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Originally published In Press as doi:10.1074/jbc.M201388200 on March 7, 2002

J. Biol. Chem., Vol. 277, Issue 21, 18266-18271, May 24, 2002
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Association of Tapasin and COPI Provides a Mechanism for the Retrograde Transport of Major Histocompatibility Complex (MHC) Class I Molecules from the Golgi Complex to the Endoplasmic Reticulum*

Kajsa M. PaulssonDagger §, Monique J. Kleijmeer, Janice Griffith, Marc Jevon§, Shangwu ChenDagger §, Per O. AndersonDagger §, Hans-Olov SjögrenDagger , Suling LiDagger ||**, and Ping Wang§DaggerDagger

From the Dagger  Institution of Tumor Immunology, Lund University, Solvegatan 21, s-223 62 Lund, Sweden, the  Department of Cell Biology, University Medical Centre, Institute of Biomembranes, 3584 CX Utrecht, The Netherlands, the || Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom, and the § Immunology Group, Department of Gastroenterology, Barts and The London School of Medicine and Dentistry, 59 Bartholomew's Close, London EC1A 7ED, United Kingdom

Received for publication, February 11, 2002, and in revised form, March 7, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta - and gamma -subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The assembly of MHC class I molecules in the endoplasmic reticulum (ER)1 is a process involving the association of MHC class I molecules with a multiprotein complex termed the loading complex (1, 2). It has been suggested that this process is required to induce the MHC class I conformation changes required for the loading of peptides (1). The transporter associated with antigen processing (TAP) is an essential component of the loading complex, interacting directly with MHC class I (1, 3). The association of TAP with MHC class I is mediated by tapasin, a type-I transmembrane protein with a cytoplasmic tail containing a double-lysine motif, which is known to retain resident proteins in the ER (4, 5). Although tapasin is not involved in peptide translocation directly, it is a subunit of the TAP trimeric complex (4, 6). Cells with a deficiency in tapasin or obtained from tapasin knockout mice have a reduced surface expression of MHC class I molecules and a deficiency in antigen presentation (6-9). Because peptide assembly is essential for the surface expression of MHC class I, it has been suggested that tapasin is involved in regulating the conformation of MHC class I molecules during peptide loading (10). Indeed, it has been reported that tapasin can stabilize the expression of TAP and increase the association of peptide with the TAP complex (11, 12). Tapasin has also been suggested to retain empty class I molecules in the ER (13, 14). The retention of unloaded MHC class I in the ER has been shown in insect cells (13), and transfection of the tapasin-deficient cell line 721.220 with H2-Kb resulted in increased transport of Kb to the cell surface compared with tapasin wild-type cells (14). Recently, studies using tapasin knockout mice have demonstrated that although MHC class I molecules are transported to the cell surface in tapasin-deficient cells a proportion of these molecules reach the cell surface without being loaded with high affinity peptides (8).

Several ER chaperones are found in the loading complex together with tapasin, including calnexin, calreticulin, and ERp57 (1). In the light of this, tapasin has been suggested to be one of the ER chaperones involved in MHC class I conformation during assembly (1). Calnexin and calreticulin are ER chaperones with lectin-like activity. They bind to several glycoproteins in the ER other than MHC class I, and their association is regulated by the glucose trimming of nascent N-linked oligosaccharides (15, 16). ERp57 shows both thiol-dependent reductase activity and cysteine protease activity toward various substrates (17, 18), and MHC class I is one of the substrates. A direct interaction of ERp57 with calreticulin and calnexin has also been revealed, suggesting that these molecules may form a tricomplex. Such a complex could modulate glycoprotein folding within the ER lumen generally (19). In contrast to the ER chaperones, tapasin selectively associates with both MHC class I and TAP indicating that it has a more definite function in class I antigen presentation.

Once a role of tapasin in the regulation of MHC class I processing had been established, it was questioned whether tapasin was an absolute requirement for MHC class I assembly. In subsequent studies, marked differences were noted between various MHC class I alleles in their assembly and antigen presentation in tapasin mutant cells (20, 21). Moreover, in contrast to TAP1 knockout mice, only a smaller part of the expressed MHC class I was found to be deficient in cells from tapasin knockout mice (8). However, despite the observed deficiency in effective surface expression, MHC class I molecules could still present some antigenic peptides, resulting in a limited positive selection of CD8 T cells and in antipathogen immune responses (8). It has also been reported that a point mutation of threonine 134 to lysine in HLA-A2.1 renders it incapable of interacting with tapasin, although high levels of the mutant HLA-A2.1 are expressed at the cell surface (22, 23). This led to the suggestion of a tapasin-independent presentation pathway (24).

