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Thermal Stability of MHC Class I-₂-Microglobulin Peptide Complexes in the Endoplasmic Reticulum Is Determined by the Peptide Occupancy of the Transporter Associated with Antigen Processing Complex¹

Barbara A. L. Owen^* and Larry R. Pease^2,*,

Departments of ^* Biochemistry and Molecular Biology, Immunology, and Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905

	Abstract

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Once MHC class I heavy chain binds ₂-microglobulin (₂m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated K^b₂m complexes within microsomes (TAP⁺/TAPasin⁺) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of K^b-₂m-peptide complexes depends on the timing of peptide binding to K^b₂m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in K^b₂m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of K^b₂m-peptide complexes.

	Introduction

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Nascent MHC class I molecules bind peptides within the endoplasmic reticulum (ER)³ after association with ₂-microglobulin (₂m). The binding of antigenic peptides to MHC class I-₂m complexes is mediated by the TAP/TAPasin complex, which is located within the ER of all cell types (1, 2, 3). The TAP protein encoded by MHC-linked genes is heterodimeric composed of TAP1 and TAP2 subunits, which are members of the ATP-binding cassette transporter family. The transport of antigenic peptides by the TAP complex in isolated microsomes has been thoroughly characterized (4, 5). The lack of either TAP1 or TAP2 results in markedly decreased cell surface expression of MHC class I proteins and retention of peptide-deficient (6), carbohydrate-immature (7) MHC class I-₂m dimers within the ER/cis-Golgi (8). The finding that TAP associates directly with newly synthesized MHC class I molecules (1, 2) has led to the speculation that TAP may play additional roles in MHC class I assembly other than providing peptide (9, 10, 11). Several MHC class I mutants have been identified that exhibit reduced binding to TAP resulting in comparable reductions in cell surface expression (12, 13, 14). These findings emphasize the importance of maintaining the ability of the class I heavy chain to interact with TAP.

TAPasin is a 48 kDa transmembrane protein (type I) that resides in the ER via a retention sequence found in its cytoplasmic tail and is a member of the Ig superfamily like the MHC class I heavy chain (3, 15, 16). TAPasin-deficient cells also exhibit reduced MHC class I surface expression; the extent appears to be allele dependent (17, 18). It has been shown that lack of TAPasin results not only in reduced TAP-MHC class I association but also calreticulin-MHC class I association (3) and that peptide binding to TAP in TAPasin-deficient microsomes is comparably reduced (19). Therefore, it appears that both TAP and TAPasin are required for efficient class I binding to TAP, peptide binding to TAP, and ultimately in stable cell surface expression of peptide-loaded class I complexes.

We have described previously the assembly of in vitro translated K^b in TAP⁺ and TAP-deficient microsomes (20). Our results indicated that TAP-dependent, peptide-specific alterations in the conformational state of the MHC class I heavy chain could be detected in isolated microsomes derived from the TAP1/TAP2-positive and TAPasin-positive RMA cell line. In contrast, K^b₂m complexes generated in TAP2-deficient, TAPasin-positive (RMA-S-derived) microsomes were not able to acquire high affinity peptide across microsomal membranes and were occupied with low affinity ligands. Using a different approach, DeSilva et al. (21) found that H-2K^b molecules expressed on RMA-S cells were not only occupied by low affinity ligands when cultured at 26°C, but became thermolabile when shifted to 37°C.

Therefore, the TAP/TAPasin complex is not only required for peptide acquisition by K^b₂m complexes, but interactions of the class I heavy chain with both TAP and TAPasin are required for proper folding of the complex. These findings support the hypothesis that TAP and TAPasin function as cochaperones for folding of MHC class I proteins within the ER (for review, see Refs. 22, 23). To further our insight into the mechanism of TAP/TAPasin-mediated folding of class I complexes, we investigated the acquisition of thermal stability of in vitro translated K^b₂m complexes within TAP/TAPasin microsomes.

	Materials and Methods

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Cell lines, mAbs, and peptides

The murine B cell line RMA was provided by the laboratory of P. Cresswell (Yale University, New Haven, CT) and maintained in 10% bovine calf serum, RPMI 1640 (Life Technologies, Grand Island, NY) at 37°C, 5% CO₂ supplemented with 2 mM L-glutamine, and 100 U penicillin/streptomycin. The mAb Y-3 (₁₂-K^b-specific, ₂m-dependent, peptide-insensitive) was purchased from American Type Culture Collection (Manassas, VA), the mAb K-10-56 (₁₂-K^b-specific, ₂m-dependent, peptide-sensitive) was originally obtained from G. Hammerling (German Institute for Immunology and Genetics, Heidelberg, Germany), and the mAb 100.3 (₁₂-K^b-specific, ₂m-dependent, peptide-sensitive) was provided by K. Hogquist (University of Minnesota, Minneapolis, MN).

