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The Journal of Immunology, 2006, 176: 7482-7488.
Copyright © 2006 by The American Association of Immunologists

The Double Lysine Motif of Tapasin Is a Retrieval Signal for Retention of Unstable MHC Class I Molecules in the Endoplasmic Reticulum1

Kajsa M. Paulsson*,{dagger}, Marc Jevon*, James W. Wang*, Suling Li2,{ddagger} and Ping Wang2,*

* Institute of Cell and Molecular Science, Barts and London School of Medicine, London, United Kingdom; {dagger} Institute for Medical Microbiology and Immunology, Panum Institute, Copenhagen, Denmark; and {ddagger} Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tapasin (tpn), an essential component of the MHC class I (MHC I) loading complex, has a canonical double lysine motif acting as a retrieval signal, which mediates retrograde transport of escaped endoplasmic reticulum (ER) proteins from the Golgi back to the ER. In this study, we mutated tpn with a substitution of the double lysine motif to double alanine (GFP-tpn-aa). This mutation abolished interaction with the coatomer protein complex I coatomer and resulted in accumulation of GFP-tpn-aa in the Golgi compartment, suggesting that the double lysine is important for the retrograde transport of tpn from late secretory compartments to the ER. In association with the increased Golgi distribution, the amount of MHC I exported from the ER to the surface was increased in 721.220 cells transfected with GFP-tpn-aa. However, the expressed MHC I were less stable and had increased turnover rate. Our results suggest that tpn with intact double lysine retrieval signal regulates retrograde transport of unstable MHC I molecules from the Golgi back to the ER to control the quality of MHC I Ag presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class I (MHC I)3 molecules present intracellular antigenic peptides to CD8-positive T cells initiating cytotoxic responses to the APCs. Therefore, it is essential that MHC I and antigenic peptides are assembled properly in the endoplasmic reticulum (ER) before transport to the cell surface. It has been suggested that the quality control of MHC I assembly in the ER is regulated by the loading complex (LC) consisting of chaperones, tapasin (tpn), and TAP (1, 2). In the LC, MHC I molecules interact with tpn until loaded with optimal peptides (3). Although it is demonstrated that proper assembly of MHC I and peptides is required for stable MHC I expression and effective Ag presentation, it is still unknown by what mechanisms unstable MHC I molecules are retained in the ER.

It was discovered previously that peptide-receptive MHC I molecules could be detected in late secretory compartments (4), indicating that unloaded or suboptimal loaded MHC I molecules could escape from the ER. This suggests a retrograde transport mechanism for retrieval of these class I molecules from post-ER compartments. Studies of GFP-tagged MHC I molecules showed that assembled MHC I are selectively exported from the ER possibly by interaction with transport receptors such as BAP31 (5). However, export of MHC I is not increased by loading with high affinity peptide, suggesting that peptide binding does not influence MHC I exit from the ER. Both properly assembled and peptide-receptive class I molecules are exported from the ER unless the latter are interacting with tpn (5, 6). In accordance with these findings, a proportion of surface-expressed MHC I molecules is unstable and has a rapid turnover in tpn-deficient cells (6), suggesting that tpn functions to retain unstable class I molecules in the ER.

In our previous studies, the association of MHC I molecules with tpn was greatly reduced in TAP-deficient cells, but the dissociation rate was extensively increased (3), suggesting that assembly with peptides may be required for MHC I to interact with tpn. Indeed, in the absence of tpn, an altered peptide profile was revealed by comparing pools of MHC I-binding peptides (7), suggesting that tpn-associated class I molecules are loaded with suboptimal peptides. The question is: how does tpn manage to retain unstable MHC I molecules in the ER? One of the possible mechanisms is that tpn interacts selectively with unstable class I molecules and retains/retrieves them in the ER until assembled with proper peptides. If this scenario is true, how does tpn prevent surface expression of peptide-receptive MHC I molecules? One important feature of tpn is the double lysine motif at its C terminus (8). Such a motif can act as a retrieval and retention signal for ER residential molecules in the ER (9). We have detected recently a small proportion of tpn in the Golgi compartment (4). In the Golgi, tpn associates with the coatomer protein complex I (COPI) coatomer, which coats vesicles active in retrograde transport of molecules from the Golgi to the ER (4, 10). Indeed, the COPI coatomer-associated tpn also interacts with MHC I molecules. This suggests tpn functions as a cargo receptor for retrograde transport of MHC I molecules (4, 11), which could be essential to prevent surface expression of unstable MHC I molecules and to maintain the homeostasis of Ag presentation.

