The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 7646 -7652
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Everett, M. W.
Right arrow Articles by Edidin, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Everett, M. W.
Right arrow Articles by Edidin, M.

Tapasin Increases Efficiency of MHC I Assembly in the Endoplasmic Reticulum but Does Not Affect MHC I Stability at the Cell Surface1

Maya W. Everett and Michael Edidin2

Department of Biology, Johns Hopkins University, Baltimore, MD 21218


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cell surface expression of MHC I molecules depends on the chaperone tapasin; how tapasin functions is not fully understood. We created three fluorescent tapasin constructs: wild-type tapasin, soluble tapasin, which does not interact with TAP, and N300 tapasin, which does not interact with MHC I. In contrast to earlier reports, all three constructs localize to the endoplasmic reticulum (ER), though soluble tapasin is more mobile than wild type and N300. Soluble tapasin does not increase MHC I surface levels to the same extent as wild type, which suggests that proximity to TAP is necessary for full tapasin function. N300 acts as a dominant-negative perhaps by blocking wild-type tapasin access to TAP. None of the constructs affects MHC I stability at the cell surface, although stability of ER resident MHC I is decreased in tapasin-negative cells. We propose that tapasin acts primarily to increase efficiency of assembly of MHC I within the ER.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cell surface expression of MHC I molecules reports the internal health of a cell by presenting an 8–10 amino acid peptide to surveillance by CD8+ T cells and NK cells. Tapasin is a chaperone dedicated to MHC I assembly within the endoplasmic reticulum (ER)3 and is critical to MHC I cell surface expression. Tapasin-knockout mice have reduced ability to mount CD8+ T cell responses against viral infections and other processes that rely on cell surface MHC I, such as CD8+ T cell development, cross-presentation by dendritic cells, and NK cell responses are also impaired (1, 2). Additionally, reduction of tapasin expression has been observed in human cancers (3).

Tapasin is a type I transmembrane protein with a large ER luminal domain, a single pass transmembrane domain, and a short cytoplasmic tail containing an ER retention-retrieval motif (4). Tapasin coordinates the formation of the MHC I peptide loading complex by tethering MHC I to the peptide transporter TAP (4) and by recruiting calreticulin and ERp57 (5, 6). Tapasin has been proposed to affect which peptide is bound by MHC I (7, 8, 9, 10, 11, 12) and recent in vitro work has shown that tapasin directly affects the peptide binding groove in collaboration with ERp57 and allows for binding of higher affinity peptides (13, 14, 15).

We and others (16, 17, 18) have shown that tapasin maintains an ER localization. However, Wang and colleagues (19, 20) have suggested that tapasin escapes the ER to retrieve unstable MHC I from the Golgi. Two deletion constructs have provided information in regards to tapasin localization and function. N300 tapasin, which lacks the majority of the ER luminal domain, is unable to bind MHC I and does not rescue MHC I surface expression in tapasin-negative cells, although it maintains ER localization and interacts with TAP as shown by pulse-chase and immunoprecipitation experiments (21). In similar experiments, soluble tapasin, which lacks the transmembrane domain and cytoplasmic tail, does not interact with TAP; indeed, some of the protein is secreted (18, 22). Despite this behavior, soluble tapasin increases MHC I surface levels when transfected into tapasin-negative cells (11, 18, 22). Although informative, these biochemical studies lack a detailed analysis of cellular localization of soluble and N300 tapasin and how they relate to tapasin function.

In an effort to clarify these findings, we created fluorescently tagged versions of the wild-type, soluble, and N300 tapasin constructs. The data collected with these constructs contrast with previous reports. We found that most soluble tapasin is localized to the ER similarly to wild type. However, soluble tapasin is more mobile than wild-type and N300 tapasin, possibly because it does not interact with TAP. These differences in mobility correlate to differences in function. Fluorescent soluble tapasin is unable to rescue MHC I surface levels in tapasin-negative cells to the same extent as wild-type tapasin. Additionally, N300 tapasin acts as a dominant-negative when coexpressed with wild-type tapasin, most likely because it blocks wild-type tapasin access to TAP. Together, our data highlight the importance of TAP proximity for full tapasin function. Although we see differences in MHC I surface expression after transfection of our constructs into tapasin-negative cells, we do not see differences in MHC I stability at the cell surface. However, we find that tapasin-negative cells have a higher amount of unstable MHC I within the ER. This suggests that tapasin enhances the assembly of stable MHC I within the ER and so increases efficiency of delivery of MHC I to the cell surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

HeLa cells were maintained in DMEM (Mediatech) supplemented with 10% heat-inactivated FBS (HyClone) and 2 mM L-glutamine (Invitrogen Life Technologies). T2 cells lack TAP whereas T1 cells are TAP positive (23). The 721.220 cell line lacks tapasin and has been stably transfected with HLA-B8 (24), HLA-B2705, and HLA-B4402 (7).

