A Role for Calnexin in the Assembly of the
MHC Class I Loading Complex in the
Endoplasmic Reticulum
This information is current as
of June 14, 2017.
Gundo Diedrich, Naveen Bangia, Mary Pan and Peter
Cresswell
J Immunol 2001; 166:1703-1709; ;
doi: 10.4049/jimmunol.166.3.1703
http://www.jimmunol.org/content/166/3/1703
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References
A Role for Calnexin in the Assembly of the MHC Class I
Loading Complex in the Endoplasmic Reticulum1
Gundo Diedrich, Naveen Bangia, Mary Pan, and Peter Cresswell2
he association of MHC class I-␤2-microglobulin (␤2m)3
dimers with peptides in the endoplasmic reticulum (ER) is
a highly regulated process involving a number of interacting components (1). The peptides are generated in the cytosol,
predominantly by proteasomal degradation, and translocated into
the ER by the TAP, a heterodimeric ATP-dependent transporter.
TAP is a component of a larger protein assembly, incorporating the
TAP1 and TAP2 subunits, the MHC-encoded glycoprotein tapasin,
the chaperone calreticulin, and the thiol oxidoreductase ERp57 as
well as the MHC class I-␤2m dimer (2–5). This assembly of proteins is often called the class I loading complex (6). Stoichiometric
analysis has suggested that each MHC class I-␤2m dimer associates with a single tapasin molecule, and that four tapasin molecules
may associate with a single TAP heterodimer (2). While the function of TAP is reasonably well understood, the roles of the additional components of the complex in MHC class I assembly are
unclear.
Before their incorporation into the loading complex, MHC class
I heavy chains can be found in association with the transmembrane
chaperone calnexin and the ER Hsp70 homologue, BiP (7). These
interactions are presumed to facilitate the initial folding of the
class I heavy chain into a form that can associate with ␤2m. After
release from these chaperones, MHC class I heavy chains are incorporated into the loading complex. The order in which the various components are introduced into the loading complex, however, is unclear. One model is that preformed TAP-tapasin
complexes act as receptors for newly assembled class I-␤2m
T
Section of Immunobiology, Howard Hughes Medical Institute, Yale University
School of Medicine, New Haven, CT 06510
Received for publication September 1, 2000. Accepted for publication November
1, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Howard Hughes Medical Institute. G.D. was supported by a Deutsche Forschungsgemeinschaft award.
2
Address correspondence and reprint requests to Dr. Peter Cresswell, Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 310
Cedar Street, New Haven, CT 06510. E-mail address: [email protected]
3
Abbreviations used in this paper: ␤2m, ␤2-microglobulin; B-LCL, B-lymphoblastoid cell line; ER, endoplasmic reticulum; TBS, 0.15 M NaCl and 0.01 M Tris, pH 7.4.
Copyright © 2001 by The American Association of Immunologists
dimers. Such complexes can exist independently of class I assembly in ␤2m-negative cell lines, which is consistent with this. However, subcomplexes containing tapasin, class I-␤2m dimers, calreticulin and ERp57 can also exist independently of TAP in TAPnegative cell lines (3, 8), suggesting an alternative model in which
tapasin binds to the class I molecule and the associated chaperones
before its association with TAP. Regardless of the order of its
assembly, however, the release of MHC class I-␤2m dimers from
the loading complex is induced when they bind peptides (9, 10).
The loaded MHC molecules satisfy the quality control criteria of
the ER and are transported to the Golgi apparatus and ultimately to
the cell surface.
Confusing the definition of the loading complex is work in the
murine system from Williams and co-workers, in which calnexin
was found to remain associated following class I-TAP interaction
(11). Conversely, others confirmed the results obtained in the human system, observing that calreticulin was a component of the
murine loading complex (12). Furthermore, although in the human
system Abs to calnexin failed to coprecipitate the other components of the loading complex, calnexin was copurified when an anti
TAP mAb was used to affinity purify the complex (2). Based on
this we speculated that calnexin could be involved in the folding
and assembly of the TAP-tapasin precursor required for generation
of the complete loading complex.
In this paper we have analyzed the order and kinetics of assembly of the various components of the class I loading complex,
investigated the interactions between them, and further investigated the role of calnexin in the assembly process.
