Direct peptide-regulatable interactions betweenMHC class I molecules and tapasinSyed Monem Rizvi and Malini Raghavan†

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620

Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved October 10, 2006 (received for review June 21, 2006)

Tapasin (Tpn) has been implicated in multiple steps of the MHCclass I assembly pathway, but the mechanisms of function remainincompletely understood. Using purified proteins, we could dem-onstrate direct binding of Tpn to peptide-deficient forms of MHCclass I molecules at physiological temperatures. Tpn also bound toM10.5, a pheromone receptor-associated MHC molecule that hasan open and empty groove and that shares significant sequenceidentity with class I sequences. Two types of MHC class I–Tpncomplexes were detectable in vitro depending on the input pro-teins; those depleted in �2m, and those containing �2m. Both werecompetent for subsequent assembly with peptides, but the lattercomplexes assembled more rapidly. Thus, the assembly rate ofTpn-associated class I was determined by the conditions underwhich Tpn–MHC class I complexes were induced. Peptide loading ofclass I inhibited Tpn–class I-binding interactions, and peptide-depletion enhanced binding. In combination with �2m, certainpeptides induced efficient dissociation of preformed Tpn–class Icomplexes. Together, these studies demonstrate direct Tpn–MHCclass I interactions and preferential binding of empty MHC class Iby Tpn, and that the Tpn–class I interaction is regulated by both�2m and peptide. In cells, Tpn is likely to be a direct mediator ofpeptide-regulated binding and release of MHC class I from the TAPcomplex.

antigen presentation � HLA � TAP transporter

Peptide products of foreign and self-proteins bind to MHCclass I heavy chains (HC) and light chains [�2-microglubulin

(�2m)] within the endoplasmic reticulum (ER) of cells and arethen transported to the cell surface, where they are available forimmune surveillance by cytotoxic T lymphocytes (1). The trans-porter associated with antigen processing (TAP) is a peptidetransporter that translocates cytosolic peptides into the ERlumen, for assembly with MHC class I HC and �2m. Other ERresident proteins that assist in MHC class I (class I) assemblyinclude the chaperones calnexin and calreticulin, the thiol-disulfide isomerase ERp57, and the MHC-encoded transmem-brane protein tapasin (Tpn). Individually or in combination,these components ensure quality control of class I-peptideassembly (1).

For many human and mouse class I allotypes, Tpn increasescell surface class I expression. How Tpn mediates this increasehas been a matter of considerable debate, and many functionshave been proposed for Tpn. Tpn stabilizes TAP, and increasessteady-state levels of TAP, thereby allowing more peptides to betranslocated into the ER (2, 3). Tpn also allows a physical linkbetween class I and the TAP (4), and by doing so, may increasethe effective concentration of peptides that are available for classI binding. Tpn may also be important for preventing ER exit ofpeptide-deficient class I (5–7), and may thereby indirectly pro-mote the accumulation of more stable class I at the cell surface.Soluble Tpn (sTpn) that does not stably bind TAP, enhance TAPexpression levels, or mediate the TAP–class I interaction, is ableto partially compensate for a defect in cellular Tpn (2), indicatingthat at least some of Tpn’s functions are independent of itseffects upon TAP. There are also indications from cell-basedexperiments, that Tpn edits/optimizes the class I peptide reper-

toire (8), or directly facilitates peptide loading of class I mole-cules (9–11).

The absence of a direct binding assay between Tpn and classI has hampered a better understanding of the mechanisms ofTpn’s function. Although cell-based experiments have demon-strated class I-Tpn binding in the absence of other componentsof the class I pathway (5, 12), there are no reports thus far ofdirect Tpn–class I-binding interactions outside the context of acell. Here, we reconstituted the Tpn–class I interaction by usingpurified proteins and investigated the effects of Tpn upon theclass I–peptide interaction, and the effects of peptide upon theclass I–Tpn interaction.

ResultsTpn Can Bind to Peptide-Deficient Class I Molecules at PhysiologicalTemperature. A FLAG-tagged sTpn was expressed in and purifiedfrom CHO cells (Fig. 6, which is published as supportinginformation on the PNAS web site), and soluble peptide-deficient class I heterodimers were purified from insect cells (13,14). Fluorescent peptides and native-PAGE-based assemblyassays (13) were used to ask whether sTpn binding influencedpeptide loading of soluble peptide-deficient HLA-A2 (A2) orHLA-B*3501 (B35). Under the conditions that were examined,no significant differences in peptide binding were observed inthe presence or absence of sTpn (Fig. 7 A–D, which is publishedas supporting information on the PNAS web site).