Recently, evidence for a role of tapasin in the optimization of peptide selection in MHC class I assembly has emerged from the study of profiles of MHC class I-associated peptides in tapasin mutant cells (25). HLA-B2705 has been found to be effective in presenting antigenic peptides and to be expressed on the cell surface of tapasin mutant cells (21). Comparison of peptides bound to HLA-B2705 expressed in tapasin mutant cells with wild-type cells revealed an overall reduction in B2705-bound peptides, with only a limited difference in the peptide profiles (25). It was suggested that the proportion of suboptimal peptides associated with B2705 in tapasin mutant cells is related to the delay found in the kinetics of B27-restricted antigen presentation (21). Similarly, an altered peptide repertoire in tapasin knockout mice was observed (9). In tapasin knockout mice, MHC class I molecules exit the ER in an unstable form (8). Taken as a whole, these results suggest that tapasin, although not required for assembly of MHC class I, provides a gating mechanism for reducing the expression of MHC class I molecules containing suboptimal peptides.

In the present study, we show that tapasin may play a role the regulation of the retrograde transport of unstable MHC class I molecules from the Golgi complex to the ER. The observed interaction of tapasin with the COPI coatomer suggests that tapasin functions as a cargo receptor, mediating the packaging of MHC class I molecules in COPI-coated vesicles for transport from the Golgi complex to the ER in a retrograde fashion. We propose that unstable MHC class I molecules recycle between the ER and the Golgi complex until the loading of optimal peptides is completed.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells-- T1, T2, .220-HLA-A2, and .221-HLA-A2 cell lines were kindly provided by Drs. S. Kvist and T. Elliott, respectively. The cell lines were cultured in RPMI 1640 medium (Invitrogen), 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-- Rabbit antiserum to human MHC class I (R425), which reacts to all forms of MHC class I molecules, was kindly provided by Dr. S. Kvist. The monoclonal antibody W6/32 specific to beta 2-microglobulin-associated human class I molecules was obtained from ATCC (Manassas, VA). Rabbit antisera against human TAP1 or tapasin were described previously (4, 26). Anti-p58 antiserum, an ER and cis-Golgi marker, was kindly provided by Dr. R. Petersson. Anti-TGN46, a trans-Golgi marker, was obtained from Serotec. Antibodies to beta - and gamma -COPI and Sec13p-COPII were provided by Drs. C. Harter and W. Hong, respectively. The polyclonal 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 [35S]methionine (Amersham Biosciences) 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 (Sigma) or 1% Nonidet P-40 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 (Pharmacia, Uppsala, Sweden). After washing, the immunoprecipitates were analyzed by SDS-PAGE as previously described (4). Western blotting and FACS analysis were performed as previously described (26). Quantification in immunoprecipitation and Western blotting was done using the Quantity One version 4.1.0 from Bio-Rad.

Peptides and Peptide Modification-- 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 PBS. The epsilon -amino group of lysine in HLA-A2-specific peptide of influenza-A matrix protein, M58-64G58YF62K (YILGKVFTL) was covalently modified by a photoreactive cross-linker as previously described (27). 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. (28). For photo-cross-linking, 125I-labeled and ANB-NOS-modified peptide was mixed with 20 µl of microsomes (absorbance 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 at 26 °C for 5 min. 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. Cross-linked microsomal proteins were immunoprecipitated with specific antiserum. The precipitates were analyzed by SDS-PAGE or quantitated using a gamma  counter.

Subfractionation of Total Microsomes by Flotation in Sucrose Gradients-- A modification of the method previously described (28) for fractionation of microsomal membranes was used. All steps were performed at 0-4 °C. Total microsomes (described above) were layered on top of 5 ml of 0.33 M sucrose containing 5 mM benzamidine, layered in turn on top of a sucrose cushion consisting of 1 ml of 2 M sucrose/5 mM benzamidine. Centrifugation in an SW41 rotor for 60 min at 110,000 × g yielded a total microsome band on top of the cushion. The total microsome band was carefully collected. Then, 2 M sucrose/5 mM benzamidine was slowly added to the microsomes to give a final concentration of 45% (w/v) sucrose. Microsomes were subfractionated by flotation using a modification of the method described previously (29). 100 µl of the total microsomes in 3 ml of 45% (w/v) sucrose was placed at the bottom of an SW41 ultracentrifuge tube and overlaid with the following sucrose solutions: 1 ml of 30% and 1.9 ml each of 27.5%, 25%, 22.5%, and 20.0% (all solutions contained 5 mM benzamidine). After centrifugation at 4 °C for 10 h at 37,000 rpm (to reach isopyknic conditions), 25 fractions of 300 µl each were collected by upward displacement.

