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INTRODUCTION |
Major histocompatibility complex
(MHC)1 class I molecules
present antigenic peptides to CD8+ T cells (1, 2). The majority of
peptides found associated with class I molecules are derived from
nuclear and cytosolic proteins, and they are generated largely by the
proteasome complex (3, 4). Peptides are transported from the cytosol
into the lumen of the endoplasmic reticulum (ER) by a peptide
transporter, which is known as the transporter associated with antigen
processing (TAP) (3, 5). Both proteins contain a hydrophobic
multimembrane spanning domain and a cytosolic nucleotide binding
domain. Comparing with other members of the ABC-transporter proteins,
which have 2 ATP binding sites and 12-16 transmembrane segments (3),
it was predicted that TAP1/2 functions as a plausible heterodimeric
complex. The requirement of TAP1/2 for peptide transport across the ER
membrane was elucidated by different assay systems (3-6). These
studies have shown that TAP preferentially transports peptides of 8-15
residues in an ATP-dependent fashion. Peptide translocation
occurs in two steps involving ATP-independent peptide binding to TAP
and peptide translocation across the ER membrane, which is ATP
dependent (7-11). In addition to functioning as a peptide transporter,
a physical association between TAP1 and class I heavy chain
(HC)/
2-microglobulin (
2m) dimer has been
demonstrated (12, 13). Because the binding of class I
HC/
2m to TAP1 is not required for the peptide
translocation, the binding of peptides to class I molecules is thought
to be facilitated by association of assembled MHC class I
HC/
2m heterodimers with the TAP complex (12-14).
Two findings suggested that TAP is not only required for peptide
transport across the ER membrane but is also required for the assembly
of peptide and MHC class I HC/
2m complex. A first finding came from the study of mutant HLA-A2.1 with a point mutation of
threonine 134 to lysine (T134K) (15, 16). This mutation makes the heavy
chain incapable of interacting with the TAP complex. This results in
decreased cell surface expression of HLA-A2.1, as well as the loss of
capacity of newly synthesized class I HC/
2m complex to
load peptide in a TAP-dependent manner (15, 16). Moreover,
direct delivery of peptide to the ER in a TAP-independent manner
restores the ability of T134K, HLA-A2.1 to present antigenic peptide to
peptide-specific CTL (15). In a human mutant cell line, .220, it has
been found that MHC class I fails to associate with TAP (17). In this
cell line, TAP1/2 are normally expressed and peptide transport into the
ER is as effective as wild type cells. The MHC class I
HC-
2m dimer in the cell appeared to lack associated
peptides (17). This finding suggests that interaction of MHC class I
HC-
2m dimer with TAP is required for the association of
peptides to class I (17) indicating the involvement of TAP in the
assembly of peptide and class I HC/
2m.
With anti-TAP1 antiserum, several proteins were coprecipitated with
TAP1/2 (13, 18). One of the coprecipitates was identified as a 48-kDa
glycoprotein (tapasin) and was found in complex with TAP1/2,
calreticulin, and MHC class I (19). Tapasin is a type I transmembrane
glycoprotein with a double lysine motif that mediates the retrieval of
proteins back from the cis-Golgi and thus maintains membrane
proteins in the ER (20, 21). Analysis of .220 cells showed that they
lacked the expression of tapasin (19). Transfection of tapasin into
.220 cells restored class I-TAP association and association of peptide
with MHC class I (21). These pieces of evidence indicate that tapasin
mediates the interaction of MHC class I HC-
2m with TAP,
and this interaction is essential for peptide loading onto MHC class I
HC-
2m. Moreover, the stability of the complex of tapasin
and TAP1/2 was analyzed in comparison with the association of MHC class
I and TAP in both normal and
2m-deficient cells (20).
Tapasin was stably present in immunoprecipitates in roughly
stoichiometric amounts with TAP1/2. However, MHC class I was rapidly
dissociated from TAP (20). A similar stable association of tapasin with
TAP1/2 was also obtained in
2m-deficient cells (20).
This indicates that TAP1, -2, and tapasin form a trimeric complex.