Absence of a significant effect of sTpn on peptide loadingcould arise because of absent or inefficient sTpn–class I-bindinginteractions under these in vitro conditions, or because ofenhanced kinetics of class I peptide loading compared with sTpnbinding. Therefore, it was important to investigate bindinginteractions between sTpn and A2. Equimolar amounts of A2heterodimers and sTpn monomers were incubated for 2 h at 4°Cor 37°C. Samples were then immunoprecipitated with the anti-FLAG antibody, proteins separated by SDS/PAGE, and visual-ized by silver-staining to observe sTpn and any A2 that bound tosTpn. A2 was visualized in the anti-FLAG immunoprecipitations(IPs) after protein incubations at 37°C (Fig. 1A, lane 6), but not4°C (Fig. 1 A, lane 3).

To examine the specificity of the A2–sTpn interaction, wetested the ability of sTpn to bind to other proteins with structuresrelated to class I. HFE (the protein mutated in hereditaryhemochromatosis) HC folds into a structure similar to class IHC, and associates with �2m, but does not bind peptides, and hasa closed peptide-binding groove (15). By IP assays similar to

Author contributions: S.M.R. and M.R. designed research; S.M.R. performed research;S.M.R. and M.R. analyzed data; and S.M.R. and M.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: HC, heavy chain; ER, endoplasmic reticulum; TAP, transporter associatedwith antigen processing; �2m, �2-microglubulin; class I, MHC class I; Tpn, tapasin; sTpn,soluble Tpn; A2, HLA-A2; B35, HLA-B*3501; IP, immunoprecipitation; M10, M10.5.

†To whom correspondence should be addressed at: Department of Microbiology andImmunology, 5641 Medical Science Building II, University of Michigan Medical School, AnnArbor, MI 48109-0620. E-mail: malinir@umich.edu.

© 2006 by The National Academy of Sciences of the USA

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those described for A2, sTpn did not interact with HFE (Fig. 1B,lane 6). M10.5 (M10), is a pheromone receptor-associatedprotein with a class I-like fold. M10 binds �2m, but the coun-terpart of its peptide-binding groove is open and unoccupied(16). Somewhat surprisingly, like A2, M10 bound sTpn at 37°C(Fig. 1C Left, lane 2), but not at lower temperatures (Fig. 1CRight, lane 7). Insect cells have previously been used to assessTAP/Tpn/class I complex formation and to reconstitute class Ipeptide loading (5, 17). Additionally, in insect cells, Tpn is theobligate mediator of the TAP–class I interaction (12). To verifythat sTpn–A2 and sTpn–M10 interactions observed in vitro werenot nonspecific binding interactions induced by protein incuba-tions at 37°C, we assessed A2 and M10 binding to Tpn in insectcells. After metabolic labeling of baculovirus-infected cells,Tpn/A2 and Tpn/M10 complexes could both be visualized by IPwith the anti-His antibody (both A2 and M10 are His tagged)(Fig. 1D, lanes 5 and 4, respectively). Together, these observa-tions indicated that Tpn–A2 and Tpn–M10 interactions were notrestricted to the in vitro incubation condition at 37°C, but werealso observable when the proteins were coexpressed in insectcells.

The observation of M10-Tpn binding suggested preferentialTpn recognition of open and empty class I molecules. Thispossibility was examined further. A2 (10 �M) was incubated with500 �M A2-specific peptide LLDVPTAAV (18) at 47°C for 2 h,to obtain a peptide-loaded version. Unbound peptide was re-moved from A2 by passage through a Biospin-30 column. Equalamounts of untreated or peptide-loaded A2 (pA2) were com-pared for tapasin binding in IP assays. In sTpn/pA2 incubations,the signal intensity in the region corresponding to A2 HC wassignificantly reduced compared with sTpn/untreated A2 incu-bations (Fig. 1E, lane 6 compared with lane 4, and quantifica-tions, Right). We also investigated Tpn binding to B35, a class Iallotype that has been described to be Tpn-dependent for its cellsurface expression (19). Soluble B35 was loaded with a specifichigh affinity peptide LPSSADVEF (20) at 47°C for 2 h. Equalamounts of untreated or peptide-loaded B35 heterodimers(pB35) were incubated with sTpn at 37°C for 2 h, and IP assaysundertaken. Peptide-deficient B35 bound to sTpn, but peptide-loaded B35 did not (Fig. 1F, lane 3 compared with lane 5).Together, these analyses indicated that Tpn preferentially rec-

ognized peptide-deficient class I molecules, as well as the emptyand open class I like protein, M10.

