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.