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The Journal of Immunology, 2003, 170: 941-946.
Copyright © 2003 by The American Association of Immunologists

Impaired Transporter Associated with Antigen Processing (TAP) Function Attributable to a Single Amino Acid Alteration in the Peptide TAP Subunit TAP11

Ulrike Ritz*, Ingo Drexler{dagger}, Dirk Sutter{ddagger}, Rupert Abele§, Christoph Huber* and Barbara Seliger2,*

* Third Department of Internal Medicine, Johannes Gutenberg-University, Mainz, Germany; {dagger} GSF Research Center, Institute for Molecular Virology, Munich, Germany; {ddagger} Technical University, Institute for Virology, Munich, Germany; and § Johann Wolfgang Goethe-University, Institute for Biochemistry, Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1-/- cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu263 was comparable to that of TAP1-/- cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe265 of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu263 of the TAP1 protein, but not the Phe265, is critical for proper TAP function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TAP belongs to the large family of ABC transporters and shares the typical three-domain structure of these transporters. TAP consists of a C terminus spanning the membrane of the endoplasmic reticulum (ER)3 several times, a substrate binding domain, and the hydrophilic nucleotide binding domain containing the Walker A and B consensus motifs (1). ABC transporters translocate a variety of substances/molecules, including ions, sugars, amino acids, peptides, and polysaccharides, but each transporter is specialized for the transport of one unique substrate or class. The best characterized human ABC transporters are the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is defective in cystic fibrosis (2), and the P-gp (3) that export hydrophobic drugs, lipids, and phospholipids out of the cell.

In the P-gp as well as in the CFTR protein, point mutations causing an amino acid substitution or deletion have been identified, which result in either a loss of function or alterations in quality and quantity of substrate transport: in CFTR point mutations of the amino acids, Phe508, Tyr109, Pro208, and Glu92 close the channel (4, 5), whereas mutations at His117, Phe334,337, and Arg347 result in a lower chloride flow rate through the channel (6). Substitutions of Phe72, 335, 522–525, and 978 to Ala of the P-gp change the transport specificity, which can be associated with a distinct substrate binding and consequently with an altered drug-resistant profile of cells (7, 8).

Although TAP is a typical ABC transporter with the highest sequence homologies to P-gp, it differs in its structure and function from other transporters of this family. TAP is the only ABC transporter with a function in the immune system as it translocates antigenic peptides length and sequence specific from the cytosol into the ER, where they are loaded onto MHC class I molecules (9). Moreover, TAP consists of two subunits, TAP1 and TAP2, and the formation of a heterodimeric complex is required for its proper function (1, 10, 11).

It is apparent that TAP is involved in numerous interactions inside the ER and the cytosol, but the exact features of TAP have not yet been defined. Based on structural and functional analyses, different topology models for TAP have been postulated. Functional analyses of C-terminal deletion mutants as well as peptide cross-linking experiments to the human TAP defined multiple regions in both subunits, which putatively contribute to the peptide binding site (12, 13). Using a series of deletion mutants in the potential peptide binding domain, the residues 366–405 of the TAP1 subunit have been identified to contain structural and/or functional relevant sequences for peptide transport (14). Furthermore, many groups characterized the ATP binding domain of TAP using a panel of deletion or chimeric mutants of TAP1 and TAP2, thereby demonstrating an influence of the NTP binding on TAP function, conformational changes, and peptide binding (15, 16, 17, 18, 19).

To provide information about the structural features of TAP that alter its function, the TAP sequence was compared with that of P-gp and CFTR and their known residues/amino acids resulting in functional changes. Using this approach, 2 aa potentially important for TAP function, the glutamic acid at aa position 263 and the phenylalanine at aa residue 265, respectively, were postulated. TAP1 mutants containing either a deletion or a substitution to alanine at Glu263 or Phe265 were subsequently characterized in terms of protein expression, ER localization, dimer formation with TAP2, peptide transport, MHC class I surface expression, and specific lysis by MHC class I-restricted CTL. By comparing the biochemical and functional properties of the cell lines expressing TAP1 mut/del 263 and TAP1 mut/del 265, we present evidence in this work that Phe265 of TAP1 has a minimal effect, whereas Glu263 of TAP1 has a striking effect on TAP function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of TAP mutants

