Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP)
Kajsa M. Paulsson,
Per O. Anderson,
Shangwu Chen,
Hans-Olov Sjögren,
Hans-Gustaf Ljunggren1,
Ping Wang2 and
Suling Li3
Tumor Immunology, Lund University, Solvegatan 21, 223 62 Lund, Sweden
1 Microbiology and Tumor Biology Center, Karolinska Institute, 171 77 Stockholm, Sweden
Correspondence to:
S. Li or P.Wang
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Abstract
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The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and ß2-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.
Keywords: antigenic peptide, MHC, TAP, tapasin
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Introduction
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The assembly of MHC class I and peptides that takes place in the endoplasmic reticules (ER) is a systematic process involving the interaction of MHC class I molecules with a multi-protein complex. This process regulates changes of conformation needed for the creation of peptide acceptable class I molecules (13). The transporter associated with antigen processing (TAP) is an essential component of the loading complex (13). The interaction of TAP with MHC class I is mediated by tapasin (4,5). Tapasin is a type I membrane protein with a cytoplasmic tail containing a double-lysine motif known to retain membrane proteins in the ER (4,5). The consistent and stoichiometric association of tapasin with TAP1/2 suggests that tapasin is a subunit of the basic TAP trimeric complex (4), although tapasin is not involved directly in peptide translocation (6). The requirement of tapasin for MHC class I antigen presentation was revealed in a tapasin-mutant cell line, 721.220, in which a deficiency in MHC class I surface expression was found (6). Presentation of antigenic peptides by the MHC class I was restored in 721.220 cells following transfection by tapasin cDNA (5). Since assembly with peptide is a prerequisite for the surface expression of MHC class I, it has been suggested that tapasin directly regulates the conformation of MHC class I during peptide loading directly (3).
The role of tapasin in the peptideTAP interaction has been demonstrated (7,8). In the absence of tapasin, peptide binding to TAP1/2 is significantly reduced. This can be corrected by transfection of the tapasin gene (7,8). Since peptides result in the dissociation of the MHC class I from tapasin, it has been suggested that the regulation of peptide loading by tapasin is associated with TAP (3,9,10). However, in a study of tapasin-mutant cells transfected with a soluble form of tapasin that associated with MHC class I but not with TAP (7), the surface expression and antigen presenting capacity of MHC class I was restored (7). Although it was suggested in that study that the tapasinMHC class I interaction was required for peptide loading, but that of tapasinTAP interaction was not, it remained unclear how loaded MHC class I molecules were released from soluble tapasin. It has been indicated that the co-export of soluble tapasin and the MHC class I complex from the ER may be due to a lack of the retention signal of soluble tapasin, which is required for the retention of MHC class I in the ER (11). The detachment of assembled MHC class I molecules from tapasin has been found recently to be dependent on the nucleotide binding to TAP (12). In that study it was suggested that a conformational change in tapasin occurs through the interaction of the nucleotide and TAP. The complex would become permissive for the peptide-induced release of the MHC class I molecules only after the binding of nucleotide to TAP has occurred (12). This nucleotide binding does not affect the interaction of MHC class I with tapasin (12). These findings indicate that the processing of peptides and their assembly with the MHC class I molecules are regulated kinetically by the function of TAP. However, studies of mutated MHC class I, in consequence with the lack of TAP association, and of the existence of MHC class I alleles that do not associate with TAP suggest that TAP is not involved in MHC class I export from the ER (1318). The interaction of TAP with MHC class I may act as a quality control rather than being directly involved in peptide loading or in release of the assembled MHC class I (3,19,20).
In order to clarify whether TAP is needed for the function of tapasin, we analyzed the association of MHC class I with tapasin, as well as peptide loading onto tapasin-associated MHC class I molecules and the peptide-induced dissociation of MHC class I molecules from tapasin in TAP-mutant cells. Our results show that the function of tapasin in relation to MHC class I assembly is not dependent on its association with TAP.
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Methods
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Cells
The TAP-mutant T2 and wild-type T1 cells were kindly provided by Dr Sune Kvist, the tapsin-mutant 220 cells were kindly provided by Dr J. C. Solheim. The cell lines were cultured in an RPMI 1640 medium (Gibco/BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine at 37°C in a 5% CO2 atmosphere.
Antibodies
The rabbit antiserum (R425) specific for both free and assembled HLA heavy chains was kindly provided by Dr Sune Kvist. The mAb W6/32 specific to ß2-microglobulin (ß2m)-associated human class I molecules was obtained from ATCC (Rockville, MD). Rabbit antisera against human TAP1 or tapasin were produced as described previously (4). The polyclonal antibodies were affinity-purified before use.
