Author response: TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway
2017-03-17

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex. https://doi.org/10.7554/eLife.23049.001 Introduction The presentation of antigenic peptides to the immune system by MHC class I molecules is crucial in generating protective responses against infection and cancer. Central to this process is the loading and optimisation of peptides onto MHC class I molecules within the peptide-loading complex (PLC) in the endoplasmic reticulum (ER) by tapasin, an MHC class I-dedicated chaperone that has been the focus of intense investigation for the past two decades (Sadasivan et al., 1996; Ortmann et al., 1997; Williams et al., 2002). It is now well established that tapasin functions as a peptide exchange catalyst for MHC class I molecules, a process that is important in the selection of high-affinity peptides onto MHC class I molecules (Chen and Bouvier, 2007; Wearsch and Cresswell, 2007). Recently, we revealed that TAPBPR, a second MHC class I-dedicated chaperone in the antigen presentation pathway, also functions as a peptide exchange catalyst for MHC class I molecules (Boyle et al., 2013; Hermann et al., 2015), a finding that was subsequently verified by Margulies and colleagues (Morozov et al., 2016). Thus, it is now clear that there are at least two MHC class I-specific chaperones in the antigen presentation pathway that are intimately involved in selecting peptides for presentation on MHC class I molecules. Although both tapasin and TAPBPR share the ability to optimise peptide selection in vitro, they cannot directly compensate for each other within a cellular environment and appear to influence peptide selection in separate yet complementary processes. In the absence of a functional tapasin molecule, inefficient peptide loading occurs, resulting in MHC class I molecules loaded with suboptimal peptide ligands (Ortmann et al., 1997; Purcell et al., 2001; Williams et al., 2002). As a consequence, the absence of tapasin produces thermolabile MHC class I complexes that are inefficiently expressed on the cell surface, although different MHC class I allomorphs differ in their dependency on tapasin (Ortmann et al., 1997; Lewis et al., 1998; Peh et al., 1998; Garbi et al., 2000; Grandea et al., 2000; Williams et al., 2002; Rizvi et al., 2014). In contrast to our understanding of tapasin, the precise role of TAPBPR-mediated peptide editing in the antigen presentation pathway has yet to be fully characterised (Hermann et al., 2015; Morozov et al., 2016). TAPBPR is not essential for the initial peptide-loading event onto MHC class I molecules (Boyle et al., 2013). Instead, TAPBPR has a more subtle, fine-tuning effect on the peptides displayed, removing some peptides of lower affinity and thus improving peptide selection and increasing the stability of MHC class I molecules (Hermann et al., 2015). We have speculated that the different effects of tapasin and TAPBPR on the peptide repertoire in cells is due, at least in part, to the environment in which the two chaperones operate. Tapasin functions within the PLC in an environment that is rich in suitable peptides for MHC class I binding (Sadasivan et al., 1996; Li et al., 1997; Ortmann et al., 1997), which helps promote efficient peptide loading onto MHC class I molecules. In contrast TAPBPR is not a component of the PLC and therefore performs peptide editing outside this complex, potentially in a more peptide-deficient environment (Boyle et al., 2013; Hermann et al., 2015), which may favour peptide dissociation from MHC class I molecules. Therefore, it seems plausible that tapasin and TAPBPR have evolved to function in distinct cellular environments. For tapasin, three regions have been identified that are essential for its localisation and function within the PLC: its transmembrane domain is responsible for its interaction with TAP (Petersen et al., 2005; Rufer et al., 2015); a free cysteine residue at position C95 is essential for its association with ERp57 (Dick et al., 2002; Peaper et al., 2005); and residues in the Ig domains interact with MHC class I (Turnquist et al., 2001; Turnquist et al., 2004, Dong et al., 2009). For TAPBPR, the only functional sites to be identified so far are those that are responsible for its interaction with MHC class