When Added in Excess, �2m Can Be Visualized as a Component ofTpn–Class I Complexes. After IP of sTpn-A2 mixtures with theanti-FLAG antibody, it was difficult to detect �2m by directstaining of gels (Fig. 2A Upper, lane 2). However, by immuno-blotting with �2m-specific antibodies, a faint signal was observedfor �2m (Fig. 2 A Lower, lane 2). We compared �2m levels in thesTpn–A2 complexes against different known amounts of puri-fied heterodimeric A2. Relative to the �2m/HC ratios in theheterodimeric A2 samples (expected to be 1:1); a significantlysubstoichiometric �2m/HC ratio was observed in sTpn-associated A2 HC (Fig. 2 A Lower, �2m blot). To examinewhether the presence of excess �2m during sTpn–class I complexformation could enhance the amount of sTpn-A2-associated

Fig. 1. Tpn can form complexes with peptide-deficient classical and nonclassical class I molecules at physiological temperatures. (A–C) sTpn–class I and sTpn–M10complexes are detectable by coimmunoprecipitation analyses. SDS/PAGE and silver staining analyses of direct protein loads of 0.2–0.5 �g A2 (A, lane 1; B, lane2), sTpn (B, lane 1, 0.5 �g), M10 (C, lanes 1 and 5, 0.5 �g) or HFE (B, lane 3, 1 �g) and anti-FLAG IP of indicated proteins or buffer that had been incubated at4°C (A, lanes 2–4), 30°C (C, lanes 6 and 7) or 37°C (A, lanes 5–8; B lanes 4–9; and C, lanes 2–4). (D) A2/Tpn and M10/Tpn complexes are detectable in insect cells.SDS/PAGE phosphorimaging analyses of indicated samples. Insect cells cultured at 26°C were infected with baculoviruses encoding full length Tpn and indicatedproteins, metabolically labeled, lysed, proteins immunoprecipitated with Tpn-specific antibody (PaSta-1, lane 1) or anti-His to detect His tagged proteins (M10or A2) (lanes 2–5). (E and F) Peptide loading of class I inhibits sTpn binding. (Left) Direct protein loads of A2 (E, lane 1, 0.25 �g), LLDVPTAAV-loaded A2 (E, lane2, 0.25 �g), or B35 (F, lane 1, 0.5 �g) and anti-FLAG IP (E, lanes 3–8; F, lanes 2–7) of indicated proteins. pA2 indicates LLDVPTAAV-loaded A2, and pB35 indicatesLPSSADVEF-loaded B35. (Right) Bar graph shows class I/sTpn intensity ratio comparisons for the indicated lanes. Binding of sTpn to peptide-deficient class I wasset at 100% to compare relative signals. Data are representative of numerous (A), two (B, C Right, E, and F) and four (C Left and D) independent analyses.

Fig. 2. Tpn can form complexes with �2m at physiological temperature.sTpn–A2 complexes were formed by incubating proteins (5 �M each) at 37°Ceither in the absence (A) or presence (B) of �2m (50 �M) and immunoprecipi-tated with anti-FLAG. (C) sTpn (5 �M) and �2m (50 �M), or �2m alone wereincubated for 2 h at 37°C, then immunoprecipitated with anti-FLAG. (A–CUpper) Silver-stained SDS/PAGE gels showing anti-FLAG IP of indicated pro-teins (A, lane 2; B, lanes 1 and 2; C lanes 1 and 2) or buffer (A, lane 1; C, lane3) and direct protein loads of indicated concentration of A2 heterodimers (Aand B, lanes 3–8). (A–C Lower) Anti-�2m blot of the above gels. Data arerepresentative of two independent analyses.

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�2m, we incubated A2 and sTpn in the presence of a ten-foldexcess of purified �2m at 37°C for 2 h, followed by IP with theanti-FLAG antibody. Indeed, when excess �2m was presentduring complex formation, the amount of sTpn-associated �2mwas enhanced and equal or higher �2m/HC ratios were observedthan in the heterodimeric A2 samples (Fig. 2B). This increase in�2m association coincided with a decrease in the overall recoveryof sTpn-associated HC.

Direct incubation of sTpn with �2m alone at 37°C for 2 h (Fig.2C Upper and Lower, lanes 1) followed by anti-FLAG IP revealeda signal for �2m that was slightly above the nonspecific control(Fig. 2C, lane 1 compared with lane 2; lane 2 was an IP of �2malone with anti-FLAG), suggesting the possibility of a weak�2m–sTpn interaction. Together, these observations indicatethat the primary site of Tpn-class I contact resides in the HC, butthat �2m can be detected in complex with Tpn when it is presentin stoichiometric excess.

Tpn-Associated Class I Is Subsequently Assembly Competent. A2–sTpn complexes were preformed by incubating both proteins (5�M each) in the presence or absence of excess �2m (50 �M) at37°C for 2 h (in 50 mM Tris/150 mM NaCl, pH 7.5). Thecomplexes were incubated with anti-FLAG beads overnight at4°C. Proteins were eluted by using 100 �M FLAG peptide, andthe eluates were found to contain both Tpn and A2 (Fig. 3A,lanes 1 and 2).

