Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules

Knittler, Michael R.; Alberts, Pēteris; Deverson, Edward V.; Howard, Jonathan C.

doi:10.1016/s0960-9822(99)80448-5

Cited by 71 publications

(96 citation statements)

References 49 publications

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“…As predicted, the HCMV and chimeric US6 proteins inhibited peptide translocation into the ER, whereas the level of peptide translocation in cells expressing CCMV US6 was comparable to that in untransfected HeLa-M cells. ATP binding and hydrolysis are essential for peptide translocation by TAP and is inhibited by HCMV US6 (16,20,26,31,33). We therefore analyzed whether CCMV US6 was able to inhibit ATP binding by TAP.…”

Section: Resultsmentioning

confidence: 99%

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Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Dugan

Hewitt

2008

J Virol

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The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.The major histocompatibility (MHC) class I antigen presentation pathway plays a central role in the immune response to viral infection. MHC class I molecules are expressed on the plasma membranes of all nucleated cells and present peptides derived from intracellular proteins to cytotoxic T lymphocytes (CTLs). CTLs monitor these MHC class I molecules for peptides derived from viral proteins and kill the infected cells. The presentation of antigenic peptides to CTLs represents the end point of a complex multistep pathway. The vast majority of peptides presented by MHC class I molecules are generated by the degradation of proteins in the cytosol by the proteasome complex (38). These peptides are then translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) (1). MHC class I molecules are assembled in the ER by a process that involves ER-resident chaperones (5). Peptide binding represents the final step of MHC class I molecule folding and is facilitated by the TAP-associated molecule tapasin, which links the nascent MHC class I molecules to TAP to form the "peptide-loading complex" (5). Only once they have bound peptides can MHC class I molecules dissociate from this complex and exit the ER for transport to the plasma membrane.TAP is a member of the ATP binding cassette (ABC) family of transporters, whose members couple ATP hydrolysis to the translocation of a broad spectrum of different substrates across cellular membranes (1,29,42). All...

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Section: Resultsmentioning

confidence: 99%

“…We therefore analyzed whether CCMV US6 was able to inhibit ATP binding by TAP. The ATP binding capacity of TAP was assayed with ATP-agarose beads (20) (Fig. 8B).…”

Section: Resultsmentioning

confidence: 99%

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Dugan

Hewitt

2008

J Virol

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“…ATP-Agarose Binding Assays-The ATP binding activity of TAP was analyzed based on published procedures (33). The inhibitory effect of US6 on TAP was analyzed as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

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

Koch

Guntrum

Heintke

et al. 2004

Journal of Biological Chemistry

154

208

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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...

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“…Single of TAP were found to influence peptide binding at the transmembrane domain (15,16,18,31). Therefore, we investigated whether these single site mutations in the C-loop have an impact on peptide binding.…”

Section: Single Site Mutants Of the C-loop-it Was Recentlymentioning

confidence: 99%

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

Chen

Abele

Tampé

2004

Journal of Biological Chemistry

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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.

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Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules

Cited by 71 publications

References 49 publications

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

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

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

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