From the
Many cell surface proteins exist as complexes of multiple
subunits.It is well established that most such complexes are
assembled within the endoplasmic reticulum (ER). However, the
mechanistic details of the assembly process are largely unknown. We
show here that
Transmembrane proteins destined for distal compartments of the
secretory pathway or the cell surface must be properly folded in order
to be competent for exit from the endoplasmic reticulum
(ER)
We have
been studying the retention of unassembled subunits in the ER using
major histocompatibility complex (MHC) class II molecules as a model
system. MHC class II molecules consist of a noncovalent complex of two
transmembrane glycoproteins,
We have previously shown that, like many misfolded or aberrantly
processed polypeptides,
In this study, we show that aggregates of newly synthesized class II
For analysis of calnexin-bound material, cells
were lysed in digitonin lysis buffer (10 mM Tris, pH 7.4, 150
mM NaCl, 1% (w/v) digitonin, 0.02% NaN
Prior analyses of spleen cells from mice deficient in Ii
expression (Ii-/- mice) showed that a large fraction of
newly synthesized MHC class II A
A similar
pulse/chase analysis of A
The large size of the HMWA
observed transiently in WT cells and more stably in Ii-/-
cells could be due either to an agglomeration of multiple newly
synthesized chains or to an association of a few chains with major ER
chaperones that were not efficiently labeled during the short metabolic
pulses used above. In order to determine whether other major proteins
were associated with A
Even though calnexin was not a component of
the aggregates observed under our solubilization conditions, we were
interested in knowing the characteristics of A
Our observations suggest that MHC class II
The HMWA that we observed are most likely composed
predominantly of unassembled or partially folded class II
Various chaperones have been shown to bind to folding
and assembly intermediates in the ER. Our data suggest that two of
these chaperones, BiP and calnexin, may have distinct and perhaps
sequential functions, as has been suggested by recent data analyzing
the folding and assembly of the vesicular stomatitis virus G protein
(36) . BiP was previously shown to bind to aggregated class II
species in the ER of transfected COS cells
(18) . We were unable
to detect BiP in our gradients of pulse-labeled spleen cells, but this
was likely due to slower synthetic and turnover rates for BiP in spleen
cells relative to those in COS cells in which overexpression of
ER-retained class II chains may have induced higher BiP expression. BiP
was observed to co-immunoprecipitate with class II chains in spleen
cells after 24 h of metabolic labeling and likely binds to transient
aggregates. Supporting this, BiP was co-precipitated in higher amounts
relative to the A
Calnexin, another ER chaperone, has been
previously shown to bind to class II assembly intermediates
(7) . Our data confirm and extend these results, suggesting that
calnexin binds primarily to more mature intermediates, such as monomers
of
Invariant chain has been suggested to play several roles in the
function of MHC class II molecules, including enhancing
In conclusion, our observations
demonstrate that aggregation of newly synthesized subunits is a dynamic
early event in the physiological assembly process for a heteromeric
protein complex, and not merely a dead-end pathway for stably
unassembled subunits. This phenomenon may be related to that reported
for the folding of several highly expressed proteins, such as
thyroglobulin
(49) and vesicular stomatitis virus G protein
(50) , which also proceeds via aggregated, BiP-associated
intermediates. Two unique aspects of the system examined in this study
are that 1) the class II chains are expressed at much lower levels than
thyroglobulin or viral proteins, showing that aggregation is not simply
a result of high expression, and 2) dissociation of the aggregated
chains cannot be completed unless all of the constituent subunits are
available in the ER. The different characteristics of proteins shown to
undergo transient aggregation in the ER suggests that this process
might represent a common mechanistic step in the folding and assembly
of many newly synthesized proteins.
We thank Drs. E. Bikoff and E. Robertson and C. Huang
for generating and generously providing Ii-/- mice; Drs. D.
Bole, M. Brenner, D. McKean, K. Schreiber, P. Romagnoli, E. Long, and
P. Roche for generous gifts of antibodies; Dr. P. Cresswell for
communication of results prior to publication; and Drs. E. Long, P.