The mAb K-10-56 can distinguish K^b bound to OVA8 with a free COOH terminus vs an amide; all experiments were performed with OVA8-amide. K-10-56 binding to K^b is down-regulated when K^b is bound to OVA8-amide, but not when it is bound to OVA8-COOH (20, 24). The binding of 100.3 is also down-regulated when K^b is bound to either OVA8 peptide (20, 25). The anti-TAP antiserum was provided by T. Hansen (Washington University, St. Louis, MO). The Mayo Peptide Synthesis Core synthesized peptides using standard f-moc synthesis and verified the sequences by both HPLC and mass spectrometric analysis. All peptides were synthesized as the free carboxyl, except OVA8 (SIINFEKL), which was also synthesized as the amide.

cDNA cloning, RNA transcription, and in vitro translation

Details can be found in Ref. 20 .

MHC class I assembly assay in TAP/TAPasin-positive (RMA) microsomes

This assay consists of isolating in vitro translated K^b from protease-resistant RMA microsomes and determining the impact that peptide added to microsomes during K^b synthesis, after microsome solubilization, or posttranslationally has on K^b₂m-peptide association. K^b was translated in the presence of the high affinity K^b-binding peptide OVA8 (SIINFEKL) or other peptide as noted at 25 µM. In experiments where microsomes and peptide were added, the microsomes were added first, followed by the peptide and then the RNA. To determine that peptide had gained access to the lumen of the microsome and to remove any leaky microsomes, all samples were treated with 50 µg/ml proteinase K (PK) for 45 min in 10 mM Tris, pH 7.5, at 0°C after microsome isolation and washing (see above). After PK treatment the microsomes were made 1 mM in PMSF and then solubilized for immunoadsorption by the mAb Y-3 or other Ab as indicated (see below).

For the posttranslational assembly assay, the following modifications were made. K^b was translated in the presence of RMA microsomes in the absence of high affinity peptide at 30°C for 90 min. After translation, the samples were moved to an ice bath for 5 min, and then peptide was added for 20 min at 0°C. At 0°C, ATP-dependent processes, such as peptide translocation, should be arrested. However, it has been shown by others (4, 26) that peptide binding to TAP is optimal at this temperature. After incubation with peptide, the microsome samples are incubated at 25°C for 5 min to allow for peptide translocation (4). The microsomes are then isolated, washed, PK-treated, solubilized, and immunoadsorbed as described below.

Immunoadsorption and SDS-PAGE

Immunoadsorptions were performed on washed and solubilized microsomes recovered from in vitro translation reactions as described above. Preadsorption consisted of a 30-min incubation at 4°C with protein A-Sepharose. In the case of immunoadsorptions using the mAbs Y-3, K-10-56, or 100.3, microsomes were solubilized with 1% Nonidet P-40 (NP40) lysis buffer for 30 min on ice (0°C) before the addition of Ab and overnight incubation at 4°C. Each Ab was added at 5 µg/ml, an excess relative to the K^b. Immunoreactive K^b complexes were recovered on at least a 2-fold excess (relative to Ab) of protein A-Sepharose beads after an additional 2-h incubation at 4°C. The entire supernatant fluid (unbound fraction) was retained for analysis by SDS-PAGE; the beads were collected by centrifugation and then washed extensively. The first wash consisted of the NP40 lysis buffer, followed by Tris-saline (0.05:0.1 M), followed by 1.0 M NaCl, 0.05 Tris, followed by Tris-saline. The washed beads were solubilized into 5% SDS and 0.1% 2-ME, heated at 100°C for 5 min, and the entire sample (bound fraction), beads included, was analyzed by SDS-PAGE. The total microsomal K^b is comprised of the fraction of K^b in the supernatant fluid, the unbound fraction, and the fraction of K^b bound to the Ab, therefore, the protein A-Sepharose. Coimmunoadsorptions with the anti-TAP Ab were performed as described above, with the following exceptions. Microsomes were not treated with PK as the epitope that the Ab binds is present on a cytoplasmic domain of the TAP1 protein. The lysis buffer consisted of 1% digitonin instead of NP40. The Ab incubation time was 2 h instead of overnight. The K^b-anti-TAP-protein A-Sepharose complexes were washed three times with the digitonin lysis buffer. SDS-PAGE (12%, reduced) was performed on the bound and unbound fractions, and the gels were fixed, enhanced with Flluoro-en³Hance (Research Products International, Boston, MA) for 45 min at room temperature, and dried before autoradiography with Bio-MAX (Kodak, Rochester, NY) film. K^b bound and unbound SDS-PAGE bands were measured by densitometry using an Ambis Systems (San Diego, CA) imaging and data analysis system. Identical, size-matched scans of lane backgrounds were subtracted from each protein band to account for film or individual lane variations. Calculated percentages (bound/total) were determined for each data set (i.e., each gel) exposed to film for the same length of time. Multiple film exposures were used to ensure that K^b protein bands were within the linear range of the analytical system.