Materials and Methods
Cells and Abs
The human cell lines HeLa M, a cervical carcinoma (13); 220.B8, a tapasin-deficient B-lymphoblastoid cell line (B-LCL), and its transfectants
(14); T1 and T2, TxB cell hybrids (15); and Daudi, a ␤2m-deficient Burkitt’s lymphoma, and the ␤2m transfectant Daudi.␤2m (8), were maintained
as previously described. The following previously described Abs were
used: 148.3, an anti-TAP.1 mAb (16); w6/32, a ␤2m-dependent anti-class
I heavy chain mAb (17); HC10, a mAb recognizing free class I heavy chain
(18); BM-63, an anti-␤2m mAb (Sigma, St. Louis, MO); AF8, an anticalnexin mAb (19); MCP21, an anti-proteasome mAb (20); R.RING4C, a
rabbit anti-peptide Ab to the C-terminal region of TAP.1 (21); rabbit anticalreticulin antiserum (Affinity Bioreagents, Golden, CO); R.gp48N and
R.gp48C, rabbit anti-peptide Abs to the N-terminal and C-terminal regions
0022-1767/01/$02.00
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Heterodimers of MHC class I glycoprotein and ␤2-microglobulin (␤2m) bind short peptides in the endoplasmic reticulum (ER).
Before peptide binding these molecules form part of a multisubunit loading complex that also contains the two subunits of the
TAP, the transmembrane glycoprotein tapasin, the soluble chaperone calreticulin, and the thiol oxidoreductase ERp57. We have
investigated the assembly of the loading complex and provide evidence that after TAP and tapasin associate with each other, the
transmembrane chaperone calnexin and ERp57 bind to the TAP-tapasin complex to generate an intermediate. These interactions
are independent of the N-linked glycan of tapasin, but require its transmembrane and/or cytoplasmic domain. This intermediate
complex binds MHC class I-␤2m dimers, an event accompanied by the loss of calnexin and the acquisition of calreticulin, generating the MHC class I loading complex. Peptide binding then induces the dissociation of MHC class I-␤2m dimers, which can
be transported to the cell surface. The Journal of Immunology, 2001, 166: 1703–1709.
1704
ASSEMBLY OF THE MHC CLASS I LOADING COMPLEX
of tapasin, respectively (8, 22); and a rabbit anti-peptide Ab against calnexin (23). The new IgG1 mAb, MaP.ERp57, was generated by immunizing
mice with recombinant ERp57 expressed in and purified from Escherichia
coli (G. Diedrich and P. Cresswell, unpublished observations), and a conventional fusion was performed using spleen cells and the myeloma cell
line, Ag.8. A rabbit antiserum recognizing ERp57, R.ERp57, was raised to
the same recombinant product. The rabbit antiserum R.SinA was generated
by immunizing rabbits with soluble tapasin expressed in and purified from
insect cells using a baculovirus expression system (G. Diedrich and P.
Cresswell, unpublished observations).
Radiolabeling, immunoprecipitation, and immunoblotting
Tapasin recruits calnexin and ERp57 to TAP-tapasin complexes
FIGURE 1. Analysis of the components of the TAP-tapasin complex in
Daudi (␤2m-negative), Daudi.␤2m, and .220. B8 cells expressing intact
(wt), N-terminally truncated (⌬300), or soluble (sol) tapasin. The indicated
cell lines were extracted in 1% Triton X-100 (A and B) or 1% digitonin
(A–C). A, The extracts were precipitated with anti-TAP1 mAb 148.3, antiERp57 mAb MaP.ERp57, or normal mouse serum as a negative control.
After separation by SDS-PAGE (10% acrylamide) and transfer to Immobilon-P membranes, the various proteins were detected with rabbit anticalnexin serum, rabbit anti-TAP1 serum (R.RING4C), rabbit anticalreticulin serum, or rabbit anti-tapasin serum (R.gp48N). B, Precipitating
Abs were R.RING4C (anti-TAP1), R.gp48C (anti-tapasin), and normal
rabbit serum as the negative control. AF8 (an anti-calnexin mAb) was used
to probe the blot. C, Precipitating Abs were Rgp48C (rabbit anti-tapasin)
and normal rabbit serum as the negative control. AF8 was again used to
probe the blot.