Metabolic labeling, immunoprecipitation and immunoblotting
Cells were washed twice with PBS and incubated for 15 min at 37°C in methionine-free RPMI 1640 medium containing 3% dialyzed FBS, where upon 0.2 mCi/ml of [35S]methionine (Amersham, Little Chalfont, UK) was added and incubation was continued for another 10 min. Subsequent to labeling, the pulse-labeled cells were chased for different periods of time at 37°C in complete medium. At the end of the chase periods, the cells were washed 3 times with ice-cold PBS and lysed in 1% digitonin (Sigma, St Louis, MO) lysis buffer containing 0.15 M NaCl, 25 mM TrisHCl, pH 7.5, 1.5 mg/ml iodoacetamide and a mixture of protease inhibitors (2 mM PMSF, 10 mg/ml leupeptin, 30 mg/ml aprotinin and 10 mg/ml pepstatin). The cleared lysates were added to antibodies previously bound to Protein ASepharose beads (Pharmacia, Uppsala, Sweden). After washing, the immunoprecipitates were analyzed by SDSPAGE as described earlier (4). For sequential immunoprecipitation and immunoblotting, the immunoprecipitates were loaded onto 10% SDSPAGE. The proteins were transferred onto a nitrocellulose filter, probed sequentially by anti-tapasin and anti-class I antisera. Detection was performed using an ECL kit (Amersham).
Peptides and peptide modification
Peptides were synthesized in a peptide synthesizer (model 431A; Applied Biosystems, Foster City, CA), using conventional F-moc chemistry. Peptides were then purified by HPLC, and dissolved in PBS. The
-amino group of lysine in HLA-A2-specific peptide of the influenza A matrix protein, M58-64G58YF62K (YILGKVFTL), was modified covalently by a photoreactive cross-linker as described previously (21). An aliquot (1 µg) of the peptide was labeled by Chloramine T-catalyzed iodination (125I). The modification and labeling experiments were performed in the dark. The cross-linker-modified and 125I-labeled peptides are referred to as [125I]MP-ANB-NOS.
Preparation of microsomes and photo-cross-linking
Microsomes from cell lines were prepared and purified according to Saraste et al. (22). For photo-cross-linking, 125I-labeled and ANB-NOS-modified peptide was mixed with 20 µl of microsomes (concentration of OD 60 A280/ml) to a final concentration of 100 nM in RM buffer (250 mM sucrose, 50 mM triethanolamineHCl, 50 mM KOAc, 2 mM MgOAc2 and 1 mM DTT) containing 0.1% digitonin for permeabilizing the membranes. This mixture was kept at 26°C for 10 min. UV irradiation was subsequently carried out for 7 min on ice at 366 nm. The 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. Cross-linked microsomal proteins were immunoprecipitated by specific antiserum. The precipitates were analyzed by SDSPAGE.
Peptide-induced dissociation of MHC class I from tapasin
The microsome permeabilization was carried out as described previously (23). In brief, microsomes from T1 and T2 cells were permeabilized for 5 min with 0.1% digitonin in the presence of anti-proteases on ice, the peptides then being added at different concentrations. After the addition of peptides, the microsomes were incubated for 6 h at 4°C. At the end of incubation, the microsomes were washed once with cold PBS and lysed in 1% digitonin buffer. The cleared lysates were immunoprecipitated by anti-tapasin antiserum. The precipitates were eluted on SDSPAGE. The co- precipitated MHC class I and tapasin were blotted by antibodies specific for them.