Thermal stability of MHC class I complexes formed in microsomes

For determinations of thermal stability of K^b₂m-peptide complexes formed in microsomes, incubation at 37°C was performed in either of two ways. The first, to determine the thermal stability of complexes solubilized away from microsomal constituents, was performed on NP40-lysed microsomes for Y-3-, K-10-56-, or 100.3-immunoadsorbed samples or digitonin lysed for coisolation by the anti-TAP antiserum. Microsomes were solubilized at 4°C for 30 min, then placed in a 37°C water bath for 15 min. The microsome samples were then returned to 4°C for 20 min before addition of Ab. Subsequent addition of protein A-Sepharose, isolation, and washing procedures were performed exactly as for immune complexes maintained at 4°C (see above). The second approach was used to determine the thermal stability of K^b₂m-peptide complexes formed and maintained in intact microsomes. For these experiments, the isolated, washed, and PK-treated microsomes were incubated at 37°C for 15 min before solubilization. Following incubation at 37°C, the microsome samples were returned to 4°C for 20 min before solubilization. Subsequent lysis and Ab and protein A-Sepharose addition steps were performed as described above.

	Results

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Cotranslational peptide binding by nascent MHC class I complexes

To determine how peptide interacts with the TAP complex and nascent MHC class I complexes, K^b was synthesized into RMA microsomes in the absence or presence of peptide. High affinity peptide was added cotranslationally, posttranslationally, or only after solubilization. The recovery of K^b₂m complexes by the Y-3 Ab, which recognizes ₂m-associated K^b or by coimmunoprecipitation with an anti-TAP antiserum was assayed in parallel. The analysis of representative microsomal fractions obtained after exposure to the SEV9 peptide as indicated is shown in Fig. 1 and a summary of replicate experiments is shown in Fig. 2.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. A, The timing of peptide addition alters TAP association with Kb. Greater than 40% of the total microsomal Kbgly fraction is associated with TAP1 in solubilized microsomes maintained at 4°C as shown in lane 1. When the OVA8 peptide is added cotranslationally (CO-T), there is an increase (>30%) in TAP association, as shown in lane 2. Peptide (OVA8) added upon solubilization of the microsomes (SOL), lane 3, and results in nearly 70% dissociation of TAP-Kb complexes compared with Lane 1. However, at 37°C TAP association is minimal and independent of peptide as shown in lanes 4–6. Replicate experiments similar to those represented here are summarized in Fig. 2. B, Kb2m-peptide complexes formed cotranslationally are not stable at 37°C after microsome solubilization. Kb was translated into RMA microsomes and the percentage of Y-3 binding was determined as described in Materials and Methods. Lanes 1–4, No peptide added; lanes 2–5, OVA8 peptide added cotranslationally (CO-T); lanes 3–6, OVA8 peptide added upon solubilization (SOL). In lanes 1–3, lysates were kept at 4°C; in lanes 4–6, the temperature was raised to 37°C for 15 min after solubilization of the microsomes. Replicate experiments similar to those represented here are summarized in Fig. 2.

View larger version (66K): [in this window] [in a new window]   FIGURE 2. The timing of peptide addition effects TAP-Kb association and the thermal stability of Kb2m-peptide complexes. Peptide added cotranslationally (CO-T) results in increased TAP association and thermal unstable Y-3-positive Kb2m-peptide complexes. Peptide added upon solubilization (SOL) results in TAP dissociation and thermal stable Y-3 Kb2m-peptide complexes. The results shown here are derived from six independent experiments and error bars depict SD.