In the ␤2m-negative cell line Daudi the assembly of the complete
loading complex is prevented by the absence of ␤2m (8, 24). TAP
and tapasin still interact, but their association with class I heavy
chain is strongly reduced (22). To identify proteins that assist the
folding of or stabilize the TAP-tapasin complex, we isolated it
from a digitonin extract of Daudi cells on an affinity column using
mAb 148.3 as previously described (2). TAP1-associated proteins
were eluted in 1% octylglucoside, which disrupts the TAP-tapasin
interaction, and were analyzed by SDS-PAGE. Three major bands
were stained with Coomassie blue and identified by N-terminal
sequencing as calnexin, ERp57, and tapasin (data not shown). Calreticulin, which has a mobility similar to that of ERp57, was not
present, in accordance with previous results (8).
To determine which components of the TAP-tapasin complex
interact with calnexin and ERp57, Daudi cells were lysed in Triton
X-100, which disrupts the TAP-tapasin interaction, or in digitonin,
which preserves it. TAP and ERp57 were immunoprecipitated
from the extracts, and the presence of associated calnexin and tapasin as well as calreticulin was analyzed by Western blotting. The
experiment shown in Fig. 1A confirms that TAP, tapasin, ERp57,
and calnexin form a complex in Daudi cells that is stable in digitonin. Calreticulin is not a component of this complex. In Triton
X-100 lysates, calnexin was coprecipitated by an anti-ERp57 Ab
(Fig. 1A), and by an anti-tapasin Ab (Fig. 1B). The ERp57 Ab also
coprecipitated tapasin (Fig. 1A). None of these proteins was coprecipitated with a TAP1-specific Ab in the presence of Triton
X-100. The results suggest that calnexin and ERp57 directly bind
to tapasin within the TAP-tapasin complex. A similar analysis
(Fig. 1A) of Daudi.␤2m was consistent with earlier results (2, 3, 8),
which showed that, in the presence of ␤2m, calreticulin and ERp57
detectably associate with TAP-tapasin complexes in digitonin and
with tapasin in Triton X-100, consistent with coassociation of
MHC class I molecules with these complexes.
Complexes of ERp57 with either calnexin or calreticulin catalyze the folding of immature glycoproteins. It is thought that
the chaperone, rather than ERp57, provides the binding specificity by interacting with the N-linked glycans of the substrates
and that the associated ERp57 catalyzes appropriate disulfide
bond formation (25–27). There is also evidence from studies of
mouse MHC class I molecules that calnexin may interact with
transmembrane regions of membrane proteins (28). Tapasin is a
transmembrane protein that also possesses an N-linked glycan.
The influence of the N-linked glycan and the transmembrane
domain of tapasin on the interaction with calnexin was analyzed
using the tapasin-negative cell line .220.B8. We used transfectants expressing intact tapasin, soluble tapasin lacking the transmembrane domain, which is readily detectable by Western blots
in cell extracts (14) (data not shown), or a truncated tapasin
mutant lacking the N-terminal 300 aa and therefore lacking the
single N-linked glycan (22). Calnexin was associated with the
membrane-integrated truncated tapasin mutant, but did not detectably bind to soluble tapasin (Fig. 1C), suggesting that a
stable interaction requires the tapasin transmembrane domain
and that the N-linked glycan is not essential for the interaction.
Consistent with this we have found that castanospermine, which
inhibits glucosidase II and prevents the generation of monoglucosylated N-linked glycans, the substrate for calnexin, does not
inhibit the calnexin-tapasin interaction (data not shown).
Results
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HeLa M cells were induced with 200 U/ml human ␥-IFN (R&D Systems,
Minneapolis, MN) for 48 h before radiolabeling. Cells were starved in
methionine- and cysteine-free medium for 60 min, pulsed with [35S]methionine and [35S]cysteine at 1.25 mCi/ml (ICN, Costa Mesa, CA), and
chased in medium containing excess methionine and cysteine (3 mM each).
At various time points, the chase was stopped by diluting cells in ice-cold
PBS. Solubilization and immunoprecipitations were performed as previously described (14). Unless otherwise indicated, the following number of
cells were used per immunoprecipitation: HeLa M, 30 ⫻ 105; Daudi or
Daudi.␤2m, 2 ⫻ 106; and T1, T2 and 220.B8 and its transfectants, 15 ⫻
106. Cells were lysed in 0.15 M NaCl and 0.01 M Tris, pH 7.4 (TBS),
containing 1% digitonin (Roche Diagnostics, Indianapolis, IN) or 1% Triton X-100 (Sigma) for 45 min. The postnuclear supernatant was precleared
for 2 h with 3 ␮l of normal mouse serum and 30 ␮l of protein G-Sepharose
(Amersham Pharmacia, Piscataway, NJ) before immunoprecipitation with
3 ␮g of Ab and 25 ␮l of protein G-Sepharose for 60 min. Precipitated
proteins were separated by SDS-PAGE and analyzed by autoradiography.