The vector pBluescript SK+ containing the human 2.3-kb TAP1A cDNA served as a template for standard PCR (1 min, 95°C; 1 min, 54°C; 1 min and 30 s, 72°C for 25 cycles) using the native Pfu polymerase (Stratagene, Amsterdam, The Netherlands) and TAP1-specific primers. The forward primers 1 contain a BbrPI restriction site (italic letters) and the deletion or the desired mutation to alanine at aa position 263 or 265 (bold letters), respectively; the reverse primer 2 contains a SpeI restriction site (italic letters; Table I). The resulting amplification products of 500 or 503 bp were then reintroduced into their original positions of the TAP1 wild-type (wt) cDNA. Upon confirmation of the integrity of the substituted or deleted amino acid by sequencing, the designed TAP1 mutants were cloned into the EcoRV site of the expression vector pIRES-neo (20).


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  		Table I. Primers used for PCR mutagenesis

 
Cell lines and culture

The murine TAP1-/- cells kindly provided by H.-G. Ljunggren (Karolinska, Stockholm) were established from a methylcholanthrene-induced tumor of a TAP1-/- C57BL/6 (B6) mouse (H-2b) (21); the human TAP1-/- BRE cells kindly provided by H. delaSalle were derived from a patient with bare lymphocyte syndrome (22). The TAP1-/- cells were either maintained in RPMI medium (mouse TAP1-/- cells; Seromed, Frickenhausen, Germany) or in DMEM (human BRE cells) supplemented with 10% FCS (Greiner, München, Germany), 2 mM L-glutamine, 100 µg streptomycin/ml, and 100 U penicillin/ml medium.

Stable transfection of TAP1-/- cells

Gene transfer of TAP1-/- cells was performed by lipofection using the wt or mutant TAP1 constructs and/or the HLA-A*0201 expression vector (23), as recently described (14). Stable neomycin-resistant (neoR) transfectants were selected in culture medium supplemented with 500 µg/ml G418.

Immunofluorescence and flow cytometric analyses

For immunofluorescence, 1 x 105 cells/well were seeded on sterile cover slips or in six-well plates for 24 h, then washed twice with PBS, followed by fixation with 4% paraformaldehyde in PBS for 10 min. After blocking, the cells were incubated with the appropriate concentration of the human anti-TAP1 (24) or anti-calnexin (Affinity, Golden, U.K.) mAb, respectively. Cells were then incubated with Hoechst stain (Roche Biochemicals, Mannheim, Germany) and using as a secondary Ab a goat anti-mouse FITC-conjugated Ig for TAP1 and a goat anti-mouse Cy-3 for calnexin, respectively. Images were obtained using a confocal laser microscope (Leica, Buffalo, NY).

Indirect immunofluorescence analysis was performed by flow cytometry, as recently described (14). Briefly, 5 x 105 cells were incubated with the appropriate concentration of the primary Ab (anti-H2 KbDb; Cedarlane Laboratories, Hornby, Ontario, Canada) or HLA-ABC (Coulter/Beckman, Krefeld, Germany) for 30 min on ice, then washed twice with PBS and additionally incubated with a goat anti-mouse FITC Ig for 30 min at 4°C. A total of 10,000 viable cells were analyzed on a flow cytometer (Coulter Epics XL MCL software, Coulter Epics XL MCL System II 3.0; Coulter/Beckman). A mouse anti-IgG2a served as control, and the mean specific fluorescence intensity was calculated by setting the results of the control and the probe in relation.

Immunoprecipitation

A total of 2 x 106 cells were lysed with buffer containing 1% Nonidet P-40 before adding a murine anti-TAP2 mAb (kindly provided by K. Früh, San Diego, CA) overnight at 4°C, followed by the addition of protein A-Sepharose for 4 h. After two washing steps, the bound proteins were eluted, precipitated with TCA, and further subjected to Western blot analysis (see below).

ATP-agarose-binding assays

ATP-agarose-binding assays were performed, as previously described (15). A total of 1 x 107 cells were disrupted by freezing and thawing, and the resulting cell suspension was centrifuged at 1,000 x g for 10 min at 4°C. The supernatant was spun at 100,000 x g for 1 h. Membrane pellets were resuspended in lysis buffer (TBS/MgCl2/2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), incubated for 30 min on ice before centrifuging at 10,000 x g for 30 min. Proteins were incubated with ATP-agarose, eluted with elution buffer, and subjected to Western blot analysis.