When solubilized microsomes are exposed to 37°C for 15 min, the TAP-K^b association drops to <5%, irrespective of conditions of peptide addition (Fig. 1A, lanes 4–6). This finding is not a failure of the immunoprecipitation experiment as the samples shown in Fig. 1, lanes 1 and 2, were isolated and washed in parallel with those in lanes 4–7. It had been determined previously in solubilized whole cells that the association of TAP with MHC class I is labile at 37°C (27). We have previously shown that K^b molecules assembled in microsomes in the absence of exogenously added peptide are not bound to high affinity peptides (20). Together, these findings indicate that the temperature-sensitive associations of class I molecules with TAP are comparable whether the class I molecules have bound to high affinity peptides or not.

The degree of TAP association in response to peptide, added cotranslationally, parallels the degree of peptide-induced assembly of K^b₂m-peptide complexes at 4°C, as measured by the increase in molecules detected using the Y-3 mAb (Fig. 1B, lanes 1 and 2). In the absence of peptide, 21% of the total microsomal K^b is in a conformation detected by mAb Y-3; with the addition of the OVA8 peptide the Y-3-positive proportion increases an additional 26% of the total microsomal K^b molecules to 47%. As shown in Fig. 2, the increase in TAP-K^b association is comparable, as an additional 32% of total microsomal K^b is bound to TAP. Similarly, when the SEV9 peptide is added cotranslationally, the gain in Y-3 conformation is 31% of microsomal K^b, as compared with a 36% increase in TAP association. However, the Y-3-positive-K^b₂m-OVA8 (or -SEV9) complexes formed when peptide is added cotranslationally are not stable when solubilized microsomes are incubated at 37°C for 15 min. Less than 15% of the total microsomal K^b remains in the Y-3 conformation (Fig. 1B, lane 5 and summarized in Fig. 2). This represents a loss of 65% of the Y-3-positive molecules assembled with OVA8 peptide and a 70% loss of MHC class I molecules assembled cotranslationally with SEV9 peptide. This loss accounts for most of the Y-3-positive K^b molecules preassociated with TAP as well as the additional molecules detected after cotranslational addition of peptide (see Fig. 1A). Therefore, it appears that the cohort of K^b molecules that associates with TAP in the presence of peptide added cotranslationally form thermally unstable K^b₂m-peptide complexes. The K^b-₂m-peptide complexes formed under these conditions have a reduced binding affinity for the peptide ligand, suggesting that the K^b heavy chain is not properly folded.

When peptide is added only after solubilization of the microsomes, 65% of the total K^b acquires the Y-3 conformation, and greater than 80% of this fraction is stable when incubated at 37°C (Fig. 1B, compare lanes 3 and 6). When K^b₂m complexes are solubilized away from ER membrane components including TAP (see Fig. 1A, lanes 3 and 6), and peptide is added concomitantly, maximal folding of K^b₂m with peptide occurs. This situation is analogous to the folding of MHC class I molecules in detergent lysates derived from either TAP⁺ or TAP^- cells. Similarly, MHC class I-₂m complexes forced to the cell surface in TAP-deficient cell lines by incubation at 26°C can fold with peptide added to the culture medium. We interpret these results to mean that posttranslational, postsolubilization assembly of MHC class I molecules with antigenic peptide is a TAP-independent event. A summary of replicate experiments is shown graphically in Fig. 2.

Thermal stable K^b₂m-peptide complexes are maintained in intact microsomes

The thermal instability of peptide-dependent conformational changes acquired when peptide is added cotranslationally indicates that K^b₂m-peptide complexes formed under these conditions differ from those found at the surface of TAP⁺ cells, which have been shown to be stable at 37°C. It is possible that K^b₂m-peptide complexes incubated at elevated temperatures lose peptide and fall apart when peptide has been acquired cotranslationally. However, when such complexes are maintained at reduced temperatures, the complexes may be sufficiently stable to be isolated by the conformationally dependent Abs, even when solubilized away from ER components. Again, the maintenance of K^b₂m-peptide complexes at 4°C that dissociate at 37°C suggests that the K^b heavy chain is binding peptide with reduced affinity.

