Conventionally, MHC class I-restricted antigen (Ag) processing requires the action of the multimolecular peptide-loading complex within the endoplasmic reticulum (ER). Here we show that early phagosomes from human dendritic cells (DCs) contain the peptideloading complex, incorporating MHC class I, ␤2 microglobulin, transporter associated with Ag processing (TAP), calreticulin, tapasin, and ERp57. Antigenic peptides could be translocated into purified phagosomes by TAP and loaded onto cognate class I molecules, inducing their specific dissociation from the loading complex. Endoglycosidase H-sensitive class I molecules were detected at the DC cell surface, suggesting that these molecules traffic there directly from phagosomes. Macropinocytosis also allowed internalized soluble Ags access to an ER-like compartment containing the class I loading complex. Blockade of TAP by endocytosis of a soluble derivative of human cytomegalovirus protein US6 confirmed that, although retrotranslocation into the cytosol is critical for processing, efficient association of class I molecules with peptides derived from exogenous Ags occurs within a compartment directly accessible to internalized proteins. Together, this evidence suggests that early phagosomes and pinosomes facilitate cross presentation of exogenous Ags by DCs.

The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 ؉ 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4 -9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.The antigen processing machinery is an important regulatory element in the cellular immune response of vertebrates. A major task is to identify infected or malignantly transformed cells. Therefore, peptides derived from proteasomal degradation of intracellular proteins are translocated via the transporter associated with antigen processing (TAP) 1 into the ER and loaded onto MHC class I molecules. Presentation of "nonself " peptides at the cell surface to CD8ϩ cytotoxic T-lymphocytes triggers elimination of the transformed cell (1). A macromolecular peptide-loading complex composed of TAP1, TAP2, tapasin, MHC class I molecules, and several auxiliary factors (e.g. calreticulin and ERp57) promotes peptide loading onto MHC molecules.Tapasin is a type I membrane glycoprotein (48 kDa) with a single transmembrane segment (TM) and a short C-terminal cytoplasmic tail (2, 3). Cells lacking tapasin display only few MHC class I molecules on their cell surface (4). The C-terminal 33 amino acids of tapasin are important for binding to TAP, suggesting that tapasin binding is mediated mainly by interaction between TM segments (5, 6). The interaction site for MHC class I molecules is located in the ER luminal domain of tapasin (7,8). Different functions have been assigned to tapasin as follows: (i) stabilization of the TAP complex (5, 6, 9 -12); (ii) anchoring of empty MHC class I molecules at TAP (2, 3, 13, 14); and (iii) coordination and modulation of peptide loading onto MHC class I molecules (15-17).Human TAP, a member of the ATP-binding cassette (ABC) protein superfamily, forms a heterodimer of TAP1 (748 amino acids) and TAP2 (686 amino acids). Each of the subunits consists of a hydrophobic transmembrane domain (TMD) and a hydrophilic, highly conserved cytoplasmic nucleotide-binding domain (NBD), which couples the chemical energy of ATP hydrolysis to translocation of peptides acros...

In dendritic cells (DCs), peptides derived from internalized particulate substrates are efficiently cross-presented by major histocompatibility complex (MHC) class I molecules. Exogenous soluble antigens are also presented by DCs but with substantially lower efficiency. Here we show that particulate and soluble antigens use different transport pathways. Particulate antigens have been shown to access peripheral endoplasmic reticulum (ER)-like phagosomes that are competent for cross-presentation, whereas we show here that soluble proteins that escape proteolysis enter the lumen of the ER. From there, they may be translocated into the cytosol by the pathway established for ER-associated degradation and their derived peptides may be transported back into the ER for binding by MHC class I molecules. MHC class I presentation involving the constitutive retrograde transport of soluble proteins to the ER by DCs may facilitate DC tolerance to components of their extracellular environment.

The human cytomegalovirus (HCMV) has evolved a set of elegant strategies to evade host immunity. The HCMV-encoded type I glycoprotein US6 inhibits peptide trafficking from the cytosol into the endoplasmic reticulum and subsequent peptide loading of major histocompatibility complex I molecules by blocking the transporter associated with antigen processing (TAP). We studied the molecular mechanism of TAP inhibition by US6 in vitro. By using purified US6 and human TAP co-reconstituted in proteoliposomes, we demonstrate that the isolated endoplasmic reticulum (ER)-luminal domain of US6 is essential and sufficient to block TAPdependent peptide transport. Neither the overall amount of bound peptides nor the peptide affinity of TAP is affected by US6. Interestingly, US6 causes a specific arrest of the peptide-stimulated ATPase activity of TAP by preventing binding of ATP but not ADP. The affinity of the US6-TAP interaction was determined to 1 M. The ER-luminal domain of US6 is monomeric in solution and consists of 19% ␣-helices, 25% ␤-sheets, and 27% ␤-turns. All eight cysteine residues are involved in forming a stabilizing network of four intramolecular disulfide bridges. Glycosylation of US6 is not required for function. These findings point to fascinating mechanistic and structural properties, by which specific binding of US6 at the ER-luminal loops of TAP signals across the membrane to the nucleotide-binding domains to prevent ATP hydrolysis of TAP.