The amounts of HC in the two anti-FLAG eluates wereestimated by titration against known amounts of A2 het-erodimers (Fig. 3A Top, lanes 3–6). sTpn-A2 eluates in whichcomplexes were formed in the absence of excess �2m contained�80 ng A2 (Fig. 3A Top, compare lane 1 with lane 5 and 6),which was reduced to �40 ng when complexes were assembledin the presence of excess �2m (Fig. 3A Top, compare lane 2 withlane 5 and 6). To assay for relative assembly competence of theeluted A2, proteins recovered from the anti-FLAG column orthe different amounts of purified A2 heterodimers were incu-

bated with excess �2m and fluorescently tagged LLDCFPTAAVat 37°C for indicated time points (Fig. 3A Middle). Peptidebinding was quantified by native-PAGE and fluorimaging anal-yses (13) (Fig. 3A Bottom). For eluates of the A2–sTpn com-plexes formed in the presence of excess �2m, the peptide-bindingsignals were only slightly lower than the corresponding signalsobtained with the 40 ng A2 standard (Fig. 3A Middle, comparelanes 2 and 5). We concluded that majority of this eluate wasassembly competent, with slightly reduced binding kinetics thanthat of free class I. For eluates of the A2–sTpn complexes thatwere formed in the absence of excess �2m (complexes that weredepleted in �2m), the peptide-binding signals obtained weresignificantly lower than the corresponding signals obtained withthe 80 ng heterodimeric A2 standard (Fig. 3A Middle, comparelanes 1 and 6). Thus, �2m binding to HC may be rate limiting forassembly of peptide-class I in these �2m-depleted sTpn–A2complexes. �2m is a critical regulator of class I-peptide assemblyrates. Peptide loading of class I heterodimers is enhanced by thepresence of excess �2m even in the absence of Tpn (Fig. 8, whichis published as supporting information on the PNAS web site).

A parallel set of results were obtained with B35. Underconditions where sTpn–B35 complexes were formed in thepresence of excess �2m, the extent of B35-peptide assemblyobserved was similar to that observed with free B35 (Fig. 3B,compare lane 2 with lane 3 and 4). Under conditions wheresTpn–B35 complexes were formed in the absence of excess �2m,B35-peptide assembly observed was significantly inhibited rela-tive to that observed with free heterodimeric B35 (Fig. 3B,compare lanes 1 and 5).

Tpn Binds Preferentially to Empty Class I Molecules. In the studiesdescribed so far, sTpn–class I complexes were observable at37°C, but not at lower temperatures (Figs. 1 A and 4 A and C).It was possible that the A2 and B35 purified from insect cellswere not completely empty, and that the 37°C incubationspromoted dissociation of peptides endogenous to insect cells. Toask whether interactions of sTpn with completely ‘‘empty’’ classI could occur at lower temperatures, A2 and B35 (100–300 �g)were dialyzed against 6 M guanidine hydrochloride (GnHCL) byusing Centricon (10-kDa membranes) to remove any associatedpeptide, and proteins refolded by gel filtration chromatography(Superose 6 column) in the presence of 100–300 �g of �2m.Fractions corresponding to heterodimers were collected andused directly in peptide-binding assays at room temperature tocompare their assembly competence in the presence or absenceof sTpn. Small enhancements, if any, were observed for peptidebinding to empty A2 and B35 in the presence of sTpn comparedwith the absence of sTpn (Fig. 9, which is published as supportinginformation on the PNAS web site).

Fig. 3. Class I isolated in complex with Tpn is subsequently assembly com-petent. (A and B Top) SDS/PAGE and silver-staining analyses of sTpn-A2 (A) orsTpn-B35 (B) complexes eluted from an anti-FLAG beads. sTpn–class I com-plexes formed in the absence or presence of excess �2m are shown in lanes 1and 2, respectively. Direct protein loads of indicated amounts of class Istandards are shown in lanes 3–6. (A and B Middle) Samples corresponding toproteins shown in Top were incubated with 1 �M LLDCFPTAAV (for A2, A) orLPSCFADVEF (for B35, B) and 1 �M �2m at 37°C for 1, 2, or 4 h. Class I–peptidecomplexes were separated from free peptide by native-PAGE, and protein–peptide complexes were visualized by fluorimaging analyses. (A and B Bot-tom) Peptide-binding signals from Middle were quantified by ImageQuantand are represented as bar graphs. Data are representative of three indepen-dent analyses.

