) of
the cellular BiP was labeled during the short pulse (Table 2).
Conversely, little or no BiP was coprecipitated in fractions which
sedimented at 5-6 S. The lightest fractions (14, 15, 16, 17) had little or no
BiP and very little Ii chain (Table 2). The Ii chain was found in
both intermediate and large DR
complexes, but interestingly,
appeared to be relatively absent from the largest DR
complexes (Table 2, fractions 1-4).
Figure 6:
The size distribution of p27 in DR-
deficient cells. 9.22.3 cells (4
10
cells) were
pulsed for 4 h and analyzed by sucrose gradient fractionation and
immunoprecipitation with HB10.A (A). Note that p27 is confined
exclusively to the densest fractions. Quantitation of p27 and total
DR
(p29 + p27) is plotted in (B). p27 was confined
exclusively to the densest functions in three independent
experiments.
Figure 7:
The degradation of p27 is rapid. 9
10
922.3 cells were labeled for 90 min with 1.5 mCi of
[
S]cysteine in RPMI medium lacking cysteine.
Cells were chased by the addition of RPMI with 1 mML-cysteine, and 1.5
10
cells were
removed at 0, 10, 20, 30, 45, and 60 min of chase. Material
immunoprecipitated with HB10.A was electrophoresed on 13.5% acrylamide
gels, the gels were dried, and radioactivity was captured by
PhosphorImaging (A). The p29 and p27 forms were resolved by
the use of the software quantitation program, ImageQuant. Note the
relative absence of Ii from cysteine labeled cells. B, linear
regression of p27 turnover. The p27 half-life in this experiment was
21.0 min and the half-life of total DR
(p27 + P29) was 54.0
min; the correlation coefficients were 0.91 and 0.98, respectively. The
p27 half-life was 17.5 ± 3.0 min in three experiments. Note that
the longer labeling period necessary to label p27 results in the
immediate onset of DR
degradation without the 15-20-min lag
normally seen. Consequently the half-life of DR
in this experiment
is shorter (54 min), by some 16-18 min than previously
reported(12) .
In this report we have demonstrated that DR chains
destined for degradation by the pre-Golgi degradative pathway enter
into an aggregated complex containing multiple copies of BiP. Our data
demonstrate that aggregation of unassembled DR
chains occurs prior
to their degradation and raises the possibility that complex formation
with BiP and aggregation may be the common fate of other
membrane-anchored substrates of the pre-Golgi degradative
pathway(25) . Aggregation appears to be a proximal event in the
degradation of DR
chains. This conclusion stems from two
observations that correlate the highly aggregated DR
state with
faster DR
turnover. The first observation is that p27, which we
have previously shown to be a more mature form of DR
, is found
exclusively in a highly aggregated state. Furthermore, p27 was found to
be degraded about three times faster than the p29 form of DR
chains. Taken together these observations suggest that aggregation is
progressive but that large DR
aggregates are continually being
removed by degradation.
It is not known whether formation of large
aggregates is a universal step for all substrates of the pre-Golgi
degradation pathway. Studies of one soluble, non-membrane anchored
substrate, a variant of -antitrypsin, indicated that although
formation of small complexes occurred, the complexes did not get
progressively larger with time (10) . The equilibrium size of
aggregates is likely to reflect the complex dynamics of their formation
and degradation. This is likely to vary depending on abundance of the
protein, its half-life and such factors as whether it is soluble or
membrane-bound. Studies comparing membrane proteins with their
anchorless variants have demonstrated differences in the kinetics of
both folding, and when observed, aggregate formation(43) .
The formation of DR aggregates reported here is similar in
nature to the slow forming aggregates previously described for a
mutated herpes gB glycoprotein (25) and the
subunit of
the acetylcholine receptor (31) . In each case, aggregate
formation was relatively slow and BiP was a prominent component of the
complex. Like DR
, both the unassembled
subunit of the
acetylcholine receptor and the mutant gB protein are aggregated in the
ER and are ultimately degraded. However, the half-lives of the these
two proteins were apparently several hours, leaving some doubt as to
whether they are degraded by the same pathway as unassembled DR
chains.
