Tapasin is required for efficient peptide binding to transporter associated with antigen processing

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.     

Mechanisms of Function of Tapasin,a Critical Major Histocompatibility Complex Class I Assembly Factor

For their efficient assembly in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class I molecules require the specific assembly factors transporter associated with antigen processing (TAP) and tapasin, as well as generic ER folding factors, including the oxidoreductases ERp57 and protein disulfide isomerase (PDI), and the chaperone calreticulin. TAP transports peptides from the cytosol into the ER. Tapasin promotes the assembly of MHC class I molecules with peptides. The formation of disulfide‐linked conjugates of tapasin with ERp57 is suggested to be crucial for tapasin function. Important functional roles are also suggested for the tapasin transmembrane and cytoplasmic domains, sites of tapasin interaction with TAP. We show that interactions of tapasin with both TAP and ERp57 are correlated with strong MHC class I recruitment and assembly enhancement. The presence of the transmembrane/cytosolic regions of tapasin is critical for efficient tapasin–MHC class I binding in interferon‐γ‐treated cells, and contributes to an ERp57‐independent mode of MHC class I assembly enhancement. A second ERp57‐dependent mode of tapasin function correlates with enhanced MHC class I binding to tapasin and calreticulin. We also show that PDI binds to TAP in a tapasin‐independent manner, but forms disulfide‐linked conjugates with soluble tapasin. Thus, full‐length tapasin is important for enhancing recruitment of MHC class I molecules and increasing specificity of tapasin–ERp57 conjugation. Furthermore, tapasin or the TAP/tapasin complex has an intrinsic ability to recruit MHC class I molecules and promote assembly, but also uses generic folding factors to enhance MHC class I recruitment and assembly.     

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

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

Targeting tumour cells with defects in the MHC Class I antigen processing pathway with CD8+ T cells specific for hydrophobic TAP- and Tapasin-independent peptides: the requirement for directed access into the ER

It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.     

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

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.     

The transporter associated with antigen processing TAP: structure and function

The transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum (ER) is an essential process for presentation to cytotoxic T-lymphocytes. The transporter associated with antigen processing (TAP) is responsible for the intracellular translocation of peptides across the membrane of the ER. Efficient assembly of MHC-peptide complex requires the formation of a macromolecular transport and chaperone complex composed of TAP, tapasin and MHC class I molecules. Therefore, structure and function of TAP is important for the understanding of the immune surveillance.     

Differential requirement for tapasin in the presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.     

Assembly and Function of the Major Histocompatibility Complex (MHC) I Peptide-loading Complex Are Conserved Across Higher Vertebrates

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.     

Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes

H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAP(o)) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAP(o) mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAP(o) cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.     

Tapasin enhances assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells.

Assembly of HLA class I-peptide complexes is assisted by multiple proteins that associate with HLA molecules in loading complexes. These include the housekeeping chaperones calnexin and calreticulin and two essential proteins, the transporters associated with antigen processing (TAP) for peptide supply, and the protein tapasin which is thought to act as a specialized chaperone. We dissected functional effects of processing cofactors by co-expressing in insect cells various combinations of the human proteins HLA-A2, HLA-B27, beta(2)-microglobulin, TAP, calnexin, calreticulin, and tapasin. Stability at 37 degrees C and surface expression of class I dimers correlated closely in baculovirus-infected Sf9 cells, suggesting that these cells retain empty dimers in the endoplasmic reticulum. Both HLA molecules form substantial quantities of stable complexes with insect cell-produced peptide pools. These pools are TAP-selected cytosolic peptides for HLA-B27 but endoplasmic reticulum-derived, i.e. TAP-independent peptides for HLA-A2. This discrepancy may be due to peptide selection by human TAP which is much better adapted to the HLA-B27 than to the HLA-A2 ligand preferences. HLA class I assembly with peptides from TAP-dependent and -independent pools was enhanced strongly by tapasin. Thus, tapasin acts as a chaperone and/or peptide editor that facilitates assembly of peptides with HLA class I molecules independently of mediating their interaction with TAP and/or retention in the endoplasmic reticulum.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

 Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and β₂-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP. Received: 20 March 1997 / Revised: 2 June 1997     

Processing of MHC class I presented antigens

The immune defences of our organism against pathogens and malignant transformation rely to a large extent on surveillance by cytotoxic T lymphocytes. This surveillance in turn depends on the antigen processing system, which provides peptide samples of the cellular protein composition to MHC (major histocompatibility complex) class I molecules displayed on the cell surface. To continuously and almost in real time provide a representative sample of the array of proteins synthesized by the cell, this system exploits some fundamental pathways of the cellular metabolism, with the help of several dedicated players acting exclusively in antigen processing. Thus, a key element in the turnover of cellular proteins, protein degradation by cytosolic proteasome complexes, is exploited as source of peptides, by recruiting a minor fraction of the produced peptides as ligands for MHC class I molecules. These peptides can be further processed and adapted to the precise binding requirements of allelic MHC class I molecules by enzymes in the cytosol and endoplasmic reticulum. The latter compartment is equipped with several dedicated players helping peptide assembly with class I molecules. These include the TAP (transporter associated with antigen processing) membrane transporter pumping peptides into the ER, and tapasin, a chaperone with a structure similar to MHC molecules that tethers class I molecules awaiting peptide loading to the TAP transporter, and mediates optimization of MHC class I ligand by a still somewhat mysterious mechanism. Additional "house-keeping" chaperones that are known to act in concert in ER quality control, assist and control correct folding, oxidation and assembly of MHC class I molecules. While this processing system handles exclusively endogenous cellular proteins in most cells, dendritic cells employ one or several special pathways to shuttle exogenous, internalized proteins into the system, in a process referred to as cross-presentation. Deciphering the cell biological mechanism creating the link between the endosomal and secretory pathways that enables cross-presentation is one of the challenges faced by contemporary research in the field of MHC class I antigen processing.     

Cellular and molecular requirements for association of the murine cytomegalovirus protein m4/gp34 with major histocompatibility complex class I molecules

The murine cytomegalovirus (MCMV) protein m4/gp34 is unique among known viral genes that target the major histocompatibility complex (MHC) class I pathway of antigen presentation in the following two ways: it is found in association with class I MHC molecules at the cell surface, and it inhibits antigen presentation without reducing cell surface class I levels. The current study was undertaken to define more clearly the structural and cellular requirements for m4/gp34 association with the MHC class I molecule K(b). We first assessed the role of the peptide-loading complex in m4/gp34-K(b) association, using cell lines lacking TAP, tapasin, or beta(2)m. m4/gp34-K(b) complexes formed in the absence of TAP or tapasin, although not as efficiently as in wild-type cells. The expression of full-length and truncation mutants of m4/gp34 in a gutless adenovirus vector revealed that the transmembrane region of m4/gp34 was required for efficient association with the K(b) heavy chain. However, the peptide-loading complex was not absolutely required for the association, since m4/gp34 readily formed complexes with K(b) in detergent lysates. The addition of K(b)-binding peptide to the detergent lysates facilitated but was not essential for the formation of the complexes. The ease of complex formation in detergent lysates contrasted with the small fractions of m4/gp34 and K(b) that form complexes in infected cells, suggesting that the endoplasmic reticulum (ER) environment restricts access of m4/gp34 to K(b). Finally, although m4/gp34-K(b) complexes could form when m4 was carried either by MCMV or by the adenovirus vector, they were only efficiently exported from the ER in MCMV-infected cells, suggesting that MCMV provides additional factors needed for transport of the complexes.     

Tapasin increases efficiency of MHC I assembly in the endoplasmic reticulum but does not affect MHC I stability at the cell surface

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.     

Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.     

A charged amino acid residue in the transmembrane/cytoplasmic region of tapasin influences MHC class I assembly and maturation

Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, beta(2)-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.     

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.