Roche, P. Cresswell, E. Bikoff, R. Klausner, and members of the
Bonifacino lab for critical review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
subunits of major histocompatibility
complex class II antigens in spleen cells of normal mice pass through a
transiently aggregated phase in the ER prior to assembly with the
invariant chain (Ii). Aggregates form immediately after synthesis and
disappear concomitantly with assembly of mature
Ii complexes.
In spleen cells lacking Ii, aggregates fail to be efficiently
dissociated over time, implicating subunit assembly as a requirement
for disaggregation. Two ER chaperones, BiP and calnexin, bind to newly
synthesized class II MHC chains but do not contribute appreciably to
the large size of the aggregates. Our observations suggest that some
subunits of multisubunit complexes pass through a transient, dynamic
high molecular weight aggregate phase during the physiological process
of assembly. The results further suggest a novel role for Ii in
promoting stable dissociation of preformed aggregates containing
and
subunits rather than in preventing their formation.
(
)(1) . For most multisubunit protein
complexes, the process of achieving transport competence requires the
assembly of the various constituents by a mechanism that probably
involves ER-resident chaperones
(1, 2) . Unassembled
subunits or incomplete complexes are generally retained within the ER
and, in some cases, degraded. The quality control mechanisms that
ensure that unassembled subunits are recognized as such and retained
within the ER prior to assembly are still poorly defined.
and
, that function in
presenting to CD4
T cells antigenic peptides derived
from proteins encountered largely within the endocytic pathway
(3) . In the ER, newly synthesized
and
chains
associate with preformed homotrimers of a third transmembrane
glycoprotein called the invariant chain (Ii)
(4) to form a
nine-subunit (
Ii)
complex
(5) . Only the
complete nonameric complex is fully transport competent; complexes that
are incomplete are retained within the ER
(6) . Recent studies
have demonstrated that an ER chaperone, calnexin (p88; IP90), remains
bound to incomplete
Ii complexes
(7, 8) ;
based on other systems
(9, 10) , calnexin is likely to
play a role in retention and/or further assembly of such complexes. In
addition to partial
Ii complexes,
and
chains that
are expressed in the absence of Ii, either by virtue of Ii-targeted
gene disruption in transgenic mice or by transfection of
and
chain cDNAs without Ii into fibroblasts, exit the ER
inefficiently at best
(11, 12, 13, 14, 15, 16, 17) .
and
chains expressed in the absence
of Ii are incorporated into large, heterogeneous aggregates that are
associated with the hsp70-like chaperone, BiP
(18) . Both the
aggregation and association with BiP, a resident ER protein, may have
contributed to retention of the incompletely assembled
and
chains in the ER. These studies thus demonstrated how, in the absence
of one subunit, the remaining subunits of a complex could accumulate in
a transport-incompetent form. Several questions, however, remained
unanswered. First, it was unclear whether aggregation would only occur
under the unusual circumstances in which
and
subunits were
inappropriately expressed in the absence of Ii or whether it could also
occur to some extent when Ii was present in normal class II-expressing
cells. Second, it was not established whether aggregates represented
irreversible, aberrant products of alternative folding side reactions
or whether aggregated subunits remained competent for assembly into
normal complexes. Finally, it was unclear whether aggregation was
related in any way to the association with the ER chaperone calnexin.
and
chains exist transiently in normal class II-expressing
spleen cells and are therefore not limited to cells in which Ii is
absent or
and
chains are overexpressed. Aggregates formed
early after synthesis and disappeared over time without concomitant
degradation of chains. This finding implicates aggregation as a dynamic
intermediate phase in the assembly of mature
Ii complexes. In
addition, Ii is shown to be required for rescue of
and
chains from aggregates, indicating that Ii does not necessarily prevent
aggregation but rather promotes the stable dissociation of previously
formed aggregates.