Tapasin serves as a docking site on the TAP complex specific for
interaction with MHC class I HC-
2m. Interaction of
tapasin with peptide in the presence of ATP suggested the function of tapasin in the loading of peptide onto MHC class I (20). More recently,
studies with transfection of soluble tapasin into .220 cells indicated
that association of tapasin with MHC class I is sufficient to
facilitate peptide loading and assembly of MHC class I molecules (22).
The lack of evidence for a tapasin analogue in mouse has suggested a
direct interaction of mouse MHC class I with TAP (23).
We have now cloned the mouse homologue of tapasin and analyzed the
interaction of mouse tapasin and TAP1/2 as well as peptide binding in
both wild type and TAP2 mutant cells. Mouse tapasin is similar in both
function and amino acid sequence to the human homologue. Interaction of
tapasin with TAP1 and MHC class I is independent of TAP2. Tapasin
associates with peptide-bound TAP1/2 and MHC class I, indicative of a
functional difference between tapasin and calreticulin.
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MATERIALS AND METHODS |
Cells--
RMA and RMA-S cell lines were kindly provided by
Professor Klas Kärre (Karolinska Institute, Stockholm, Sweden).
The cell lines were cultured in RPMI 1640 medium (Life Technologies,
Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM
glutamine, at 37 °C in a 5% CO2 atmosphere.
Antibodies--
The conformational specific antibody to
H-2Kb (Y3) was obtained from American Type Culture
Collection (Manassas, VA). Rabbit antiserum against mouse TAP1 was
kindly provided by Dr. Y. Yang (The R.W. Johnson Pharmaceutical
Research Institute, San Diego). Anti-calreticulin antiserum was
obtained from Affinity Bioreagents, New Jersey. The anti-mouse tapasin
antiserum was generated by immunization with a peptide
(CATAASLTIPRNSKKSQ) derived from the C terminus of mouse tapasin. The
antibodies were affinity-purified before use.
Metabolic Labeling, Immunoprecipitation, and
Immunoblotting--
Cells were washed twice with phosphate-buffered
saline and incubated for 15 min at 37 °C in methionine-free RPMI
1640 medium containing 3% dialyzed fetal bovine serum. Then, 0.2 mCi/ml of [35S]methionine (Amersham Pharmacia Biotech)
was added, and the incubation was continued for 60 min. At the end of
labeling, cells were washed three times with ice-cold
phosphate-buffered saline and lysed in 1% digitonin (repurified from
digitonin purchased from Sigma) lysis buffer containing 0.15 M NaCl, 25 mM Tris-HCl, pH 7.5, 1.5 mg/ml
iodoacetamide, and a mixture of protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 30 mg/ml aprotinin, 10 mg/ml pepstatin). The cleared lysates were added to antibodies previously bound to protein-A-Sepharose beads (Amersham Pharmacia Biotech). After washing, the immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis as described previously (20).
For immunoblotting, aliquots of cell lysates were loaded onto a 10%
SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto
a nitrocellulose filter, which was probed with the anti-tapasin
antiserum at a dilution of 1:1,000. Detection was performed according
to Kaltoft et al. (24).
cDNA Cloning of Mouse Tapasin--
A cDNA library
constructed into a
gt10 phage vector (CLONTECH)
from poly(A)-selected RNA prepared from mouse liver was screened with a
probe prepared from cDNA of human tapasin. Twenty positive plaques
contained overlapping inserts. Ten of these inserts were sequenced and
reconstructed in a 1.8-kilobase pair fragment into a pGM3f(-) vector
under T7 promoter. The nucleotide sequence reported in this paper has
been submitted to the EMBL GenBankTM with accession number
AF106278.
Peptides and Peptide Modification--
All peptides were
synthesized in a peptide synthesizer (Applied Biosystems, Model 431A),
using conventional Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Peptides were subsequently purified by high pressure liquid
chromatography, and dissolved in phosphate-buffered saline. The
H-2Kb binding peptide of OVA peptide (residues 257-264,
SIINFEKL), was covalently modified by coupling a phenyl azide with a
nitro group to the e-amino group of lysine (position 7) to allow for photoactivation and by substitution of the isoleucine at position 3 with tyrosine to allow for iodination as described previously (10, 11).