To examine whether the double lysine motif is essential for retaining MHC I in the ER and for retrograde transport of unstable MHC I molecules from the Golgi back to the ER, GFP was inserted between the ER targeting signal sequence and the N-terminal domain of tpn. In addition, the C-terminal double lysine was substituted with two alanines generating a GFP-tpn-aa mutant. By comparing MHC I expression and intracellular distribution of GFP-tpn-aa or GFP-tpn in 721.220 transfectants, respectively, we found that although the predominant localization of GFP-tpn-aa is in the ER, there is an increase in the Golgi distribution tpn, which is accompanied with an increased MHC I surface expression in GFP-tpn-aa transfectants. However, the surface-expressed MHC I molecules were unstable and turned over rapidly. Moreover, mutation of the double lysine abolished the interaction of tpn with COPI coatomer. We propose that the double lysine motif of tpn is important for the retrieval of unstable MHC I molecules back to the ER.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

The 721.221-A2, 721.220, and 721.221 cell lines (provided by T. Elliott, University of Southampton, Southampton, U.K.) were grown in RPMI 1640 medium. The cell line 721.221 was derived from .184TGr cells, selected for loss of HLA after {gamma} irradiation, and lacks HLA-A, -B, and -C molecules and transcripts (12). The 721.221 cells are still capable of expressing normal amounts of transgene-encoded MHC I molecules at the cell surface. Tapasin-mutated 721.220 cell line was isolated in the same experiment that yielded 721.221, does not express HLA-A or -B molecules, but has retained partial expression of HLA-Cwl (12). The 721.221-A2 is stably transfected with a HLA-A2 (12). NIH-3T3 cells were grown in DMEM. All cell lines were cultured in medium supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO2 atmosphere.

Antibodies

Rabbit antiserum to human MHC I (R425), which reacts to all conformations of MHC I molecules, was provided by S. Kvist (Scandinavian Gene Synthesis AB, Köping, Sweden). The conformation-specific mAb W6/32, which reacts specifically with beta2-microglobulin-associated human class I molecules, was obtained from American Type Culture Collection. Rabbit antisera against human TAP1 or tpn were described previously (13). Ab to beta-COPI were provided by C. Harter (Biochemie-Zentrum Heidelberg Ruprecht-Karls-Universität, Heidelberg, Germany). The polyclonal Abs were affinity purified before use. The cis-Golgi marker GM130 was obtained from BD Transduction Laboratories.

Constructs and transfection

cDNA encoding wild-type tpn (tpn-wt) and tpn with replacement of the double lysine motif with two alanines (tpn-aa) were tagged with enhanced GFP (EGFP). The EGFP was inserted between the signal sequence and the N-terminal domain of tpn. The insertion of the EGFP-tag after the signal peptide of tpn allows for proper insertion of the expressed protein into the endoplasmic reticulum. The GFP-tpn-wt and GFP-tpn-aa constructs, containing a neomycin resistance gene, were transfected into 721.220 cells by electroporation with Nucleofactor Device and transfection kit (Amaxa). Transfected cells were either analyzed for class I expression after 24 h or selected by neomycin-containing medium. The selected clones were expanded after sorting of GFP-positive cells by FACS. Expression of GFP-tpn-wt, GFP-tpn-aa was determined by SDS PAGE and metabolic labeling. Clones expressing equal amounts of GFP-tpn-wt and GFP-tpn-aa were selected for further studies.

Flow cytometry

Cells were washed once in PBS containing 3% FBS, 0.2% sodium azide before incubation with or without W6/32. After washing, cells were incubated with PE-labeled anti-mouse IgG Ab. After removing free Ab, the intensity of PE was analyzed on gated GFP-positive cells using flow cytometry with CellQuest software (BD Biosciences). For analysis of class I expression after brefeldin A (BFA; Sigma-Aldrich) treatment, cells were washed twice with 1% FBS complete medium, and the cell pellet was suspended in 10% FBS complete medium (4 x 106 cells/ml) supplemented with 5 µg/ml BFA. BFA is a fungal metabolite that has been shown to block protein transport to the cell surface. Cells were then cultured at 37°C for various lengths of time, as indicated. At the end of incubation, cells were washed twice with PBS. The expression of surface MHC I was examined by staining the cells with W6/32 Ab and analyzed by flow cytometry.

