The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAPspecific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V max of 2 mol͞min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.membrane proteins ͉ transport mechanism ͉ ATPase ͉ antigen processing A n essential step in the major histocompatibility complex (MHC) class I-mediated cellular immune defense is the transport of antigenic peptides from the cytoplasm into the endoplasmic reticulum (ER) (reviewed by ref. 1). The transporter associated with antigen processing (TAP) translocates peptides that are mainly derived from the proteasomal degradation into the ER (reviewed by ref. 2). Subsequently, peptides bind to MHC class I molecules and are presented at the cell surface, where they are recognized by cytotoxic T lymphocytes.TAP belongs to the family of ATP-binding cassette (ABC) transporters that, under consumption of ATP, translocate a vast spectrum of substrates across membranes (3). On the basis of sequence comparison, TAP fits in the B subfamily of human ABC transporters (www.gene.ucl.ac.uk͞users͞hester͞abc.html), including the well-characterized P-glycoprotein (ABCB1). The TAP complex forms a heterodimer of TAP1 (ABCB2) and TAP2 (ABCB3), where each subunit comprises a hydrophobic transmembrane domain and a cytoplasmic nucleotide-binding domain (NBD) containing the Walker A͞B and C-loop consensus sequences. The transmembrane domains are thought to form a channel by which peptides are transported across the ER membrane driven by ATP hydrolysis. The coupling between NBDs, the substrate-binding site, and the translocation pathway is not understood and is a central topic to be studied for all ABC transporters.TAP is part of a macromolecular peptide translocation and loading complex that comprises TAP1, TAP2, tapasin, MHC class I hea...

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.