The studies described here demonstrate that weak intrinsic interactions between the lumenal domains of tapasin and MHC class I are strongly enhanced by the presence of the transmembrane and cytosolic domains of tapasin, and also enhanced by tapasin-ERp57 binding ( and ). The inability to recruit ERp57 strongly impairs the functional activity of soluble tapasin(C95A), which is a weak inducer of MHC class I assembly (). While the C95A mutation also reduces the functional activity of full-length tapasin, significant ERp57 binding-independent functional activity is measurable with full-length tapasin(C95A), which is not strongly correlated with tapasin-mediated stabilization of TAP ( and ). The tapasin(C95A) mutation also impacts the efficiency of calreticulin recruitment to MHC class I molecules (). While ERp57 and calreticulin binding to TAP are strongly dependent on or linked to tapasin(C95), PDI association with TAP is tapasin-independent, and in fact appears to be enhanced by the absence of tapasin or a fully-functional tapasin molecule. These findings indicate distinct modes of binding and distinct functions for ERp57 and PDI within MHC class I assembly complexes. Significant C95-dependent conjugation of PDI to tapasin is only detectable in the context of soluble tapasin, studies that point to the importance of full-length tapasin for enhancing specificity of interactions within MHC class I assembly complexes.
The panel of mutants described here were also characterized in a recent report from the Cresswell laboratory (24
), published while this manuscript was under revision. While the two studies come to similar conclusions about the importance of both tapasin-ERp57 binding and the transmembrane/cytosolic regions of tapasin in its interactions with MHC class I and functional activities, there are some notable differences between the studies. Our studies in M553 cells suggest that the deletion of the transmembrane/cytosolic domains of tapasin had a more significant impact on the efficiency of tapasin-MHC class I binding than did ERp57 conjugation to tapasin ( and ). Immunoprecipitations with the anti-tapasin antibody by the Cresswell laboratory suggested a more critical role for tapasin-ERp57 binding in HLA-B*4402 recruitment to tapasin, following analyses of various tapasin mutants in 721.220/HLA-B*4402 cells (24
). Additionally, despite the lower efficiency of soluble tapasin-class I complex formation, we were able to detect complexes between soluble tapasin(C95A) and MHC class I heavy chains ( and ), whereas these complexes were not detectable in the other study (24
). The observed differences may relate to the use of IFN-γ in our assays, which upregulates TAP as well as MHC class I subunits, promoting several of the interactions that are dependent on one or both of these proteins. TAP expression levels are very relevant to interactions mediated by full-length tapasin, and thus analyses under conditions where TAP expression is not limiting are important. It is alternatively possible that there are allele-specific or other cell type-specific differences in requirements of ERp57 associations vs. the tapasin transmembrane/cytoplasmic domains for efficient tapasin-MHC class I binding. Our analyses examined tapasin binding to endogenously-expressed MHC class I allotypes of M553 cells, which include HLA-A28, HLA-B*5701 and HLA-B*5001 (16
). The Cresswell laboratory study examined interactions of exogenously expressed HLA-B*4402 in 721.220 cells which lack endogenous HLA-A and HLA-B, but express an endogenous HLA-C. It is possible that binding of tapasin to HLA-B*4402, a highly tapasin-dependent allotype (25
), has more stringent requirements for accessory ER chaperones.
Our data indicate that tapasin promotes MHC class I assembly by two major mechanisms, involving (i) an ERp57-independent mode of tapasin function that requires the transmembrane and cytosolic domains of tapasin and involves associations with TAP () and (ii) an ERp57-dependent mode of tapasin function requiring the ER luminal domains of tapasin and associations with calreticulin (). The ERp57-independent mode of tapasin function may involve the editing functions of tapasin which optimizes its peptide repertoire, as suggested using fos-jun tethered tapasin-MHC class I complexes (11
). Furthermore, localization of MHC class I in the vicinity of the peptide source, TAP, could account for the ERp57-independent activity of tapasin. In some species such as chicken, which lack the counterpart of C95, the ERp57-independent mode of tapasin function may be the sole mechanism of tapasin function (26
). Importantly, while the ERp57-independent mode of function requires the transmembrane and cytosolic domains of tapasin (), the role of these domains extends beyond the previously suggested functions for these domains in TAP stabilization (4
) (, , ).
Two modes of tapasin function Panel A: ERp57-independent mode of tapasin function
Tapasin-dependent recruitment of ERp57 enhanced the ability of human tapasin to mediate MHC class I assembly (), even under conditions where tapasin-MHC class I interactions were relatively weak (soluble tapasin; and ). ERp57 and calreticulin interact outside of the MHC class I assembly complex (19
), and higher representation of ERp57 in conjugation with purified tapasin correlated with increased calreticulin binding in vitro
(). Tapasin-C95-mediated recruitment of ERp57 and enhanced recruitment of calreticulin contributed to enhanced calreticulin-MHC class I binding and increased stabilities of MHC class I heavy chains (, and ). Thus calreticulin is likely an important player in the ERp57-dependent functional activity of tapasin, but further studies are needed to understand how calreticulin interacts with MHC class I and tapasin, and the extent of its contribution to the functional activity of tapasin. It is widely believed that binding monoglucosylated glycans is sufficient to recruit calreticulin to substrates. However, the findings reported here indicate that multiple tapasin-dependent interactions rather than glycan binding alone contribute to efficient recruitment of calreticulin to the MHC class I assembly complex. Further understanding of the mechanisms of calreticulin recruitment into the MHC class I assembly complex will allow insights into whether similar mechanisms could be used promote recruitment of ER chaperones to enhance assembly of other multi-protein complexes, particularly of viruses.