An aliquot (100 ng) of the peptide was labeled by chloramine
T-catalyzed iodination (125I). The modification and
labeling experiments were performed in the dark. The modified peptide
is referred to as 125I-OVA-ANB-NOS.
Preparation of Microsomes and Photo-crosslinking--
Microsomes
from RMA or RMA-S cells were prepared and purified according to Saraste
et al. (25). For photo-crosslinking, 125I-OVA-ANB-NOS peptide was mixed with 20 µl of
microsomes (concentration of 60 A280/ml) to the final concentration of
100 nM, in RM buffer (250 mM sucrose, 50 mM triethanolamine-HCl, 50 mM KOAc, 2 mM MgOAc2, 1 mM dithiothreitol). This mixture
was then kept for 10 min at 26 °C. UV irradiation was subsequently
carried out for 5 min on ice at 366 nm. Microsomal membranes were
recovered by centrifugation through a 0.5 M sucrose cushion
in RM buffer containing 1 mM cold peptide (unlabeled
peptide without ANB-NOS modification). The microsomal membranes were
washed once with cold RM buffer, lysed by 1% digitonin, and subjected
to immunoprecipitation. Crosslinked microsomal proteins were
immunoprecipitated with specific antiserum and analyzed by
SDS-polyacrylamide gel electrophoresis. Crosslinking reactions with 1 mM ATP were performed as described previously (10, 11). For
peptide competition, 100 nM of the
125I-OVA-ANB-NOS peptide was mixed with a 10-fold molar
excess of unlabeled and unmodified OVA peptide before the crosslinking reaction.
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RESULTS |
cDNA Cloning of Mouse Tapasin--
To obtain the mouse
tapasin, a mouse liver cDNA library was screened with human tapasin
cDNA as a probe. Ten positive clones were sequenced. A full-length
clone with a 1.8-kilobase pair insert accommodated an open reading
frame encoding a polypeptide of 465 amino acids as compared with the
448 amino acids of human tapasin (Fig.
1). Amino acid sequence comparison of
mouse and human tapasin showed 78% identity including identical
consensus sequences of signal peptide, N-linked
glycosylation site and double lysine motif at the C-terminal end (Fig.
1). Both polypeptides showed the same hydrophobic profile and a similar
amino acid long hydrophobic stretch of amino acids, (position 395 to
427 in human and position 402 to 427 in mouse tapasin) predicated to be
a transmembrane domain (Fig. 1). Despite the close homology in most
parts of mouse and human tapasin, significant differences in the
predicted cytosolic domain were revealed. Sequence identity between
mouse and human cytosolic domains was less than 50% (Fig. 1). In
addition, mouse tapasin has a longer cytosolic domain, with 35 amino
acids compared with 21 amino acids of the human homologue (Fig. 1).
This may indicate the species specificity of the interaction of tapasin with MHC class I or TAP1/2.

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Fig. 1.
Comparison of the deduced amino acid
sequences of mouse- and human-tapasin. Mouse and human sequences
share 78% identity. The predicted N-terminal signal sequence
(continuous lines), and the predicted C-terminal
transmembrane domain (discontinuous lines) are indicated.
The single N-glycosylation site is marked in
bold, as is the double lysine motif toward the C
terminus.
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Interaction of Tapasin with TAP1 Is
TAP2-independent--
Previously, we have shown that human tapasin is
a subunit of the TAP complex demonstrated by stable association of
tapasin and TAP1/2 in stoichiometric amounts even after a 3 h
chase (20). To further characterize the properties of mouse tapasin, we
prepared an anti-mouse tapasin antiserum specific for the C-terminal
end (see "Materials and Methods"). The antiserum was specific for mouse tapasin and did not bind to human tapasin (Fig.