DNA constructs

We previously made N-terminal-tagged cyan fluorescent protein (CFP)-wild-type and yellow fluorescent protein (YFP)-wild-type tapasin (17). We generated monomeric CFP and YFP by introducing A243K, which corresponds to the A206K mutation that abolishes dimerization of these fluorophores at high concentrations (25). Fluorescent deletion tapasin constructs were designed by PCR amplification of the desired regions of the tapasin construct and reinsertion into the original plasmid with the tapasin sequence removed via restriction digest. The tandem tapasin construct was engineered by PCR amplification of the CFP plus linker sequence from the CFP-tapasin construct and then inserted into the YFP-tapasin construct so that the YFP is linked to the CFP, which is then linked to the tapasin sequence. All primers were synthesized by Integrated DNA Technologies. All constructs were sequenced by Johns Hopkins University Core DNA Analysis Facility. YFP-labeled HLA-A2 has been described (26).

Microscopy and colocalization measurements

HeLa cells were plated on coverslips and transfected using Fugene 6 transfection reagent (Roche). Cells were fixed and stained as described (17). The Golgi complex was visualized with a rabbit polyclonal Ab against the Golgi marker giantin (Covance Research Products) and a goat anti-rabbit IgG conjugated to Alexa Fluor 633 (Molecular Probes). Cells were mounted using the SlowFade antifade kit (Molecular Probes).

Images were collected using a Zeiss LSM 510 confocal microscope with a 63x objective. Colocalization analyses were performed with MIPAV software (27). Background subtraction was performed before colocalization analysis using the orthogonal regression tool. Percent colocalization refers to the percentage of the population of one fluorophore, which is located in the same pixels as the opposite fluorophore. All images were taken with the same microscope settings. All cells were of comparable fluorescence intensities as measured by the glow scale indicator on the microscope.

Fluorescence recovery after photobleaching (FRAP) data collection and analysis

HeLa cells were plated on coverslips and transfected using Fugene 6 transfection reagent (Roche). T1 and T2 cells were transfected by electroporation (BTX Electro Cell Manipulator 600). HeLa cells were mounted in HBSS on microscope slides and sealed with nail polish. T1 and T2 cells suspended in HBSS were imaged in Vitrotubes (Vitrocom). All data were taken within 30 min of sample preparation. Our FRAP setup and data collection has been previously described (28). Statistical analyses were performed using GraphPad software.

Flow cytometry, MHC I surface expression, and stability measurements

721.220 cell lines were transiently transfected by electroporation (BTX Electro Cell Manipulator 600). MHC I surface molecules were labeled with the mouse mAbs KE2 (29) or W6/32 (30), purified from hybridoma supernatants using Protein-A affinity chromatography, and directly conjugated to Cy5 with the Amersham Cy5 mono-reactive dye pack (GE Healthcare). Cells were washed in cold PBS before staining with saturating levels of Ab (determined by titration curves). Cells were washed again in cold PBS to remove excess Ab. For measurements with a secondary Ab, after staining cells with saturating levels of unlabeled W6/32, cells were washed with cold PBS and then stained with goat anti-mouse IgG conjugated to Alexa Fluor 633 (Molecular Probes) and then washed in cold PBS. Data were collected on a FACSCalibur (BD Biosciences). For MHC I stability measurements, cells were treated with 5 µg/ml brefeldin A (BFA) (Sigma-Aldrich). Release of unstable and/or empty MHC I to the cell surface was induced by incubating the cells at 25°C for 5 h. Curve fits and statistical analyses were performed using GraphPad software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Wild-type and soluble tapasin colocalize in the ER and neither construct colocalizes with a Golgi complex marker

It has been reported that soluble tapasin is secreted whereas wild-type tapasin maintains an ER localization (16, 17, 18, 22). Using our fluorescent tapasin constructs, we found no evidence for secretion of soluble tapasin. Both colors of wild-type and soluble tapasin colocalize in the ER (Fig. 1) and have the same colocalization coefficients (80–85%) as tandem tapasin, which has CFP and YFP physically linked (data not shown). Neither wild-type nor soluble tapasin colocalized with a Golgi complex marker (Fig. 2, A and B). In contrast, there was significant colocalization of a YFP-tagged MHC I molecule HLA-A2 with the Golgi (Fig. 2C), consistent with the traffic of this molecule through the Golgi to the cell surface. Whereas most YFP-tagged HLA-A2 expressing cells have 20–30% of the YFP localized to the Golgi, there is <10% colocalization of YFP-wild-type or YFP-soluble tapasin with the Golgi marker (Fig. 2D). Thus most soluble tapasin is retained in the ER.