Western blot analysis

For the detection of TAP proteins, samples from both the immunoprecipitation and the ATP-binding assays were size fractionated by 10% SDS-polyacrylamide gels, visualized by Ponceau S, and transferred onto nitrocellulose membranes (Schleicher & Schüell, Dassel, Germany). The membranes were blocked with TBS/10% horse serum/5% milk powder/0.1% Tween 20 for 2 h, briefly rinsed with TBS/0.1% Tween 20, and additionally incubated with the anti-human TAP1 mAb 148.3 recognizing the C terminus of TAP1 for 2 h. After rinsing twice in TBS/0.1% Tween 20, membranes were incubated with either alkaline phosphatase- or HRP-conjugated rabbit anti-mouse Ig-specific Ab (DAKO, Hamburg, Germany) for 1 h, followed by two washing steps. Then, the membranes were developed with the ECL determination kit (Amersham Pharmacia, Freiburg, Germany) or using nitroblue tetrazolium/X phosphate as substrate (Roche Biochemicals).

Membrane preparation and peptide-binding assays

Cells were thawed on ice in 10 mM Tris buffer (pH 8.0) supplemented with protease inhibitors and 1 mM DTT and homogenized with a glass homogenizer. Lysed cells were centrifuged at 1000 x g for 10 min. For collection of crude membranes, supernatant was centrifuged at 200,000 x g for 30 min and pellets were resuspended in PBS. The protein concentration of the crude membranes was determined by micro bicinchoninic acid protein assay. For peptide binding, the peptide RRYQKSTEL was labeled by the chloramine-T method using 1.5 nmol peptide and 100 µCi 125I. To determine the amount of functional TAP, the membranes were incubated with radiolabeled peptide (200 nM) in a total volume of 50 µl in PBS, 5 mM MgCl2 for 15 min on ice. Unbound peptide was removed by adding 500 µl PBS, 5 mM MgCl2, and centrifuged for 8 min at 20,000 x g. The supernatant was discarded, and the washing procedure was repeated once. The membrane-associated radioactivity was directly quantified by gamma counting.

Peptide transport assay

Peptide translocation assays were conducted on streptolysin O-permeabilized cells using the reporter peptides TNKTRIDGQY and RRLYQNSTEL, as described by Neefjes et al. (25). Shortly, the peptides were radiolabeled with 125I by the chloramine T method using 10 nmol peptide, 1.0 mCi 125I, and Sephadex G-10 columns for removal of unincorporated iodine. After trypsinization, 2.5 x 106 TAP1-/- cells or transfectants were permeabilized with 2 IU streptolysin O (Welcome Reagent, Beckenham, U.K.) in 50 µl incubation buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA, 2 mM MgCl2, and 5 mM HEPES, pH 7.3) for 5 min at 37°C, followed by the addition of 10 µl ATP (10 mM; Roche) and 2.5 µl of radioiodinated peptides (0.5 µM) in a final volume of 160 µl, and incubated at 37°C for 20 min. Subsequent to cell lysis in 1% Nonidet P-40 (Sigma-Aldrich, St. Louis, MO), glycosylated peptides were recovered by adding Con A-Sepharose beads (Pharmacia, Uppsala, Sweden) overnight, and after extensive washing the radioactivity associated with the beads was measured by gamma counting. Translocation efficiencies were calculated according to the formula: cpm (glycosylated labeled peptides bound to Con A beads)/cpm (total label of peptides) x 100.

Cytotoxicity assay of TAP1 transfectants

Human BRE-/- cells and their wt or mutTAP1/HLA-A2-transfected derivatives were used as targets for HLA-A2-restricted tyrosinase-specific CTL using the standard chromium release assay (26). Cells were infected with 10 U/cell of vaccinia-tyrosinase virus and incubated at 37°C, as described (27). After 3 h, cells were labeled with 51Cr and further incubated at 37°C. Twelve hours postinfection, cytotoxicity assays were performed. 51Cr-labeled targets were mixed with different concentrations of effectors (E:T ratio) in triplicates and incubated for 4 h at 37°C. Supernatants were then harvested, and radioactivity was counted in a gamma counter. Percent specific lysis was calculated as 100 x (experimental release - spontaneous release)/(maximal release - spontaneous release).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of potentially important amino acids of TAP by sequence comparison with other ABC transporters