Fig. 4. Tpn preferentially binds empty class I molecules. (A–C) Silver-stainedSDS/PAGE showing direct protein loads of A2 (A, lane 1, 0.3 �g), B35 (C, lane2, 0.2 �g), or sTpn (A, lane 2, 0.05 �g; B, lane 1, 0.2 �g; C, lane 1, 0.1 �g) oranti-FLAG IP (A, lanes 3 and 4; B, lanes 2 and 3; C, lanes 3–6) of indicatedproteins (3 �M each) after incubations at 30°C for 6 h. Empty class I moleculesare represented as eA2 and eB35, and untreated class I molecules are repre-sented as A2 and B35. Data are representative of two (A and B) or three (C)independent analyses.

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Empty class I molecules were concentrated in the presence ofexcess �2m (300 �g) and analyzed for binding to sTpn at lowertemperature (Fig. 4). Empty A2 and B35 but not the precursoruntreated proteins were observed to form complexes with sTpnat 30°C (Fig. 4B, lane 3, and Fig. 4C, lane 6). The ability ofdenatured/refolded class I (but not the precursor class I) to bindsTpn at 30°C also supports the possibility that sTpn preferentiallybinds to a conformation found in empty class I molecules. EmptyA2 and B35 that were complexed to Tpn at 30°C were purifiedby using anti-FLAG beads, and proteins eluted with the FLAGpeptide as described in Fig. 3. Eluates containing A2 and B35were both found to be assembly competent (Fig. 10, which ispublished as supporting information on the PNAS web site).

Regulation of the Tpn–Class I Interaction by Peptide and �2m. In thebinding analyses of Figs. 3 and 10, peptide-loaded class Imolecules migrated at the same position on a native gel regard-less of whether the protein used in the assembly assays was freeclass I or Tpn-class I eluates from the anti-FLAG column. Theseresults suggested that sTpn had dissociated from the peptide-loaded class I either as a result of peptide loading, or as a resultof protein dilutions after protein elutions from anti-FLAGbeads. To directly investigate the effect of peptide on disassem-bly of sTpn-class I, sTpn and A2 were incubated for 2 h in theabsence of excess �2m after which, the mixture was divided intothree parts. One part was stored on ice and the remaining twoparts were each incubated with buffer or excess �2m and A2specific peptides (LLDVPTAAV) at 37°C for an additional 2 hand IP analyses were undertaken (Fig. 5A). Compared with theadditional incubation with buffer (Fig. 5A, lane 5), and com-pared with samples that were incubated for just 2 h (Fig. 5A, lane4), additional incubation with peptide and �2m resulted in areduction in the amount of coimmunoprecipitating A2 (Fig. 5A,lanes 6). The FLPSDDFPSV peptide that bound to A2 with2.5-fold higher affinity than LLDVPTAAV (Fig. 11 A, which ispublished as supporting information on the PNAS web site) alsoinduced dissociation of A2 from Tpn to similar levels as LLD-VPTAAV (Fig. 11B). However, parallel analyses revealed moreefficient dissociation of Tpn-B35 induced by the high affinitypeptide LPSSADVEF compared with the lower affinity YPL-HEQHGM (Fig. 5 B and C, compare lanes 6). LPSSADVEF wasestimated to bind B35 with �6-fold higher affinity than YPL-HEQHGM (Fig. 11C).

To further investigate and compare conditions for dissociationof class I–Tpn, complexes were isolated by using anti-FLAGbeads, and then subject to different elution conditions. The firstset of elutions with the FLAG peptide verified that class Ibinding to the anti-FLAG beads was indeed dependent on thepresence of tapasin (Fig. 5 D and E). In the next set of elutions(Fig. 5F), anti-FLAG beads containing sTpn–class I complexesformed in the presence of �2m were incubated at room temper-ature for 4 h with 0.1 ml buffer containing specific peptidesalone, �2m and specific peptides, �2m and nonspecific peptides,or buffer alone, as indicated. Supernatants were collected andanalyzed for the presence of class I by immunoblotting analyseswith HC-specific antibodies. The analyses showed that specificpeptides alone could elute B35 to some extent, and low levels ofA2 (Fig. 5F, lanes 3 and 4 compared with lanes 7 and 8). �2m plusnonspecific peptides eluted more A2 and B35 compared with thespecific peptides alone or buffer (Fig. 5F, lanes 5 and 6). The �2mplus specific peptide combination was most efficient at elution ofboth A2 and B35 (Fig. 5F, lanes 1 and 2); however the differencesbetween elutions with �2m/nonspecific peptide and �2m/specificpeptide were more pronounced for A2 than for B35, andindicated more efficient elution of B35 with �2m alone.