Figure 1
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 1. Wild-type and soluble tapasin fluorescent constructs colocalize in the ER. HeLa cells were transiently transfected with YFP-soluble and CFP-wild-type tapasin or YFP-wild-type and CFP-soluble tapasin. Yellow indicates colocalization in the overlay.

 

Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 2. YFP-wild-type tapasin and YFP-soluble tapasin do not colocalize with a Golgi marker. HeLa cells were transiently transfected with YFP-labeled wild-type tapasin (A), soluble tapasin (B), or HLA-A2 (C). YFP is shown in green and Ab staining is shown in red. The distribution of percent colocalization of YFP with Golgi is shown in D as a histogram. Cell numbers are normalized to percentage of total cells analyzed. Data are pooled from two experiments with N {approx} 30.

 
Soluble tapasin is more mobile than wild type and N300 in the presence of TAP

Because soluble tapasin does not bind TAP (22), we expected the mobility of soluble tapasin in the ER to differ from that of wild type and N300, which do bind TAP (4, 21). Although soluble tapasin is not a membrane protein, measurements of its mobile fraction should be independent of membrane attachment. We measured mobility by FRAP. YFP-soluble tapasin had a higher mobility than YFP-wild type and YFP-N300 in the TAP-positive cell lines HeLa and T1, but all three constructs had the same mobility in the TAP-negative cell line T2 (Table I). Although mobile fraction values were different between the cell lines, the trend was the same in TAP-positive cells and different in TAP-negative cells. Although YFP-N300 had a lower mobility than YFP-wild-type tapasin in HeLa cells, this trend was not seen in T1 or T2 cells and may be a cell-specific effect.


View this table:
[in this window]
[in a new window]

  		Table I. YFP-wild-type and YFP-N300 tapasin have limited mobility due to interactions with TAPa

 
Tapasin proximity to TAP increases MHC I surface expression

The mobility differences we observed for the YFP-wild-type and YFP-soluble tapasin constructs corresponded to differences in their function. YFP-wild-type, YFP-soluble, or YFP-N300 tapasin were transfected into the tapasin-negative cell line 721.220, stably expressing the tapasin-dependent allele HLA-B4402. YFP-wild-type and YFP-soluble tapasin transfections increased MHC I surface levels in proportion to the amount of tapasin expressed. At higher levels of expression, both constructs reached a level of saturation in which increased expression did not result in increased MHC I surface levels (Fig. 3A). This saturating level of YFP expression was used for all additional experiments. YFP-soluble tapasin did not increase MHC I surface levels to the same extent as YFP-wild-type tapasin (Fig. 3A). As expected from previous work, transfection with YFP-N300 tapasin did not significantly increase MHC I surface levels (Fig. 3A) (21). We repeated this experiment with two other MHC I alleles: B2705 and B8 (Fig. 3B). We observed the same trends for B2705 and B8 as for B4402 (Fig. 3B). Consistent with its reported tapasin independence (7), B2705 showed the smallest increase in MHC I surface expression after transfection of YFP-wild-type tapasin and had the highest levels of surface MHC I after transfection of YFP-N300 or mock transfection (Fig. 3B).


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 3. YFP-soluble tapasin does not increase MHC I surface levels to the same extent as YFP-wild-type tapasin. 721.220.B4402 cells were transiently transfected with YFP-wild-type, YFP-soluble, or YFP-N300 tapasin and MHC I surface molecules were stained with Cy5-conjugated KE2 and measured by flow cytometry (A). MHC I surface levels in 721.220.B4402, 721.220.B2705, and 721.220.B8 cells transiently transfected with YFP-wild-type, YFP-soluble, or YFP-N300 were compared (B). Data are combined from two independent experiments. (***, p < 0.0001).