I (Hermann et al., 2013), and as yet, no association partners that function with TAPBPR have been characterised. Our aim here was to investigate whether any other co-factors interacted with TAPBPR in cells, which would explain the ability of TAPBPR to optimise peptide selection. Results TAPBPR binds to UDP-glucose:glycoprotein glucosyltransferase 1 To identify potential cellular binding partners for TAPBPR, we transiently transfected HeLaM cells with a construct in which the cDNA encoding amino acids 22–468 of human TAPBPR (i.e. the mature protein) was cloned downstream of a generic ER leader sequence, two protein A cassettes and a myc tag (ZZ-TAPBPR). This resulted in the expression of ZZ-TAPBPR at an approximately five-fold greater level than that observed for endogenous TAPBPR in IFN-γ-treated HeLaM. Affinity chromatography with IgG-sepharose beads was subsequently used to isolate ZZ-TAPBPR and any associated proteins. As expected, tandem mass spectrometry (MS/MS) analysis identified the MHC class I heavy chain and β2m in the ZZ-TAPBPR immunoprecipitate (Table 1) (Boyle et al., 2013). Interestingly, UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) was also isolated in the immunoprecipitates from ZZ-TAPBPR-expressing cells, but not in control HeLaM cells transfected with an empty vector (Table 1), suggesting that UGT1 could be a novel binding partner for TAPBPR. UGT1 was ranked as the third specific hit in the ZZ-TAPBPR pulldown after the MHC class I heavy chain and TAPBPR (Table 1). UGT1 is an ER/cis-Golgi resident enzyme that monitors glycoprotein folding (Arnold et al., 2000; Tessier et al., 2000; Zuber et al., 2001; D'Alessio et al., 2010). In 2011, Cresswell and colleagues showed that UGT1 plays an important role in the MHC class I antigen processing and presentation pathway (Wearsch et al., 2011; Zhang et al., 2011). Although no direct association was demonstrated between MHC class I and UGT1 within a cellular environment, they found that MHC class I maturation and assembly was delayed, surface expression of MHC class I was reduced, and there was an impairment of peptide selection in UGT1-deficient cells (Zhang et al., 2011). Furthermore, recombinant UGT1 was found to reglucosylate MHC class I molecules associated with suboptimal ligands and permitted their re-engagement with the PLC, thus providing direct evidence for the role of UGT1 in the antigen presentation pathway (Wearsch et al., 2011). To confirm that the interaction between TAPBPR and UGT1 was not an artefact of tagging or TAPBPR overexpression, immunoprecipitation of TAPBPR was performed in IFN-γ-induced HeLaM and KBM-7 cells, followed by western blotting for UGT1. An association between endogenous TAPBPR and UGT1 was observed in both IFN-γ-treated HeLaM and KBM-7 cells (Figure 1A and B respectively). The association was not observed in cells that were knocked out (Figure 1A) or depleted (Figure 1B) of TAPBPR, demonstrating that the co-precipitation of UGT1 was a direct consequence of TAPBPR presence. Furthermore, the association between TAPBPR and UGT1 was observed in the absence of tapasin (Figure 1A and B), suggesting that a functional PLC is not required for TAPBPR to associate with UGT1. As shown in Figure 1A and B, we observed that the association between TAPBPR and MHC class I was increased in the absence of tapasin, an observation that is consistent with our previously published findings (Hermann et al., 2013). In contrast to the association observed between TAPBPR and UGT1, no association was observed between tapasin and UGT1 in IFN-γ-induced KBM-7 cells (Figure 1C). These results confirm that UGT1 is a novel binding partner for TAPBPR. However, as human TAPBPR lacks an N-linked glycan and UGT1 monitors glycoprotein folding by recognising hydrophobic patches near a Man9GlcNAc2 moiety (Trombetta et al., 1989; Caramelo et al., 2003, Caramelo et al., 2004; Ritter et al., 2005), it is highly unlikely that UGT1 functions directly in the quality control of TAPBPR. Figure 1 Download asset Open asset TAPBPR associates with UDP-glucose:glycoprotein glucosyltransferase 1 (A and B) TAPBPR or (C) tapasin were immunoprecipitated using PeTe4 or Pasta1, respectively, from (A) IFN-γ-treated HeLaM, TAPBPR KO, tapasin KO, and double tapasin KO/TAPBPR KO HeLaM cells or (B and C) IFN-γ-treated