Anti-FLAG beads containing sTpn–class I complexes formedin the absence of excess �2m were incubated at room tempera-ture for 4 h with 0.1 ml �2m and specific peptides, �2m and

nonspecific peptides, �2m alone, or buffer alone, and the eluateanalyzed for class I by immunoblotting (Fig. 5G). Although someelution was observed with �2m alone or �2m in combination withnonspecific peptides (Fig. 5G, lanes 1–2 and 5–6), the�2m�specific peptide combinations were more effective atdissociating class I from tapasin (Fig. 5G, lanes 3 and 4).

If Tpn associates preferentially with the peptide-free form of

Fig. 5. Dissociation of sTpn–A2 and sTpn–B35 complexes by peptides and�2m. (A–C Left) Silver-stained SDS/PAGE gels of indicated proteins (lanes 1 and2) or anti-FLAG IP (lanes 3–7). (A and B) Indicated class I and sTpn (5 �M each)or class I alone were incubated for 2 h at 37°C (lanes 3 and 4, respectively),followed by additional 2 h incubation with buffer (lanes 5), or with 50 �M �2mand 500 �M LLDVPTAAV (LLD) or LPSSADVEF (LPS) (lanes 6). (C) B35 and sTpnwere incubated for 2 h at 37°C (lane 5), followed by additional 2 h incubationwith buffer (lane 3) or with 50 �M �2m and 50 �M YPLHEQHGM (YPL) (lane 6).Samples were then processed for IP as described in Experimental Procedures.(A–C Right) Bar graphs show class I/sTpn intensity ratio comparisons for theindicated lanes, with the highest ratio in each gel set at 100%. Data in Rightpanels are averaged over two independent analyses. (D and E) Class I bindingto anti-FLAG beads depends on Tpn. Class I (5 �M) was incubated at 37°C in thepresence or absence of sTpn (5 �M) and �2m (50 �M) as indicated at 37°C for4 h. Proteins isolated with anti-FLAG beads were eluted with 100 �M FLAGpeptide and analyzed by immunoblotting with anti-His (D; A2) or HC10 (E;B35). (F and G) Class I–sTpn complexes were formed as described in D and E inthe presence (F) or absence (G) of excess �2m, isolated with anti-FLAG beads,and class I was eluted with combinations of specific peptide (S), nonspecificpeptide (NS), and �2m as indicated, or buffer alone. Immunoblotting of A2 andB35 elutions is indicated in the Upper and Lower panels, respectively. Volumesof eluates used in the immunoblotting analyses are indicated (10, 20, or 40 �l).These data are representative of three and four independent analyses, re-spectively, for F and G.

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class I, the expectation is that different peptides would inhibitTpn–class I complex formation to an extent that is determinedby the intrinsic affinity of the particular peptide–class I inter-action. Indeed, this was the observed result. When presentduring sTpn–class I complex formation, LLDVPTAAV was abetter inhibitor of the sTpn–class I interaction than the loweraffinity LLDVPTAAVQ (Fig. 7 and Fig. 12 A, which is publishedas supporting information on the PNAS web site; see Fig. 7 forcomparisons of binding of the two peptides). Likewise for B35peptides, the higher affinity LPSCFADVEF peptide was a betterinhibitor of the sTpn–class I interaction than the lower affinityYPLHEQHGM peptide (Fig. 12 B and C).

DiscussionOur data suggest two types of Tpn–class I complexes: thosecontaining predominantly HC and those containing both HCand �2m. �2m seems to destabilize Tpn–HC interactions, asindicated by the observations of inhibited HC-Tpn binding in thepresence of excess �2m (Fig. 3), as well as �2m-induced elutionof class I HC from Tpn (Fig. 5 F and G). Excess �2m may simplyreduce the exposure of Tpn-binding residues by stabilization ofheterodimeric HC–�2m interactions (in the same manner thatpeptides do), rendering HC–Tpn interaction weaker, but alsomore dynamic and assembly competent (compared with theHC–Tpn interaction formed in the absence of excess �2m; Fig.3). The observations that B35 was more easily dissociated by�2m/peptide combinations and �2m alone compared with A2(Fig. 5) is reminiscent of previous findings that HLA-B*3501/TAP interactions were not detectable in cell lysates underconditions where A2/TAP interactions were readily detectable(21). The stabilities of peptide-deficient forms of class I allotypesmay be variable, which in turn could be reflected in the extentof steady-state Tpn/TAP binding. Additionally, for some class Imolecules such as A2, assembly of HC/�2m/peptide trimers maybe a cooperative event, whereas the heterodimeric intermediatesmay be more stable for other class I allotypes.

That �2m destabilizes HC–Tpn interactions may seem sur-prising in light of reports that Tpn–HC interactions were signif-icantly reduced in �2m-deficient cells compared with �2m-sufficient cells (22). However, in cells, the absence of �2mmarkedly decreases levels of HLA class I HC, making it difficultto directly quantify relative propensities of free HC and �2m-associated HC for Tpn binding (as was the case in insect cells; C.Perria and M.R., unpublished observations). Alternatively/additionally, in cells, in the absence of �2m, free HC may besequestered from Tpn binding by other chaperones such ascalnexin.