 
N300 is a dominant-negative when coexpressed with wild-type tapasin

N300 tapasin does not interact with MHC I but does interact with TAP (21). This interaction with TAP correlates with our observation of its reduced mobility in TAP-positive cells (Table I). We expected that binding of N300 to TAP would block binding of wild-type tapasin and interfere with its function. YFP-N300 tapasin transfection of 721.220 cells stably transfected with unlabeled wild-type tapasin decreased surface expression of B4402, B2705, and B8 (Fig. 4). Transfection of YFP-N300 does not alter MHC I surface expression in tapasin-negative cells (Fig. 3). Expression of B2705, which is reported to be the least tapasin-dependent of the three HLA alleles (7), was least affected when YFP-N300 was coexpressed with wild-type tapasin (Fig. 4).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. YFP-N300 tapasin is a dominant-negative when coexpressed with wild-type tapasin. 721.220.B4402, 721.220.B8, and 721.220.B2705 cells with stably transfected unlabeled wild-type tapasin were transiently transfected with YFP-N300. MHC I surface molecules were stained with Cy5-conjugated KE2 and MHC I surface levels were measured by flow cytometry. MHC I surface levels were normalized to non-YFP expressing cells. Data are pooled from two experiments and shown with SEs. (*, p < 0.05, **, p < 0.01, ***, p < 0.0001).

 
Tapasin does not affect stability of surface MHC I molecules

Though the wild-type, soluble, and N300 tapasin constructs have different effects on levels of surface MHC I expression, we found that they do not affect the surface lifetimes of these molecules at 37°C (Fig. 5). Although B4402 has low expression levels in the absence of tapasin (Fig. 3), these levels were still significantly above background and allowed for accurate measurements of decay curves (data not shown). These stability measurements were in contrast to previous work which has reported that MHC I surface lifetimes are reduced in the absence of wild-type tapasin or the presence of soluble tapasin (1, 8, 9, 11, 18, 31). This discrepancy may lie in the fact that previously reported data were collected using indirect immunofluorescence, whereas we used directly conjugated primary Ab. Additionally, we used the KE2 mouse mAb, which we have found to bind the same epitope as the W6/32 mouse mAb that was primarily used in previous work with human cell lines. Repeating our stability measurements using directly conjugated W6/32 showed no difference in surface stabilities between YFP-wild-type tapasin and mock-transfected 721.220 cells (data not shown). However, use of unlabeled W6/32 followed by fluorescent secondary Ab verified that this technique could qualitatively yield opposite results, although we observed higher experimental error, which revealed these results as not statistically significant (data not shown).


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 5. YFP-tapasin constructs do not affect MHC I surface stability. 721.220.B4402 (A), 721.220.B8 (B), or 721.220.B2705 (C) cells were transiently transfected with YFP-wild-type, YFP-soluble, or YFP-N300 tapasin. Cells were treated with 5 µg/ml BFA for varying lengths of time. MHC I surface molecules were stained with Cy5-conjugated KE2 and surface levels were measured by flow cytometry. Decay curves are standardized to each transfection at time zero. Rate constants are shown with SEs (D).

 
Tapasin reduces the fraction of empty and/or unstable MHC I molecules in the ER

Incubation of cells at 25°C stabilizes empty MHC I molecules and allows them to reach the cell surface (32). We used this treatment to investigate whether tapasin reduced the fraction of empty MHC I molecules in the ER. After transfection with YFP-wild-type tapasin, cells were cooled to 25°C for 5 h before measuring the stability of the MHC I surface molecules over time at 37°C. All three MHC I alleles had longer lifetimes on the cell surface when assembled in the presence of YFP-wild-type tapasin than when assembled in the absence of tapasin (Fig. 6). These data show that tapasin-negative cells have a larger population of poorly assembled and/or empty MHC I within the ER compared with tapasin-positive cells. At biological temperatures, these molecules do not reach the cell surface because there is no difference between lifetimes of surface MHC I molecules from tapasin-positive or tapasin-negative cells that were not previously treated at 25°C (Fig. 5).