Stripping of precipitated proteins and reimmunoprecipitation with different
Abs were performed as previously described (14). For the elution of TAP.1
from mAb148.3 or ERp57 from mAb MaP.ERp57, the immune complexes
were incubated for at least 12 h in the presence of the peptide (0.1 mM in
1% digitonin/TBS) to which the mAb was raised or in recombinant ERp57
(100 ␮g/ml in 1% digitonin/TBS). Immunoblotting was performed as previously described (8).
The Journal of Immunology
The binding of calnexin and MHC class I to TAP-tapasin
complexes is mutually exclusive
Newly synthesized class I heavy chains rapidly associate with
TAP1 and ERp57
To analyze the kinetics with which the various components are
integrated into the class I loading complex, we performed pulsechase experiments using IFN-␥-induced HeLa M cells. These cells
were used because the various components of the complex incorporate label more efficiently than in B-LCL. The cells were metabolically labeled for 3 min and chased in an excess of nonlabeled
methionine for up to 75 min. TAP- and ERp57-associated proteins
were recovered from digitonin lysates with mAbs 148.3 and
MaP.ERp57, respectively (Fig. 3). Newly synthesized tapasin and
class I heavy chains were found to associate rapidly with TAP.
About 50% of the maximal level of each protein was bound to
TAP at the beginning of the chase (0 min), and ⬎90% was asso-
FIGURE 2. Calnexin is not a component of the complete class I loading
complex. Daudi or Daudi.␤2m cells were lysed in 1% digitonin, and the
TAP complex was immunoprecipitated with the mAb 148.3. Bound complexes were eluted by competition with specific peptide and reprecipitated
with mAbs specific for ERp57 (MaP. ERp57) or ␤2m (BM-63) or with a
control mAb (MCP21). After separation by SDS-PAGE, proteins were
transferred to an Immobilon-P membrane and probed with rabbit antisera
to calnexin, ERp57 (R.ERp57), or tapasin (R.SinA).
ciated after a 15-min chase (Fig. 3D). It appears, therefore, that
both proteins rapidly fold into a conformation that allows their
association with TAP. The rates of dissociation of the different
class I heavy chain isoforms from TAP differed significantly. The
lower class I heavy chain band (HC2), which reacts predominantly
with the mAb HCA2, disappeared with a half-time of approximately 90 min, whereas the upper band (HC1), which reacts predominantly with the mAb HC10, remained stably bound for ⬎5 h
(data not shown).
The signals for calreticulin and ERp57 were weak in a precipitation with the TAP1-specific mAb because the fraction of these
proteins that is labeled is small compared with the already existing
pools of nonlabeled proteins. To enhance the signal for these proteins, a 10-fold greater number of labeled HeLa M cells was used
for the primary precipitation with the mAb 148.3, and the isolated
TAP complexes were eluted with the peptide to which the mAb
was raised. The complexes were then reprecipitated with
MaP.ERp57 (Fig. 3B). The rates of association of calreticulin and
ERp57 (unresolved on this gel) with TAP proved to be similar to
the tapasin and class I heavy chain association rates, with ⬎80%
of the proteins bound to TAP after a 15-min chase (Fig. 3D).
The interaction of class I heavy chain with ERp57 followed
kinetics similar to those of its interaction with TAP1 (Fig. 3, C and
E). However, after a 15-min chase only 60% of labeled TAP and
40% of labeled tapasin were bound to ERp57. The delayed kinetics
with which TAP and tapasin associate with ERp57, and the fact
that the TAP-tapasin interaction occurs faster than the ERp57-tapasin interaction suggest that TAP and tapasin form a precomplex
that has to fold into a specific conformation before ERp57 can be
bound. The association rates of TAP, tapasin, and class I heavy
chain with calreticulin and ␤2m were very similar to their association rates with ERp57, i.e., the binding of TAP and tapasin was
delayed compared with the binding of class I heavy chain (data not
shown).
