Functional Role of C-Terminal Sequence Elements in the Transporter Associated with Antigen Processing

Ehses, Sarah; Leonhardt, Ralf M.; Hansen, Guido; Knittler, Michael R.

doi:10.4049/jimmunol.174.1.328

Cited by 13 publications

(9 citation statements)

References 56 publications

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“…After incubation for 30 min at 4°C, the lysates were analyzed in Western blots probed for TAP1 and TAP2. Immunoprecipitations and Western blotting were performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation and Purification of Microsomes-Microsomes from 2 ϫ 10 8 T2 cells were generated by sucrose gradient fractionation as described previously (10). To strip the microsomes off their bound ribosomes, 400 l of the microsomal suspension (concentration of 0.4 equivalent/l corresponding to A 280 ϭ 20) were incubated with 4 ml of high salt buffer (50 mM Hepes, pH 7.5, 250 mM sucrose, 1 mM dithiothreitol, 1 M KCl, and protease inhibitor mixture (Roche Applied Science)) and then centrifuged at 100,000 ϫ g for 30 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

Keusekotten

Leonhardt

Ehses

et al. 2006

Journal of Biological Chemistry

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The transporter associated with antigen processing (TAP) is essential for the delivery of antigenic peptides from the cytosol into the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility complex class I molecules. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2. As for many other oligomeric protein complexes, which are synthesized in the ER, the process of subunit assembly is essential for TAP to attain a native functional state. Here, we have analyzed the individual requirements of TAP1 and TAP2 for the formation of a functional TAP complex. Unlike TAP1, TAP2 is very unstable when expressed in isolation. We show that heterodimerization of TAP subunits is required for maintaining a stable level of TAP2. By using an in vitro expression system we demonstrate that the biogenesis of functional TAP depends on the assembly of preexisting TAP1 with newly synthesized TAP2, but not vice versa. The pore forming core transmembrane domain (core TMD) of in vitro expressed TAP2 is necessary and sufficient to allow functional complex formation with pre-existing TAP1. We propose that the observed assembly mechanism of TAP protects newly synthesized TAP2 from rapid degradation and controls the number of transport active transporter molecules. Our findings open up new possibilities to investigate functional and structural properties of TAP and provide a powerful model system to address the biosynthetic assembly of oligomeric transmembrane proteins in the ER.The antigen processing machinery plays an important role in the cellular immune response of vertebrates for the identification of infected or malignantly transformed cells. Peptides derived from proteasomal degradation of intracellular proteins are translocated via the transporter associated with antigen processing (TAP) 3 into the ER lumen and loaded onto major histocompatibility complex class I molecules (1). Presentation of "nonself " peptides at the cell surface to cytotoxic T-cells triggers the elimination of the infected or transformed cell. Lack of TAP expression e.g. in tumor tissue is associated with a dramatic loss of surface major histocompatibility complex class I and consequently promotes strongly the evasion of malignant cells from a proper cellular immune response (2). The ATP-binding cassette (ABC) transporter TAP is a heterodimer that comprises the two homologous subunits, TAP1 and TAP2, each of which consists of a hydrophobic transmembrane domain (TMD) and a hydrophilic, highly conserved cytoplasmic nucleotide-binding domain (NBD). The TMDs of both TAP subunits contribute to the formation of the peptide-binding site and the translocation pore (1). The binding and hydrolysis of ATP is believed to power the transport process by inducing conformational changes in the NBDs. These structural changes alter the conformation of the TMDs and cause the binding and movement of peptides across the ER membrane. The TMDs of TAP1 and TAP2 have unique N-terminal domains (N-domains with three to f...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

Keusekotten

Leonhardt

Ehses

et al. 2006

Journal of Biological Chemistry

Self Cite

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“…While it has been suggested that TAP2 only displays a preference for ADP when isolated from TAP1 and that the physiological TAP1/2 heterodimer has no such nucleotide preferences [47], another possibility is that the nucleotide specificity of TAP2 is regulated during a transport cycle. Using chimeras between the TAP1 and TAP2 proteins, it was observed that a short stretch of amino acids near the C-terminus is responsible for the unique nucleotide specificities [48]. In the TAP1-NBD structure [39,40], these amino acids are distant from the ATP-binding site and instead form a hinge-like loop that bridges two subdomains ( Figure 2B).…”

Section: Asymmetry In the Tap Atpase Sites Refines A Model For Peptidmentioning

confidence: 99%

Antigen processing and presentation: TAPping into ABC transporters

Procko

Gaudet

2009

Current Opinion in Immunology

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Adaptive, cell-mediated immunity involves the presentation of antigenic peptides on class I MHC molecules at the cell surface. This requires an ABC transporter associated with antigen processing (TAP) to transport antigenic peptides generated in the cytosol into the endoplasmic reticulum (ER) for loading onto class I MHC. Recent crystal structures of bacterial ABC transporters suggest how the transmembrane domains of TAP form a peptide-binding cavity that acquires peptides from the cytosol, and following ATP-induced conformational changes, the peptide-binding cavity closes to the cytosol and instead opens to the ER lumen for peptide release. Extensive biochemical studies show how transport is driven by ATP binding and hydrolysis on an asymmetric pair of cytosolic nucleotide-binding domains, which are physically coupled to the peptidebinding site to propagate conformational changes through the protein.

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“…30 More recently, it was suggested that the so-called α6/β10-loops close to the C termini of TAP1 and TAP2 determine the non-synonymous nucleotide binding of the two NBDs. 38 However, there is no indication from structural or biochemical studies that the α6/β10-loop region makes direct contact with bound ATP or ADP, suggesting that the α6/β10-loops contribute to the regulation of nucleotide binding in a conformational rather than a sequence-specific manner.…”

Section: Discussionmentioning

confidence: 98%

Modulation of the Antigenic Peptide Transporter TAP by Recombinant Antibodies Binding to the Last Five Residues of TAP1

Plewnia

Schulze

Hunte

et al. 2007

Journal of Molecular Biology

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Functional Role of C-Terminal Sequence Elements in the Transporter Associated with Antigen Processing

Cited by 13 publications

References 56 publications

Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

Antigen processing and presentation: TAPping into ABC transporters

Modulation of the Antigenic Peptide Transporter TAP by Recombinant Antibodies Binding to the Last Five Residues of TAP1

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