Metabolic labeling, immunoprecipitation, and immunoblotting

Cells were washed twice with PBS and incubated for 30 min at 37°C in methionine-free RPMI 1640 medium containing 3% dialyzed FBS. Then 0.2 mCi/ml [35S]methionine (Amersham Biosciences) was added, and the incubation was continued for 30 min. At the end of labeling, cells were washed three times with ice-cold PBS and lysed in 1% digitonin 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 PMSF, 10 mg/ml leupeptin, 30 mg/ml aprotinin, and 10 mg/ml pepstatin). The cleared lysates were added to Abs previously bound to protein A-Sepharose beads. After washing, the immunoprecipitates were analyzed by SDS-PAGE, as previously described (14). SDS gels with samples subjected to metabolic labeling were put in fixation solution for 30 min and then in enhancer solution for 1 h. After drying the gels, they were exposed to autoradiography using intensifying screen. For pulse-chase and Endo-H experiments, cells were pulsed with 0.2 mCi/ml [35S]methionine for 15 min, and then chased for different periods of time. At the end of chase, cells were lysed in 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. The cleared lysates were precipitated with anti-tapasin or anti-MHC I Ab R425. The precipitated immune complex was washed three times in Nonidet P-40 lysis buffer. Endo-H (New England Biolabs) digestion was conducted according to the manufacturer’s suggestion. After Endo-H treatment, the precipitates were separated on 10% SDS gel. Amount of Endo-H-resistant and Endo-H-sensitive class I molecules was quantified by phospho imaging analysis. Endo-H experiments were done in triplicates, and SD was calculated.

Intracellular staining and confocal microscopy

NIH-3T3 cells were grown on sterile coverslips placed in six-well plates. The cells were transfected at 95% confluence with Fugene (Roche) and incubated at 37°C, 5% CO2 for 24 h to allow expression of the transgene. Cells were washed once with PBS, fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, and permeabilized with 0.5% Triton X-100 in PBS. Blocking was done in blocking buffer 10% FBS and 0.5% Tween 20 in PBS. Staining was done with Gm130 Golgi marker used in dilution 1/500, and secondary Ab Alexa Fluor 568 goat anti-mouse IgG was used in dilution recommended by manufacturer (catalogue A-11031; Molecular Probes). Confocal analyses and colocalization measurement were done with the Zeiss LSM 510 microscope and LSM 5 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mutation of the tpn double lysine motif did not affect tpn interactions with TAP or MHC I molecules

To investigate the function of the double lysine on tpn, we substituted the double lysine motif (KKXX), a canonical ER retrieval signal at the C terminus of tpn with double alanine (AAXX). In addition, a GFP was inserted between the membrane signal sequence and the N terminus of tpn or double lysine-mutated tpn in the final construct of GFP-tpn and GFP-tpn-aa, respectively. The interaction with TAP and MHC I molecules was tested after transfection of these two fusion constructs, respectively, into tpn-deficient 721.220 cells. Both GFP-fusion tpn proteins interacted efficiently with TAP and endogenous MHC I molecules (Fig. 1). There was no significant difference in the interaction with TAP or MHC I between these two transfectants. The results show no alteration of tpn function after addition of the GFP tag and are consistent with previous studies of YFP-tagged tpn (15). The association GFP-tpn-aa with TAP and MHC I suggests that the KKXX motif is not involved directly in the function of tpn regarding its effect on TAP peptide transport (16) and MHC I binding to the LC (17), which supports previous findings that KKXX is a putative retrieval signal and does not affect other functions of KKXX-carrying molecules (18, 19).


Figure 1
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  		FIGURE 1. Functional interaction of GFP-tagged tpn with MHC I and TAP. The 721.220 cells were transfected with GFP-tpn or GFP-tpn-aa. Transfection with empty plasmid served as negative control. Incorporation of GFP-tpn and GFP-tpn-aa into the LC was examined by coprecipitation with anti-tpn antiserum. Both GFP-tpn fusion proteins coprecipitated and interacted efficiently with TAP and MHC I.