Class I heavy chain and ERp57 do not associate detectably in
the absence of tapasin
The observation that newly synthesized class I heavy chains associate faster with ERp57 than with newly synthesized TAP and
tapasin could be explained in two ways. First, class I heavy chain
could interact with ERp57 independently of its association with the
TAP-tapasin precomplex. Alternatively, the ERp57-class I heavy
chain complex seen in the early time points of the chase period
(when no or weak bands for TAP and tapasin are observed) could
contain unlabeled TAP and tapasin. To address this question we
looked for an ERp57-class I heavy chain interaction in TAP- and
tapasin-negative cells. An interaction between ERp57 and class I
heavy chain in a TAP-negative cell line was previously reported
(3, 4). The existence of an ERp57-class I heavy chain complex in
tapasin-negative cells is controversial. Lindquist et al. (4) described such a complex, whereas our laboratory failed to detect it
(3). We used MaP.ERp57 (or a polyclonal antiserum, R.ERp57;
data not shown) to precipitate ERp57 from digitonin extracts of
metabolically labeled tapasin-negative cell lines, i.e., 220.B8,
220.B27 (Fig. 4), 220.A2, and 220.B44 (data not shown), and
looked for coprecipitation of class I heavy chain. We could not
detect any interaction between class I heavy chain and ERp57 in
the absence of tapasin, whereas the interaction was easily detectable in the tapasin-transfectant 220.B8.tapasin (Fig. 4). Farmery et
al. (29), using an in vitro translation system, observed that ERp57
interacts with class I heavy chains before complete oxidation of
disulfide bonds. This may be difficult to observe in a conventional
pulse-chase experiment such as that employed here.
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The data in Fig. 1 show that calnexin is associated with the TAPtapasin complex in Daudi cells. It is also clear that calnexin still
can be found in association with TAP when ␤2m is introduced into
Daudi or when tapasin is transfected into .220.B8 cells. These data
and the earlier demonstration that calnexin copurified with the
TAP complex from normal B-LCL (2) could be explained if a
mixture of TAP-tapasin complexes, some containing MHC class
I-␤2m dimers and some lacking class I but containing calnexin,
were present in the purified material. To test this hypothesis,
TAP1-containing complexes were affinity isolated from digitonin
extracts of Daudi or Daudi.␤2m cells using the mAb 148.3. After
release from the mAb by competitive peptide elution, the complexes were reprecipitated with mAbs specific for ERp57, ␤2m or,
as a negative control, the proteasome. The ERp57-specific mAb
coprecipitated calnexin and tapasin from both Daudi-derived and
Daudi.␤2m-derived TAP complexes, as analyzed by Western blotting (Fig. 2). The ␤2m-specific mAb coprecipitated ERp57 and
tapasin only from Daudi.␤2m cells, as expected. However calnexin
was not coprecipitated with ␤2m even from Daudi.␤2m-derived
TAP complexes. Similar data were obtained using TAP complexes
purified from HeLa M cells (not shown). Assuming that binding of
TAP-associated class I-␤2m dimers with the ␤2m-specific Ab is
not affected by calnexin association, this suggests that two kinds
of TAP-tapasin complexes exist in ␤2m-expressing cells. One is
calnexin free and contains class I molecules, and one contains
calnexin and may correspond to complexes either in the process of
folding and assembly or after dissociation of MHC molecules.
1705
1706
ASSEMBLY OF THE MHC CLASS I LOADING COMPLEX
The data suggest that all complexes we observed in the pulsechase experiments that contain ERp57 and class I heavy chain
must also contain TAP and tapasin. At the early time points nonlabeled TAP and tapasin predominate in the complexes, while at
later time points labeled TAP and tapasin are incorporated. To
verify this assumption we wanted to determine whether all the
ERp57-associated class I heavy chain could be precipitated with
Abs to TAP or tapasin. The pulse-chase experiment and the subsequent precipitation with the ERp57-specific mAb were repeated,
but instead of analyzing the precipitated complexes directly, they
were first eluted from the mAb with recombinant ERp57. The
complexes were then reprecipitated with mAb 148.3 (against
TAP1; Fig. 5) or with a rabbit antiserum against tapasin (R.gp48C;
data not shown). All complexes between ERp57 and class I heavy
chain recognized by MaP.ERp57 in the primary immunoprecipitation (Fig. 3C) were also recognized by the TAP1- and tapasinspecific Abs and therefore must contain TAP and tapasin.