Single amino acids have been described in the CFTR as well as in the P-gp, which influence their binding specificities and/or function. These include the Glu92 of CFTR and the Phe72 of P-gp. Using computer-based sequence analyses, an overlapping stretch of CFTR and P-gp sequences was identified in the TAP1 subunit, postulating a potential importance of these 2 aa: the Glu263 and the Phe265 (Fig. 1). To explore the role of Glu263 and Phe265 in TAP function, both amino acids were either deleted (TAP1 del263 and TAP1 del265) or substituted to alanine (TAP1 mut263 and TAP1 mut265) by PCR mutagenesis. Wt and mutant TAP1 constructs were stably expressed in both murine (TAP1-/- C57BL/6) and human TAP1-negative cells (BRE fibroblasts). Upon selection in G418, stable integration of wt and mutant TAP1 cDNA was confirmed in neoR clones by genomic PCR using construct-specific primers (data not shown).



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  		FIGURE 1. Sequence comparison of three ABC transporters. Sequences from two ABC transporters, CFTR and P-gp, which influence their function and/or binding specificity, were compared with that of the TAP1 subunit. Two amino acids potentially essential for proper TAP function, Glu263 and Phe265, were identified and either deleted or mutated to Ala for further analyses.

 
The different mutTAP1 proteins are localized in the ER membrane, bind ATP, and form complexes with TAP2

Expression of human wt and mutant TAP1 proteins in murine and human TAP1-/- transfectants was determined by Western blot analysis. Murine and human TAP1-/- cells served as controls. A similar protein expression pattern of human wt and mutant TAP1 proteins was detected in murine and human cell lines analyzed (data not shown). To define the biological properties, ER localization, ATP-binding ability, and heterodimer formation of the different TAP mutants and the wt TAP1 transfectants were evaluated. Immunostaining with the anti-TAP1 mAb 148.3 revealed ER localization of all mut/del TAP1 proteins, as representatively shown for the murine cells transfected with the human TAP1 del263 mutant, which was comparable to that of wt TAP1 and calnexin (Fig. 2, a, c, and d). As expected, no TAP1-specific staining was detected in TAP1-/- cells (Fig. 2b). As representatively shown for the human BRE TAP1-/- cells, this was also associated with lack of TAP1/TAP2 complex formation (Fig. 3a, lane 1) and deficient ATP binding (Fig. 3b, lane 1). In contrast, the human cells transfected with human wt TAP1 and the different mut/del TAP1 constructs possess the capability to form heterodimers with TAP2 and to bind ATP (Fig. 3, a and b). Thus, both the deletions and amino acid replacements of Glu263 or Phe265 of the TAP1 subunit seem not to influence the structure of the peptide transporter because a protein product of the expected size, ER localization, heterodimer formation with TAP2, and ATP binding were detected.



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  		FIGURE 2. ER localization of mutTAP1 proteins. Immunfluorescence was performed as described in Materials and Methods. Calnexin (a) and wt TAP1 (c) served as positive controls for ER localization, whereas the TAP1-/- cells served as negative control (b). TAP1 del263 is clearly localized in the ER membrane at a position comparable to that of wt TAP1 and calnexin (d).

 


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  		FIGURE 3. Heterodimer formation with TAP2 and ATP binding of the TAP1 mut/del proteins expressed in human BRE cells. Lysates of TAP1 mut/del transfectants and control cells were analyzed for the TAP complex formation by immunoprecipitation (a) and ATP-binding capacity (b), as described in Materials and Methods.

 
Deletion or mutation of the glutamic acid at aa position 263 results in loss of TAP function and MHC class I surface expression

Human BRE cells expressing TAP1 mutants as well as TAP1-negative and wt TAP1-transfected control cells were investigated for peptide binding to TAP. As representatively shown in Fig. 4, peptide binding was abolished in the TAP1-negative control cells and in the human cells expressing TAP1 mutants with a point mutation at Glu263. In contrast, peptide binding was demonstrated in the TAP1 mut265 cells (13,600 cpm), which was comparable to that of wt TAP1 BRE transfectants (13,400 cpm).



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  		FIGURE 4. Peptide binding to wt TAP1 and TAP1 mut 263/265 transfectants. BRE cells were analyzed for peptide binding to TAP, as described in Materials and Methods. The results are expressed as recovered {gamma}-cpm.