Mice and Antibodies
Normal C57BL/6 mice
(H-2; purchased from Charles River Laboratories, Raleigh,
NC) and Ii-deficient mice bred to the C57BL/6 background (maintained at
BioQual, Rockville, MD) were maintained as described previously
(11) . Cell suspensions of freshly isolated spleens from
2-6-month-old mice were prepared by disruption in DMEM containing
10% fetal bovine serum and used immediately for metabolic labeling
experiments. Rabbit antisera specific for the cytoplasmic tails of
A
and A
chains
(19) and for murine calnexin
(8) , and monoclonal antibodies specific for the Ii cytoplasmic
tail (In-1
(20) ) and lumenal domain (P4H5
(21) ), for
H-2K
(Y3
(22) and AF6-88.5.3
(23) ),
for CD45 (M1/89.18.7.HK
(24) ), and for BiP
(25) have
been described. The calnexin-specific antiserum and the anti-BiP
monoclonal antibody were generous gifts of Drs. D. McKean (Mayo Clinic)
and D. Bole (University of Michigan), respectively.
Metabolic Labeling
Spleen cell suspensions were
washed once with DMEM, 10% fetal bovine serum and then incubated in
60-mm dishes for 60-90 min at 37 °C in 10 ml/4 spleen
equivalents of leucine-free DMEM containing 5% dialyzed fetal bovine
serum and antibiotics. Cells were harvested and then incubated with
periodic agitation at 37 °C for various times in the same medium
containing 2-3 mCi/ml [3,4,5-H]leucine
(DuPont NEN). For pulse-chase experiments, a 10-fold (v/v) excess of
ice-cold DMEM containing 10% fetal bovine serum and a 15-fold (w/v)
excess of unlabeled leucine was added to samples, cells were harvested
and divided into aliquots, and chase was continued by the addition of
warm DMEM, 10% fetal bovine serum with excess leucine. In some
experiments, methionine/cysteine-free DMEM, Tran
S-label
(ICN Radiochemicals, Irvine, CA), and excess methionine and cysteine
were substituted for the respective leucine and leucine-free reagents.
For 24-h labeling, spleen cells were cultured in methionine-free DMEM
supplemented with 10% fetal bovine serum, antibiotics, and 50 µg/ml
lipopolysaccharide containing 0.6 mg/liter unlabeled methionine and 62
µCi/ml [
S]methionine for 24 h. At the end of
the labeling/chase periods, cells were harvested and resuspended in
phosphate-buffered saline containing 20 mM N-ethylmaleimide and a mixture of protease inhibitors (see
below) for 15-20 min on ice
(18) . Cells were harvested
and either extracted immediately or frozen on dry ice.
Cell Lysis, Preclearing, and Preprecipitation with
Anti-calnexin Antibody
Detergent lysates were prepared as
described previously
(18) using lysis buffer (50 mM
Tris, 300 mM NaCl, 1% (w/v) Triton X-100) containing 20
mM iodoacetamide and a mixture of protease inhibitors (0.25
mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mM N- p-tosyl-L-lysine
chloromethyl ketone, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, 5
µg/ml pepstatin A, 5 µg/ml E-64, 10 µg/ml leupeptin, 33
µg/ml aprotinin). In some experiments, 5 mM ATP and 5
mM MgCl
were included in the lysis buffer
(26) , as indicated. Lysates were clarified by high speed
centrifugation and precleared twice with rabbit anti-mouse
immunoglobulin coupled to protein A-Sepharose, once with normal rabbit
serum coupled to Pansorbin, and once with protein A-Sepharose alone
prior to either specific immunoprecipitation or sedimentation on
sucrose gradients.
, 20
mM iodoacetamide with protease inhibitors). After preclearing,
lysates were subjected to two rounds of immunoprecipitation with
antiserum to calnexin bound to protein A-Sepharose. Immunoprecipitates
were washed 3 times with digitonin wash buffer (10 mM Tris, pH
7.4, 150 mM NaCl, 0.1% (w/v) digitonin) and then resuspended
in lysis buffer containing 1% (w/v) Triton X-100 and incubated at 25
°C for 30 min, as described previously
(7) . Unbound lysate
fractions were similarly incubated with 1% Triton X-100. Following a
final round of pre-clearing with protein A-Sepharose, eluate and
unbound material were fractionated and analyzed by sedimentation. Only
the results of fractionation of the bound and eluted material is shown;
results with the unbound fraction were not significantly different from
those obtained using lysates that had not been incubated with the
antiserum to calnexin, perhaps due to inefficient immunodepletion.