2). After metabolic labeling, RMA and
RMA-S cells were lysed by digitonin and precipitated with anti-mouse
tapasin antiserum. Analogously to antiserum against human tapasin,
anti-mouse tapasin coprecipitated both TAP1/2 and MHC class I of RMA
cells (Fig. 3, lane 1). From RMA-S cells, anti-tapasin recovered tapasin, TAP1, and MHC class I
(Fig. 3, lane 2). Because RMA-S has a mutation of TAP2
causing premature termination of the TAP2 protein (26), the
coprecipitation of tapasin and TAP1 from RMA-S indicates direct
association of tapasin with TAP1. This finding is in line with the
previous observation that interaction of MHC class I with TAP was
TAP2-independent (12, 13). In addition to TAP1/2 and MHC class I,
anti-tapasin antiserum precipitated a 65-kDa protein in RMA cells (Fig.
3, lane 1). The nature of this molecule has not yet been
identified.

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Fig. 2.
Specificity of anti-mouse tapasin
antiserum. Lysates of RMA or LCL cells were solubilized in
SDS-loading buffer. Proteins were separated on an SDS gel and
transferred onto a nitrocellulose filter, which was probed with the
anti-mouse tapasin antiserum. The position of mouse tapasin is
indicated. The C-terminal antigenic peptide competed the binding of the
antiserum (lane 3).
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Fig. 3.
Association of tapasin with TAP1 and MHC
class I in RMA-S cells. RMA (lane 1) and RMA-S cells
(lane 2) were labeled for 1 h with
[35S]methionine and lysed with 1% digitonin. Aliquots of
the lysates were immunoprecipitated with anti-tapasin antiserum. The
positions of TAP1, TAP2, tapasin, and MHC class I HC are
indicated.
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Association of Tapasin with Peptide-bound Class I--
It has been
reported that the MHC class I HC-
2m dimer interacts with
both calreticulin and tapasin (19). In the absence of tapasin, assembly
of peptide with MHC class I was defective, which is indicative of
involvement of tapasin, but not calreticulin, in the process of peptide
loading (19). To clarify whether binding of peptides can bring the
interaction of tapasin, TAP1/2, and MHC class I into effect, we
investigated the association of tapasin with class I and/or TAP1/2 in
the presence of peptides. As described previously, we used a
crosslinker modified and 125I-labeled H-2Kb
binding OVA peptide (residues 257-264, SIINFEKL) with a substitution of isoleucine at position 3 with a tyrosine to allow iodination (11).
This modified peptide is referred to as 125I-OVA-ANB-NOS.
For analysis of interaction of tapasin to peptide-bound MHC class I and
TAP1/2, the 125I-OVA-ANB-NOS was incubated with microsomes
purified from RMA cells in the absence or presence of ATP. This will
allow the binding of peptides to TAP1/2 and/or MHC class I (11). After
crosslinking, the microsomes were washed and lysed by 1% digitonin
lysis buffer. The clear lysates were precipitated with anti-TAP1 and
anti-tapasin antiserum, respectively. In the absence of ATP,
peptide-bound TAP1/2, but not MHC class I, was recovered by both
anti-TAP1 and anti-tapasin antisera from RMA microsomes (Fig.
4, lanes 3 and 4).
In the presence of ATP, both anti-TAP1 and anti-tapasin precipitated peptide-bound TAP1/2 and MHC class I (Fig. 4, lanes 1 and
2). In RMA-S microsomes, neither peptide-bound TAP1/2 nor
MHC class I were detected by anti-TAP1 and anti-tapasin in the presence of ATP (Fig. 4, lanes 6 and 7), suggesting a
defective peptide transport in RMA-S microsomes. The evidence of
tapasin binding to peptide-loaded MHC class I may indicate that peptide
transport and peptide loading are very transient processes and that
tapasin directly facilitates peptide loading onto MHC class I. The
specificity of peptide binding was indicated by competition of cold
peptide for the binding of 125I-OVA-ANB-NOS (Fig. 4,
lane 5).

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Fig. 4.