Immunoelectron Microscopy-- .220A2 and .221A2 cells were prepared for immunoelectron microscopy as described previously (30). Briefly, cells were fixed with 0.1 M phosphate buffer, 2% paraformaldehyde, and 0.2% glutaraldehyde for 2 h at room temperature. The cells were embedded in 10% gelatin, incubated for 4 h in 2.3 M sucrose at 4 °C, and frozen in liquid nitrogen. Ultrathin cryosections were immunolabeled with tapasin antibody and 10 nm of protein A gold particles. To quantitate gold particles representing tapasin five times 5 × 200 gold particles were randomly counted at an instrumental magnification of 10,000×. Only gold particles within 20 nm of a membrane were counted. The background labeling on .220A2 cells was negligible.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COPI Coatomer Interacts with Tapasin in Association with MHC Class I and TAP-- The exact role of tapasin in the regulation of MHC class I peptide loading has not yet been resolved. We anticipated that the double lysine motif at the C-terminal end of tapasin might be important for tapasin function. Proteins containing the characteristic KKXX retrieval motif interact with COPI-coatomers (31), which mediate retrograde transport from the Golgi complex back to the ER (32). To examine the possible interaction between tapasin and COPI, microsomes from both the tapasin wild-type cell line 721.221 and the tapasin mutant cell line 720.220 were lysed and precipitated using the anti-tapasin antibody. The tapasin precipitates were analyzed further by Western blot with antibodies specific to COPI coatomer subunits. We found that anti-tapasin co-precipitated both beta -COPI and gamma -COPI coatomer subunits (Fig. 1). In addition, anti-tapasin also precipitated MHC class I and TAP, indicative of a functional association of COPI-coated vesicles with the MHC class I loading complexes (Fig. 1). The specific interaction of tapasin and the COPI coatomer was likewise confirmed by precipitation with anti-MHC class I and anti-TAP1 antisera. Similar amounts of MHC class I and TAP were precipitated by anti-MHC class I and anti-TAP1 antibodies, respectively, in both wild-type cells and tapasin mutant cells (Fig. 1). However, both antibodies were found to co-precipitate the COPI coatomer in wild-type cells only (Fig. 1). The specific interaction of tapasin and the COPI coatomer suggests that tapasin packs TAP and MHC class I into the COPI vesicles and mediates the retrograde transport of these molecules. There are two major types of coated vesicles, COPI and COPII, regulating intracellular transport of membrane proteins and secretory proteins (32). In contrast to the COPI-coated vesicles, the COPII-coated vesicles regulate the anterograde transport of newly synthesized proteins from the ER to the Golgi network (33). Interaction of the COPII coatomer and tapasin was also examined, but no detectable association of tapasin with the COPII coatomer was found (data not shown).


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  		Fig. 1.   Association of tapasin with COPI coatomer. .221A2 (lanes 1, 3, 5) or .220A2 cells (lanes 2, 4, 6) were immunoprecipitated with anti-tapasin (lanes 1 and 2), with anti-TAP1 (lanes 3 and 4), and with anti-MHC class I (lanes 5 and 6). The precipitates were sequentially blotted with anti-coatomer beta -subunit, anti-coatomer gamma -subunit, anti-TAP1, anti-tapasin, and anti-MHC class I, respectively. The position of coatomer subunits, TAP1, tapasin, and MHC class I are indicated.

Subcellular Localization of Tapasin in Both the ER and the Golgi Complex-- Tapasin was predicted to be an ER residential protein on the basis of its double lysine motif at the C terminus. The KKXX motif is one of the ER retrieval signals (34). To investigate the intracellular localization of tapasin, we performed immunoelectron microscopy on the .221A2 and .220A2 cells using the tapasin antiserum and 10 nm of protein A gold particles. Specific labeling for tapasin could be detected on membranes of the ER and the Golgi complex in the .221A2 cells. Semiquantitative analysis showed that 95% ± 2 of the total tapasin labeling was associated with ER membranes and 5% ± 2 with membranes in the Golgi area (Fig. 2). There was no specific tapasin staining in the .220A2 cells (data not shown). Because tapasin is an ER resident protein having a double lysine motif at the C terminus, the Golgi localization suggests that tapasin is retrieved from the Golgi back to the ER.