Sedimentation Velocity Analysis
Precleared lysates
(or calnexin-eluted material) (0.5 ml) were applied to the top of 12-ml
linear 5-20% (w/v) sucrose gradients, subjected to centrifugation
for 16 h at 4 °C in a SW41 rotor at 39,000 rpm (188,000
g), and fractionated as described previously
(18) .
Each of the 15 fractions collected from each gradient was sequentially
immunoprecipitated with antisera or monoclonal antibodies prebound to
protein A-Sepharose or Gammabind G Sepharose (Pharmacia Biotech Inc.).
Generally, anti-A
and anti-A
immunoprecipitations were
performed first, followed by immunoprecipitations with anti-Ii, where
appropriate. Peak fractions for the migration of H-2K
(
60 kDa with
-microglobulin) and CD45
(
220 kDa) molecules were identified by a final round of
immunoprecipitation to provide internal size standards for integral
membrane proteins. Estimates of molecular mass were calculated as
described previously
(27) .
Immunoprecipitation and SDS-PAGE
Sequential
immunoprecipitations were performed for 2 h at 4 °C with
protein A-Sepharose or Gammabind G Sepharose that had been prebound to
antisera or monoclonal antibody supernatants and washed 2 times with
wash buffer (50 mM Tris, 300 mM NaCl, 0.1% (w/v)
Triton X-100). Immunoprecipitates were washed 4
with wash
buffer and once with phosphate-buffered saline prior to elution with
SDS sample buffer at 95 °C for 10 min. SDS-PAGE was as described
previously
(28) using 13% acrylamide gels cross-linked to
GelBond with AcrylAide, and fluorography was with 1 M sodium
salicylate. Quantitation of bands was done by scanning laser
densitometry of autoradiographs or by PhosphorImager analysis on a
Molecular Dynamics system. Calculations of percent A
and A
chains present in aggregate fractions was based on densitometry from
three independent experiments.
and A
chains remained unassociated
(11) and were incorporated
into high molecular weight aggregates (HMWA) as determined by
sedimentation on sucrose density gradients (see Ref. 18 and below). In
contrast, most A
and A
chains
synthesized in the presence of Ii in spleen cells from wild-type
C57BL/6 (WT) mice were assembled into discrete complexes that migrated
as a 200-300 kDa species
(18) , as expected for a
nonameric
Ii complex of
30-35-kDa subunits
(5) . Interestingly, prolonged exposure of autoradiograms of
size-fractionated class II chains from metabolically labeled WT spleen
cells revealed a small amount of unassembled A
(Fig. 1 a, bracket) or A
(Fig. 1 b, bracket) chain in fractions that
corresponded to M
of 300,000-1,000,000.
These high molecular weight forms of A
and A
chains, although
present in much smaller amounts, were reminiscent of the HMWA observed
in spleen cells that do not express Ii.