 
Mutation of the KKXX motif results in an increase of tpn in the Golgi

Tpn is a type I transmembrane protein with KKXX ER retrieval signal and is detected in the ER at steady state (4, 15). Although most of the tpn is localized in the ER, subcellular fractionation and immunoelectron microscopy revealed that a proportion can be detected in the Golgi (4). However, there is no evidence that tpn is expressed on the cell surface, suggesting a mechanism for retrograde transport of tpn from the Golgi back to the ER. The double lysine motif has been reported to be the retrieval signal to activate this retrograde transport (4). We reasoned that tpn may play a role in retrieval of MHC I molecules from the Golgi back to the ER and/or the retention of MHC I molecules in the ER. To examine the intracellular distribution of KKXX mutant tpn, GFP-tpn-aa and GFP-tpn were transfected into NIH-3T3 cells, respectively. Although most of the GFP-tpn-aa was located in the ER in GFP-tpn-aa-transfected NIH-3T3 cells, the amount of GFP-tpn-aa overlapping with the Golgi marker was increased, in comparison with that in the GFP-tpn transfectants (Fig. 2). There was no detectable surface expression of either wild-type or mutant tpn. In addition, the level of Endo-H-resistant GFP-tpn-aa was higher than GFP-tpn (Fig. 3), indicating an increased distribution of KKXX mutant tpn in post-ER secretory compartments. Detection of Endo-H-resistant tapasin at the end of labeling in both transfectants may indicate that a small proportion of tapasin is rapidly transported to the post-ER compartment, while the level of Endo-H-resistant tapasin was unchanged in GFP-tpn-wt transfectants throughout the chase, suggesting recycling of the exported tapasin between the ER and the post-ER compartment. The mutation of the KKXX motif did not notably alter the localization to the ER of tpn, suggesting that the ER retention of tpn is not solely dependent on the KKXX motif. The retention of tpn in the ER may largely be due to its clustering with TAP and other ER chaperones, as exemplified by other ER proteins, which lack known ER retention signals (20). These results suggest that the retrieval of tpn from the post-ER compartments, but not ER retention, is regulated by the KKXX motif.


Figure 2
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  		FIGURE 2. Mutation of the tpn KKXX motif alters the intracellular distribution of tpn. NIH-3T3 cells were transfected with GFP-tpn or GFP-tpn-aa. The transfectants were stained by anti-Golgi marker Gm130 and Alexa Fluor 568 secondary Ab. Intracellular localization of GFP-tpn or GFP-tpn-aa and GM130 was analyzed by Zeiss LSM 510 confocal microscopy. Green shows tpn or tpn-aa, red GM130, and yellow shows the colocalization. A predominate green staining was observed in both transfectants. However, a significant proportion of GFP-tpn-aa, but not GFP-tpn, was colocalized with GM130. The transfection and confocal analysis were repeated several times with identical results.

 

Figure 3
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  		FIGURE 3. An increased proportion of GFP-tpn-aa is Endo-H resistant compared with GFP-tpn. GFP-tpn- and GFP-tpn-aa-transfected 721.220 cells were pulsed with 35S-methionine for 30 min and chased for different periods of time, as indicated. After chase, the cleared lysates were precipitated with anti-tpn Ab. The precipitates were treated with Endo-H, and then separated on SDS-PAGE. The labeled precipitates were visualized by autoradiography (a). The Endo-H-sensitive and -resistant molecules were quantified and shown in percentage (b).

 
Mutation of the KKXX motif of tpn results in increased surface expression of MHC I molecules

Previously, we found that tpn and associated MHC I could be detected in the Golgi membrane fraction (4). Evidence of tpn and COPI coatomer interaction suggests that tpn may act as a MHC I-cargo receptor for COPI vesicles. To investigate whether increased Golgi distribution of tpn affects surface expression of MHC I, 721.220 transiently transfected with GFP-tpn or GFP-tpn-aa were incubated with W6/32 Ab, and subsequently fluorescent secondary Ab. After the staining, the expression of MHC I was analyzed by FACS on gated GFP-positive cells. Cells incubated with normal mouse Ig served as background control. The expression of MHC I molecules on the 721.220 GFP-tpn-aa-transfected cells was increased >20% compared with 721.220 GFP-tpn (Fig. 4b) despite a similar level of class I and tpn in both transfectants (Fig. 4a). The background signals were identical. These results suggest that the lack of the tpn retrieval signal increases the surface expression of MHC I.