wild-type, TAPBPR-depleted (+ shTAPBPR), and tapasin KO KBM-7 cells. In (A), immunoprecipitation with an isotype control antibody was used as a control (labelled 'Ctrl'). Western blot analysis was performed for UGT1, TAPBPR (using R021), tapasin (Rgp48N), MHC class I HC (using 3B10.7), or calnexin as a loading control on immunoprecipitates or lysates as indicated. Note: the amounts of TAPBPR present in the lysates were below limits of detection and were detectable after concentration via immunoprecipitation. In (C), * denotes the tapasin protein from the previous blot, while the lower band is the MHC class I HC. The data shown are representative of three independent experiments. In (A and B), an increased association between TAPBPR and MHC class I molecules in the absence of tapasin can be observed. While this is not immediately obvious in (A) with HeLaM cells, a significant loss of MHC class I expression is observed in the absence of tapasin in this cell line. Therefore, a relative increase in the association between MHC class I molecules and TAPBPR is observed in the absence of tapasin. KO: knockout; UGT1: UDP-glucose:glycoprotein glucosyltransferase 1; IP: immunoprecipitation; WB: western blot. https://doi.org/10.7554/eLife.23049.002 Table 1 Selected proteins identified in IgG-sepharose pulldowns on ZZ-TAPBPR Affinity chromatography with IgG-sepharose was performed on HeLaM cells expressing a protein-A-tagged TAPBPR molecule (ZZ-TAPBPR) or HeLaM cells transduced with an empty vector (control). Immunoprecipitates were analysed by in gel tryptic digest followed by liquid chromatography-tandem mass spectrometry and data were processed using Scaffold. Identified proteins are shown with their exclusive unique peptide count, percentage coverage, and exclusive unique spectrum count as determined by Scaffold. Rank denotes the position when data are sorted by exclusive unique peptide count with all proteins present in the control removed. Pep: exclusive unique peptide count; Cov: percentage coverage; Count: exclusive unique spectrum count. https://doi.org/10.7554/eLife.23049.003 ProteinGene nameControlZZ-TAPBPRRankPep (Cov)CountPep (Cov)CountTapasin-related proteinTAPBPL––8 (16)112HLA class 1, A-68HLA-A––14 (35)211β-2-microglobulinβ2M––1 (8.4)195UDP-glucose:glycoprotein glucosyltransferase 1UGGT1––10 (7.3)103 Residue C94 in TAPBPR is not involved in an intramolecular disulphide bond Since the extracellular domain of TAPBPR contains seven cysteine residues (Teng et al., 2002), we were intrigued as to the probable existence, and functional relevance, of an unpaired cysteine residue. For tapasin, intramolecular disulphide bonds exist between C7 and C71 in the N-terminal domain and C295 and C363 in the membrane proximal Ig-like domain, while C95 is known not to be involved in an intramolecular disulphide bound, but instead forms an intermolecular disulphide bond with C57 of ERp57 (Herberg et al., 1998; Dick et al., 2002; Dong et al., 2009)(Figure 2A and B). Our Fold and Functional Assignment System (FFAS) model of TAPBPR (Hermann et al., 2013) now supported by small-angle X-ray scattering (SAXS) data (Morozov et al., 2016), predicts that C18 and C101 in the N-terminal domain and C300 and C361 in the IgC domain of TAPBPR form similar intramolecular disulphide bonds as those found in tapasin, and that C191 and C262 form an additional intramolecular disulphide bond (Figure 2C). This analysis predicts that C94 in TAPBPR is very unlikely to be involved in an intramolecular disulphide bond (Figure 2C). To verify this, all cysteine residues were individually changed to alanine, cloned into a lentiviral vector containing a bicistronic GFP, and then successfully transduced into HeLaM cells (Figure 2—figure supplement 1). Mutation of C300 or C361 in the IgC domain led to significantly reduced steady-state expression of TAPBPR (Figure 2D), suggesting that these cysteines are essential for TAPBPR stability. Under non-reducing conditions, TAPBPRC18A and TAPBPRC101A exhibited lower electrophoretic mobility relative to TAPBPRWT (Figure 2D), suggesting that a disulphide bond exists between C18 and C101 that is important for the structural integrity of the N-terminal domain. Mutation of C191 and C262 did not significantly affect steady-state TAPBPR expression; however, a very subtle change in electrophoretic