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Results
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Proper association of MHC class I with tapasin in TAP-mutant cells, but in reduced quantity
In an earlier study of the association of tapasin with TAP and MHC class I molecules, the interaction of tapasin and TAP was found to be independent of the MHC class I (4). To address the requirement of TAP for the association of tapasin and MHC class I, titrated T2 microsome lysates were immunoprecipitated using an anti-tapasin antibody and subsequently blotted by anti-MHC class I antibody. The anti-tapasin co-precipitated MHC class I in the T2 cells was at reduced levels compared to the T1 cells despite similar amounts of tapasin detected in both cell lines (Fig. 1
). The reduced association of MHC class I molecules with tapasin in T2 cells could be due either to the absence of TAPtapasin interaction or to the presence of fewer dimerized MHC class I molecules in the ER, which is a prerequisite for MHC class I to associate with tapasin (4). In TAP-mutant T2 cells, the surface expression of MHC class I molecules is deficient (24). However, the dimerization of MHC class I heavy chain and ß2m was not significantly impaired, but the affinity was low (24). Since tapasin interacts with the MHC class I heavy chainß2m dimer, changes in the stability of MHC class I dimer in TAP-mutant cells may affect the association of tapasin with MHC class I. To elucidate the quantitative differences in the MHC class Iß2m dimer in microsomes derived from the TAP-mutant T2 and the wild-type T1 cells, the microsomes were lysed and immunoprecipitated by antibody R425, which reacts with all forms of MHC class I molecules, or by the antibody W6/32, which reacts specifically with the MHC class Iß2m dimer. The precipitates were detected by Western blotting, using R425 antiserum. Similar amounts of total class I molecules were recovered by R425 antiserum in both the T1 and T2 cells (Fig. 2
). This confirms earlier findings of high steady-state levels of class I in T2 cells (24). When using the dimer-specific antibody W6/32, the amount of retrieved MHC class I molecules in the T2 cells was less, being only ~60% that of the dimer level observed in the T1 cells (Fig. 2
). The ratio of the tapasin-associated MHC class I to the total MHC class I dimer in the T2 cells was equivalent to that in the T1 cells (Figs 1 and 2
). Thus, the reduced level of tapasin-associated MHC class I in the T2 cells can be accounted for by the low level of the dimer of MHC class I heavy chain and ß2m, but not by absence of the TAPtapasin association. This suggests the stability of the MHC class I dimer, which is regulated by peptides, affects the association between tapasin and the MHC class I molecules.

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Fig. 1. Quantitation of the tapasin-associated MHC class I in T1 and T2 cells. Microsome samples (10 µl) of T1 (lanes 15) and T2 (lanes 610) cells were lysed in 1% digitonin lysis buffer. The cleared lysates were diluted by the dilution factor indicated and were precipitated by anti-tapasin antiserum. The precipitates were separated by SDSPAGE, and were blotted sequentially by anti-class I antiserum R425 and anti-tapasin antiserum. The tapasin-mutant 220 cells were treated in the same way and served as a negative control (lane 11).
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Fig. 2. Analysis of total MHC class I and class I heavy chainß2m dimer in T1 and T2 cells. Aliquots (10 µl) of microsome lysates from T1 (lanes 15) and T2 (lanes 610) cells were diluted as indicated by the dilution factor. Each lysate was divided in two, one part being precipitated with R425 antiserum, reactive to the free and the assembled class I heavy chain, and the other with W6/32, specific for the class I heavy chainß2m dimer. The precipitated class I molecules were identified by Western blotting using R425 antiserum.
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Retention of MHC class I molecules in association with tapasin in TAP-mutant cells
Peptides can release MHC class I molecules from tapasin (9,10). This could indicate that the off-rate of MHC class I from tapasin is lower in the absence of TAP, i.e. in the T2 cells than in the T1 cells. To examine this, T1 and T2 cells were pulse-labeled for 10 min with [35S]methionine-containing medium and then chased for up to 40 min, followed by immunoprecipitation with anti-tapasin antiserum. After 30 min chase, most of the newly synthesized MHC class I molecules were dissociated from tapasin in the T1 cells (Fig. 3
, lanes 4 and 5). Due to the short labeling time and long half-life of TAP1/2 molecules, TAP was not visualized on the gel precipitated from the T1 cells (data not shown). In contrast to the dissociation rate of MHC class I from tapasin in the T1 cells, the amount of tapasin-associated MHC class I molecules had increased in the T2 cells during the periods of chase (Fig. 3
, lanes 68). The low rate of dissociation of MHC class I from tapasin in the TAP-mutant cells demonstrates the function of tapasin in the retention of unloaded class I molecules in the ER. This corresponds to similar findings obtained for tapasin and class I co-transfected insect cells (11). The paradoxical result of a reduced association of MHC class I to tapasin and a low rate of dissociation of class I from tapasin in the TAP-mutant cells indicates that tapasin might have an important function in controlling the quality of the assembly of MHC class I molecule.

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Fig. 3. Pulsechase analysis of the rate of dissociation of MHC class I molecules from tapasin in T1 and T2 cells. T1 (lanes 15) and T2 (lanes 610) cells were labeled metabolically with [35S]methionine for 10 min, and were chased in an excess of unlabeled methionine for the indicated periods of time prior to lysis by 1% digitonin. Aliquots were immunoprecipitated with anti-tapasin antiserum. The precipitates were analyzed by SDSPAGE and autoradiography. The positions of tapasin and MHC class I heavy chain are indicated.