Figure 1:
A portion of newly synthesized
A and A
chains are found in high
molecular weight aggregates in normal spleen cells. Spleen cells from
WT C57BL/6 mice were metabolically labeled for 30 min with
[
H]leucine, and detergent lysates were
fractionated by sedimentation in sucrose density gradients. Individual
gradient fractions were immunoprecipitated with antisera to the
cytoplasmic tails of A
( a) or A
( b) and
analyzed by SDS-PAGE and fluorography. As internal size standards for
integral membrane proteins, the MHC class I antigen
H-2K
-microglobulin complex (
60
kDa) and the CD45 antigen (
220 kDa) were also immunoprecipitated
from gradient fractions. Gradient fraction numbers, from top to bottom, are indicated at the bottom. The peak
fractions for the H-2K
(fractions 2 and
3) and CD45 (fraction 5) markers are indicated at the
top. The bracket indicates fractions defined as
HMWA. The migration of protein molecular weight standards
(10
M
) by SDS-PAGE is
indicated on the left, and the positions of
,
, and
Ii chains and a degradation product of Ii ( Ii`) are indicated
on the right. The
Ii complex migrates as a single
peak in fractions 4- 6 in this gradient. Bands
present in fraction 1 (and most likely some in fractions 2 and 3) represent material that did not enter or partially
entered the gradient. Fig. 1 a is an overexposure of an
autoradiogram similar to one shown in Fig. 8 of Ref.
18.
The HMWA observed in WT
cells could have represented either the product of a distinct dead-end
pathway for a minor amount of unassembled and/or misfolded A or
A
chains, or alternatively, an intermediate in the normal assembly
process for MHC class II molecules. To distinguish between these
possibilities, WT spleen cells were pulse-labeled with
[
H]leucine for only 5 min and chased for various
times prior to detergent lysis and analysis by sedimentation and
immunoprecipitation. As shown in Fig. 2, a large fraction (42.1
± 0.7%) of A
chains ( left, bracket) and a
smaller but significant fraction (10.6 ± 3.3%) of A
chains
( right, bracket) were present in HMWA after the 5-min
pulse. In contrast, less than 1% of Ii directly immunoprecipitated from
the same gradient was observed in fractions corresponding to HMWA
(Fig. 3); rather, Ii was found predominantly in fractions 4 and
5, corresponding in size to Ii trimers and partial
Ii
complexes. Similarly, mature H-2 K
and CD45, used as
molecular size markers in our experiments, were never observed in HMWA
(data not shown). These observations suggested that aggregation was an
intrinsic characteristic of unassembled class II chains.
Figure 2:
Pulse-chase analysis of aggregation and
disaggregation. Spleen cells from WT mice were pulse-labeled for 5 min
with [H]leucine and then chased with excess
unlabeled leucine for 10, 30, or 180 min at 37 °C. Lysates were
fractionated by sucrose density gradient centrifugation, individual
fractions were immunoprecipitated sequentially with antibodies to
A
( left) and A
( right), and
immunoprecipitates were separated by SDS-PAGE. Only the portion of the
gels corresponding to the relevant chains are shown; no specific bands
were visible elsewhere in the gels. The bracket indicates fractions
defined as HMWA. The positions of the
,
, and Ii chains and
of a degradation product of Ii ( Ii`) are indicated. The band
above
chain in fractions containing Ii corresponds to Iip41, the
product of an alternatively spliced form of Ii (51). The Ii observed in
fraction 6 at the 180-min chase most likely represents excess
Ii that was synthesized in the pulse and associated with A
and
A
chains synthesized during the chase.
Figure 3:
Pulse-chase analysis of the aggregation
state of Ii. Ii was isolated by immunoprecipitation from the same
gradient fractions described in Fig. 2 after sequential
immunoprecipitation of A and A
. The positions of the Ii
chain, a degradation product of Ii ( Ii`) and Iip41 are
indicated. Notice the absence of aggregated species of Ii in fractions
that contain aggregated A
and A
chains
( bracket).
With
increasing chase times, the fraction of A and A
chains in
HMWA decreased, such that by 30 min no detectable A
chains and a
small fraction (
5%) of A
chains remained aggregated
(Fig. 2). Concomitantly, there was an increase in the level of
chains detected in fractions corresponding to the mature
Ii
complex (fractions 5-7). Quantitation of direct
immunoprecipitates from lysates in multiple experiments indicated no
decrease in the amount of labeled
or
chains from the pulse
to the 30-min chase period (data not shown), suggesting that the chains
were not significantly degraded over the course of the experiment.
