Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

Hulpke, Sabine; Tomioka, Maiko; Kremmer, Elisabeth; Ueda, Kazumitsu; Abele, Rupert; Tampé, Robert

doi:10.1007/s00018-012-1005-6

Cited by 30 publications

(40 citation statements)

References 50 publications

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“…Even more interesting, when using a TAP-specific Ab for IP, equal amounts of tpn and TPSN Ex3 were co-precipitated. Since our HLA-B*44:02 stability analysis never showed a complete loss in the presence of TPSN Ex3, the data indicate, that more than one active MHC-I loading site per TAP molecule exist, which has recently been proposed also by others [15,16]. Furthermore, since TPSN Ex3 was not able to displace tpn completely from the TAP complex, it suggests, in a functional sense, that there are at least two differing binding sites for tpn on TAP, which has been observed previously for rat TAP subunits [11].…”

Section: Discussionsupporting

confidence: 81%

“…It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al [17].In addition to connecting the components of the PLC, tpn is crucial for the optimal loading and selection of peptide ligands onto . In the process of peptide editing, a stable disulfide bond formed between tpn (Cys-95) and the oxidoreductase ERp57 (Cys-57) has been suggested to play an important role [21].…”

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confidence: 73%

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A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

et al. 2013

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To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the Exon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the Exon3 variant with ERp57 was strongly impaired. Although the Exon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the Exon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the Exon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the Exon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8 + T-cell responses.Keywords: MHC class I peptide loading r PLC r TAP r Tapasin Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Anne Halenius e-mail: anne.halenius@uniklink-freiburg.de * These authors contributed equally to this work. Eur. J. Immunol. 2013Immunol. . 43: 1459Immunol. -1469 Introduction Presentation of endogenous peptides by MHC class I (MHC-I) to CD8 + T cells is critical to controlling viral infections and tumor growth. Peptides originating from cytosolic proteasomal degradation are translocated into the ER lumen by the heterodimeric peptide transporter TAP1/2 (transporter associated with Ag processing). Optimally TAP transports peptides with a length of 8-16 amino acids [1,2] and these can be further trimmed in the ER to fit the peptide binding groove of MHC class I (MHC-I) molecules [3], yielding a stable peptide-MHC-I complex that is transported through the secretory pathway for cell surface expression. Proper peptide loading of MHC-I is controlled by several chaperones that form the peptide loading complex (PLC) in the ER [4]. In the PLC the MHC-I heterodimer associates with tapasin (tpn), calreticulin, ERp57, and indirectly with TAP; tpn and TAP interact via their transmembrane segments (TMSs), thereby stabilizing the structure and function of TAP [5][6][7][8][9]. It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al...

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Section: Discussionsupporting

confidence: 81%

mentioning

confidence: 73%

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

et al. 2013

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“…8B, all three TAP2 mutants co-isolated less TPN per immunoprecipitated TAP2 when compared with TAP2wt (i.e., the TAP2/TPN ratio increased from 1.0 to values of 1.3-1.5). This suggests that the central TM leucine residues L20 (TM1) and L66 (TM2) are indeed relevant for proper stoichiometric complex formation between TAP2 and TPN (7,15). Although the observed TPN-binding differences between wt and TAP2 mutants are small, we could replicate this interesting result by performing quantitative immunodepletion experiments (Fig.…”

Section: Resultsmentioning

confidence: 74%

“…Although both N-domains of TAP independently contribute to MHC I/TPN binding, various studies have provided strong evidence for a functional asymmetry with greater significance of the TAP2/TPN interaction for PLC function (7,15). In line with this, only one TPN bound to TAP2 is sufficient for functional PLC assembly, efficient quality control, and stable peptide loading (7).…”

Section: Discussionmentioning

confidence: 97%

“…Most interestingly, we and others also showed that one TPN molecule bound either to TAP1 or TAP2 is sufficient for efficient MHC I Ag presentation (3,12). However, although both N-domains of TAP contribute independently to MHC I/TPN binding, different studies provided strong evidence for a functional asymmetry resulting in a greater signi-ficance of the TAP2/TPN interaction for PLC function (7,15). Consistently, in all avian MHC loci sequenced so far, only TAP2 harbors an N-domain, whereas TAP1 has no such domain and is therefore equivalent to core TAP1 (1DN) (16,17).…”

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confidence: 78%

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Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2

Rufer

Kägebein

Leonhardt

et al. 2015

The Journal of Immunology

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The transporter associated with Ag processing (TAP) translocates proteasomally derived cytosolic peptides into the endoplasmic reticulum. TAP is a central component of the peptide-loading complex (PLC), to which tapasin (TPN) recruits MHC class I (MHC I) and accessory chaperones. The PLC functions to facilitate and optimize MHC I–mediated Ag presentation. The heterodimeric peptide transporter consists of two homologous subunits, TAP1 and TAP2, each of which contains an N-terminal domain (N-domain) in addition to a conserved transmembrane (TM) core segment. Each N-domain binds to the TM region of a single TPN molecule, which recruits one MHC I molecule to TAP1 and/or TAP2. Although both N-domains act as TPN-docking sites, various studies suggest a functional asymmetry within the PLC resulting in greater significance of the TAP2/TPN interaction for MHC loading. In this study, we demonstrate that the leucine-rich hydrophobic sequence stretches (with the central leucine residues L20 and L66) in the first and second TM helix of TAP2 form a functional unit acting as a docking site for optimal TPN/MHC I recruitment, whereas three distinct highly conserved arginine and/or aspartate residues inside or flanking these TM helices are dispensable. Moreover, we show that the physical interaction between TAP2 and TPN is disrupted by benzene, a compound known to interfere with hydrophobic interactions, such as those between pairing leucine zippers. No such effects were observed for the TAP1/TAP2 interaction or the complex formation between TPN and MHC I. We propose that TAP/TPN complex formation is driven by hydrophobic interactions via leucine zipper–like motifs.

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Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes

Trowitzsch

Tampé

2015

Methods in Enzymology

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Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

Cited by 30 publications

References 50 publications

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2

Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes

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