Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta- and gamma-subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.     

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.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum

Tapasin (tpn), an essential component of the MHC class I (MHC I) loading complex, has a canonical double lysine motif acting as a retrieval signal, which mediates retrograde transport of escaped endoplasmic reticulum (ER) proteins from the Golgi back to the ER. In this study, we mutated tpn with a substitution of the double lysine motif to double alanine (GFP-tpn-aa). This mutation abolished interaction with the coatomer protein complex I coatomer and resulted in accumulation of GFP-tpn-aa in the Golgi compartment, suggesting that the double lysine is important for the retrograde transport of tpn from late secretory compartments to the ER. In association with the increased Golgi distribution, the amount of MHC I exported from the ER to the surface was increased in 721.220 cells transfected with GFP-tpn-aa. However, the expressed MHC I were less stable and had increased turnover rate. Our results suggest that tpn with intact double lysine retrieval signal regulates retrograde transport of unstable MHC I molecules from the Golgi back to the ER to control the quality of MHC I Ag presentation.     

Major histocompatibility complex class I molecules expressed with monoglucosylated N-linked glycans bind calreticulin independently of their assembly status

The assembly of major histocompatibility complex (MHC) class I molecules with peptides in the endoplasmic reticulum (ER) is a critical step in the presentation of viral antigens to CD8+ T cells. This process is subject to quality control restrictions that prevent free class I heavy chains (HCs) and peptide-free HC-beta(2)-microglobulin (beta(2)m) dimers from exiting the ER. The lectin-like chaperone calreticulin associates with HC-beta(2)m heterodimers prior to peptide binding, but its precise role in regulating the subsequent events of peptide association and ER to Golgi transport remains undefined. In vitro analysis of the assembly process has been limited by the specificity of calreticulin for monoglucosylated N-linked glycans, which are transient biosynthetic intermediates. To address this problem, we developed a novel expression system using Saccharomyces cerevisiae glycosylation mutants to produce class I HC bearing N-linked oligosaccharides with the specific structure Glc(1)Man(9)GlcNAc(2). The monoglucosylated glycan proved to be both necessary and sufficient for in vitro binding of calreticulin to MHC class I molecules. Calreticulin bound as efficiently to peptide-loaded MHC class I complexes as it did to folding intermediates created in vitro, namely free class I HC and empty HC-beta(2)m heterodimers. Thus, calreticulin is unable to discriminate between native and non-native MHC class I conformations and therefore unlikely to play a role in the recognition and release of peptide-loaded complexes from the ER. Furthermore, the recombinant expression system developed in this study can be used to produce a broad range of calreticulin substrates to elucidate its general mechanism of activity in vitro.     

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.     

Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.     

Crystal structures of two rat MHC class Ia (RT1-A) molecules that are associated differentially with peptide transporter alleles TAP-A and TAP-B

Antigenic peptides are loaded onto class I MHC molecules in the endoplasmic reticulum (ER) by a complex consisting of the MHC class I heavy chain, beta(2)-microglobulin, calreticulin, tapasin, Erp57 (ER60) and the transporter associated with antigen processing (TAP). While most mammalian species transport these peptides into the ER via a single allele of TAP, rats have evolved different TAPs, TAP-A and TAP-B, that are present in different inbred strains. Each TAP delivers a different spectrum of peptides and is associated genetically with distinct subsets of MHC class Ia alleles, but the molecular basis for the conservation (or co-evolution) of the two transporter alleles is unknown. We have determined the crystal structures of a representative of each MHC subset, viz RT1-A(a) and RT1-A1(c), in association with high-affinity nonamer peptides. The structures reveal how the chemical properties of the two different rat MHC F-pockets match those of the corresponding C termini of the peptides, corroborating biochemical data on the rates of peptide-MHC complex assembly. An unusual sequence in RT1-A1(c) leads to a major deviation from the highly conserved beta(3)/alpha(1) loop (residues 40-59) conformation in mouse and human MHC class I structures. This loop change contributes to profound changes in the shape of the A-pocket in the peptide-binding groove and may explain the function of RT1-A1(c) as an inhibitory natural killer cell ligand.     

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.     

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.     

Distinct but dispensable N-glycosylation of human CD69 proteins.

Human CD69 is uniquely glycosylated at typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) motifs, which represents the molecular basis for the formation of CD69 homodimers and heterodimers. Here we examined the importance of N-glycosylation for the assembly and intracellular transport of CD69 proteins using mutant CD69 molecules that specifically lack typical and atypical N-glycan attachment motifs. These studies verify the importance of Cys residues in atypical triplet sequences for N-glycan addition to human CD69 proteins in the endoplasmic reticulum (ER). In addition, these data demonstrate that monoglycosylated CD69 proteins (bearing N-glycans exclusively at atypical or typical sites) and aglycosylated CD69 molecules (lacking N-glycans) efficiently dimerize in the ER and have similar stability as wild-type CD69 molecules. Finally, these results show that CD69 proteins lacking atypical or typical N-glycan addition sites are transported to the plasma membrane.     

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.     

The quality control of MHC class I peptide loading

The assembly of major histocompatibility complex (MHC) class I molecules is one of the more widely studied examples of protein folding in the endoplasmic reticulum (ER). It is also one of the most unusual cases of glycoprotein quality control involving the thiol oxidoreductase ERp57 and the lectin-like chaperones calnexin and calreticulin. The multistep assembly of MHC class I heavy chain with beta(2)-microglobulin and peptide is facilitated by these ER-resident proteins and further tailored by the involvement of a peptide transporter, aminopeptidases, and the chaperone-like molecule tapasin. Here we summarize recent progress in understanding the roles of these general and class I-specific ER proteins in facilitating the optimal assembly of MHC class I molecules with high affinity peptides for antigen presentation.     

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 is required for efficient peptide binding to transporter associated with antigen processing

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.     

N-glycoproteomics in plants: perspectives and challenges

In eukaryotes, proteins that are secreted into the ER are mostly modified by N-glycans on consensus NxS/T sites. The N-linked glycan subsequently undergoes varying degrees of processing by enzymes which are spatially distributed over the ER and the Golgi apparatus. The post-ER N-glycan processing to complex glycans differs between animals and plants, with consequences for N-glycan and glycopeptide isolation and characterization of plant glycoproteins. Here we describe some recent developments in plant glycoproteomics and illustrate how general and plant specific technologies may be used to address different important biological questions.     

The transporter associated with antigen processing TAP: structure and function

The transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum (ER) is an essential process for presentation to cytotoxic T-lymphocytes. The transporter associated with antigen processing (TAP) is responsible for the intracellular translocation of peptides across the membrane of the ER. Efficient assembly of MHC-peptide complex requires the formation of a macromolecular transport and chaperone complex composed of TAP, tapasin and MHC class I molecules. Therefore, structure and function of TAP is important for the understanding of the immune surveillance.     

An extensive region of an MHC class I alpha 2 domain loop influences interaction with the assembly complex.

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and beta 2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the beta 2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I alpha 2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.     

Powering the peptide pump: TAP crosstalk with energetic nucleotides

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with āntigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.     

Bap29/31 influences the intracellular traffic of MHC class I molecules

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.