These data were consistent with a large fraction of the A
chains,
and perhaps A
chains, in the
Ii complexes originating
from precursor HMWAs. Similar results were obtained upon pulse-chase
and sedimentation analysis of HLA-DR
and
chains from a
HLA-DR5-expressing human B-lymphoblastoid cell line (data not shown),
suggesting that transient aggregation is a general phenomenon during
assembly of class II chains. By 180 min of chase, no A
or
A
-containing aggregates were observed, and Ii had dissociated from
most of the
Ii complexes, leaving predominantly
dimers that migrate as a discrete
60 kDa species (Fig. 2).
This is consistent with proteolysis and removal of Ii upon transport
into a late endosomal compartment, releasing Ii-free peptide-bound
dimers for transport to the cell surface
(29, 30, 31, 32) .
and A
chains from Ii-/-
spleen cells (Fig. 4) showed that while aggregates also formed
immediately, disaggregation was much less efficient than in WT cells.
Even by 180 min of chase, the vast majority of A
and A
chains
remained in HMWA, rather than in fractions expected for mature
A
A
dimers (fraction 3, co-migrating with H-2K
).
These data suggest that in the absence of Ii, newly synthesized
and
chains are incorporated into HMWA as they do in the presence
of Ii, but become arrested at the aggregate phase and fail to be
efficiently disaggregated.
Figure 4:
Aggregates containing A and A
fail to be dissociated in the absence of Ii. Spleen cells from
Ii-/- mice were pulse-labeled for 5 min with
Tran
S-label and then chased with excess unlabeled
methionine and cysteine for 30 or 180 min at 37 °C. Lysates were
fractionated by sucrose density gradient centrifugation; individual
fractions were immunoprecipitated sequentially with antibodies to
A
, A
, H-2K
and CD45; and immunoprecipitates were
separated by SDS-PAGE. The positions of
and
chains and of a
degradation product of
(
`) chain are indicated.
Identification of
` as a proteolytic fragment of A
, possibly
generated subsequent to solubilization, was by reprecipitation with
anti-A
antibodies following elution with SDS and boiling
(18).
The higher amount of chain relative
to
chain present in aggregates may be a consequence of a larger
pool of free
chains than that of free
chains. This
difference in pool sizes could be due to the fact that spleen cells
synthesize A
chains in excess of A
chains (data not shown;
the same phenomenon was noted for HLA-DR5
and
chains in a
human B lymphoblastoid cell line). In pulse-chase experiments, this
uneven rate of synthesis results in the assembly of labeled
chains with
chains synthesized prior to the pulse and of labeled
chains with
chains synthesized during the chase (results
not shown). It should also be noted that co-precipitation of aggregated
chains with
chain was observed in some experiments
( e.g. see Fig. 4) but not others ( e.g. see
Fig. 2
). Co-aggregation of
with
was reminiscent of
results in transfected fibroblasts in which the chains were
overexpressed
(18) and may reflect heterogeneity of aggregate
content (see ``Discussion'').
or A
chains under the conditions of
our experiments, spleen cells from WT mice were metabolically labeled
for 24 h prior to immunoprecipitation. Only a 78-kDa protein was
specifically co-precipitated in significant amounts with A
chain
(Fig. 5, lane4). A similar band was observed
in anti-A
chain immunoprecipitates and in anti-A
chain
immunoprecipitates from Ii-/- spleen cell lysates (data not
shown). This band corresponded to BiP/GRP78 since it co-migrated with a
protein precipitated by an anti-BiP antibody ( lane3)
and was absent in anti-A
immunoprecipitates from lysates treated
with 5 mM ATP ( lane6), consistent with the
well characterized ATP-dependent dissociation of BiP from substrate
proteins
(2, 33) . Since no other bands were observed,
it is unlikely that the large size of the HMWA is due to a contribution
from some other major bound protein.
