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Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 3, 4, 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 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 TAP-negative 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 anti-calnexin 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 anti-calreticulin antiserum
(Affinity Bioreagents, Golden, CO); R.gp48N and R.gp48C, rabbit
anti-peptide Abs to the N-terminal and C-terminal regions 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
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 x 105;
Daudi or Daudi.2m, 2 x
106; and T1, T2 and 220.B8 and its transfectants,
15 x 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 µl of
normal mouse serum and 30 µl of protein G-Sepharose (Amersham
Pharmacia, Piscataway, NJ) before immunoprecipitation with 3 µg of Ab
and 25 µl 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 µg/ml in 1% digitonin/TBS). Immunoblotting
was performed as previously described (8).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
A 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.
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C),
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). The binding of calnexin and MHC class I to TAP-tapasin complexes is mutually exclusive
The data in Fig. 1 show that calnexin is associated with the
TAP-tapasin 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.
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To analyze the kinetics with which the various components are
integrated into the class I loading complex, we performed pulse-chase
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 associated 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).
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B). 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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) 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 tapasin-specific Abs and therefore must contain TAP and
tapasin.
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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 TAP-negative cells (3, 8), indicating that
lack of Ab reactivity with such complexes is not a problem.
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, 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 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). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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.
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C). 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 molecules 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 (22) that the individual components of the
loading complex interact in multiple ways in a highly cooperative
manner. There may be transient interactions between members on the way
to assembling the complete loading complex, but if they do occur they
apparently do not survive detergent solubilization. The free class I
heavy chains that partially fold before 2m
association and/or association with the loading complex have previously
been shown to associate with calnexin and/or BiP (7, 8, 30).
After assembly of the complete loading complex, release of the
associated MHC class I- 2m dimers can be
induced by the translocation of specific peptides into the ER by the
TAP component (9). This indicates that peptide binding
promotes class I release and disassembly of the loading complex,
leaving the TAP-tapasin component available for the binding of new MHC
class I- 2m dimers. In Daudi cells, i.e., in
the absence of 2m, the TAP-tapasin complex
contains calnexin and ERp57. The presence of these components at steady
state is inconsistent with a simple role for the chaperones in assembly
of the TAP-tapasin complex. It seems likely, rather, that the
association of calnexin and ERp57 represents a transient intermediate
in a loading cycle, and that this accumulates in Daudi cells because of
the absence of 2m. We suggest, as indicated in
Fig. 7, that association of MHC class I molecules together with
calreticulin with the TAP-tapasin complex induces the release of
calnexin and further suggest that the class I molecules, in turn, are
released when appropriate peptides are translocated and bind to them.
ERp57 may dissociate and reassociate along with calnexin dissociation
and calreticulin binding or remain associated, as discussed above.
Although complexes containing class I, 2m
dimers, tapasin, calreticulin, and ERp57 can be found in TAP-negative
cells (3, 8), they are not detectable in
TAP-positive cells. The limiting component for the formation of such
complexes may be tapasin, which rapidly associates with TAP under these
circumstances.
Recent data from Howard and co-workers (10) suggests that
ATP binding and hydrolysis by the cytoplasmic domains of TAP1 and TAP2
are required for the peptide-mediated release of class I molecules from
the loading complex. Precisely how peptide translocation, peptide
binding, and release of loaded class I molecules are coordinated is
unclear. MHC class I molecules apparently not associated with TAP can
be loaded with peptides (31), and HLA-A2 molecules in
particular can bind signal sequence-derived peptides in TAP-negative
cells (32). Soluble tapasin molecules, which bind class
I-2m dimers, but do not appear to mediate the
class I-TAP interaction, nevertheless permit class I peptide loading
(14). It is also clear that although the experiments
reported here point to a key role for calnexin in the assembly of the
loading 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.
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Acknowledgments |
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Footnotes |
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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.
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.