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 6. Tapasin-negative cells have a higher percentage of unstable MHC I that do not reach the cell surface at steady state. 721.220.B4402 (A), 721.220.B8 (B), or 721.220.B2705 (C) cells were transiently transfected with YFP-wild-type tapasin or mock transfected. Cells were cooled to 25°C for 5 h before treatment with 5 µg/ml BFA for varying lengths of time. MHC I surface molecules were stained with Cy5-conjugated KE2, and surface levels were measured by flow cytometry. Decay curves are standardized to each transfection. The rate constants for the decay curves are shown with SEs (D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We used fluorescent constructs to investigate how tapasin localization correlates with its function. Our data highlight the importance of tapasin proximity to TAP and its local peptide supply for optimal MHC I surface expression. Tapasin increases TAP stability and TAP peptide translocation (2, 21, 22, 33, 34), which indicates that tapasin contributes to increased peptide concentrations near the tethered MHC I. Wild-type tapasin, which tethers MHC I to TAP, enhanced MHC I surface expression. N300 tapasin, which binds TAP but not MHC I, reduced the effect of wild-type tapasin and acted as a dominant-negative apparently by competing for TAP binding sites. Soluble tapasin, which binds MHC I but not TAP, also enhances MHC I surface expression, although not to the same extent as wild type. Consistent with this finding, mutations in TAP that diminished tapasin binding are reported to reduce surface expression of MHC I (35). Other laboratories have also found that soluble tapasin seems to work less efficiently by taking more time to either assemble equivalent amounts of thermally stable MHC I molecules (31) or rescue MHC I surface levels after acid strip of existing surface MHC I (11). Recombinant tapasin in collaboration with ERp57 has been shown to act on the peptide binding groove to allow for peptide binding and editing (13, 14, 15); because soluble tapasin is ER localized, it may be that binding of soluble tapasin to MHC I allows for peptide binding, although this process is limited due to lower concentrations of local peptide supply.

Our imaging analyses of tapasin and its mutants differ from earlier reports. We find that a significant population of soluble tapasin is in the ER, and we cannot detect soluble tapasin in the Golgi. Previous work using pulse-chase methods reported secretion of soluble tapasin (18, 22). Pulse-chase measurements are more sensitive than microscope image analysis, and a small portion of the soluble tapasin population escaping into the secretory pathway could be missed by colocalization experiments. Wang and colleagues (19) have reported on similar work with a fluorescent tapasin construct in which the ER retention-retrieval motif was mutated by alanine substitution and showed that their construct colocalizes with the Golgi. Our contrasting observations may be due to differences in cell line, Golgi marker used, expression levels of the fluorescent construct, or the need for a transmembrane domain and cytoplasmic tail for ER export. We also note that image collection with a confocal microscope using large pinhole settings can lead to spurious colocalization due to thickness of optical sections (F. Abe and M. Edidin, unpublished observations); we maintained the smallest pinhole sizes possible for all colocalization measurements.

We also present the novel finding that tapasin does not affect the lifetime of surface MHC I molecules. This work contradicts a number of studies that have reported reduced cell surface lifetime of MHC I molecules assembled in the absence of tapasin or presence of soluble tapasin (1, 8, 9, 11, 18, 31). We propose that the difference in observations may be a result of use of a secondary Ab for measuring levels of MHC I surface expression. We found that labeling with directly conjugated primary Abs yielded more accurate measurements of MHC I surface levels. Additionally, although one report noted similar MHC I surface expression levels between tapasin-positive and -negative cells (9), it has been shown that 6–12 times expression levels of MHC I in tapasin-deficient cells are needed to achieve similar MHC I surface levels in tapasin-positive cells (36). This level of MHC I expression may cause the peptide pool to be limiting and affect MHC I stability; hence, differences in expression levels of MHC I may also have contributed to the observed discrepancies between our data and some previous reports.

Another method that is used to measure stability of MHC I is immunoprecipitation of the native MHC I peptide complex from heated cell lysates (1, 18, 31). In these experiments, molecules from both the cell surface and the ER are captured. If tapasin acts to assemble the MHC I subunits, assembly would be limited in tapasin-negative cells, and a large portion of unstable heterodimers lacking peptide would be present in the ER. These molecules would contribute to thermal instability in immunoprecipitation experiments, but they do not represent MHC I stability at the surface. Consistent with this idea, we found that when we stabilized ER resident MHC I complexes by cooling cells to 25°C and allowed their export to the cell surface, we observed a difference in MHC I surface stabilities at 37°C between tapasin-positive and tapasin-negative cells, indicating that there is a larger population of unstable MHC I in the ER of tapasin-negative cells than in the ER of tapasin-positive cells.