Association of tapasin with peptide-bound
TAP1/2 and MHC class I. The 125I-OVA-ANB-NOS peptide
was mixed with microsomes purified from RMA (lanes
1-5) or RMA-S cells (lanes 6 and
7) in the presence (lanes 1, 2,
5, 6, and 7) or absence (lanes
3 and 4) of ATP. In lane 5, the
125I-OVA-ANB-NOS peptide was mixed with a 10-fold molar
excess of unlabeled and unmodified native OVA peptide. The mixtures
were incubated for 10 min at 26 °C and then transferred to ice and
exposed to UV light for 5 min to allow for crosslinking. After
crosslinking, microsomes were collected by centrifugation and lysed in
1% digitonin buffer. The cleared lysates were precipitated with
anti-TAP1 (lanes 2, 4, 5, and
7) or anti-tapasin (lanes 1, 3, and
6) antiserum. The immunoprecipitates were analyzed on an SDS
gel. Positions of TAP1/2 and MHC class I HC are indicated.
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Dissociation of Peptide-bound MHC Class I from
Tapasin--
Earlier, we have shown (20) that tapasin binds
constitutively to TAP1/2 but transiently to MHC class I. To explore the
kinetics of the interaction between peptide-loaded MHC class I and
tapasin, 125I-OVA-ANB-NOS peptide was mixed with RMA
microsomes in the presence of ATP. After incubation for 15 min at
25 °C, the free peptides were washed off, and microsomes were
resuspended in ATP containing RM buffer (see "Materials and
Methods"). The resuspended microsomes were incubated for 0 or 15 min
at 25 °C (Fig. 5). After incubation, the microsomes were pelleted again and lysed with 1% digitonin buffer.
The cleared lysates were precipitated with antibody to MHC class I (Y3)
(Fig. 5, lane 2) or tapasin (Fig. 5, lanes 3 and
4). The 15 min chase resulted in dissociation of
peptide-bound MHC class I from tapasin (Fig. 5, lane 3).
These results demonstrated that MHC class I molecules were loaded with
peptides when they were part of the TAP complex and then released from
this complex.

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Fig. 5.
Dissociation of peptide-bound MHC class I
from TAP complexes. The 125I-OVA-ANB-NOS peptide was
mixed with purified microsomes from RMA cells and incubated for 10 min
at 25 °C in the presence of ATP. After incubation, microsomal
membranes were pelleted and free peptides were washed off. The pellets
were resuspended in RM buffer in the presence of ATP for 0 (lanes
1 and 4) or 15 min (lanes 2 and
3) at 25 °C. The membranes were pelleted again and
crosslinked (see Fig. 4 legend). Cleared lysates were
immunoprecipitated with conformation specific antibody (Y3) (lane
2) or anti-tapasin (lanes 3 and 4),
respectively. Precipitation with normal serum (lane 1)
served as a control. The precipitates were then analyzed by
SDS-polyacrylamide gel electrophoresis. Positions of TAP1/2, tapasin,
and MHC class I are indicated.
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Calreticulin Interacts Only with Peptide-free Class I--
Both
calreticulin and tapasin bind to MHC class I HC-
2m dimer
(19). Moreover, a complex of calreticulin, tapasin, TAP1/2, and MHC
class I was also found (19, 21). For investigation of binding of
calreticulin to peptide-loaded MHC class I,
125I-OVA-ANB-NOS was incubated with microsomes derived from
RMA cells in the presence of ATP. After crosslinking, microsomes were
lysed with 1% digitonin and precipitated with anti-calreticulin
antibody or anti-tapasin antiserum. The anti-tapasin readily recovered peptide-bound MHC class I and TAP1/2 (Fig.
6B, lane 8). Anti-calreticulin did not precipitate peptide associated MHC class I, nor peptide-bound TAP1/2 (Fig. 6B, lane 7). After first run precipitation, the
supernatant precipitated with anti-tapasin was reprecipitated with
anti-calreticulin, and the supernatant precipitated with
anti-calreticulin was reprecipitated with anti-tapasin. The results
showed again that peptide-bound MHC class I interacted with tapasin but
not calreticulin (Fig. 6B, lanes 3 and 4). To
confirm the binding of calreticulin to peptide-free MHC class I, RMA
and RMA-S cells were metabolically labeled and lysed with 1%
digitonin. The cleared lysates were precipitated with anti-calreticulin
antibody. A significant amount of MHC class I molecules was
coprecipitated with calreticulin from both cell lines (Fig. 6A,
lanes 1 and 2), which suggested that calreticulin
interacted with peptide-free MHC class I molecules.