The recent crystal structure analysis of a soluble tapasin-ERp57 heterodimer showed that both catalytic domains of ERp57 (a and a′ domain) are involved in tapasin binding (7
). ERp57 residues involved in binding to tapasin are conserved in PDI (7
). Specificity of tapasin-ERp57 heterodimer formation was attributed to differences in inter-domain orientations between different PDI family members. A shorter distance was noted between Cα atoms of C57 and C406 of ERp57 in the tapasin-ERp57 heterodimer (7
), compared to counterpart PDI residues in the yeast PDI structure which was crystallized as free protein rather than in a substrate-conjugated form (27
). Our studies indicate that soluble tapasin per se
is not fully specific for ERp57 conjugation as conjugates between soluble tapasin and PDI were also readily detectable, although at a level approximately 7.5 fold lower than that of ERp57 (, and Supplementary Figure 1
How might TAP binding inhibit tapasin-PDI binding and promote tapasin-ERp57 interaction specificity? Cooperative interactions that stabilize the heptameric complexes involving TAP1/TAP2/tapasin/MHC class I/calreticulin/ERp57 via multimodal binding between the different components, must impose a strong structural constraint on the complete complex, that is absent in the corresponding soluble tapasin complexes. The lack of complex stability in soluble tapasin complexes is likely directly responsible for the reduction in oxido-reductase conjugation specificity. Thus, structural constraints of the complete MHC class I assembly complex contribute to the specificity of tapasin-ERp57 binding.
We show that PDI is likely a bonafide component of the TAP complex not by its ability to conjugate with tapasin, but rather via its ability to bind to TAP (). TAP-associated PDI has been implicated in the maintenance of oxidized forms of MHC class I heavy chains, as higher levels of reduced heavy chains were found in PDI-depleted cells (13
). However, the majority of TAP-associated heavy chains were oxidized in PDI-depleted cells (13
), which suggests that PDI did not have a major influence on maintaining oxidized forms of TAP-associated heavy chains. The possibility of a function for TAP-associated PDI in mediating tapasin oxidation is suggested by the findings that TAP-associated forms of tapasin are more oxidized compared to Pasta-1 associated forms of tapasin (data not shown); however, the oxidized form of tapasin found in complex with TAP may also simply result from steric constraints of reductase access to TAP-associated forms of tapasin, rather than PDI-dependent catalytic oxidation. The relevant role of TAP-associated PDI may relate to the sequestration of TAP-translocated peptides, to prevent their premature retro-translocation, as previously suggested (13
). Alternatively, since the presence of functional forms of TAP-associated tapasin reduced the extent of PDI-TAP binding (), it is also possible that PDI functions in the retro-translocation and degradation of components of the TAP complex, including TAP itself, when not stabilized by a complete repertoire of associated factors. PDI binding to TAP may involve interactions with surface hydrophobic patches on PDI (27
). Further functional studies will be needed to assess whether TAP-associated PDI represents a specific mechanism for mediating peptide retention in the ER, or PLC component degradation. It is a formal possibility that the observed TAP-PDI complex represents a more non-specific hydrophobic interactions-based binding.
Our studies also allowed a direct assessment of the effects of ERp57 and TAP binding on tapasin folding. Abrogation of C95-mediated conjugate formation did not induce significant soluble tapasin(C95A) aggregation in cell lysates, and soluble tapasin (C95A) migrated predominantly as monomers (data not shown). However, soluble wild type tapasin was aggregated when purified from cell supernatants, even when co-expressed with ERp57 (), suggesting that in addition to oxido-reductase binding, interactions with ER lumenal factors such as calreticulin and GRP78 may be important for maintaining tapasin stability and inhibiting its aggregation ().
In summary, we show that tapasin functions by two major mechanisms, involving (i) binding of MHC class I molecules via interactions involving its ER lumenal and transmembrane/cytosolic domains and (ii) recruitment of ERp57 and calreticulin into the MHC class I assembly complex (). Additional studies are required to understand whether the tapasin transmembrane and cytosolic domains directly impact tapasin-MHC class I binding, or whether TAP/tapasin binding impacts the efficiency of tapasin-MHC class I binding. Alternatively, the transmembrane domain of tapasin could induce binding merely by increasing the local concentrations of tapasin in membranes containing MHC class I. Each of these possibilities remains to be further investigated. Additionally, whether the ERp57-independent functional activity of tapasin results in part from its ability to promote physical interactions between MHC class I and TAP (thus achieving a higher local concentration of peptides) also remain to be investigated. Furthermore, modes of calreticulin interaction and function remain to be defined, as also the precise function of TAP-associated PDI.