Function of the transport complex TAP in cellular immune recognition

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155

208

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...

Section: Resultsmentioning

confidence: 99%

“…Although conserved among TAP from other vertebrates, the first 175 amino acids of TAP1 and 140 amino acids of TAP2 show no sequence homology to other proteins (19). The role of these unique N-terminal domains in TAP transport function has not been elucidated yet.…”

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confidence: 99%

Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP)

Koch

Guntrum

Heintke

et al. 2004

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155

208

“…The TAP complex is a heterodimer composed of TAP1 (ABCB2) and TAP2 (ABCB3). Both subunits consist of a transmembrane domain followed by an NBD (3). The peptide-binding pocket is located in the transmembrane domain (4) and preferentially binds peptides with a length of 8 -16 amino acids (5).…”

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confidence: 99%

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

Chen

Abele

Tampé

2004

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The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.

“…TAP is comprised of two related subunits, TAP1 and TAP2, which are necessary and sufficient for peptide translocation from the cytosol into the endoplasmic reticulum (2,3). Both proteins contain an N-terminal membrane-spanning region (MSR) and a C-terminal nucleotide-binding domain (NBD).…”

mentioning

confidence: 99%

Nucleotide Interactions with Membrane-bound Transporter Associated with Antigen Processing Proteins

Lapinski¹,

Raghuraman²,

Raghavan

2003

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4°C using the photo-cross-linkable nucleotide analogs 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.The transporter associated with antigen processing (TAP) 1 complex is an integral part of the major histocompatibility complex class I antigen presentation pathway (1). TAP is comprised of two related subunits, TAP1 and TAP2, which are necessary and sufficient for peptide translocation from the cytosol into the endoplasmic reticulum (2, 3). Both proteins contain an N-terminal membrane-spanning region (MSR) and a C-terminal nucleotide-binding domain (NBD). Peptide translocation by TAP complexes is preceded by peptide binding to the cytosolic face of TAP1/TAP2 complexes, a step that appears to be nucleotide binding-independent, at least at low temperatures (4, 5). However, the presence of ATP or ADP is critical for maintaining the structural stability of TAP complexes at physiological temperatures (6), and TAP mutants that are defective in nucleotide binding at the TAP2 site lose their ability to bind peptide at 37°C but not at lower temperatures (7). ATP hydrolysis is critical for peptide translocation across the endoplasmic reticulum membrane (8), and a peptide-stimulated ATPase activity has been described for TAP complexes (9).The role of each NBD during peptide translocation is not well understood. Mutations in both NBD have been shown to affect peptide translocation efficiency, although mutations on TAP2 NBD generally have more severe consequences. Based upon mutagenesis studies at structurally analogous residues on TAP1 and TAP2, experiments from several labs have resulted in the postulation of functional distinctions between a TAP1 and a TAP2 NBD during t...