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  		Fig. 2.   Intracellular localization of tapasin. A, shows the ER area, B shows the Golgi areas. The gold particles for tapasin were directly counted at an instrumental magnification of 10,000. The background labeling as determined for nucleus and mitochondria was less than 3%.

Evidence for Recycling of Tapasin-associated MHC Class I from the Trans-Golgi to the ER in TAP Mutant Cells-- In TAP mutant cells, though most of the MHC class I heavy chain and the beta 2m dimers are unstable and surface expression of MHC class I is deficient, it was found that similar amounts of MHC class I molecules could be detected in the trans-Golgi network compared with wild-type cells (35). In the TAP mutant cells, tapasin could still associate with MHC class I molecules (36). If most of the class I molecules in the TAP mutant cells are unstable and tapasin facilitates the retrieval of unstable class I from the secretory pathway back to the ER, tapasin-associated class I molecules should accumulate in the ER of TAP mutant cells. To examine the off-rate of the MHC class I-tapasin complexes in the TAP mutant T2 cells, we performed pulse-chase experiments with T2 and T1 cells before precipitation with the anti-tapasin anti-serum. In T2 cells, the amount of tapasin-associated class I molecules is reduced compared with T1 cells (Fig. 3A) (36). Following the chase, class I molecules dissociated from tapasin in T1 cells but accumulated in T2 cells for more than 30 min before dissociation. The low off-rate of tapasin-associated MHC class I in the TAP mutant cells is due to a lack of optimal peptides for dissociating the class I molecules from tapasin. Analysis of tapasin and MHC class I molecules in subcellular fractionated microsomes showed that the amount of MHC class I and tapasin in the Golgi complex of .221A2 and T2 cells is similar (Fig. 4A). The association of the tapasin-MHC class I complexes with the COPI coatomer was also demonstrated in T2 cells (Fig. 3B). These results indicate that in the absence of peptides, the tapasin-associated MHC class I molecules are not released but are instead recycled back to the ER.


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  		Fig. 3.   Pulse-chase analysis of the association of MHC class I with tapasin and the association of coatomer and tapasin in T1 and T2 cells. A, T1 (lanes 1-6) and T2 (lanes 7-12) were labeled for 15 min with [35S]methionine and chased for the indicated periods of time before lysis. Lysates were precipitated with anti-tapasin antiserum. The position of MHC class I is indicated. The histogram shows the relative intensity of the lanes during the chase. B, T2 cells were lysed and precipitated with anti-tapasin or anti-MHC class I antiserum R425. The precipitates were sequentially blotted by anti-coatomer, anti-tapasin, and anti-MHC class I antisera, respectively. The positions of coatomer beta - and gamma -subunits, tapasin, and MHC class I are indicated.


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  		Fig. 4.   Peptide-MHC class I binding assay of subfractionated microsomes from .221A2, T2, and .220A2 cells. Purified microsomes of .221A2, T2, and .220A2 cells were fractionated by sedimentation on sucrose gradients. A, 20 µl of each fraction was blotted with anti-p58 (an ER and cis-Golgi marker), anti-TGN46 (a trans-Golgi network marker), anti-MHC class I, anti-tapasin and anti-TAP1, respectively. The positions of detected molecules are indicated. B, identified ER and Golgi fractions of .221A2, T2, and .220A2 microsomes were permeabilized with 0.1% digitonin. The permeabilized microsomes were mixed with cross-linker-modified HLA-A2 binding peptide MP. After cross-linking, microsomes were lysed and precipitated with anti-tapasin antiserum. Position of cross-linked MHC class I is indicated.