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Peptide loading onto tapasin-associated MHC class I molecules is independent of TAP
We have shown recently that peptide can be loaded onto the tapasin-associated MHC class I molecules (25), indicating the presence of a peptide-receptive form of MHC class I molecules after interaction with tapasin. To examine the involvement of the TAPtapasin interaction in the peptide loading of tapasin-associated MHC class I, microsomes purified from T2 cells were permeabilized and subsequently incubated with the cross-linker-modified and 125I-labeled HLA-A2 binding peptide MP, as described previously (21). This modification makes it possible to detect the peptide-bound HLA-A2 molecules in the microsomal membranes. After incubation for 10 min with peptides, excess peptides were washed away. The microsomes were then UV irradiated for 7 min on ice and lysed with digitonin lysis buffer. The loading of tapasin-associated MHC class I molecules was then analyzed by immunoprecipitation with anti-tapasin antiserum. As we had found previously (4), both peptide cross-linked TAP and HLA-A2 were precipitated by the anti-tapasin antibody in the T1 microsomes (Fig. 4
, lane 1) indicative of formation of tapasin complex with TAP and with MHC class I, and of the peptides loading onto the tapasin-associated MHC class I molecules. In the T2 microsomes, effective loading of peptides onto the tapasin-associated MHC class I was also detected (Fig. 4
, lane 2). This shows that the tapasinTAP interaction is not required for the peptide loading of tapasin-associated MHC class I.

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Fig. 4. Binding of MP peptides to HLA-A2 in permeabilized microsomes of T1 and T2 cells. Purified microsomes from T1 (lane 1) and T2 (lane 2) cells were permeabilized by 0.1% digitonin, and mixed with the 125I-labeled and cross-linker-modified MP peptides. After incubation for 10 min at 26°C, the mixtures were exposed to UV light for cross-linking. The cross-linked microsomal membranes were lysed in 1% digitonin and precipitated with anti-tapasin antiserum. The precipitated protein complexes were analyzed by SDSPAGE, the cross-linked TAP and HLA-A2 being visualized by autoradiography. The precipitates from the microsomes of tapasin-mutant 220 cells served as a negative control (lane 3).
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High-affinity peptides but not TAP are required for the dissociation of MHC class I from tapasin
We have shown here that, in the absence of TAP, tapasin is capable of binding to MHC class I, and that for exogenous peptides the loading of tapasin-associated MHC class I molecules was intact. It was found recently that nucleotide binding to TAP is required for the dissociation of assembled MHC class I from TAP complex (12). To examine the necessity of TAP for the peptide-induced dissociation of MHC class I from tapasin, microsomes from T2 cells were permeabilized and incubated with different concentrations of HLA-A2 binding peptides. After the free peptides had been washed away, the microsomes were lysed and were precipitated by anti-tapasin. The immunoprecipitates were dissolved on SDSPAGE and subsequently blotted by anti-MHC class I antibody. The peptides dissociated the MHC class I from tapasin in a dose-dependent manner in the TAP-deficient T2 cells (Fig. 5
, lanes 610). The dissociation rate was similar to that of T1 cells (Fig. 5
, lanes 15). This result demonstrates that the tapasinTAP interaction is not required for the dissociation of peptide loaded MHC class I from tapasin.

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Fig. 5. Dissociation of MHC class I from tapasin by use of peptides in permeabilized microsomes obtained from T1 and T2 cells. Microsomes derived from T1 (lanes 15) and T2 (lanes 610) cells were permeabilized as described in the legend to Fig. 4 , and were mixed with HLA-A2 binding peptides at the concentrations indicated. The mixtures were incubated for 6 h at 4°C. After the removal of access peptides, the microsomes were lysed in 1% digitonin buffer. The cleared lysates were precipitated with anti-tapasin antiserum. The precipitated complexes were separated by SDSPAGE, and were analyzed sequentially by Western blotting with anti-tapasin and with anti-MHC class I antisera respectively.