mobility of these two mutants was observed under non-reducing conditions compared to TAPBPRWT (Figure 2D). This may reflect disruption of a disulphide bond within a stable protein domain that does not lead to sufficient unfolding as to be detected by electrophoresis, as is the case for MHC class II molecules (Kaufman and Strominger, 1982, 1983). Another possibility is that, under the conditions used, the sample was reoxidised prior to loading. In contrast, mutation of C94 in TAPBPR did not affect steady-state protein expression or electrophoretic mobility (Figure 2D). As an additional check to determine whether TAPBPRC94A was stable and folded, this mutant was cloned into the pHLsec expression vector and transiently transfected into HEK293F cells. As observed with TAPBPRWT (Figure 2E) (Hermann et al., 2015), TAPBPRC94A was efficiently expressed and purified using Ni affinity and size exclusion chromatography (Figure 2F). Using differential scanning fluorimetry, the melting temperature of purified TAPBPRWT and TAPBPRC94A were the same, demonstrating that mutation of C94 to alanine did not affect protein stability (Figure 2G). Figure 2 with 1 supplement see all Download asset Open asset C94 in TAPBPR is not involved in an intramolecular disulphide bond (A) Amino acid sequences of human TAPBPR (NP_060479.3) and tapasin (AAC20076.1) were aligned using ClustalW. Cysteine residues are marked in yellow boxes, with the cysteine in tapasin that interacts with ERp57 and the predicted unpaired cysteine in TAPBPR highlighted in red. The positions of the cysteine residues in TAPBPR and tapasin are labelled above and below, in blue or green, respectively. (B) Structure of tapasin (Protein Data Bank ID: 3F8U), with the cysteines involved in intramolecular disulphide bonds highlighted in yellow and the free cysteine C95 highlighted in red. (C) FFAS model for TAPBPR (Hermann et al., 2013), with potential disulphide bridges highlighted in yellow and the predicted free cysteine C94 highlighted in red. (D) Lysates from a HeLaM cell panel expressing cysteine-mutant TAPBPR molecules were resolved under non-reducing (no 2-ME) or reducing (+2-ME) conditions, then blotted for TAPBPR (using mouse anti-TAPBPR), MHC class I heavy chain (using HC10), or calnexin as a loading control. The antibody used to detect TAPBPR was raised against the membrane distal domain (aa 23–122); therefore, it is unlikely that the lack of detection of C300 and C361 is due to a lack of antibody recognition of these IgC domain mutants. Data shown in (D) are representative of three independent experiments. (E and F) Size exclusion chromatogram of TAPBPRWT and TAPBPRC94A purified from cell culture supernatant. The protein peaks were analysed by SDS-PAGE followed by Coomassie staining. (G) Differential scanning fluorimetry of TAPBPRWT and TAPBPRC94A demonstrates equivalent thermal denaturation profiles. WT: wild-type; WB: western blot; RFU: relative fluorescence units. https://doi.org/10.7554/eLife.23049.004 C94 in TAPBPR is essential for its association with UGT1 To explore the functional importance of the unpaired cysteine at position 94, we reconstituted the TAPBPR-knockout HeLaM cell line (HeLaMKO) we characterised previously (Hermann et al., 2015) with either TAPBPRWT or TAPBPRC94A. First, we asked whether the interactions between TAPBPR and other proteins were altered when C94 was mutated to alanine. Comparison of TAPBPR immunoprecipitates from HeLaMKOTAPBPRWT and HeLaMKOTAPBPRC94A by MS/MS analysis revealed that TAPBPR still associated with both the MHC class I heavy chain and β2m in the absence of C94 (Table 2). However, the most striking difference between the immunoprecipitates was the absence of UGT1 in the HeLaMKOTAPBPRC94A sample (Table 2). These findings were confirmed by western blot analysis on TAPBPR immunoprecipitates, where the amount of UGT1 that was co-immunoprecipitated was drastically reduced following mutation of C94 (Figure 3A). These results suggest that the C94 residue in TAPBPR is important for its association with UGT1. Given that all lumenal cysteine residues are conserved in TAPBPR from different species (Figure 3—figure supplement 1), it is possible that the association between TAPBPR and UGT1 is conserved from humans to fish. Figure 3 with 2 supplements see all Download asset Open