Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.     

Mechanisms of Function of Tapasin,a Critical Major Histocompatibility Complex Class I Assembly Factor

For their efficient assembly in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class I molecules require the specific assembly factors transporter associated with antigen processing (TAP) and tapasin, as well as generic ER folding factors, including the oxidoreductases ERp57 and protein disulfide isomerase (PDI), and the chaperone calreticulin. TAP transports peptides from the cytosol into the ER. Tapasin promotes the assembly of MHC class I molecules with peptides. The formation of disulfide‐linked conjugates of tapasin with ERp57 is suggested to be crucial for tapasin function. Important functional roles are also suggested for the tapasin transmembrane and cytoplasmic domains, sites of tapasin interaction with TAP. We show that interactions of tapasin with both TAP and ERp57 are correlated with strong MHC class I recruitment and assembly enhancement. The presence of the transmembrane/cytosolic regions of tapasin is critical for efficient tapasin–MHC class I binding in interferon‐γ‐treated cells, and contributes to an ERp57‐independent mode of MHC class I assembly enhancement. A second ERp57‐dependent mode of tapasin function correlates with enhanced MHC class I binding to tapasin and calreticulin. We also show that PDI binds to TAP in a tapasin‐independent manner, but forms disulfide‐linked conjugates with soluble tapasin. Thus, full‐length tapasin is important for enhancing recruitment of MHC class I molecules and increasing specificity of tapasin–ERp57 conjugation. Furthermore, tapasin or the TAP/tapasin complex has an intrinsic ability to recruit MHC class I molecules and promote assembly, but also uses generic folding factors to enhance MHC class I recruitment and assembly.     

A role for calnexin in the assembly of the MHC class I loading complex in the endoplasmic reticulum

Heterodimers of MHC class I glycoprotein and beta(2)-microglobulin (beta(2)m) bind short peptides in the endoplasmic reticulum (ER). Before peptide binding these molecules form part of a multisubunit loading complex that also contains the two subunits of the TAP, the transmembrane glycoprotein tapasin, the soluble chaperone calreticulin, and the thiol oxidoreductase ERp57. We have investigated the assembly of the loading complex and provide evidence that after TAP and tapasin associate with each other, the transmembrane chaperone calnexin and ERp57 bind to the TAP-tapasin complex to generate an intermediate. These interactions are independent of the N:-linked glycan of tapasin, but require its transmembrane and/or cytoplasmic domain. This intermediate complex binds MHC class I-beta(2)m dimers, an event accompanied by the loss of calnexin and the acquisition of calreticulin, generating the MHC class I loading complex. Peptide binding then induces the dissociation of MHC class I-beta(2)m dimers, which can be transported to the cell surface.     

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol-oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide-linked to ERp57 within the cell. Tapasin upregulation by interferon-gamma induces sequestration of the vast majority of ERp57 into the MHC class I peptide-loading complex. The rate of tapasin-ERp57 conjugate formation is unaffected by the absence of beta2-microglubulin (beta2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N-linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N-terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide-bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide-loading complex.     

The TAP translocation machinery in adaptive immunity and viral escape mechanisms

The adaptive immune system plays an essential role in protecting vertebrates against a broad range of pathogens and cancer. The MHC class I-dependent pathway of antigen presentation represents a sophisticated cellular machinery to recognize and eliminate infected or malignantly transformed cells, taking advantage of the proteasomal turnover of the cell's proteome. TAP (transporter associated with antigen processing) 1/2 (ABCB2/3, where ABC is ATP-binding cassette) is the principal component in the recognition, translocation, chaperoning, editing and final loading of antigenic peptides on to MHC I complexes in the ER (endoplasmic reticulum) lumen. These different tasks are co-ordinated within a dynamic macromolecular peptide-loading complex consisting of TAP1/2 and various auxiliary factors, such as the adapter protein tapasin, the oxidoreductase ERp57, the lectin chaperone calreticulin, and the final peptide acceptor the MHC I heavy chain associated with β2-microglobulin. In this chapter, we summarize the structural organization and molecular mechanism of the antigen-translocation machinery as well as various modes of regulation by viral factors and in genetic diseases and tumour development.     

Major histocompatibility complex class I-ERp57-tapasin interactions within the peptide-loading complex

The endoplasmic reticulum-located multimolecular peptide-loading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8(+) T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.     

Molecular architecture of the TAP-associated MHC class I peptide-loading complex

Tapasin organizes the peptide-loading complex (PLC) by recruiting peptide-receptive MHC class I (MHC-I) and accessory chaperones to the N-terminal regions of the TAP subunits TAP1 and TAP2. Despite numerous studies have shown that the formation of the PLC is essential to facilitate proper MHC-I loading, the molecular architecture of this complex is still highly controversial. We studied the stoichiometry of the PLC by blue native-PAGE in combination with Ab-shift assays and found that TAP/tapasin complexes exist at steady state as a mixture of two distinct oligomers of 350 and 450 kDa. Only the higher m.w. complex contains MHC-I and disulfide-linked tapasin/ER60 conjugates. Moreover, we show for the first time to our knowledge that the fully assembled PLC comprises two tapasin, two ER60, but only one complex of MHC-I and calreticulin. Based hereon we postulate that the TAP subunits alternate in the recruitment and loading of a single MHC-I.     

Recruitment of MHC class I molecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading

The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/beta(2)-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410-->F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient.220.B*4402 cells. Consistent with suboptimal peptide loading, the t(1/2) of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.     

Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules

Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β(2)m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deficient H chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain-β(2)m heterodimers, for which tapasin-ERp57 or tapasin-CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deficient PLCs.     

Assembly and Function of the Major Histocompatibility Complex (MHC) I Peptide-loading Complex Are Conserved Across Higher Vertebrates

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.     

Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly

The assembly and peptide loading of major histocompatibility complex Class I molecules within the endoplasmic reticulum are essential for antigen presentation at the cell surface and are facilitated by the peptide-loading complex. The formation of a mixed disulfide between the heavy chain of Class I and components of the loading complex (ERp57, protein disulfide isomerase, and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is coincident with the stabilization of the alpha2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57, and tapasin was observed and shown to be stabilized by a disulfide between both tapasinheavy chain and tapasin-ERp57. No disulfides were observed between ERp57 and heavy chain within the loading complex. The results provide a detailed evaluation of the various transient disulfides formed within the peptide-loading complex during biosynthesis. In addition, the absence of the disulfide between tapasin and heavy chain in TAP-deficient cells indicates that a change in the spatial organization of tapasin and heavy chain occurs following assembly into the loading complex.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Association of ERp57 with mouse MHC class I molecules is tapasin dependent and mimics that of calreticulin and not calnexin

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta(2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of L(d) mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient.220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.     

Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP)

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.     

Cellular and molecular requirements for association of the murine cytomegalovirus protein m4/gp34 with major histocompatibility complex class I molecules

The murine cytomegalovirus (MCMV) protein m4/gp34 is unique among known viral genes that target the major histocompatibility complex (MHC) class I pathway of antigen presentation in the following two ways: it is found in association with class I MHC molecules at the cell surface, and it inhibits antigen presentation without reducing cell surface class I levels. The current study was undertaken to define more clearly the structural and cellular requirements for m4/gp34 association with the MHC class I molecule K(b). We first assessed the role of the peptide-loading complex in m4/gp34-K(b) association, using cell lines lacking TAP, tapasin, or beta(2)m. m4/gp34-K(b) complexes formed in the absence of TAP or tapasin, although not as efficiently as in wild-type cells. The expression of full-length and truncation mutants of m4/gp34 in a gutless adenovirus vector revealed that the transmembrane region of m4/gp34 was required for efficient association with the K(b) heavy chain. However, the peptide-loading complex was not absolutely required for the association, since m4/gp34 readily formed complexes with K(b) in detergent lysates. The addition of K(b)-binding peptide to the detergent lysates facilitated but was not essential for the formation of the complexes. The ease of complex formation in detergent lysates contrasted with the small fractions of m4/gp34 and K(b) that form complexes in infected cells, suggesting that the endoplasmic reticulum (ER) environment restricts access of m4/gp34 to K(b). Finally, although m4/gp34-K(b) complexes could form when m4 was carried either by MCMV or by the adenovirus vector, they were only efficiently exported from the ER in MCMV-infected cells, suggesting that MCMV provides additional factors needed for transport of the complexes.     

Membrane topology of the transporter associated with antigen processing (TAP1) within an assembled functional peptide-loading complex

The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized "living" cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading complex.     

Tapasin and other chaperones: models of the MHC class I loading complex

MHC (major histocompatibility complex) class I molecules bind intracellular virus-derived peptides in the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T lymphocytes. Peptide-free class I molecules at the cell surface, however, could lead to aberrant T cell killing. Therefore, cells ensure that class I molecules bind high-affinity ligand peptides in the ER, and restrict the export of empty class I molecules to the Golgi apparatus. For both of these safeguard mechanisms, the MHC class I loading complex (which consists of the peptide transporter TAP, the chaperones tapasin and calreticulin, and the protein disulfide isomerase ERp57) plays a central role. This article reviews the actions of accessory proteins in the biogenesis of class I molecules, specifically the functions of the loading complex in high-affinity peptide binding and localization of class I molecules, and the known connections between these two regulatory mechanisms. It introduces new models for the mode of action of tapasin, the role of the class I loading complex in peptide editing, and the intracellular localization of class I molecules.     

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.     

The varicellovirus UL49.5 protein blocks the transporter associated with antigen processing (TAP) by inhibiting essential conformational transitions in the 6+6 transmembrane TAP core complex

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.     

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.