Figure 4
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  		FIGURE 4. The surface expression of MHC I is increased on cells expressing KKXX mutant tpn. The 721.220 cells were transiently transfected with GFP-fusion products of either wild-type tpn or a mutant tpn with substitution of the double lysine motif with a double alanine motif. Cells were stained with or without the MHC I dimer-specific Ab W6/32, followed by secondary PE-labeled anti-mouse IgG. The surface expression of class I was measured by FACS on gated GFP-expressing cells. The transfected cells incubated with normal mouse Ig served as a negative control. The expression of MHC I molecules on the 721.220 GFP-tpn-aa-transfected cells was increased with >20% compared with 721.220 GFP-tpn. b, Despite a similar level of class I and tpn in both transfectants (a). The results shown are representatives of three experiments with similar results.

 
Next, we examined the turnover of the surface-expressed MHC I by blocking intracellular protein transport. The cells were treated without or with BFA for different periods of time. At the end of treatment, cells were incubated with W6/32 Ab for 5 min on ice. After removing free Ab, cells were stained with PE-labeled secondary Ab and analyzed by FACS. The labeled MHC I molecules detected from the cell surface were increased in untreated 721.220 GFP-tpn-aa cells (Fig. 5), indicating again an increased export of MHC I in tpn double lysine mutants. However, following BFA treatment, the level of surface class I molecules decreased faster on 721.220 GFP-tpn-aa, suggesting that class I molecules are turned over faster on 721.220 GFP-tpn-aa cells than 721.220 GFP-tpn cells (Fig. 5). These results resemble those of MHC I expression at the surface of tpn-deficient cells (6, 21) and suggest that an active tpn KKXX motif is important to retain unstable MHC I molecules in the ER.


Figure 5
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  		FIGURE 5. A faster turnover of surface MHC I molecules on cells expressing double lysine mutant tpn compared with cells expressing wild-type tpn. To detect the surface class I stability, cells were treated without or with BFA for different periods of time, as indicated. Before and after treatment, cells were stained with W6/32 Ab and stained class I molecules were quantified by FACS (a), and the ratio of main intensity of GFP-TPN-aa vs GFP-TPN-wt was shown in b. Consistent with the results in Fig. 4, surface expression of class I was increased in GFP-tpn-aa cells before the treatment. After BFA treatment, surface class I was more significantly reduced on GFP-tpn-aa cells than on the GFP-tpn-wt. The presented results are from one of three replicated experiments.

 
MHC I is increased in the Golgi compartment in the 721.220 GFP-tpn-aa transfectants

Tpn-deficient 721.220 cells have reduced surface expression of endogenous MHC I molecules (22). In the absence of double lysine motif, tpn was increased in the Golgi (Fig. 3). To examine whether the lack of double lysine motif on tpn influences the transport of MHC I, we pulsed 721.220 GFP-tpn and 721.220 GFP-tpn-aa transfectants with [35S]methionine for 15 min and chased for different periods of time. After chase, the cleared lysates were subjected to precipitation of MHC I molecules and then treated with Endo-H. The Endo-H-sensitive or -resistant class I molecules were quantified after SDS-PAGE and autoradiography. At the beginning of the chase, the amount of pulse-labeled class I molecules was similar in both cell lines (Fig. 6). A small fraction of Endo-H-resistant class I molecules suggests a rapid export of MHC I molecules during a 15-min labeling period. However, the proportion of Endo-H-resistant MHC I molecules was increased in 721.220 GFP-tpn-aa transfectants following chase. Although we could not determine whether the increase was due to increased rate of MHC I transported with tpn into the Golgi compartment or due to the lack of retrograde transport of suboptimal class I molecules back to the ER, the increased cell surface expression of unstable MHC I molecules supports the later. Nevertheless, the increase of Endo-H-resistant class I molecules is associated with mutation of the tpn KKXX motif. Thus, the KKXX motif of tpn may have an important function to retrieve unstable MHC I molecules from the late secretory pathway back to the ER.


Figure 6
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  		FIGURE 6. The amount of MHC I in the Golgi is increased in 721.220 GFP-tpn-aa transfectants. The 721.220 GFP-tpn and 721.220 GFP-tpn-aa transfectants were pulsed with [35S]methionine and chased for different periods of time. After the chase, the cleared lysates were precipitated with the anti-MHC I Ab W6/32. The Endo-H-sensitive or -resistant class I molecules were quantified after SDS-PAGE and autoradiography (a). Before the chase, the amount of labeled class I molecules was similar in both cell lines. However, the proportion of Endo-H-resistant MHC I molecules was increased in 721.220 GFP-tpn-aa transfectants following the chase. The percentage of Endo-H-resistant and Endo-H-sensitive MHC I is shown in a graph based on a quantitative analysis of the labeled MHC I molecules (b). The results are from one of two replicated experiments.

