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...
The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.
Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.
TAP, an ABC transporter in the ER membrane, provides antigenic peptides derived from proteasomal degradation to MHC class I molecules for inspection by cytotoxic T lymphocytes at the cell surface so as to trace malignant or infected cells. To investigate the minimal number of transmembrane segments (TMs) required for assembly of the TAP complex based on hydrophobicity algorithms and alignments with other ABC transporters we generated N-terminal truncation variants of human TAP1 and TAP2. As a result, a 6 + 6 TM core-TAP complex represents the minimal functional unit of the transporter, which is essential and sufficient for heterodimer assembly, peptide binding, and peptide translocation into the ER. The TM1 of both, core-TAP1 and core-TAP2 are critical for heterodimerization of the complex.
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