FIGURE 4. The interaction between class I heavy chain and ERp57 is
tapasin dependent. The tapasin-deficient cell lines 220.B8 and 220.B27 and
the tapasin transfectant 220.B8.tapasin were radiolabeled for 15 min and
lysed in 1% digitonin, and the extracts were immunoprecipitated with
mAbs against class I heavy chain (HC10) or ERp57 (MaP.ERp57). When
indicated, ERp57-associated proteins were eluted in SDS and DTT, and
class I heavy chains (HC) were reprecipitated with HC10 or with normal
mouse serum (ctrl) as a negative control. Isolated proteins were separated
by SDS-PAGE and detected by fluorography.
Class I heavy chain does not form TAP-independent complexes
with ERp57, calreticulin, or tapasin in wild-type cells
We took a more general approach to look for TAP-independent
and ERp57-independent complexes that might contain class I
heavy chain. HeLa M cells were labeled for 3 min and chased for
either 7 min (Fig. 6, A–C) or 75 min (Fig. 6, D and E). The digitonin lysates were precleared with the TAP1-specific mAb 148.3
(Fig. 6, B and D) or with MaP.ERp57 (Fig. 6, C and E). To confirm
the quantitative removal of TAP1-containing or ERp57-containing
complexes, the precleared lysates were reprecipitated with the
mAbs used for preclearing: no residual coprecipitated class I heavy
chain or other bands were detected. Immunoprecipitations of the
precleared lysates with mAb w6/32, which recognizes heavy
chain-␤2m dimers that are not bound to TAP, confirmed that only
TAP-associated or ERp57-associated proteins had been removed,
since the intensities of the w6/32-precipitated proteins did not significantly change. Immunoprecipitations of the 148.3-precleared
lysate with Abs against the individual components of the loading
complex demonstrated that class I heavy chains were only precipitated by the ␤2m-specific Ab (Fig. 6, B–E). Thus, except for the
mature class I molecules recognized by the ␤2m antiserum and
those in the loading complex itself, no additional class I-containing
complexes were found. If class I-containing complexes involving
calreticulin, ERp57, or tapasin but lacking TAP exist, they must be
much less abundant than the complete loading complex. Complexes containing these components are readily detectable in TAPnegative cells (3, 8), indicating that lack of Ab reactivity with such
complexes is not a problem.
In Fig. 6, the amount of ERp57 and calreticulin present in the
lysate after the 148.3-preclear did not decrease significantly, indicating that only a small fraction of the cellular pool of these proteins is associated with TAP. Because these molecules are housekeeping proteins, this is not unexpected. In contrast, tapasin was
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FIGURE 3. Newly synthesized class I heavy chain appears to associate faster than newly synthesized TAP and tapasin with ERp57. IFN-␥-induced HeLa
M cells were labeled with [35S]methionine for 3 min and chased for the indicated times. Extracts in 1% digitonin were immunoprecipitated with mAb 148.3
(TAP1 specific; A and B) or MaP.ERp57 (C), and the immunoprecipitates were separated by SDS-PAGE. B, The TAP complexes were eluted by specific
peptide and reprecipitated with MaP.ERp57 before SDS-PAGE analysis. The intensities of the precipitated proteins were quantitated by image analysis (D
and E). Values are the average of at least three independent experiments.
The Journal of Immunology
1707
FIGURE 5. All complexes of class I heavy chain
and ERp57 contain TAP1. A, Lysates of HeLa M
cells in 1% digitonin were immunoprecipitated with
the mAb MaP.ERp57. Isolated complexes were
competitively eluted with recombinant ERp57, reprecipitated with the anti TAP1 mAb 148.3, and
subjected to SDS-PAGE. B, The intensities of the
class I heavy chain bands were quantitated by image
analysis. The ERp57-associated HC data are taken
from Fig. 3C. Values are the average of two independent experiments.