 
KKXX motif is required for tpn to interact with COPI coatomer

COPI-coated vesicles are important for retrograde transport of molecules from the Golgi back to the ER. Proteins leaving the ER are a mixture of escaped ER resident proteins and mature membrane proteins destined for various cellular locations, and as a consequence the proteins incorporated into COPI vesicles have to be selected by specific cargo receptors. Previously, we have shown that tpn could interact with COPI coatomer and that this complex could be detected in the Golgi (4). The COPI coatomer-associated tpn was also associated with MHC I molecules, indicating that tpn acts as a cargo receptor for MHC I molecules. The presence of a KKXX motif is known to allow cargo receptors to mediate retrograde transport (9, 23). To investigate the importance of the KKXX motif for tpn interaction with COPI coatomer, tpn coprecipitation experiments were performed on GFP-tpn and GFP-tpn-aa transfectants. The precipitates were subsequently blotted by anti-beta-COPI coatomer Ab (Fig. 7a). Consistent with our previous findings, coprecipitation of COPI coatomer with tpn was found in 721.220 GFP-tpn transfectants; however, this interaction could not be detected in tpn precipitates of GFP-tpn-aa 721.220 transfectants. This result clearly indicates that the KKXX motif is required for tpn to interact with COPI coatomer. To further investigate whether tpn acts as cargo receptor for MHC I in COPI vesicle-mediated transport, lysates of GFP-tpn-aa or GFP-tpn were precipitated by W6/32 and the precipitates were blotted by anti-beta-COPI coatomer Ab (Fig. 7b). Indeed, consistent with our previous findings, COPI coatomer was detected in the MHC I precipitates. Such interaction was not detected in 721.220 GFP-tpn-aa transfectants.


Figure 7
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  		FIGURE 7. The double lysine motif of tpn is essential for the interaction with COPI coatomer. The 721.220 cells transfected with GFP-tpn or with GFP-tpn-aa were lysed in digitonin lysis buffer. Cleared lysates were precipitated with anti-tpn (a) and W6/32 Ab (b), respectively. Coprecipitates were separated on SDS-PAGE and subsequently blotted by anti-beta-COPI coatomer Ab. The blots were reblotted with anti-tpn and anti-MHC I Abs, respectively. beta-COPI could be precipitated with both the W6/32 and anti-tpn Abs, but only in cells expressing wild-type tpn. The 721.221-A2 served as positive control. The 721.220 was negative control for tpn coprecipitation, and 721.221 cells were negative control for MHC I coprecipitation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Tpn, which interacts with both MHC I and TAP, was initially suggested to facilitate assembly of stable MHC I with proper peptides in the ER (13, 24). However, studies of cell lines deficient in tpn, cell lines expressing MHC I mutants that do not interact with tpn, and tpn knockouts revealed that in the absence of tpn-MHC class interaction, MHC I molecules can still bind peptides in the ER, be exported to the cell surface, and present Ags to CD8+ T cells (6, 25, 26, 27). Thus, tpn is not involved in peptide loading directly.

From studies using fluorescence-tagged MHC I molecules, it was reported that the export of MHC I molecules from the ER might not be controlled by tpn (5, 15). However, the interaction with tpn increases the ER retention time of MHC I molecules, suggesting that tpn may selectively retain a certain proportion of the MHC I molecules, which are unstable due to lack of optimal peptide. Analysis of peptide profiles from MHC I expressed on tpn-deficient cells demonstrates a different peptide repertoire in comparison with MHC I on wild-type cells. Moreover, such difference is associated with a rapid turnover of MHC I molecules on the cell surface (7). These results provide further support that tpn-associated MHC I molecules are indeed unstable and loaded with suboptimal peptides.

The tpn-associated MHC I molecules retained in the ER could be either targeted for degradation or processed for an optimal peptide assembly. Peptide-receptive MHC I molecules are found early as well as late in the secretory pathway (4), but much less as at the cell surface (28, 29). It is not known whether the peptide-receptive MHC I molecules exported to late secretory compartments are degraded or transported in a retrograde manner back to the ER.

