Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules

Momburg, Frank; Hämmerling, Günter J.

doi:10.1016/s0065-2776(08)60560-x

Cited by 96 publications

(65 citation statements)

References 440 publications

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“…Furthermore, based upon limited proteolytic digestion analysis, the proteolysis profiles observed for TAP1⅐TAP2(K509M) complexes closely parallel the profiles seen for TAP1⅐TAP2 complexes. 2 Thus, nucleotide hydrolysis by TAP complexes containing mutant TAP2 appears to be impaired. Likewise, the TAP1(K544M) mutation reduces peptide translocation efficiency of TAP complexes even 2 S. Arora and M. Raghavan, unpublished observations.…”

Section: Expression and Atp Binding By Tap1 And Tap2mentioning

confidence: 99%

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Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

Lapinski¹,

Neubig

Raghavan³

2001

Journal of Biological Chemistry

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We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1⅐TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)⅐TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.The transporter associated with antigen processing (TAP) 1 is a critical component of the major histocompatibility complex (MHC) class I antigen presentation (1-3). TAP functions to translocate peptides from the cytosol to the ER. Binding of peptides to newly synthesized MHC class I molecules in the ER stabilizes the MHC class I heterodimer and allows transit of MHC class I-peptide complexes to the cell surface for immune surveillance by T cells (4). Two structurally related subunits of the TAP transporter, TAP1 and TAP2, form a complex on the ER membrane that is necessary and sufficient for peptide translocation from the cytosol into the ER. The cytosolic face of TAP1⅐TAP2 complexes contains a binding site for peptides (5), which can function in the sequestration of peptides derived from proteasomal proteolysis. A recently discovered protein called tapasin is associated with the TAP1⅐TAP2 complex (6, 7). Tapasin has been shown to enhance the expression level of TAP1 and increase peptide transport by TAP complexes (8). However, tapasin is not required for peptide binding by TAP1⅐TAP2 complexes or for translocation per se, since TAP1⅐TAP2 complexes expressed heterologously in insect cells can bind and transport peptides (9).TAP is a member of the ATP-binding cassette (ABC) family of transmembrane transport proteins (10). Members of this family transport various substrates acro...

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Section: Expression and Atp Binding By Tap1 And Tap2mentioning

confidence: 99%

“…The transporter associated with antigen processing (TAP) 1 is a critical component of the major histocompatibility complex (MHC) class I antigen presentation (1)(2)(3). TAP functions to translocate peptides from the cytosol to the ER.…”

mentioning

confidence: 99%

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

Lapinski¹,

Neubig

Raghavan³

2001

Journal of Biological Chemistry

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“…Involvement of the Ub pathway for the generation of MHC class I-ligands is also supported by the finding that the suppression of ubiquitinylation resulted in a marked loss of presentation of endogenous antigens associated with MHC class I-molecules. The resultant antigenic peptides generated by the Ub-proteasome pathway are transported by a transporter associated with antigen processing (TAP) into the ER where they become associated with MHC class I molecules and are then delivered to the cell surface to activate cytotoxic T lymphocytes (CTLs) (reviewed in Momburg & Hämmering 1998).…”

Section: Biological Roles Of the Ubiquitinylationproteasome Directed mentioning

confidence: 99%

The proteasome: a protein‐destroying machine

Tanaka

Chiba

1998

Genes to Cells

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Most cellular proteins are targeted for degradation by the proteasome, a eukaryotic ATPdependent protease, after they have been covalently attached to ubiquitin (Ub) in the form of a poly Ub chain functioning as a degradation signal. The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (called the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations, to form enzymatically active proteasomes. The large assembled proteasome acts as a protein-destroying machine responsible for the selective breakdown of numerous ubiquitinylated cellular proteins and certain nonubiquitinylated proteins. To date, proteolysis mediated by the Ub-proteasome pathway has been shown to be involved in a wide variety of biologically important processes, such as the cell cycle, apoptosis, metabolism, signal transduction, immune response and protein quality control, implying that it functions as a previously unrecognized regulatory system for determining the final fate of protein factors involved in these biological reactions.

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“…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

Journal of Biological Chemistry

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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...

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Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules

Cited by 96 publications

References 440 publications

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

The proteasome: a protein‐destroying machine

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

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