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ATP is required for peptide translocation, but not for the dissociation of assembled MHC class I from tapasin in the human cell line
It was reported recently that nucleotide binding to TAP triggers the release of assembled MHC class I from tapasin in T2 cells transfected by rat TAP (12). To examine the need of nucleotides for the dissociation of assembled MHC class I from the human TAP complex, we analyzed peptide translocation across the ER and the dissociation of assembled MHC class I from tapasin in the T1 cells. The addition of apyrase, which degrades the nucleotides, inhibited peptide translocation into the T1 microsomes (Fig. 6a
), which is consistent with earlier findings (23). Whereas apyrase failed to prevent the dissociation of MHC class I from tapasin, as indicated by the dissociation rate of MHC class I from tapasin in the presence and in the absence of apyrase being similar (Fig. 6b
). This somewhat controversial result suggests that the interactive structure of TAP and tapasin for rat and human may differ. Compelling evidence for this is to be seen in findings that mouse tapasin was not able to replace human tapasin for restoring MHC class I expression in tapasin-mutant human cells (18).


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Fig. 6. Apyrase inhibits peptide translocation but not the dissociation of assembled MHC class I from tapasin. (a) Apyrase inhibits peptide translocation across the microsomal membranes of T1 cells. Intact microsomes from T1 cells were incubated with 100 nM HLA-A2 binding peptide modified by a cross-linker and labeled with 125I (see Methods) in ATP-containing transport buffer (21), with and without 12.5 U/ml apyrase. After cross-linking, the microsomes were washed and lysed in 1% digitonin lysis buffer. The cleared lysates were precipitated with anti-TAP1 antiserum and ran on 10% SDSPAGE. Peptide-cross-linked TAP and MHC class I molecules are indicated. (b) Dissociation of peptide-loaded MHC class I molecules from tapasin in the presence or absence of apyrase. T1 microsomes were lysed with 1% digitonin lysis buffer and incubated with apyrase 12.5 U/ml for 30 min at 30°C. Peptides at different concentrations were then added into the lysates and kept for 6 h at 4°C in the presence or absence of 12.5 U/ml apyrase. After incubation, the lysates were submitted to precipitation by anti-tapasin antiserum. The precipitates were analyzed on 10% SDSPAGE and were subsequently blotted by anti-MHC class I antiserum, R425 or anti-tapasin.
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Discussion
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The association of MHC class I and TAP appears to be important for the assembly of MHC class I molecules (3). This association is mediated by tapasin, an ER-resident protein which has binding sites for both TAP and MHC class I (35,26). The importance of the association of MHC class I with tapasin for the surface expression of MHC class I has been revealed in studies of tapasin-mutant cells (3,5,6). Cells lacking tapasin show a deficient surface expression of MHC class I, a deficiency that can be corrected by the transfection of wild-type tapasin cDNA (5). Apart from being important for the regulation of MHC class I expression, tapasin is also important for efficient peptideTAP interaction (7,8). In the present study, we investigated the requirement of TAP for the function of tapasin by studying the association of MHC class I with tapasin, the loading of peptides onto MHC class I and the release of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, tapasin associates properly with the MHC class I dimer. The reduced quantity of tapasin-associated MHC class I in the T2 cells is due to the instability of the MHC class I dimer in the absence of peptides. This suggests that pre-loading with low-affinity peptides may be essential for the interaction of MHC class I with tapasin. This interaction precedes the optimization of the MHC class I assembly by the replacement of pre-loaded low-affinity peptide with high-affinity peptide, as indicated by the retention of MHC class I in association with tapasin in the T2 cells.
The demonstration of a prolonged association of newly synthesized class I molecules with tapasin in TAP-mutant cells also indicates that tapasin has an important function in the retention of immature MHC class I molecules in the ER. Indeed, in a reconstituted insect antigen-presentation system, the transfection of tapasin was found to maintain unloaded class I in the ER (11). Taken together, these findings suggest a sequential process of the peptide-regulated assembly of MHC class I, since the low-affinity peptides initiate the MHC class I dimer and the association of MHC class I with tapasin. After association of MHC class I with the loading complex, high-affinity peptides replace the low-affinity peptides and dissociate the MHC class I from tapasin. By addition of exogenous peptides, we found that peptide loading onto the tapasin-associated MHC class I and the peptide-induced dissociation of MHC class I from tapasin were as efficient in the T2 cells as in the wild-type T1 cells, indicating that the physical interaction between TAP and tapasin is not required either for the assembly of tapasin-associated MHC class I or for the dissociation of optimized MHC class I from tapasin. Since TAP functions as a peptide transporter, the association of MHC class I with the TAP complex may be essential for the optimization of peptide loading by allocation of the MHC class I that is close to the peptide transporter.