Cell surface expression of many class I allotypes is markedlyincreased by the presence of Tpn, and Tpn increases MHC class Iexport from the ER (11), class I thermostability (8), and cell surfacestability (11). How might the data described here explain theseassembly-promoting effects of Tpn? Our studies indicate prefer-ential recognition of empty class I by Tpn. Furthermore, samplingof the peptide environment by the MHC class I seems to bepermissive under conditions where Tpn binding was observed (Fig.5), either because MHC class I is in dynamic equilibrium betweenTpn-free and Tpn-associated conformations, or because a Tpn-bound conformation of class I is also directly permissive for peptideloading. Higher affinity peptides were better inducers of Tpn-classI dissociation (Fig. 5 B compared with C) and stronger inhibitors ofthe Tpn class I binding (Fig. 12). It seems likely based on these datathat the presence of Tpn could allow for a more stringent classI-peptide affinity checkpoint, consistent with models of Tpn-mediated editing/optimization (8). Only peptides that are able toefficiently dissociate class I from tapasin will be found in associationwith class I on the surface of Tpn-sufficient cells. Zarling et al. (11)have suggested a role for Tpn as a peptide loading facilitator ratherthan editor. This type of activity might have been reflected by an

increase in peptide loading by class I in Tpn-associated complexes,but our studies revealed very small effects if any (Fig. 10). Addi-tional conditions may need to be explored, varying protein con-centrations and including Tpn-associated factors. Compared withthe in vitro experiments shown here, within the ER, assembly anddegradation of peptide-deficient class I are likely to be stronglycompeting processes. It has been shown recently that Tpn efficientlyrecruits ERp57 into the class I assembly complex (23), and it is wellknown that ERp57 is found in physical association with the lectinchaperones calreticulin/calnexin. Thus, the presence of Tpn mayserve to efficiently recruit a chaperone network around the assem-bling empty class I, which is supported by previous observations thatinteractions within the assembly complex are all cooperative tosome extent (reviewed in ref. 1). Assembly within this protectivechaperone network environment may be more highly favored thanoutside this environment, where proteases and ER degradationpathways could compete more effectively with assembly of class Iwith peptide.

By preferential recognition of the empty conformation of classI, Tpn could also actively enhance dissociation of low affinitypeptides and promote peptide exchange, as previously observedfor HLA-DM in the MHC class II antigen presentation pathway(reviewed in ref. 24). A detailed analysis of this possibility willbe important by using real time measurements of class I-peptidedissociation and exchange in the presence and absence of Tpn.

Although our data indicate preferential recognition of emptyclass I by Tpn (Fig. 4), Tpn may not be essential for ER retentionof empty MHC class I in all cases (9, 10, 25). For example, humanMHC class I molecules seem to be retained efficiently in the EReven in the absence of Tpn (25). However, Tpn seems to be quiteimportant for ER retention of murine MHC class I (5–7). In theabsence of Tpn, increased ER escape and cell surface expressionof empty and suboptimally loaded class I could also, at least inpart explain the reduction in MHC class I surface expression insome Tpn-deficient cells.

The interaction of Tpn with M10 was initially surprising inlight of the report that TAP is not expressed in cells that expressM10 (26), but subsequent comparison of the sequences of M10and class I molecules revealed 40%, 40%, and 74% sequenceidentity in the �1, �2, and �3 domains, respectively. The extentof sequence identity between HFE and class I was considerablyless significant (31%, 25%, and 41% sequence identity in the �1,�2, and �3 domains, respectively). Thus, whereas Tpn may notbe a bona fide assembly factor for M10 in cells, the significantsequence identity between class I and M10 HC, taken togetherwith its ‘‘open and empty groove’’ structure may promoteTpn-M10 cross-reactivity in vitro (Fig. 1C), or under conditionsof coexpression (Fig. 1D). Residues that have been implicated inthe Tpn–class I interaction include residues 132 (27) and 134 (28,29) in the �2 domain of human class I. These residues areconserved between A2 and M10, but not between HFE and A2,supporting the possibility that this region forms a contact site forTpn binding. Also conserved in M10 are D227 and E229 in the�3 domain, additional residues implicated in Tpn binding byhuman MHC class I (27). It will be important to investigatewhether mutations within both the �2 and �3 domains disruptor destabilize Tpn-MHC class I binding interactions by the assaydescribed here.