One of the mechanisms for intracellular retrograde transport is COPI-coated vesicles (10). These vesicles transport cargo molecules from the Golgi back to the ER. The cargo molecules have been suggested to be selected by receptors that have KKXX (transmembrane proteins) or KDEL (soluble proteins) retrieval signals at the C terminus (4, 30). Tpn carries a KKXX motif at its cytoplasmic C terminus and selectively interacts with unstable MHC I molecules, suggesting that tpn could be a cargo receptor for MHC I molecules for transport in COPI-coated vesicles. Like other proteins with KKXX motifs, tpn is predominantly located in the ER (Fig. 2) (4). Consistent with recent reports, we could not detect an active recycling of tpn between the ER and the Golgi by time-lapse photo bleaching of GFP-tpn transfectants (data not shown) (15). This suggests that only a limited amount of tpn is recycling between the ER and the post-ER compartment. The phenotype of increased tpn-aa in the Golgi, but with predominant ER distribution, suggests that the retention of tpn depends on the formation of complex of tpn with a number of ER chaperones and TAP, but not on the KKXX motif. However, the increase in Golgi distribution of KKXX-mutated tpn indicates that the retrieval signal may be still important for retrograde transport of tpn that escaped from the ER.

The exit of membrane proteins from the ER has been suggested to be mainly mediated by bulk flow at ER exit sites (31). The transport receptor molecules function to enrich cargo molecules at specialized ER exit sites for coatomer protein complex II vesicle-mediated export (10). If the cargo molecules are immature, they will be clustered with ER proteins, e.g., chaperones, and retained in the ER (4, 32, 33, 34). ER residential molecules, such as tpn, rely on molecular clustering and retrograde transport to be retained in the ER (15, 35). Studies from a number of KKXX motif-containing ER molecules indicate that how far down the secretory pathway KKXX mutants escape varies widely in the range from plasma membrane expression to limited increase in post-ER compartments (18, 36). This variation is largely due to differences in interaction with other ER retention proteins and the maturation process in its own, e.g., glycosylation (37). However, most of the studied ER proteins with KKXX mutations have been shown to increase their distribution in post-ER compartments (18, 37). Although tpn is an ER residential molecule, a proportion of tpn is detected in the Golgi (4) and tpn interacts with an ER export receptor (38), supporting the notion that tpn could escape from the ER. The association of COPI coatomer with tpn indicates that these escaped tpn are retrograde transported back to the ER by COPI-coated vesicles (4). In the absence of tpn, class I expression is considerably reduced (25). However, it is unknown whether the reduction of class I expression is due to the deficiency in peptide loading or to the lower stability of class I molecules. The reduced surface expression of class I can be restored by a soluble form of tpn, suggesting that the interaction between tpn and class I is essential for class I expression (39). However, further study showed that class I expression induced by soluble tpn was less stable and loaded with different peptide profile, similar to class I from tpn-deficient cells (40), indicating that tpn does not change the quality of class I directly, but controls the quality of class I expression by retaining unstable class I in the ER. Together with our previous findings, we have now provided evidence that tpn, mediated by its double lysine motif, may play important role for the retrograde transport of unstable class I molecules back to the ER. The interaction of double lysine motif with COPI coatomer suggests that such retrograde transport is mediated by COPI-coated vesicles. Thus, tpn retains unstable MHC I molecules in the ER by clustering them away from the ER exit sites and by regulating the retrograde transport of unstable class I molecules from post-ER compartments in the secretory pathway for a stable presentation of optimal antigenic peptides.


   Acknowledgments
 
We thank Drs. M. T. Barel and V. McDonald for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Biotechnology and Biological Sciences Research Council and Medical Research Council. K.M.P. is supported by the Swedish Research Council (project K2003-99PK-14896-01A) and Alfred Benzon Foundation. Back

2 Address correspondence and reprint requests to Dr. Suling Li, Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K.; E-mail address: su-ling.li{at}brunel.ac.uk or Dr. Ping Wang, Institute of Cell and Molecular Science, Barts and London School of Medicine, Turner Street, London E1 2AD, U.K.; E-mail address: p.wang{at}qmul.ac.uk Back

3 Abbreviations used in this paper: MHC I, MHC class I; ER, endoplasmic reticulum; BFA, brefeldin A; COPI, coatomer protein complex I; EGFP, enhanced GFP; LC, loading complex; tpn, tapasin; tpn-aa, tpn with replacement of double lysine motif with two alanines; tpn-wt, wild-type tpn. Back

Received for publication August 3, 2005. Accepted for publication March 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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