Although it has been proposed that retention of MHC I molecules in the ER is an important aspect of tapasin function (2, 8, 16), H2-M3, a MHC class Ib molecule, is efficiently retained in the ER in the absence of tapasin (37, 38). Similarly, we have found a large population of unstable ER resident MHC I in tapasin-negative cells that fail to reach the cell surface, which argues against tapasin functioning to retain MHC I in the ER. A number of past reports have provided evidence that tapasin expression alters the peptide repertoire bound by MHC I (7, 8, 9, 10, 11, 12). As previously mentioned, recent work has provided insight that tapasin, along with ERp57, directly affects peptide loading and editing of the MHC I molecule (13, 14, 15). Interestingly, although peptide editing should confer higher stability of the resulting MHC I complex, we do not observe differences in MHC I lifetimes at the cell surface between tapasin-positive and tapasin-negative cells. It may be that in the absence of tapasin, unloaded or suboptimally loaded MHC I are rapidly degraded before or during ER export (2). Tapasin function may also be compensated by other proteins that assist in MHC I peptide assembly and/or prevent unstable MHC I from reaching the cell surface. Protein disulfide isomerase may stabilize a peptide-receptive state of the MHC I heterodimer and allow for peptide editing (39). Additionally, we have identified Bap31 as a cargo receptor for export of MHC I to the secretory pathway (40). Recent work suggests that Bap31 contributes to quality control of MHC I peptide complexes by selectively retrieving MHC I destabilized by the lower pH of the ER-Golgi intermediate compartment (41). Although Williams and colleagues (42) have shown that Bap31 does not affect MHC I surface lifetimes, these measurements were performed after treatment at 26°C, which would allow the release of unstable MHC I and may bypass our proposed mechanism for Bap31. Protein disulfide isomerase, Bap31, and other unidentified proteins may ensure that only stable MHC I molecules reach the cell surface. Although our data do not show that tapasin increases MHC I stability at the cell surface, the efficiency of stable MHC I peptide assembly and subsequent cell surface expression cannot be matched by other proteins in the absence of tapasin. As mentioned, loss of tapasin has many detrimental effects on immune system mechanisms dependent on MHC I in mice and humans (1, 2, 3). Our data suggest that these effects are caused by a decrease in MHC I surface levels and not by a decrease in MHC I stability.


   Acknowledgments
 
We thank Dr. Peter Cresswell (Yale University) for providing the 721.220 cell lines, Edward Perkins (Johns Hopkins University Integrated Imaging Center), and Drs. David Fooksman, Saame Raza Shaikh, and Jason Weil for helpful discussions and technical expertise.


   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 National Institutes of Health Grant AI14584. Back

2 Address correspondence and reprint requests to Dr. Michael Edidin, Johns Hopkins University, 144 Mudd Hall, 3400 North Charles Street, Baltimore, MD 21218. E-mail address: edidin{at}jhu.edu Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; BFA, brefeldin A; FRAP, fluorescence recovery after photobleaching. Back