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Fig. 6.
Calreticulin associates with peptide-free MHC
class I. For detection of association of calreticulin with MHC
class I, RMA (panel A, lane 1) and
RMA-S cells (panel A, lane 2) were labeled for
1 h with [35S]methionine and lysed with 1%
digitonin. Aliquots of the lysates were immunoprecipitated with
anti-calreticulin antiserum. The positions of calreticulin and MHC
class I HC are indicated. For detection of interaction of calreticulin
and tapasin with peptide-bound MHC class I, the
125I-OVA-ANB-NOS peptide was mixed with microsomes purified
from RMA in the presence of ATP (panel B, lanes
3-8). The mixtures were incubated for 10 min at
26 °C and then transferred to ice and exposed to UV light for 5 min
to allow for crosslinking. After crosslinking, microsomes were
collected by centrifugation and lysed in 1% digitonin buffer. The
cleared lysates were first precipitated with anti-tapasin
(panel B, lane 8) or anti-calreticulin
(panel B, lane 7) antiserum. The supernatant
precipitated with anti-tapasin was reprecipitated with
anti-calreticulin (panel B, lane 4) and
supernatant precipitated with anti-calreticulin was reprecipitated with
anti-tapasin (panel B, lane 3). The
immunoprecipitates were analyzed on an SDS gel. Positions of
peptide-bound TAP1/2 and MHC class I HC are indicated. Panel
B, lanes 5 and 6 showed peptide-bound MHC class I
precipitated with anti-H-2Kb antibody (Y3) as
control.
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DISCUSSION |
As a peptide transporter, TAP functions to translocate peptides
from the cytosol into the lumen of the ER (3-5). In addition to
translocating peptides, interaction of MHC class I with TAP has been
found to be important in the assembly of peptide and MHC class I (12,
13). Recently, it has been discovered that tapasin, a subunit of the
TAP complex, mediates the association of MHC class I and TAP. Cells
lacking tapasin have a deficient expression of surface MHC class I as a
result of reduced assembly of MHC class I and peptides in the ER (17,
19). Transfection of tapasin restored the assembly and surface
expression of class I (21). Human tapasin has been cloned and the amino
acid sequence showed a type I transmembrane glycoprotein with a strong
ER retention signal at the C terminus (20, 21). Because a mouse
analogue of tapasin had not been identified from coprecipitated TAP
complex, it was suggested that mouse MHC class I may not need tapasin
for interaction with TAP (23). We have now cloned mouse tapasin. The
predicted amino acid sequence reveals 78% identity to human tapasin
with identical signal peptide, N-glycosylation site,
transmembrane domain, and double lysine motif at the C-terminal end,
indicative of a similar function of human and mouse tapasin. An
interesting feature of the sequence of mouse tapasin is the predicted
cytosolic domain, which showed less than 50% homology with the human
protein. In addition, mouse tapasin has 14 extra amino acids at the C
terminus compared with human tapasin, suggestive of species specificity of the tapasin function. In line with the sequence results, it was
recently found that the intracellular maturation and surface expression
of HLA-B*4402 in murine cells required co-expression of human tapasin
but not human TAP1/2 (37). The cytosolic domain of tapasin may
determine its interaction with MHC class I or TAP1.
In studies of the interaction between MHC class I and TAP, it was found
that TAP1, but not TAP2, is required for the association of TAP with
class I molecules (12, 13). Because tapasin is essential for the
association of MHC class I to TAP (19), tapasin may directly interact
with TAP1. We have now shown that tapasin interacts with TAP1 and MHC
class I in RMA-S cells, which have a mutation that causes premature
termination of the TAP2 protein. Thus, the interaction between TAP1 and
MHC class I appears to be mediated by the tapasin. From these results,
the predicted order of interaction between different molecules in the
TAP complex is TAP2 to TAP1, TAP1 to tapasin, and tapasin to MHC class
I. By these linked molecules, the translocation and loading of peptides are rapidly and efficiently processed in the same microenviroment.