A significant finding of these studies is that conditions thatpromoted heterodimer assembly induced dissociation of MHCclass I-Tpn. Other investigators have reported difficulties indemonstrating peptide-induced dissociation of class I from theloading complex (30). Our data indicate that both �2m (Figs. 3and 5) and the type of peptide used (Fig. 5) are likely to beimportant determinants of the extent of dissociation of class Ifrom a Tpn-associated complex. Furthermore, as illustrated inFig. 3, the specific conditions under which peptide loading

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complexes were isolated could determine the assembly kineticsof Tpn-associated MHC class I molecules.

In summary, our studies demonstrate that Tpn binds topeptide-deficient class I, that physiological temperatures pro-mote binding, and that conditions favorable for class I assemblydissociate Tpn–class I complexes. In cells, Tpn interaction withempty class I may serve to recruit nascent class I into a chaperonenetwork comprising Tpn, ERp57, and the lectin chaperones,which together, may create an environment that favors peptideloading and assembly over degradation.

Experimental ProceduresProteins and Peptides. Soluble A2/�2m, B35/�2m, and full-lengthTpn expression in insect cells has been previously described (13,14, 31). Expression and purification of soluble tapasin and �2mand detection of class I/Tpn complexes in insect cells aredescribed in Supporting Materials and Methods, which is pub-lished as supporting information on the PNAS web site. Fluo-rescently labeled versions of class I-binding peptides were ob-tained by cysteine substitutions at positions 4 or 5 of the parentsequences, and labeling with iodoacetamidofluorescein, as pre-viously described (13, 14). Purified human HFE (15), mouseM10.5 (16), and a baculovirus encoding mouse M10.5/human�2m (16) were obtained from the laboratory of P. J. Bjorkman(California Institute of Technology, Pasadena, CA).

Detection of Tpn–Class I Complexes Using Purified Proteins. Solubleclass I, HFE, or M10 (2.5–5 �M each) was incubated with sTpn(2.5–5 �M) in 50 mM Tris, 150 mM NaCl, pH 7.5, at indicatedtemperatures for indicated time. After incubations, the sampleswere centrifuged, diluted in the same buffer containing 1% Triton-X-100 to 1 ml, and incubated with anti-FLAG antibody (3–4 �g perimmunoprecipitation) overnight at 4°C. The supernatants werefurther centrifuged and incubated for 2–4 h at 4°C with protein Gbeads. The beads were washed three times with buffer containing0.25% gelatin (10 mM Tris/10 mM phosphate buffer/130 mMNaCl/1% Triton X-100, pH 7.5) and once with buffer without

gelatin. The beads were boiled in SDS/PAGE buffer, samples wereresolved by SDS/PAGE, and immunoprecipitated proteins werevisualized by silver staining. For �2m detection, the anti-FLAGimmunoprecipitated samples or direct proteins loads were trans-ferred to PVDF membrane, immunoblotted with anti-�2m antisera(Roche, Nutley, NJ), HRP-conjugated secondary antibody, anddeveloped by ECL plus kit (Amersham Biosciences, Piscataway,NJ).

Isolation of Tpn–Class I Complexes with Anti-FLAG Beads. sTpn–classI complexes were formed by incubation of proteins (5 �M) in thepresence or absence of 50 �M �2m in a total volume of 40 �l at37°C for 2–4 h. Proteins were then diluted to 1 ml buffer (50 mMTris/150 mM NaCl/1% Triton X-100), incubated overnight at4°C with 100-�l FLAG-beads, loaded onto an empty column,and washed with 5–15 ml buffer to remove free class I. For someanalyses, beads were eluted with FLAG peptide (100 �M in 100�l). For other analyses, beads were then incubated with 100 �lof buffer (50 mM Tris/150 mM NaCl) containing 50 �M �2m, 50�M �2m plus 500 �M specific or nonspecific peptides, or bufferalone at room temperature for 4 h. Specific peptide used for A2was FLPSDDFPSV and for B35 was LPSSADVEF and nonspe-cific peptide for both was RRYQKSTEL. After incubations,supernatants were collected and analyzed by SDS/PAGE andimmunoblotting analyses with anti-His (for A2) or HC10 (forB35) antibodies.

We thank Chris Perria for conducting the experiment shown in Fig. 1D;Dr. Peter Cresswell (Yale University School of Medicine, New Haven,CT) for the PaSta-1 antibody; Dr. Pamela Bjorkman (California Instituteof Technology, Pasadena, CA) for M10 and HFE constructs; theUniversity of Michigan Biomedical Research Core Facilities for DNAsequencing and peptide syntheses and purification; the Hybridoma Corefor ascites production; and the Rheumatic Diseases Core Center and theMichigan Diabetes Research and Training Centers for financial support.This work was supported by National Institutes of Health Grant AI-44115 and a Cancer Research Institute Investigator Award (to M.R.).

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