Peptide-Receptive MHC Class I Molecules Are Present in Both the ER and Golgi Complexes-- When tapasin retrieval unloads class I molecules from the trans-Golgi network back to the ER, the tapasin-associated MHC class I molecules in the Golgi should potentially be receptive molecules for optimal peptides. To examine this in more detail, microsomes of .220A2, .221A2, and T2 cells were fractionated in ER and trans-Golgi fractions as shown in Fig. 4A. The fractionated microsomes were permeabilized with 0.1% digitonin and incubated with the 125I-labeled and cross-linker-modified HLA-A2 binding peptide MP. After incubation, the digitonin lysates were precipitated with anti-tapasin antiserum. If the tapasin-associated class I molecules are peptide-receptive, they will be cross-linked with reporter peptides. A significant amount of tapasin-associated class I molecules were shown to be bound to the reporter peptides in both the Golgi and ER fractions of the .221A2 and T2 cells (Fig. 4B). This indicates that tapasin-associated class I molecules in the Golgi network are indeed peptide-receptive and suggests that they are transported back to the ER in a retrograde manner for the assembly of optimal peptides.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Studies of TAP mutant cells showed that assembly of MHC class I molecules with peptides in the ER is essential for their surface expression (35). It was suggested that peptide loading in the ER and the subsequent export of mature MHC class I from the ER to the Golgi network is a rapid process (37). Recent reports of studies on intracellular trafficking, however, have revealed a complex process of MHC class I assembly and exit from the ER (2, 38). MHC class I assembly is mediated by the interaction of MHC class I with the TAP complex and with other ER chaperones (10). The MHC class I-TAP interaction is mediated by tapasin, a type I transmembrane protein with a double lysine motif as a retrieval signal at its C terminus (4). Although tapasin has been suggested to facilitate MHC class I assembly in the ER (5), there is still no clear evidence for a direct involvement of tapasin in peptide loading. Despite a quantitative defect in class I antigen presentation in tapasin mutant cell lines and in the cells of tapasin knockout mice (6, 8), findings concerning the role of tapasin directly in class I assembly are not compelling (14, 21, 22, 25, 39). Using cross-linker-modified reporter peptides, we could not detect any significant defect in peptide binding to class I in tapasin mutant cells (12). This suggests that tapasin indirectly regulates the quality of the class I expression but not the catalytic step in the assembly between MHC class I and peptides. Immunoelectron microscopy showed tapasin to be distributed in both the Golgi network and the ER, suggesting a retrograde transport of tapasin and tapasin-associated MHC class I molecules from the Golgi network back to the ER. Compelling support for this model is provided by the discovery of a direct interaction of tapasin with the COPI coatomer. The COPI coatomer consists of seven COPI subunits preassembled in the cytosol to form a complex called the coatomer (40). The function of the coatomer is to drive the formation of retrograde transport vesicles responsible for the retrieval of ER resident proteins from the Golgi complex (41, 42). The association of tapasin with COPI-interacting MHC class I molecules demonstrates a novel function of tapasin in mediating the packing of MHC class I molecules into the COPI vesicles as cargo proteins. Peptide loading experiments demonstrated that the tapasin-associated MHC class I molecules in the Golgi are indeed peptide-receptive molecules that are to be loaded with optimal peptides. Together with the finding that tapasin is indeed located on Golgi membranes, these data suggest that tapasin-mediated COPI vesicle transport provides a control mechanism for retrieval of unstable MHC class I molecules back to the ER.

In tapasin mutant cells MHC class I can exit from the ER, although a proportion of exported class I molecules are unstable and rapidly degraded (8, 9, 14). The lack of association of class I with the retrograde transport vesicles in tapasin mutant cells could very well be a reason for the expression of unstable MHC class I molecules in these cells. In TAP mutant cells, peptide-receptive class I molecules also exit from the ER but with a severe deficiency in their surface expression. This suggests that there is an intact retrograde transport pathway mediated by tapasin-associated COPI vesicles for recycling peptide-receptive class I molecules back to the ER.

Compelling support for the control of optimized MHC class I expression by tapasin has been provided by the analysis of peptide loading-profiles in tapasin mutant cells (25). In that study, the total amount of recoverable class I-associated peptides was found to be reduced in tapasin mutant cells (25). Moreover, despite overlap of the peptide profiles eluted from MHC class I molecules in tapasin mutant and wild-type cells, subsets of peptides have been recovered exclusively from wild-type cells, and a few peptides were eluted only from MHC class I molecules of tapasin mutant cells, suggestive of changes in the peptide repertoire occurring during the process of optimized addition of peptides (25). Because every MHC class I allele can assemble with a diverse set of peptides of restricted length and hydrophobic C termini, tapasin is unlikely to specifically chaperone each class I allele for the loading of specific sets of peptides. The change of suboptimal to optimal peptides may be due largely to the competition of high affinity peptides with preloaded low affinity peptides in the ER. A long retention time for TAP-dissociated MHC class I in the secretory pathway has been demonstrated (2), and H2Kb molecules retained intracellularly are loaded with new peptides up to 3 h after synthesis (43). We have demonstrated that tapasin-associated MHC class I molecules from both the Golgi and the ER are peptide-receptive molecules, suggesting that these class I molecules can be either unloaded or loaded with suboptimal peptides. The tapasin-associated MHC class I can be dissociated with high affinity peptides (36). The interaction of tapasin with COPI-coated vesicles, retains unloaded or suboptimally loaded MHC class I in the ER for the refined loading with high affinity peptides as suggested in Fig. 5. Moreover, it has been shown that both high and low affinity peptides could be eluted from the class I molecules present in tapasin mutant cells. This suggests that tapasin is not a prerequisite for loading of high affinity peptides.