asset TAPBPR binds to UDP-glucose:glycoprotein glucosyltransferase 1 in a C94-dependent manner, but not via a disulphide bond and bridges UDP-glucose:glycoprotein glucosyltransferase 1 to MHC class I molecules (A–C) TAPBPR or (A) HC10-reactive MHC class I were isolated by immunoprecipitation from (A and B) HeLaM-TAPBPRKO cells (HeLaMKO) and HeLaMKO cells reconstituted with either TAPBPRWT, TAPBPRC94A, or TAPBPRTN5 or (C) WT, TAPBPR-depleted (+ shTAPBPR), and tapasin KO KBM-7 cells, with or without IFN-γ treatment, as indicated under (A) reducing or (B and C) non-reducing conditions. (D) HC10 and W6/32-reactive MHC class I molecules were isolated from IFN-γ-treated HeLaM and HeLaMKOcells and resolved under reducing conditions. Western blot analysis was performed for UGT1, TAPBPR (using R021), MHC class I HC (using 3B10.7), or calnexin as a loading control on immunoprecipitates or lysates as indicated. The lane labelled ‘none’ in (B) indicates PeTe4 antibody only, with no cellular lysate in immunoprecipitation. All data shown are representative of three independent experiments. KO: knockout; UGT1: UDP-glucose:glycoprotein glucosyltransferase 1; WT: wild-type; WB: western blot; IP: immunoprecipitation. https://doi.org/10.7554/eLife.23049.006 Table 2 Selected proteins identified in TAPBPR co-immunoprecipitates TAPBPR was immunuoprecipitated using PeTe4 from IFN-γ-treated HeLaM-TAPBPRKO(HeLaMKO) cells reconstituted with either TAPBPRWT or TAPBPRC94A. Immunoprecipitates were analysed by in gel tryptic digest followed by liquid chromatography-tandem mass spectrometry and data were processed using Scaffold. Identified proteins are shown with their exclusive unique peptide count, total percentage coverage, and exclusive unique spectrum count as determined by Scaffold. Pep: exclusive unique peptide count; Cov: percentage coverage; Count: exclusive unique spectrum count. https://doi.org/10.7554/eLife.23049.009 ProteinGene nameTAPBPRWTTAPBPRC94APep (Cov)CountPep (Cov)CountTapasin-related proteinTAPBPL32 (43)5429 (47)50HLA class 1, A-68HLA-A50 (64)8841 (59)70β-2-microglobulinβ2M4 (46)71 (8.4)1UDP-glucose:glycoprotein glucosyltransferase 1UGGT119 (11)25–– TAPBPR does not form a disulphide-linked dimer with UGT1 Having revealed that the association between TAPBPR and UGT1 occurred in a C94-dependent manner, we tested whether a disulphide bond formed between the two proteins. Under non-reducing conditions, there was no evidence of a disulphide-linked heterodimer between TAPBPR and UGT1 (Figure 3B and C). Both UGT1 and TAPBPR isolated from TAPBPR immunoprecipitates from HeLaMKOTAPBPRWT cells resolved at the predicted size of the monomeric proteins (~175 kDa for UGT1 and <52 kDa for TAPBPR) (Figure 3B). Furthermore, no difference in electrophoretic mobility was observed if the cells expressed TAPBPRWT or TAPBPRC94A (Figure 3B). In addition to suggesting that TAPBPR does not form a disulphide-linked dimer with UGT1, the results also indicate that TAPBPR does not form a covalent bond with any other as-yet unidentified binding partner of measurable size in these cells. There was also no evidence of a disulphide-linked dimer between endogenously expressed TAPBPR and UGT1 in IFN-γ-induced KBM-7 cells (Figure 3C). Based on our FFAS model of TAPBPR (Hermann et al., 2013), additional residues (I83, E87, L90, H91, and D93) predicted to form an α-helix on the surface of TAPBPR near C94 were also anticipated to contribute to the UGT1 interface. Mutation of these residues (UBS1: I83K, E87K, or UBS2: E87K, L90K, H91S, and D93R) decreased the association of UGT1 with TAPBPR (Figure 3—figure supplement 2). This suggests that the interaction of TAPBPR with UGT1 may involve an extended interface. TAPBPR acts as a between UGT1 and MHC class I Our results thus far suggest that distinct regions of TAPBPR are responsible for binding its interaction with residues in and IgC domains being for binding MHC class I molecules (Hermann et al., 2013) and C94 in the N-terminal domain being important for with UGT1. we to determine whether a complex between TAPBPR, UGT1, and MHC class I formed in cells by our to the of MHC class I to the First, we asked whether an association between TAPBPR and MHC class I was required for UGT1 to efficiently associate with TAPBPR. In TAPBPR immunoprecipitates from HeLaMKO cells transduced