Figure 5:
Immunoprecipitation of class II chains
from WT spleen cells labeled for 24 h. Spleen cells from WT mice were
labeled for 24 h with TranS-label. Detergent lysates were
prepared with or without 5 mM ATP ( ± ATP), and
immunoprecipitated sequentially with nonspecific rabbit serum
( NS) and then either a monoclonal antibody to BiP, an
antiserum to calnexin ( Cnx), or an antiserum to A
, as
indicated. Because of the limited availability of anti-BiP and
anti-calnexin antibodies, immunoprecipitation of BiP and calnexin was
not quantitative. The migration of protein molecular weight standards
(10
M
) by SDS-PAGE is
indicated at the left, and the positions of
,
, Ii,
BiP and calnexin are indicated at the right. Similar results
were obtained when cells were labeled for 16 h with
[
H]leucine (not shown). The lower band
co-precipitated with A
is most likely the Ii` degradation product
of Ii.
In order to assess a possible
contribution of bound BiP to the size of the HMWA, we analyzed the
sedimentation characteristics of newly synthesized A and A
chains before and after treatment of lysates with 5 mM ATP. As
shown in Fig. 6, the sedimentation of A
and A
chains
from pulse-labeled WT spleen cells was virtually unchanged by ATP
treatment. Identical results were obtained with A
and A
chains from Ii-/- spleen cells (data not shown). These data
suggested that BiP did not substantially contribute to the size of the
- and
-containing HMWA and was therefore most likely present
in substoichiometric amounts.
Figure 6:
Dissociation of BiP does not affect
migration of HMWA. Spleen cells from WT mice were metabolically labeled
with TranS-label for 5 min and extracted in lysis buffer
that did or did not contain 5 mM ATP. Lysates were
fractionated on sucrose density gradients, and fractions were
sequentially immunoprecipitated with antibodies to A
( left), A
( right), H-2K
, and CD45;
separated by SDS-PAGE; and analyzed by fluorography. Only the portion
of the gels containing relevant bands are shown. The peak fractions of
migration for CD45 and H-2K
are indicated at the
top; fraction numbers are indicated at the bottom;
the positions of the
,
, and Ii chains and a proteolytic
product of Ii ( Ii`) are indicated to the left and
right.
Two recent publications have described
the association of another ER-resident chaperone, calnexin (Ip90; p88),
with assembling MHC class II chains
(7, 8) . We did not
detect a band co-migrating with calnexin in immunoprecipitates from
long term labeled cells (Fig. 5), suggesting that calnexin was
not responsible for the large size of the HMWA. The inability to detect
bound calnexin in our system was not surprising, since the detergent
and salt conditions used during cell solubilization in our experiments
would be expected to have largely dissociated calnexin from class II
subunits
(7) .
and A
chains
that bound to calnexin under more favorable conditions. To this end, we
took advantage of the reported stability of the calnexin/class II
interaction in digitonin at 4 °C and its instability in Triton
X-100 at room temperature
(7) . Pulse-labeled proteins from WT
spleen cells were immunoprecipitated from digitonin-solubilized cells
with an anti-calnexin antiserum. Calnexin-associated proteins were then
eluted with Triton X-100 at room temperature, fractionated on sucrose
gradients, and reimmunoprecipitated with antibodies to A
or
A
. As shown in Fig. 7, the A
and A
chains that had
been bound to and eluted from calnexin were enriched in species that
sedimented even more slowly than the mature
Ii complex, as
would be expected for
or
chain monomers or small oligomers.
These species may include partially assembled
Ii complexes as
previously observed
(7) , particularly those containing
radiolabeled A
chain associated with unlabeled pre-existing A
and Ii chains, both of which are synthesized in excess in normal spleen
cells. These data suggest that calnexin interacts primarily with a pool
of monomeric class II chains or incomplete complexes. Due to the low
sensitivity of the assay, however, we cannot exclude that calnexin
binds to the aggregates to some extent.
Figure 7:
Calnexin binds primarily to low molecular
weight class II species. Spleen cells from WT mice were metabolically
labeled for 20 min with [H]leucine and then lysed
in buffer containing 1% (w/v) digitonin as described previously (7).