Previous reports have indicated the existence of several ER chaperones,
which interact with MHC class I (4, 19, 27-30). Among them, calnexin
and calreticulin were best characterized (4, 31). By using conformation
specific antibodies and a
2m mutant cell line, a
distinct difference between calreticulin and calnexin in their mode of
association with MHC class I was found (19, 29, 32). Calnexin binds
only to
2m-free heavy chain in human cells (29, 33),
whereas, calreticulin binds only to the MHC class I
HC-
2m dimer (19, 32). Similarly to calreticulin,
interaction of tapasin with MHC class I is
2m-dependent (19, 20), despite the fact that
calreticulin and tapasin are quite different molecules. Like calnexin,
calreticulin is an ER chaperone with lectin-like activity, and it binds
to several other glycoproteins in the ER besides MHC class I (34, 35).
The binding is regulated by glucose trimming of nascent
N-linked oligosaccharides (34, 35). Tapasin so far was found
only in the complexes where MHC class I and/or TAP1/2 are present (19,
20). Because tapasin is also an N-linked glycoprotein, it
has not yet been proven whether tapasin can directly bind to
calreticulin. In addition, tapasin is a subunit of the TAP complex, but
calreticulin does not directly associate with TAP (19, 20). Assembly of
MHC class I and peptides was defective in the absence of tapasin, and
the presence of calreticulin (19), indicating that either calreticulin
is not required for the loading of peptides to MHC class I or both
tapasin and calreticulin are essential for the association of peptide
with MHC class I. In this study, a distinct difference in association
of these two molecules with peptide-bound MHC class I molecules was
found. Tapasin, but not calreticulin, bound to peptide-loaded MHC class I before this complex dissociated from TAP. This may indicate that
tapasin directly catalyzes the loading of peptides onto MHC class I,
and calreticulin controls the conformation of
2m-heavy chain dimer before it interacts with TAP. Lehner et al. (22) have shown that TAP1/2-free soluble tapasin can restore MHC class I
expression in tapasin-negative cell line .220. This finding, together
with the demonstrated interaction between tapasin and peptide-loaded
MHC class I, suggests that tapasin functions not only to form a bridge
between TAP1/2 and MHC class I but also directly to facilitate the
assembly of MHC class I with peptides.
The interaction of TAP with MHC class I molecules was found to be
polymorphic (36). Some HLA-B alleles associate less or not at all with
TAP (36). The binding of different peptides to TAP-free MHC class I may
differ from that of TAP associated MHC class I molecules. Polymorphism
in mechanisms of peptide loading has been found recently in studies of
HLA-B*2705, which can present antigenic peptides in the absence of
tapasin (37). In comparison, expression of B*4402 was
tapasin-dependent (37), although tapasin mediated
interaction of both B*2705 and B*4402 with TAP1/2. The question is
whether the difference in peptide loading is dependent on the peptides
selected by these alleles or on the native structure of different
alleles resulting in different stability of the MHC class I
HC-
2m dimer in the ER. It is important now to determine the molecular structure of the binding between MHC class I and tapasin
as well as between tapasin and TAP1. This will form a basis for
understanding both the function of tapasin and the selectivity of
peptide assembly with different MHC class I.
Based on our results and the data previously published, we conclude
that mouse tapasin is similar to the human analogue both in amino acid
sequence and function. The difference between the mouse and human
molecules in the cytosolic domain may indicate a species specificity of
tapasin. Tapasin interacts with TAP1 and MHC class I in the absence of
TAP2, raising the possibility of sequential interaction of
TAP2-TAP1-tapasin-MHC class I in the TAP complex. Interaction of
peptide-bound MHC class I with tapasin, but not calreticulin suggests
that tapasin may directly influence the loading of peptides onto MHC
class I.