Major histocompatibility complex class I molecules bind antigenic peptides in the endoplasmic reticulum (ER) and transport them to the cell surface for recognition by cytotoxic T lymphocytes. The peptides are predominantly generated from cytoplasmic proteins, probably by the action of the multicatalytic proteinase complex, or proteasome. They are transported into the ER by the transporters associated with antigen processing (TAP), a complex formed from two subunits, TAP.1 and TAP.2 (refs 3-5). Here we show that the TAP molecules are intimately involved in the assembly of the class I/beta 2-microglobulin (beta 2m) peptide complex. Free class I heavy chains are associated in the ER with the chaperone calnexin. In human B-cell lines, however, class I/beta 2m dimers in the ER are physically associated with TAP molecules rather than calnexin. Our results suggest that calnexin mediates class I/beta 2m dimerization, and subsequent binding of the dimers to TAP molecules facilitates their association with TAP-transported peptides.

We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and P2-microglobulin light chain. The poreforming toxin streptolysin 0 was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides.A basic requirement for the presentation ofantigenic peptides by major histocompatibility complex (MHC) class I molecules to T cells is the transport of these peptides to the site of class I assembly. The breakdown of protein antigens to peptides destined for presentation by class I molecules is thought to occur in the cytosol through the degradative action of proteasomes (1). Two proteins encoded within the MHC, termed LMPs for low molecular weight proteins, share homology with proteasome subunits and may play a role in the specific degradation of MHC class I-associated antigens (2). Once the peptide is generated, it must be targeted to the correct compartment where it can bind to class I molecules. The available evidence suggests that this compartment is the endoplasmic reticulum (ER) (3). The discovery of two other proteins encoded within the MHC, termed TAP-1 and TAP-2 for transporters associated with antigen processing because of their homologies to the ATP-binding cassette family of transporters (for review, see refs. 2 and 4), has led to speculation that these proteins are responsible for transporting peptides across the ER membrane. Restoration of normal class I processing and presentation occur in mutant cell lines that lack one or both of the TAP proteins when functionalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. TAP-encoding genes are introduced by transfection (5, 6).Two such mutant cell lines, .174 a...

Major histocompatibility complex (MHC) class I molecules bind peptides that are delivered from the cytosol into the endoplasmic reticulum by the MHC-encoded transporter associated with antigen processing (TAP). Peptide capture by immature heterodimers of class I heavy chains and beta 2-microglobulin may be facilitated by their physical association with TAP. A genetic defect in a human mutant cell line causes the complete failure of diverse class I heterodimers to associate with TAP. This deficiency impairs the ability of the class I heterodimers to efficiently capture peptides and results from loss of function of an unidentified gene or genes linked to the MHC.

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Antigen processing provides major histocompatibility complex (MHC) class I molecules with short peptides, which they selectively bind and present to cytotoxic T lymphocytes. The proteolytic system generating these peptides in the cytosol is unidentified, but their delivery into the endoplasmic reticulum is mediated by the TAP1-TAP2 transporter encoded in the MHC class II region. Closely linked to TAP1 and TAP2 are genes for the LMP2 and LMP7 proteins, which resemble components of proteasomes, proteolytic complexes known to degrade cytosolic proteins. This association has led to the common assumption that proteasomes function in this immunological pathway (discussed in ref. 15). We now show that the expression of stably assembled class I molecules and apparently normal peptide processing can be completely restored in the absence of LMP2 and LMP7 in the human lymphoblastoid cell line mutant 721.174 (refs 16, 17). The identity of LMP7 is directly confirmed by reconstitution of a proteasomal subunit after gene transfer. These results therefore dispute the hypothetical involvement of proteasomes in antigen processing, although a more subtle effect of LMP2 and LMP7 cannot be ruled out.

The transporter proteins associated with antigen processing (TAP proteins) transport antigenic peptides across the endoplasmic reticulum membrane where they can assemble with newly synthesized maijor histocompatibility complex (MHC) class I/p2-microglobulin (j32m) dimers. We have shown previously that TAP possesses a peptide-recognition site with broad specificity and that MHC class I/p2m dimers physically associate with TAP. Here, we further characterize the nature of the peptide-binding site on TAP, and the site of interaction of TAP with MHC class I/fi2m dimers. TAP photoaffinity labeling experiments revealed that both TAPi and TAP2 are photolabeled by two distinct photopeptide analogues, suggesting that elements of both TAPN and TAP2 compose the peptide-recognition site. TAP photolabeling analysis on transfectant cell lines that express TAPi and TAP2 both individually and together revealed that efficient formation of the peptide-binding site occurs only when TAPN and TAP2 are coexpressed, which correlates with the finding that peptide translocation via TAP occurs only in the presence of both TAPN and TAP2. These data strongly support the notion that TAP functions as a heterodimer. MHC class I/132m dimers were shown to associate with individual TAP1 chains but were not detectable with individual TAP2 chains. This result suggests that the site of interaction for MHC class I/A2m dimers with TAP is on TAP1.Traffic ATPases or ABC transporters (for ATP-binding cassette) constitute a large family of related molecules that are responsible for translocating a variety of compounds across membranes (for review, see ref. 1). Examples include the oligopeptide (OppABCDF) and hemolysin (HlyB) transporters in bacteria (2, 3), the STE6 (sterile 6) a-factor transporter in yeast (4), the eukaryotic P-glycoprotein associated with multidrug resistance (5, 6), and the cystic fibrosis transmembrane conductance regulator (7). Although many such transporters have been identified in prokaryotes and eukaryotes, limited information is available on the actual mechanism of translocation. Members of the ABC transporter family possess a characteristic structure consisting of two hydrophobic domains (each with six putative membrane-spanning segments) and two hydrophilic ATP-binding domains. The transporter proteins associated with antigen processing (TAP proteins) are recently identified members of the traffic ATPases (8-10). TAP molecules transport antigenic peptides across the endoplasmic reticulum (ER) membrane (11-13), an essential step in the major histocompatibility complex (MHC) class I antigen-presentation pathway. Through the action of TAP, peptides generated in the cytosol gain entry into the ER, where they can associate with MHC class I/02-microglobulin (f32m) heterodimers. TAP molecules are made up of two distinct polypeptides, termed TAP1 and 3!AP2, each containing single hydrophobic and ATP-binding domains. Normal peptide loading and class I transport can be reconstituted in mutant cells that lack both TAPl-and TAP2-...

We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza-specific CTL response to an epitope in the nucleoprotein that overlaps the B8-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8.