Received for publication August 7, 2007. Accepted for publication September 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Garbi, N., P. Tan, A. D. Diehl, B. J. Chambers, H. G. Ljunggren, F. Momburg, G. J. Hammerling. 2000. Impaired immune responses and altered peptide repertoire in tapasin-deficient mice. Nat. Immunol. 1: 234-238. [Medline]
  2. Grandea, A. G., III, T. N. Golovina, S. E. Hamilton, V. Sriram, T. Spies, R. R. Brutkiewicz, J. T. Harty, L. C. Eisenlohr, L. Van Kaer. 2000. Impaired assembly yet normal trafficking of MHC class I molecules in Tapasin mutant mice. Immunity 13: 213-222. [Medline]
  3. Seliger, B., K. Schreiber, K. Delp, M. Meissner, S. Hammers, T. Reichert, K. Pawlischko, R. Tampe, C. Huber. 2001. Downregulation of the constitutive tapasin expression in human tumor cells of distinct origin and its transcriptional upregulation by cytokines. Tissue Antigens 57: 39-45. [Medline]
  4. Ortmann, B., J. Copeman, P. J. Lehner, B. Sadasivan, J. A. Herberg, A. G. Grandea, S. R. Riddell, R. Tampe, T. Spies, J. Trowsdale, P. Cresswell. 1997. A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes. Science 277: 1306-1309. [Abstract/Free Full Text]
  5. Solheim, J. C., M. R. Harris, C. S. Kindle, T. H. Hansen. 1997. Prominence of β 2-microglobulin, class I heavy chain conformation, and tapasin in the interactions of class I heavy chain with calreticulin and the transporter associated with antigen processing. J. Immunol. 158: 2236-2241. [Abstract]
  6. Hughes, E. A., P. Cresswell. 1998. The thiol oxidoreductase ERp57 is a component of the MHC class I peptide-loading complex. Curr. Biol. 8: 709-712. [Medline]
  7. Peh, C. A., S. R. Burrows, M. Barnden, R. Khanna, P. Cresswell, D. J. Moss, J. McCluskey. 1998. HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading. Immunity 8: 531-542. [Medline]
  8. Barnden, M. J., A. W. Purcell, J. J. Gorman, J. McCluskey. 2000. Tapasin-mediated retention and optimization of peptide ligands during the assembly of class I molecules. J. Immunol. 165: 322-330. [Abstract/Free Full Text]
  9. Barber, L. D., M. Howarth, P. Bowness, T. Elliott. 2001. The quantity of naturally processed peptides stably bound by HLA-A*0201 is significantly reduced in the absence of tapasin. Tissue Antigens 58: 363-368. [Medline]
  10. Purcell, A. W., J. J. Gorman, M. Garcia-Peydro, A. Paradela, S. R. Burrows, G. H. Talbo, N. Laham, C. A. Peh, E. C. Reynolds, J. A. Lopez De Castro, J. McCluskey. 2001. Quantitative and qualitative influences of tapasin on the class I peptide repertoire. J. Immunol. 166: 1016-1027. [Abstract/Free Full Text]
  11. Zarling, A. L., C. J. Luckey, J. A. Marto, F. M. White, C. J. Brame, A. M. Evans, P. J. Lehner, P. Cresswell, J. Shabanowitz, D. F. Hunt, V. H. Engelhard. 2003. Tapasin is a facilitator, not an editor, of class I MHC peptide binding. J. Immunol. 171: 5287-5295. [Abstract/Free Full Text]
  12. Howarth, M., A. Williams, A. B. Tolstrup, T. Elliott. 2004. Tapasin enhances MHC class I peptide presentation according to peptide half-life. Proc. Natl. Acad. Sci. USA 101: 11737-11742. [Abstract/Free Full Text]
  13. Chen, M., M. Bouvier. 2007. Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection. EMBO J. 26: 1681-1690. [Medline]
  14. Kienast, A., M. Preuss, M. Winkler, T. P. Dick. 2007. Redox regulation of peptide receptivity of major histocompatibility complex class I molecules by ERp57 and tapasin. Nat. Immunol. 8: 864-872. [Medline]
  15. Wearsch, P. A., P. Cresswell. 2007. Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer. Nat. Immunol. 8: 873-881. [Medline]
  16. Schoenhals, G. J., R. M. Krishna, A. G. Grandea, III, T. Spies, P. A. Peterson, Y. Yang, K. Fruh. 1999. Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells. EMBO J. 18: 743-753. [Medline]
  17. Pentcheva, T., E. T. Spiliotis, M. Edidin. 2002. Cutting edge: tapasin is retained in the endoplasmic reticulum by dynamic clustering and exclusion from endoplasmic reticulum exit sites. J. Immunol. 168: 1538-1541. [Abstract/Free Full Text]
  18. Tan, P., H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, F. Momburg. 2002. Recruitment of MHC class I molecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading. J. Immunol. 168: 1950-1960. [Abstract/Free Full Text]
  19. Paulsson, K. M., M. Jevon, J. W. Wang, S. Li, P. Wang. 2006. The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum. J. Immunol. 176: 7482-7488. [Abstract/Free Full Text]
  20. Paulsson, K. M., M. J. Kleijmeer, J. Griffith, M. Jevon, S. Chen, P. O. Anderson, H. O. Sjogren, S. Li, P. Wang. 2002. 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. J. Biol. Chem. 277: 18266-18271. [Abstract/Free Full Text]
  21. Bangia, N., P. J. Lehner, E. A. Hughes, M. Surman, P. Cresswell. 1999. The N-terminal region of tapasin is required to stabilize the MHC class I loading complex. Eur. J. Immunol. 29: 1858-1870. [Medline]