 
To confirm the different TAP function of TAP1 mut/del 263/265 cells, TAP-mediated peptide translocation into the ER was determined using two different iodinated reporter peptides. Murine NIH3T3 fibroblasts, human cervical carcinoma cells (HT3), wt transfectants, and TAP1-/- cells served as controls. As expected, murine and human cells transfected with human wt TAP1 transport peptides with a high efficiency of 2.9, 2.1, and 4.4%, respectively (Fig. 5a). In contrast, a heterogeneous peptide transport rate was demonstrated in the different mut/del TAP1 proteins: the human cells expressing human TAP1 mutants with either a deletion or point mutation at Glu263 lack peptide transport and show a transport efficiency similar to that of TAP1-/- cells (transport rate: 0.01–0.1%). In contrast, human cells containing TAP1 mutants with a deletion or point mutation at Phe265 were capable of translocating peptides with an efficiency ranging from 1.2 to 2.5%, depending on the peptide used. This efficiency was comparable to that of murine NIH3T3 fibroblasts and the cervical carcinoma cell line (HT3) exhibiting a peptide transport rate of 1.1 and 3.2%, respectively, but represents only 50% of the level of translocation of wt TAP1 transfectants.



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  		FIGURE 5. Peptide translocation and MHC class I surface expression of wt TAP1 and TAP1 del/mut-transfected TAP1-/- cells. A series of murine and human cells expressing human TAP1 del/mut 263/265 mutants and control cells (NIH3T3, HT3, and murTAP1-/-) was analyzed for peptide transport utilizing the peptide translocation assay using two peptides (a) and MHC class I surface expression by flow cytometry (b), as described in Materials and Methods. The results are expressed as percentage of transported peptides (a) and as mean specific fluorescence intensity (b; MFI). All experiments were performed at least twice.

 
Peptides translocated by TAP are loaded onto the {beta}2-microglobulin ({beta}2m)/MHC class I dimer, which is then transported to the cell surface and presented to CTL. TAP deficiencies result in the expression of unstable {beta}2-microglobulin/MHC class I dimers. To assess the functional consequences of the different peptide transport rate of TAP1 mut/del 263 and 265, MHC class I surface expression was analyzed by flow cytometry using MHC class I-specific Abs. As shown in Fig. 5b, the murine and human cell lines transfected with TAP1 mut/del 263 showed no MHC class I surface expression (Fig. 5b), whereas the TAP1 mut/del 265 cell lines expressed significant levels of MHC class I surface Ags that were comparable to that of wt TAP1 transfectants, murine NIH3T3, and human HT3 control cells.

Cells transfected with TAP1 del or mut 263 are not recognized by specific CTL

To define whether there exists a correlation between MHC class I surface expression and immune recognition, vaccinia tyrosinase-infected parental BRE TAP1-/- cells as well as BRE wt and TAP1 mut/del 263/265 variants were used as targets for HLA-A2-restricted tyrosinase-specific CTL. Before cytotoxicity assays, all cell lines were stably transfected with an HLA-A2 expression vector.

As expected, the HLA-A2-positive BRE TAP1 -/- cells were not lysed by tyrosinase-specific CTL. Similarly, the HLA-A2-expressing BRE TAP1 mut/del 263 cells were not recognized by these CTL. In contrast, BRE wt and TAP1 mut/del 265 transfectants as well as tyrosinase-transfected NA8 melanoma control cells were efficiently lysed (Fig. 6), thereby confirming the data from the peptide translocation assay and MHC class I surface expression.



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  		FIGURE 6. TAP1 del/mut 263 are not recognized by tyrosinase-specific CTL. Vaccinia tyrosinase-infected BRE TAP1 mut/del cells served as targets in a standard chromium release assay with tyrosinase-specific CTL using different E:T ratios. Tyrosinase-positive NA8 melanoma cells served as controls. All experiments were performed at least three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been postulated that the Glu263 and Phe265 of the TAP1 subunit are involved in the function of the peptide transporter. This assumption was based on sequence comparisons between TAP and two other members of the ABC transporter superfamily, the CFTR and P-gp, in which these amino acid residues alter the substrate specificity and function of the transporters, respectively (4, 7, 28, 29). The Glu263 and Phe265 of human TAP1 were either deleted or substituted to alanine, and the constructs were transfected into murine and human TAP1-/- cells. The TAP1 variants were analyzed regarding their individual contributions to TAP function. The experimental data concerning MHC class I surface expression of both the human and murine TAP1 del/mut-transfected cell lines were comparable. These data confirm the absence of species-specific differences between the human TAP1/TAP2 complex and the TAP heterodimer consisting of human TAP1 and murine TAP2, as described earlier (14, 30).