To investigate whether K^b molecules assembled with cotranslationally added peptide are unstable in situ, isolated, washed, and PK-treated microsomes containing K^b₂m-OVA8 or -SEV9 complexes were incubated at 37°C before solubilization of the microsomes. Under these conditions, K^b₂m-peptide complexes formed cotranslationally are stable at the elevated temperature, as measured by the K^b-specific conformational epitopes detected by Y-3, K-10-56, and 100.3 Abs. We had shown previously that the K-10-56 and 100.3 Abs recognize unique epitopes on K^b only when bound to specific peptides. Specifically, the K-10-56 and 100.3 epitopes on K^b are lost upon binding of the OVA8 peptide and increase when K^b binds the SEV9 peptide. As shown in Table I, the decreased K-10-56 and 100.3 binding to K^b is directly proportional to OVA8 binding K^b. Similarly, the increased K-10-56 and 100.3 binding to K^b is directly proportional to K^b binding the SEV9 peptide. The finding that these Ab profiles are maintained at 37°C with either peptide indicates that K^b₂m complexes remain bound to high affinity peptide within intact microsomes. These results imply that microsomal components stabilize K^b₂m-peptide complexes that in isolation become denatured at 37°C.

View this table: [in this window] [in a new window]   Table I. Kb2m-peptide complexes formed cotranslationally are maintained at 37°C within intact microsomes1

The observed thermal instability of K^b₂m-OVA8 (or -SEV9) complexes and the increase in TAP-K^b association with peptide added cotranslationally contrasts with measurements of MHC class I complexes derived from permeabilized cells after the addition of peptide (1, 2). When peptide is added to metabolically labeled and permeabilized cells, the MHC class I-₂m-peptide complex dissociates from TAP concomitantly with acquisition of thermal stability. Therefore, a fundamental difference in subunit associations must occur under the different conditions of peptide addition. Both conditions yield assembly of MHC class I-₂m-peptide complexes; however, in one case thermally stable complexes are generated, whereas in the other case they are not. Although there are considerable differences in the two types of experiments, one parameter that is addressable is the timing of peptide addition. Adding peptide 20–30 min before the chase in the permeabilized cell studies could explain these differences. MHC class I-₂m complexes that were detected at "0 time" chase associated with TAP in the absence of peptide. Therefore, in these experiments, class I molecules dissociated from TAP in response to peptide added posttranslationally.

Assembly of K^b₂m-peptide complexes in microsomes posttranslationally

To address the issue of posttranslational and cotranslational assembly, K^b was first translated in the presence of RMA microsomes without high affinity peptide. Translation was arrested by moving the reactions to an ice bath (0°C), and high affinity peptide was then added for 20 min. ATP-dependent processes, such as peptide translocation, should be arrested at 0°C, and in vitro translation is essentially halted at this temperature (28). However, peptide binding to TAP has been shown to be optimal at this temperature (4, 26). Following posttranslational addition of peptide, the microsomes were immediately diluted into a translation inhibition buffer at 25°C and maintained at this temperature for 15 min to allow for ATP-dependent processes, such as TAP-mediated peptide translocation, to proceed. Isolation of K^b₂m-peptide complexes was performed exactly as described previously for K^b₂m-peptide complexes formed cotranslationally. As shown in Fig. 3A, recovery of K^b molecules with Y-3 Ab is increased 30% when peptide is added posttranslationally to microsomes compared with the 26% increase detected when peptide was added cotranslationally. However, in contrast to the increase in TAP-K^b association detected when peptide is added cotranslationally, peptide added posttranslationally results in a 40% decrease in TAP- K^b association (Fig. 3B). Furthermore, 80% of the Y3-positive-K^b₂m-peptide complexes formed when peptide is added posttranslationally is stable after microsome solubilization and incubation at 37°C (Fig. 3A, and summarized in Fig. 4). These results imply that when MHC class I molecules encounter TAP, the peptide-binding status of TAP determines, in part, the stability of the MHC class I-₂m-peptide complex.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. A, Peptide added posttranslationally (POST-T) to Kb2m complexes yield 37°C stable Kb2m-peptide complexes in RMA microsomes. The OVA8 peptide was added POST-T to Kb2m complexes formed in RMA microsomes as indicated and the percentage of Y-3 binding was determined as described in detail in Materials and Methods. After solubilization of the microsomes, the temperature was maintained at 4°C, lanes 1 and 2, or incubated at 37°C for 15 min after solubilization of the microsomes, lanes 3 and 4. The results shown here are summarized in Fig. 4. B, Kb2m complexes dissociate from TAP when peptide is added POST-T. The OVA8 peptide was added POST-T as indicated to Kb translated into RMA microsomes and the percentage of TAP binding was determined. Lanes 1 and 2 were maintained at 4°C, whereas lanes 3 and 4 were incubated at 37°C for 15 min after solubilization of the microsomes. The results shown here are summarized in Fig. 4.