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  		Fig. 5.   A model illustrating the retrograde transport of unloaded or suboptimal loaded MHC class I molecules back to the ER by tapasin-mediated COPI vesicles.

In addition to the association of the tapasin-class I complex with the COPI-coated vesicles, an accumulation of class I-tapasin complexes in TAP mutant cells following a chase time was revealed (Fig. 3A). This points to recycling of the tapasin-associated class I molecules in the secretory pathway when peptides are absent, a result consistent with the earlier observation that class I molecules can exit from the ER in TAP mutant cells in an unstable form but cannot be expressed on the cell surface (35, 44) (Fig. 3, A and B). Apart from the association of tapasin with MHC class I and COPI coatomer subunits, TAP was also detected in the COPI coatomer complex, indicative of a retrograde transport of unstable MHC class I molecules associated with a peptide transporter leading to increased chances for MHC class I to assemble with optimal peptides.

It has been found that COPI-coated vesicles mediate both the anterograde transport of secretory proteins between the Golgi stacks and the retrograde transport of ER resident proteins from the Golgi back to the ER (42). In a recent report, evidence from a study of green fluorescence protein-tagged class I molecules suggests a selective export of class I molecules from the ER to the Golgi by the COPII transport vesicles (38). Whether tapasin-associated COPI vesicles mediate bi-directional transport of unstable MHC class I molecules from the Golgi to the ER and of properly loaded MHC class I molecules from within the Golgi network remains to be investigated.

Although there is no correlation between tapasin dependence and either the expression of HLA-B2705 or affinity for B2705 (25), the differential association of various MHC class I alleles with tapasin and their differences in tapasin-dependent presentation suggest that tapasin may differentially regulate class I molecules (45). This may possibly be a result of the differences both in the diversity of the loadable peptides and in the stability of the various alleles after loading (46). Association with tapasin may chaperone the suboptimal MHC class I molecules until they are stabilized by optimal peptides. Membrane-free tapasin can thus increase the stable expression of class I molecules at the surface of tapasin mutant cells (47).

The findings presented in this study not only reveal a novel function of tapasin in antigen presentation but, together with the finding of vesicle-mediated export of class I molecules (38), also reveal co-evolution of antigen presentation and the general cell biological mechanisms of intracellular protein transport. Future studies will clarify the role of tapasin-dependent antigen presentation in the selection of optimal peptides for the stable expression of MHC class I alleles.

    ACKNOWLEDGEMENTS

We thank Drs. S. Kvist, R. Petersson, C. Harter, and W. Hong for the antisera. We thank Ralf F. Pettersson for valuable help reading the manuscript.

    FOOTNOTES

* This study was supported by Crafoordska Stiftelsen Grant 990589, the Swedish Cancer Society Grants 3975-B99-03XAB and 4504-B01-01RAA, the Medical Faculty, Lund University, the Swedish Foundation for Strategic Research (Infection and Vaccinology, 36/98 and from Inflammation Research Program/99), STINT The Swedish Foundation for International Cooperation in Research and Higher Education Grant 2001-6099, the Emma Ekstrands, Hildur Teggers, and Jan Teggers Fundation, and Grant 805-48-014 from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence may be addressed. Tel.: 44-20-76018469; Fax: 44-20-76005901; E-mail: su-ling.li@wblab.lu.se.

Dagger Dagger To whom correspondence may be addressed. Tel.: 44-20-76018469; Fax: 44-20-76005901: E-mail: p.wang@qmul.ac.uk.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201388200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; beta 2m, beta 2-microglobulin; Tapasin, TAP-associated glycoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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