On the basis of previous studies, the involvement of TAP in tapasin-associated MHC class I assembly appeared controversial. A soluble form of tapasin, that lacks the transmembrane and cytosolic domains as well as the association with TAP was found able to restore MHC class I assembly and surface expression in tapasin-mutant cells (7). This suggests that the function of tapasin is independent of TAP. Since soluble tapasin lacks an ER retention signal, which has been suggested to be necessary for the retention of MHC class I in the ER (11), the increased surface expression of MHC class I may at least partially be due to the failure of the control for MHC class I excition from the ER. Efforts to determine how the nucleotide binding site of the TAP functions led recently to the finding that in T2 cells transfected by rat TAP molecules with mutated nucleotide-binding site the MHC class I can no longer dissociate from TAP (12). These results suggest sequential conformational changes first in TAP and then in tapasin after binding of the nucleotide to TAP, that result in the release of peptide-loaded MHC class I (12). In our study, we also examined the involvement of nucleotides in MHC class I association with and dissociation from tapasin by the addition of apyrase, which degrades nucleotides, in an assay using T1 microsomes. No difference in the association of MHC class I to tapasin nor in the dissociation of MHC class I from tapasin could be observed. Since the finding of nucleotide being required for the release of MHC class I from tapasin was obtained for T2 cells transfected with rat TAP, one possibility is that there is a species specificity of TAP function as indicated by the earlier finding that mouse tapasin cannot fully replace human tapasin in regulating the MHC class I assembly (18). The very similar results obtained for the TAP-mutant and wild-type cells concerning the association of MHC class I with tapasin, the loading of peptides onto tapasin-associated MHC class I, and the dissociation of MHC class I from tapasin by peptides provide no support for TAP association being required for tapasin to function. The TAP-independent function of tapasin demonstrated in the present study reveals tapasin to have a distinct function in the loading complex.
The function of the interaction between MHC class I and tapasin here suggests that tapasin may have a specific chaperone-like role, protecting the dimer of MHC class I heavy chainß2m from degradation and facilitating peptide loading (3). Studies of MHC class I assembly in the absence of tapasin interaction have yielded inconsistent results (1316). Some MHC class I alleles, although not associating demonstrably with tapasin, are able to assemble with peptides (17,18). These findings can be interpreted either as indicating an allelic preference or as being due to experimental limitations in the ability to demonstrate a weak biological association. One study of MHC class I assembly in TAP-mutant cells showed a portion of the MHC class I in TAP-mutant cells to be reactive toward a conformational-specific antibody, indicating these class I molecules to be loaded with peptides (28). These stable class I molecules could also be re-loaded with high-affinity peptides (28). This finding emerges from the observation that an HLA-A2 mutant, T134K, is deficient in the association of MHC class I with tapasin and that it expresses a paradoxical peptide assembly in which the assembly is normal in the ER, but is deficient in the presenting of viral peptides. This suggests that the loading of MHC class I occurs in two steps, the first being the loading of low-affinity peptides for stabilization of the MHC class I dimer and the second optimization by the replacement of low-affinity by high-affinity peptides. Such a view is supported by a recent study of GFP-tagged MHC class I which shows that peptide-loaded MHC class I can also be retained in the ER for optimizing peptide loading (20).
The paradoxical finding that in TAP-mutant T2 cells there is less binding of the MHC class I dimer to tapasin, at the same time as the dissociation rate of MHC class I from tapasin is low, suggests that low-affinity peptides may be essential for the formation of the MHC class I heavy chainß2m dimer. The association of the dimer with tapasin retains both unloaded and low-affinity peptide-loaded MHC class I in the ER. This precedes the optimization of MHC class I by high-affinity peptides. The interaction of tapasin and the MHC class I complex with TAP is not required directly for the function of tapasin. However, the close association of MHC class I with TAP may be essential for the selection of high-affinity peptides.
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Acknowledgments
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We thank Dr S. Kvist for the antisera. This study was supported by grants from Crafoodska stiftelsen (990589), the Swedish Cancer Society (3975-B99-03XAB), the Medical Faculty, Lund University and the Foundation for Strategic Research (SFF, from Infection & Vaccinology, 36/98 and from Inflammation Research Program/99).
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Abbreviations
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ß2m ß2-microglobulin |
ER endoplasmic reticulum |
TAP transporter associated with antigen processing |
tapasin TAP-associated glycoprotein |
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Notes
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2 Present address: Immunology Group, Department of Pediatric Gastroenterology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, UK 
3 Present address: Department of Biological Science, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK 
Transmitting editor: H. Wigzell
Received 1 June 2000,
accepted 22 September 2000.
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