Because of the rapid turnover of DR chains in
DR
-deficient cells (T
of 70 min) the high
molecular weight BiP
DR
complexes do not accumulate and can
only be observed during a relatively narrow kinetic window. However,
several properties of the late forming BiP
DR
complexes allow
their detection and the independent examination of their role as
intermediates in the DR
degradative pathway. The large
BiP
DR
complexes observed at 30-120 min after DR
synthesis are seen only in the DR
-deficient cells. Second, the
stoichiometry of BiP
DR
in the late forming complexes is high
(approximately 2). Finally, p27 is found exclusively in large
aggregates (7-16 S) and is degraded much more rapidly than the
p29 form of DR
. These findings suggest that the late forming
complexes represent DR
chains destined for degradation.
The
kinetics of BiP association with DR provide insights into the way
DR
monomers may be targeted for degradation. BiP interacts with a
given DR
monomer not once, but in multiple cycles. Shortly after
DR
synthesis, BiP interacts transiently with DR
monomers and
then dissociates (Fig. 2A). At later times, the ratio
of the BiP to DR
increases so that at steady state it is
approximately 2. This suggests that the dissociation rate of BiP from
DR
has decreased, probably because DR
chains, in the absence
of DR
chains, cannot achieve a stable, native conformation. As a
consequence, they increasingly enter into a misfolded state, probably
exposing hydrophobic surfaces with which BiP is known to tightly
interact(35, 36) . BiP can exist as a
homodimer(44) , but it is not known whether BiP is functionally
divalent and capable by itself of cross-linking misfolded DR
chains. Our data suggest that the binding of multiple BiP molecules
results in the incorporation of DR
into a supramolecular complex.
Thus the signal which targets DR
monomers for degradation may be a
physical change of state, the cross-linking of the monomer or their
aggregation into large, oligomeric complexes with BiP. BiP is the only
protein (other than Ii) which we have identified in DR
complexes,
but other, unidentified proteins of M
of
90,000-94,000 and 68,000-72,000 are also present in various
amounts in the high molecular weight complexes. The identity of these
other proteins is currently under study.
The selective aggregation of misfolded proteins may constitute a means by which misfolded proteins can be concentrated and selectively sorted from other properly folded secreted and membrane proteins. Selective aggregation of misfolded proteins might be a necessary step in segregating proteins destined for degradation and transporting them to either a specialized subcompartment of the ER or possibly for vesicular transport to another pre-Golgi location. It is interesting to note that in pancreatic exocrine cells and other regulated secretory systems, sorting of regulated secreted proteins into dense granules is thought to involve their selective aggregation(45, 46) . Moreover, the delay in degradation which is characteristic of the pre-Golgi degradative system is a distinctive feature of many aggregation or nucleation-dependent biological systems(47) . Thus aggregation is a general biological phenomenon which may play a necessary role in both the recognition and differential sorting of misfolded proteins within the endoplasmic reticulum.
It is surprising that the endoplasmic reticulum should be both a site of protein folding/subunit assembly and of protein degradation. The potential for disruption of subunit assembly by proteolytic fragments would seem to be great if such fragments were allowed to exist in a freely soluble state at the site of assembly. Therefore, the cell must take measures to ensure that either proteolysis takes place at a spatially segregated site or that proteolytic fragments do not become soluble. The ER resident chaperones such as BiP are a class of proteins which have been demonstrated to interact with a range of newly synthesized protein subunits and therefore are the likeliest candidates to function in recognizing misfolded proteins. BiP and other chaperones bind to a newly synthesized protein and prevent it from aggregating until it achieves a folded state(33, 34, 35, 36) . However, the failure of a protein to fold correctly results in its stable association with BiP, and ultimately, its incorporation into large aggregates(19, 25) . Thus BiP and other chaperones are well suited to act at the decision point in a protein's fate, either by temporarily preventing the misfolding of newly synthesized proteins or by facilitating the aggregation of misfolded proteins.