The deletion or mutation of aa 263 or 265 resulted in various effects on TAP function, despite their normal properties of TAP1 in terms of protein expression, ER localization, and ATP binding. These data suggest that the deletion or mutation of the aa residues 263 or 265 in TAP1 causes no conformational changes. However, a deletion or mutation to Ala at Glu263 of TAP1 causes a loss of transporter function, as measured by both peptide binding and translocation using model peptides, which have been demonstrated to be optimal substrates for TAP. This impaired TAP function was directly associated with the lack of MHC class I surface expression and CTL-mediated immune recognition. The peptide transport is proposed to be a two-step process that includes peptide binding, followed by the peptide transport cycle (19). Because alterations at Glu263 of TAP1 affect peptide binding, the mut 263 cells lack peptide transport. In contrast, a deletion or mutation to Ala in residue Phe265 of TAP1 does neither affect MHC class I surface expression nor immune recognition when compared with that of wt TAP1 transfectants or control cells. Concerning TAP function, peptide binding was not impaired in these cells, whereas a reduced transport activity of the TAP1 mut/del 265 transfectants in comparison with wt TAP1 control cells was detected with both peptides used. These data suggest that alteration at Phe265 of TAP1 affects the peptide transport rather than peptide binding to TAP. This altered peptide transport specificity in comparison with wt TAP1 transfectants might be attributable to changes in the transport kinetic for these peptides. In this view, the amino acid Phe265 may play a role in the pore-forming activity of TAP and not as earlier proposed in peptide binding (12, 13, 14). Similar effects have been described for different amino acid substitutions in the P-gp (7).

The deletion or mutation of Glu263 abolishes TAP function, whereas the deletion or mutation of Phe265 influences peptide transport specificities rather than peptide binding, proposing functional differences between both amino acids. In contrast to previous studies, recent data show that the TAP1-mediated ATP hydrolysis is not essential for peptide translocation, but is involved in later stages of the translocation cycle or in controlling TAP activity (19). Furthermore, there exist functional differences between the nucleotide binding domains of the TAP1 and TAP2 subunits regarding the distinct steps of the TAP transport cycle (18, 19). The early events in the peptide transport produce conformational changes in TAP. This may be associated with a coordinated crosstalk among ATP binding, ATP hydrolysis, peptide binding, and translocation (16, 31). Further studies aimed at confirming this assumption by both peptide translocation and binding assays using a series of model peptides and by functional analyses of Glu263 and Phe265 replacements by different amino acids are currently performed. The distinct biological properties of Glu263 and Phe265 of the TAP1 subunit further support the hypothesis that structural features of members of the ABC transporter family provide information about amino acids or domains critical for TAP function. Thus, sequence similarities may lead at least in some cases to the prediction of motifs important for substrate specificity and transporter function.


   Acknowledgments
 
We thank Dr. T. Wölfel and T. Kreer (Johannes Gutenberg-University, Third Department of Internal Medicine) for kindly providing us with the tyrosinase-specific CTL and the pcDNA3-HLA-A*0201 construct; Dr. Soldano Ferrone (Roswell Park Cancer Institute, Department of Immunology, Buffalo, NY), for the HLA-A2 Ab; and Dr. Dennis Strand (Johannes Gutenberg-University, First Department of Internal Medicine) for his help in performing the immunofluorescence analysis.


   Footnotes
 
1 This work was supported by a grant from the Sonderforschungsbereich SFB 432, Project A5. Back

2 Address correspondence and reprint requests to Dr. Barbara Seliger, Johannes Gutenberg University, Third Department of Internal Medicine, Langenbeckstr. 1, 55101 Mainz, Germany. E-mail address: B. Seliger{at}3-med.klinik.uni-mainz.de Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; CFTR, cystic fibrosis transmembrane conductance regulator; wt, wild type. Back

Received for publication December 17, 2001. Accepted for publication November 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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