Received for publication September 1, 2000. Accepted for publication November 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin binding to the transporters associated with antigen processing (TAP1 and TAP2). Proc. Natl. Acad. Sci. USA 91:12716.2-microglobulin complexes associate with TAP transporters before peptide binding. Nature 368:864.[Medline]
2-microglobulin, class I heavy chain conformation, and tapasin in the interactions of class I heavy chain with calreticulin and the transporter associated with antigen processing. J. Immunol. 158:2236.[Abstract]
2 microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules. J. Immunol. 142:2796.[Abstract]
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K. A. Kennedy, E. G. Gachelet, and B. Traxler Evidence for Multiple Pathways in the Assembly of the Escherichia coli Maltose Transport Complex J. Biol. Chem., August 6, 2004; 279(32): 33290 - 33297. [Abstract] [Full Text] [PDF]
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M.-E. Paquet, M. Cohen-Doyle, G. C. Shore, and D. B. Williams Bap29/31 Influences the Intracellular Traffic of MHC Class I Molecules J. Immunol., June 15, 2004; 172(12): 7548 - 7555. [Abstract] [Full Text] [PDF]
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Z. Frenkel, M. Shenkman, M. Kondratyev, and G. Z. Lederkremer Separate Roles and Different Routing of Calnexin and ERp57 in Endoplasmic Reticulum Quality Control Revealed by Interactions with Asialoglycoprotein Receptor Chains Mol. Biol. Cell, May 1, 2004; 15(5): 2133 - 2142. [Abstract] [Full Text] [PDF]
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S. Ford, A. Antoniou, G. W. Butcher, and S. J. Powis Competition for Access to the Rat Major Histocompatibility Complex Class I Peptide-loading Complex Reveals Optimization of Peptide Cargo in the Absence of Transporter Associated with Antigen Processing (TAP) Association J. Biol. Chem., April 16, 2004; 279(16): 16077 - 16082. [Abstract] [Full Text] [PDF]
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T. M. Tran, N. Satumtira, M. L. Dorris, E. May, A. Wang, E. Furuta, and J. D. Taurog HLA-B27 in Transgenic Rats Forms Disulfide-Linked Heavy Chain Oligomers and Multimers That Bind to the Chaperone BiP J. Immunol., April 15, 2004; 172(8): 5110 - 5119. [Abstract] [Full Text] [PDF]
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 A. L. Ackerman, C. Kyritsis, R. Tampe, and P. Cresswell Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens PNAS, October 28, 2003; 100(22): 12889 - 12894. [Abstract] [Full Text] [PDF]
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 H. L. Wilson and H. C. O'Neill Identification of differentially expressed genes representing dendritic cell precursors and their progeny Blood, September 1, 2003; 102(5): 1661 - 1669. [Abstract] [Full Text] [PDF]
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C. M. Radcliffe, G. Diedrich, D. J. Harvey, R. A. Dwek, P. Cresswell, and P. M. Rudd Identification of Specific Glycoforms of Major Histocompatibility Complex Class I Heavy Chains Suggests That Class I Peptide Loading Is an Adaptation of the Quality Control Pathway Involving Calreticulin and ERp57 J. Biol. Chem., November 22, 2002; 277(48): 46415 - 46423. [Abstract] [Full Text] [PDF]
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S.-J. Kang and P. Cresswell Calnexin, Calreticulin, and ERp57 Cooperate in Disulfide Bond Formation in Human CD1d Heavy Chain J. Biol. Chem., November 15, 2002; 277(47): 44838 - 44844. [Abstract] [Full Text] [PDF]
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 M.-E. Paquet and D. B. Williams Mutant MHC class I molecules define interactions between components of the peptide-loading complex Int. Immunol., April 1, 2002; 14(4): 347 - 358. [Abstract] [Full Text] [PDF]
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T. Pentcheva, E. T. Spiliotis, and M. Edidin Cutting Edge: Tapasin Is Retained in the Endoplasmic Reticulum by Dynamic Clustering and Exclusion from Endoplasmic Reticulum Exit Sites J. Immunol., February 15, 2002; 168(4): 1538 - 1541. [Abstract] [Full Text] [PDF]
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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, anti-ERp57 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 anti-calnexin serum, rabbit anti-TAP1
serum (R.RING4C), rabbit anti-calreticulin 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.