Following pre-clearing, lysates were immunoprecipitated with an
antiserum to calnexin, and calnexin-bound polypeptides were eluted with
1% Triton X-100 at 25 °C (7). Eluted material was fractionated on
sucrose density gradients. Fractions were sequentially
immunoprecipitated with antibodies to A
, A
, H-2K
,
and CD45, and immunoprecipitates were analyzed by SDS-PAGE and
fluorography. Relevant bands and peak fractions are
indicated.
(and perhaps
) chains pass through a transient physiological intermediate
phase, prior to assembly, in which they are present in high molecular
weight aggregates. These transient aggregates are observed in normal
spleen cells expressing typically low levels of the class II
polypeptides and thus are not a result of overexpression or a property
of transformed cells. The fact that no degradation of chains is
observed over the time course in which aggregates are dissembled favors
the argument that aggregates are a preassembly intermediate rather than
a predegradation intermediate. The eventual release of class II chains
over time demonstrates that aggregates can be dynamic complexes, the
assembly and disassembly of which must be regulated by resident ER
proteins.
or
chains, bound to substoichiometric quantities of BiP and perhaps
other ER chaperones
(15, 17) . Aggregation is likely to
occur in vivo, based on our earlier observations with
transfected COS cells. In the COS cell system, A
and A
chains
were found to aggregate together, but only when co-expressed in the
same cells; no association of aggregated A
and A
chains was
observed when cells that expressed each chain individually were mixed
prior to cell lysis. In either case, the transient aggregation observed
in our experiments minimally represents a transient tendency to
aggregate, most likely due to an immature folding state. It is not
clear whether the aggregates are products of homotypic associations
between like chains or represent an agglomeration of multiple newly
synthesized polypeptides, as would be consistent with the previously
observed co-aggregation of two misfolded proteins in vivo(34) or of unassembled subunits in vitro(35) .
Indeed, the aggregates may contain, besides A
and A
chains,
complex mixtures of additional polypeptides such as other folding
and/or assembly intermediates; the heterogeneity of these polypeptides
might render them undetectable by SDS-PAGE. This hypothesis is
consistent with the ability to co-precipitate aggregated
and
chains in some experiments, particularly those in which their
level of expression may have been elevated ( e.g. co-precipitation of excess aggregated DR
chain with DR
chain was observed consistently in an Epstein-Barr virus-transformed
human B cell line in which class II chains are relatively highly
expressed).
chain in spleen cells from Ii-/-
mice than from normal mice (data not shown), in which aggregates would
be expected to constitute a higher proportion of the steady state level
of class II chains. Based on these observations and our previous
observations in COS cells
(18) , BiP may bind to aggregated
species and function either in blocking exposed patches on the surface
of the aggregates or in promoting their dissociation
(2, 37) .
and
chains, in addition to small oligomers that may
include partially assembled
Ii complexes as described
previously
(7) . This finding is consistent with observations
for other calnexin-bound proteins
(38, 39, 40) .
We speculate that calnexin binds to free class II chains that have
dissociated from aggregates or assists in dissociation from aggregates,
playing a role in folding or stabilizing the chains in a conformation
that is permissive for assembly with cognate chains
(41) .
assembly and ER egress
(11, 12, 13, 14, 15, 16, 17, 42) ,
blocking peptide binding
(43, 44) , and transporting
dimers to a late endosomal compartment
(45, 46, 47) . The fact that aggregation of
and
chains is transient in WT cells and more stable in
Ii-/- cells suggests that Ii enhances assembly by
facilitating the stable release of
and
chains from
aggregates rather than by preventing aggregation from occurring. This
function might involve promoting proper folding
(15) or
sequestering properly folded monomers of
and
subunits. In
either case, Ii would relieve aggregation that results from an
incompletely or incorrectly folded structure of newly synthesized
chains. Alternatively, it is possible that aggregation of
and
chains occurs by interaction of partial peptide-binding domains
of individual subunits
(48) with exposed peptide-like regions
of other proteins within the ER; the ability of Ii to inhibit peptide
binding by class II molecules could thus sequester dissociated chains
away from the aggregates.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.