ecules assemble. This involves the association of the class I heavy
chain, ␤2m, and calreticulin with the TAP-tapasin complex and the
loss of calnexin. The association of calnexin and class I molecules
with TAP appears to be mutually exclusive (Fig. 2). ERp57 is still
found in the complex after class I association, but whether new
ERp57 molecules associate together with newly introduced calreticulin molecules or whether the ERp57 shifts from a calnexin
interaction to a calreticulin interaction within the complex is unclear. No class I heavy chains associated with other individual
components of the complex, except ␤2m, can be detected during
assembly (Fig. 6). This is consistent with our previous suggestion
Discussion
The data presented here argue for an assembly pathway for the
class I loading complex as schematized in Fig. 7. The first step
involves the assembly of a complex containing TAP, tapasin, calnexin, and ERp57. The TAP.1 and TAP.2 subunits first associate
with tapasin. Calnexin together with ERp57 then associates with
the TAP-tapasin complex. Tapasin is required for the interaction of
ERp57 with the complex (3), and differential detergent solubilization experiments indicate that tapasin is responsible for both
ERp57 and calnexin association with the TAP-tapasin complex
(Fig. 1B). The interaction does not require the N-linked glycan of
tapasin based on studies with tapasin mutants (Fig. 1C). Soluble
tapasin failed to detectably associate with calnexin, while a deletion mutant lacking the N-linked glycan did associate. Such a mutant could theoretically bind calnexin as a result of aggregation in
the ER. However, we also found that castanospermine, which inhibits the enzyme glucosidase II and prevents generation of the
monoglucosylated N-linked glycan substrate for calnexin, failed to
inhibit the interaction of calnexin with the TAP-tapasin complex
(data not shown). These data also argue that the N-linked glycan is
not required for the interaction.
Association of the TAP subunits with each other or with tapasin
does not appear to require ERp57, as kinetic experiments suggest
that TAP-tapasin association is detectable before their interaction
with ERp57 (Fig. 6C). Whether there is an association with calnexin independent of ERp57 is difficult to determine, because no
cell lines expressing calnexin but lacking ERp57 exist, and none of
the Abs to calnexin we have used react with the chaperone while
it is associated with the TAP-tapasin complex (8). Both calnexin
and ERp57 can readily be found associated with the TAP-tapasin
complex in the cell line Daudi (Fig. 1), where the absence of ␤2m
prevents the strong association of MHC class I molecules (22).
The TAP-tapasin complex containing calnexin and ERp57 appears to serve as the scaffold on which empty MHC class I mol-
FIGURE 6. Class I heavy chain does not form TAP1-independent complexes with ERp57, calreticulin, or tapasin in wild-type cells. IFN-␥- induced HeLa M cells were labeled with [35S]methionine for 3 min and
chased for either 7 min (A–C) or 75 min (D and E). Extracts in 1% digitonin were precleared with normal rabbit serum and divided into three
samples, which were either directly used for immunoprecipitation (A) or
were extensively precleared with 148.3 (TAP1-specific; B and D) or
MaP.ERp57 (C and E) coupled to Sepharose beads. The latter samples
were then immunoprecipitated with mAbs against TAP1 (148.3), ERp57
(MaP.ERp57), calreticulin (rabbit antiserum), tapasin (R.gp48c), ␤2m (rabbit antiserum), or MHC class I-␤2m dimers (w6/32). The immunoprecipitates were analyzed by SDS-PAGE followed by fluorography.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
quantitatively removed after the 148.3-preclear (Fig. 6, B and D),
suggesting that tapasin rapidly associates with TAP after synthesis
and that there is no pool of free tapasin. Preclearing with the
ERp57-specific mAb after a long chase removed the signal for
class I heavy chain in subsequent immunoprecipitations with Abs
against TAP1, calreticulin, or tapasin, indicating that there is no
significant pool of class I heavy chain bound to TAP, calreticulin,
or tapasin that does not contain ERp57 (Fig. 6, C and E). However,
after a 7-min chase, tapasin could still be precipitated with a
TAP1-specific mAb after the ERp57-preclear (Fig. 6C), supporting
the conclusion from the pulse-chase experiments that TAP and
tapasin form a precomplex without ERp57. At later times residual
tapasin was not precipitated with the TAP.1-specific Ab after the
ERp57 preclear, indicating that the labeled tapasin was now incorporated into complete ERp57-containing loading complexes
(Fig. 6E).
1708
ASSEMBLY OF THE MHC CLASS I LOADING COMPLEX
FIGURE 7. Model for the order of assembly of the
MHC class I loading complex
complex, MHC class I peptide loading can occur independently of
calnexin (33, 34). The mechanisms that regulate MHC class I-peptide association and release from TAP-tapasin complexes after the
loading complex is formed, and even the requirement for its formation, are far from clear.
Acknowledgments
We thank Dr. Tobias Dick for critically reading the manuscript and Nancy
Dometios for its preparation.
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