with which is to to MHC class I molecules (Hermann et al., 2013), the amount of UGT1 to TAPBPR was significantly reduced compared to cells expressing TAPBPRWT (Figure 3A). Interestingly, IFN-γ treatment, which MHC class I TAPBPRTN5 was to interact with MHC class I molecules, and the amount of UGT1 to TAPBPRTN5 increased (Figure 3A). these results suggest that the association between TAPBPR and MHC class I is important for the association of UGT1 with TAPBPR, the that a complex forms between TAPBPR, UGT1, and MHC class We whether TAPBPR was as a or between UGT1 and MHC class which is to be peptide free or loaded with findings (Hermann et al., 2015; Morozov et al., 2016). To this, we determined whether TAPBPR was required for UGT1 to associate with MHC class In HeLaMKO cells, we observed a association between MHC class I and UGT1 (Figure 3A). However, in HeLaMKOTAPBPRWT cells, this association was significantly (Figure 3A). was observed in cells expressing the mutant forms TAPBPRC94A or in the of IFN-γ (Figure 3A). This suggests that the association between MHC class I molecules and UGT1 is by TAPBPR. We also tested the for TAPBPR in the association between MHC class I molecules and UGT1 when endogenous TAPBPR was in HeLaM cells by UGT1 was found to associate with both and W6/32-reactive MHC class I molecules in IFN-γ-treated HeLaM cells (Figure However, in IFN-γ-treated HeLaMKO cells, a significant was observed between and W6/32-reactive MHC class I molecules (Figure These results are consistent with TAPBPR as a between UGT1 and MHC class I and suggest that UGT1 recognition of MHC class I molecules can in a (i.e. for the of HC10-reactive MHC class or in a with W6/32-reactive MHC TAPBPRC94A still functions as a peptide in Recently, we and have shown that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules (Hermann et al., 2015; Morozov et al., 2016). Having identified an interaction between UGT1 and TAPBPR, we asked important this association was for peptide optimisation on MHC class I molecules in a cellular First, we tested whether TAPBPRC94A was a MHC class I peptide in Using fluorescence we observed a ability of TAPBPRC94A and TAPBPRWT to peptide dissociation and association on in (Figure and B). Furthermore, when we tested the ability of TAPBPRC94A to between high-affinity labelled peptide and peptides for binding to Figure supplement 1 for a of the affinity of these two peptides for in the of TAPBPRC94A, a as observed with TAPBPRWT (Figure (Hermann et al., 2015). These results suggest that the function of TAPBPR is not altered as a direct consequence of mutation of the C94 residue. Figure with 1 supplement see all Download asset Open asset TAPBPRC94A still functions as a peptide in (A) of the peptide from in the absence or of TAPBPRWT or TAPBPRC94A. molecules with as previously in Hermann et al., 2015) were to at for The protein was then with denotes at The complexes were then and with with either (no protein) or with either TAPBPRWT or TAPBPRC94A. The data shown are representative of three experiments. (B) of the peptide with in the absence or of TAPBPRWT or TAPBPRC94A. molecules with were with human β2m and to at for and then was in the absence or of TAPBPRWT or TAPBPRC94A immediately fluorescence were representative of three is (C) between peptides for binding with peptide-receptive molecules in the or absence of TAPBPRWT or TAPBPRC94A as by fluorescence molecules with were with human β2m and to at for and then with high-affinity peptide and of the peptide in the or absence of TAPBPRWT or TAPBPRC94A Figure supplement 1 for a of the affinity of or for were after at representative of three is WT: UGT1 to TAPBPR helps peptide selection on MHC class I molecules we compared the surface expression of MHC class I molecules on HeLaMKO cells reconstituted with TAPBPRC94A or TAPBPRWT, we found that both molecules total MHC class as detected by to a similar (Figure However, we observed lower surface expression of in cells expressing TAPBPRC94A compared to TAPBPRWT (Figure and B) of steady-state expression of TAPBPR in cell transduced with TAPBPRWT and TAPBPRC94A (Figure 3A). in to determine the that the UGT1 to TAPBPR has on peptide selection in a cellular environment, we compared the MHC class I peptide repertoire on HeLaMKOTAPBPRWT with HeLaMKOTAPBPRC94A cells,