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The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing (TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8؉ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6.The transporter associated with antigen processing (TAP) 3 represents a bottleneck in the MHC class I antigen-presenting pathway. TAP translocates 8 -16-mer peptides generated in the cytosol across the ER membrane into the ER lumen for loading onto MHC class I molecules (1, 2). Upon anchoring of the peptide, the MHC class I heavy chain and ␤ 2 -microglobulin form a stable ternary complex that is released from the peptide loading complex consisting of TAP, tapasin, calreticulin, and the thioreductase ERp57 (3, 4). MHC class I molecules carry the peptide to the cell surface for monitoring by CD8ϩ cytotoxic T lymphocytes. TAP-deficient cells show drastically reduced levels of MHC class I surface expression indicating that TAP represents the principal source of peptide ligands.TAP is a member of the superfamily of ATP-binding cassette (ABC) transporters, which utilize ATP energy for translocation of their substrates across membrane barriers. TAP is built by two subunits, TAP1 and TAP2, each consisting of one transmembrane domain (TMD) and one nucleotide binding domain (NBD). Peptide binding to TAP is dependent on the formation of heterodimers (5). It was shown that the TMDs, and not the NBDs, of TAP1 and TAP2 encompass the peptide binding domain (6), which has been mapped to the C-terminal cytosolic loops on both subunits (7). The NBDs harbor conserved sequence motifs required for ATP binding and hydrolysis (8). According to a re...

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