  22. Lehner, P. J., M. J. Surman, P. Cresswell. 1998. Soluble tapasin restores MHC class I expression and function in the tapasin-negative cell line 220. Immunity 8: 221-231. [Medline]
  23. Salter, R. D., P. Cresswell. 1986. Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J. 5: 943-949. [Medline]
  24. Grandea, A. G., III, M. J. Androlewicz, R. S. Athwal, D. E. Geraghty, T. Spies. 1995. Dependence of peptide binding by MHC class I molecules on their interaction with TAP. Science 270: 105-108. [Abstract/Free Full Text]
  25. Zacharias, D. A., J. D. Violin, A. C. Newton, R. Y. Tsien. 2002. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296: 913-916. [Abstract/Free Full Text]
  26. Pentcheva, T., M. Edidin. 2001. Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer. J. Immunol. 166: 6625-6632. [Abstract/Free Full Text]
  27. McAuliffee, M. J., F. Lalonde, D. P. McGarry, W. Gandler, K. Csaky, B. L. Trus. 2001. Medical image processing, analysis and visualization in clinical research. 14th IEEE Symposium on Computer-Based Medical Systems (CBMS) 381-386.
  28. Marguet, D., E. T. Spiliotis, T. Pentcheva, M. Lebowitz, J. Schneck, M. Edidin. 1999. Lateral diffusion of GFP-tagged H2Ld molecules and of GFP-TAP1 reports on the assembly and retention of these molecules in the endoplasmic reticulum. Immunity 11: 231-240. [Medline]
  29. Schreiber, A. B., J. Schlessinger, M. Edidin. 1984. Interaction between major histocompatibility complex antigens and epidermal growth factor receptors on human cells. J. Cell Biol. 98: 725-731. [Abstract/Free Full Text]
  30. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F. Williams, A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 14: 9-20. [Medline]
  31. Williams, A. P., C. A. Peh, A. W. Purcell, J. McCluskey, T. Elliott. 2002. Optimization of the MHC class I peptide cargo is dependent on tapasin. Immunity 16: 509-520. [Medline]
  32. Ljunggren, H. G., N. J. Stam, C. Ohlen, J. J. Neefjes, P. Hoglund, M. T. Heemels, J. Bastin, T. N. Schumacher, A. Townsend, K. Karre. 1990. Empty MHC class I molecules come out in the cold. Nature 346: 476-480. [Medline]
  33. Raghuraman, G., P. E. Lapinski, M. Raghavan. 2002. Tapasin interacts with the membrane-spanning domains of both TAP subunits and enhances the structural stability of TAP1 x TAP2 complexes. J. Biol. Chem. 277: 41786-41794. [Abstract/Free Full Text]
  34. Garbi, N., N. Tiwari, F. Momburg, G. J. Hammerling. 2003. A major role for tapasin as a stabilizer of the TAP peptide transporter and consequences for MHC class I expression. Eur. J. Immunol. 33: 264-273. [Medline]
  35. Procko, E., G. Raghuraman, D. C. Wiley, M. Raghavan, R. Gaudet. 2005. Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I. Immunol. Cell Biol. 83: 475-482. [Medline]
  36. Myers, N. B., M. R. Harris, J. M. Connolly, L. Lybarger, Y. Y. Yu, T. H. Hansen. 2000. Kb, Kd, and Ld molecules share common tapasin dependencies as determined using a novel epitope tag. J. Immunol. 165: 5656-5663. [Abstract/Free Full Text]
  37. Chun, T., A. G. Grandea, III, L. Lybarger, J. Forman, L. Van Kaer, C. R. Wang. 2001. Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes. J. Immunol. 167: 1507-1514. [Abstract/Free Full Text]
  38. Lybarger, L., Y. Y. Yu, T. Chun, C. R. Wang, A. G. Grande, III, L. Van Kaer, T. H. Hansen. 2001. Tapasin enhances peptide-induced expression of H2–M3 molecules, but is not required for the retention of open conformers. J. Immunol. 167: 2097-2105. [Abstract/Free Full Text]
  39. Park, B., S. Lee, E. Kim, K. Cho, S. R. Riddell, S. Cho, K. Ahn. 2006. Redox regulation facilitates optimal peptide selection by MHC class I during antigen processing. Cell 127: 369-382. [Medline]
  40. Spiliotis, E. T., H. Manley, M. Osorio, M. C. Zuniga, M. Edidin. 2000. Selective export of MHC class I molecules from the ER after their dissociation from TAP. Immunity 13: 841-851. [Medline]
  41. Ladasky, J. J., S. Boyle, M. Seth, H. Li, T. Pentcheva, F. Abe, S. J. Steinberg, M. Edidin. 2006. Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules. J. Immunol. 177: 6172-6181. [Abstract/Free Full Text]
  42. Paquet, M. E., M. Cohen-Doyle, G. C. Shore, D. B. Williams. 2004. Bap29/31 influences the intracellular traffic of MHC class I molecules. J. Immunol. 172: 7548-7555. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
F. Abe, N. Van Prooyen, J. J. Ladasky, and M. Edidin
Interaction of Bap31 and MHC Class I Molecules and Their Traffic Out of the Endoplasmic Reticulum
J. Immunol., April 15, 2009; 182(8): 4776 - 4783.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Everett, M. W.
Right arrow Articles by Edidin, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Everett, M. W.
Right arrow Articles by Edidin, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS