Influence of the tapasin C terminus on the assembly of MHC class I allotypes

Several endoplasmic reticulum proteins, including tapasin, play an important role in major histocompatibility complex (MHC) class I assembly. In this study, we assessed the influence of the tapasin cytoplasmic tail on three mouse MHC class I allotypes (H2-K^b, -K^d, and -L^d) and demonstrated that the expression of truncated mouse tapasin in mouse cells resulted in very low K^b, K^d, and L^d surface expression. The surface expression of K^d also could not be rescued by human soluble tapasin, suggesting that the surface expression phenotype of the mouse MHC class I molecules in the presence of soluble tapasin was not due to mouse/human differences in tapasin. Notably, soluble mouse tapasin was able to partially rescue HLA-B8 surface expression on human 721.220 cells. Thus, the cytoplasmic tail of tapasin (either mouse or human) has a stronger impact on the surface expression of murine MHC class I molecules on mouse cells than on the expression of HLA-B8 on human cells. A K408W mutation in the mouse tapasin transmembrane/cytoplasmic domain disrupted K^d folding and release from tapasin, but not interaction with transporter associated with antigen processing (TAP), indicating that the mechanism whereby the tapasin transmembrane/cytoplasmic domain facilitates MHC class I assembly is not limited to TAP stabilization. Our findings indicate that the C terminus of mouse tapasin plays a vital role in enabling murine MHC class I molecules to be expressed at the surface of mouse cells.     

A charged amino acid residue in the transmembrane/cytoplasmic region of tapasin influences MHC class I assembly and maturation

Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, beta(2)-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.     

Tapasin increases efficiency of MHC I assembly in the endoplasmic reticulum but does not affect MHC I stability at the cell surface

Cell surface expression of MHC I molecules depends on the chaperone tapasin; how tapasin functions is not fully understood. We created three fluorescent tapasin constructs: wild-type tapasin, soluble tapasin, which does not interact with TAP, and N300 tapasin, which does not interact with MHC I. In contrast to earlier reports, all three constructs localize to the endoplasmic reticulum (ER), though soluble tapasin is more mobile than wild type and N300. Soluble tapasin does not increase MHC I surface levels to the same extent as wild type, which suggests that proximity to TAP is necessary for full tapasin function. N300 acts as a dominant-negative perhaps by blocking wild-type tapasin access to TAP. None of the constructs affects MHC I stability at the cell surface, although stability of ER resident MHC I is decreased in tapasin-negative cells. We propose that tapasin acts primarily to increase efficiency of assembly of MHC I within the ER.     

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.     

Tapasin enhances peptide-induced expression of H2-M3 molecules, but is not required for the retention of open conformers

H2-M3 is a class Ib MHC molecule that binds a highly restricted pool of peptides, resulting in its intracellular retention under normal conditions. However, addition of exogenous M3 ligands induces its escape from the endoplasmic reticulum (ER) and, ultimately, its expression at the cell surface. These features of M3 make it a powerful and novel model system to study the potentially interrelated functions of the ER-resident class I chaperone tapasin. The functions ascribed to tapasin include: 1) ER retention of peptide-empty class I molecules, 2) TAP stabilization resulting in increased peptide transport, 3) direct facilitation of peptide binding by class I, and 4) peptide editing. We report in this study that M3 is associated with the peptide-loading complex and that incubation of live cells with M3 ligands dramatically decreased this association. Furthermore, high levels of open conformers of M3 were efficiently retained intracellularly in tapasin-deficient cells, and addition of exogenous M3 ligands resulted in substantial surface induction that was enhanced by coexpression of either membrane-bound or soluble tapasin. Thus, in the case of M3, tapasin directly facilitates intracellular peptide binding, but is not required for intracellular retention of open conformers. As an alternative approach to define unique aspects of M3 biosynthesis, M3 was expressed in human cell lines that lack an M3 ortholog, but support expression of murine class Ia molecules. Unexpectedly, peptide-induced surface expression of M3 was observed in only one of two cell lines. These results demonstrate that M3 expression is dependent on a unique factor compared with class Ia molecules.     

Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.     

A region of tapasin that affects L(d) binding and assembly

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.     

Distinct functions of tapasin revealed by polymorphism in MHC class I peptide loading

Peptide assembly with class I molecules is orchestrated by multiple chaperones including tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the tapasin-deficient human cell line 721.220. These findings indicate distinct roles for tapasin in class I peptide loading. First, tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.     

Differential requirement for tapasin in the presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

 Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and β₂-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP. Received: 20 March 1997 / Revised: 2 June 1997     

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.     

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.     

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.     

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly

 Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products. Received: 1 February 1998 / Revised: 23 March 1998     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC 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.