View larger version (50K): [in this window] [in a new window]   FIGURE 4. Thermal stability of Kb2m-peptide complexes is dependent upon the timing of peptide addition. Peptide added posttranslationally (POST-T) results in Kb dissociation from TAP, and the resulting Kb2m-peptide complexes are thermally stable. The results shown here are derived from six independent experiments, and error bars depict SD.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. A schematic view of the co- and posttranslational in vitro assembly of Kb2m-peptide complexes is shown. Left, Posttranslational pathway in which the intermediate complex (indicated by the 5-point star) is generated composed of Kb2m associated with TAP, TAPasin, and calreticulin. Upon addition of peptide to the microsomes, TAP (and most likely TAPasin and calreticulin) dissociates and the Kb2m complex binds peptide. The complex isolated in digitonin lysis buffer is indicated by the (*) symbol. This complex of Kb2m-peptide (indicated by the (**) symbol) as measured by Y-3 reactivity is then stable after solubilization of the microsomes in NP40 lysis buffer and incubation at 37°C. Right, Cotranslational pathway in which the intermediate complex, indicated by the 4-point star, is generated composed of peptide-TAP- Kb2m, which may or may not be associated with TAPasin and calreticulin. Upon solubilization of the microsomes in digitonin lysis buffer, the Kb2m complex, indicated by the (#) symbol, binds peptide but remains associated with TAP. TAPasin and calreticulin may or may not be associated with this complex. After solubilization in NP40 buffer and incubation at 37°C, TAP dissociates and peptide is lost from the Kb2m complex (##), resulting in dissociation of 2m and Kb as detected by the loss of Y-3 binding.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Because the TAP/TAPasin complex has been shown to modulate the folding pathway of the MHC class I-₂m-peptide complex, it has recently been referred to as a MHC class I-specific chaperone complex (10, 22, 23, 30). This hypothesis is reinforced by the evidence that direct TAP- and TAPasin-MHC class I heavy chain interactions have been shown to be essential for assembly of MHC class I-₂m-peptide complexes. Class I molecules that have point mutations within positions 128–137 and 220–230 exhibit reduced interactions with TAP, TAPasin, and calreticulin (13, 14). To delineate the importance of the interactions of the TAP/TAPasin complex in moderating stable peptide acquisition by the class I heavy chain in the ER, assembly of in vitro translated K^b₂m-peptide complexes in TAP/TAPasin-positive microsomes was studied by TAP-dependent peptide delivery under several different conditions.

We previously demonstrated that MHC class I assembly with ₂m and peptide could be measured quantitatively in an isolated microsome system (20). Nascent K^b molecules were shown to fold within microsomes derived from RMA cells into conformations recognized by ₂m-dependent and peptide-specific Abs. Such complex formation required both TAP1 and TAP2 and ₂m at the time of MHC class I synthesis. In contrast, TAP2-deficient microsomes derived from RMA-S cells formed conformationally aberrant K^b-₂m complexes in the absence of high affinity peptide. Additionally, peptide added cotranslationally could not access MHC class I molecules in sealed, TAP2-deficient, TAPasin-positive microsomes. These results demonstrated that TAP, in addition to supplying peptide across a sealed microsome membrane, influenced the assembly of MHC class I complexes. An intact TAP1/TAP2 heterodimer was required to prevent premature assembly of MHC class I-₂m dimers with low affinity, microsomally derived ligands in the absence of high affinity peptide.

In this manuscript we report that the K^b₂mOVA8 (or SEV9) complexes formed cotranslationally are not stable at 37°C once the complexes have been solubilized away from microsomal components. This is not due to a fundamental difference in in vitro translated MHC class I molecules because K^b₂m complexes formed in vitro could fold into thermal stable complexes when the peptide was added immediately upon solubilization of the microsomes. However, binding of peptides to class I molecules outside of the microsome is a TAP-independent process, as class I molecules assembled in this manner from TAP-positive or TAP-negative cells are equivalent.

While nascent class I molecules assembled cotranslationally with peptide reside within sealed microsomes they remain heat stable only exhibiting the heat-labile phenotype after solubilization. Although a 15-min incubation of intact microsomes at 37°C containing the K^b₂m-peptide complexes was not sufficient to dissociate peptide and cause the class I molecules to lose conformational dependent Ab epitopes, the class I molecules in heat-pulsed microsomes became dissociated from TAP. Consequently, it appears that a microsomal component other than TAP may be responsible for maintaining the stability of otherwise heat-labile class I molecules.

MHC class I heavy chain association with TAP has been demonstrated previously by coimmunoadsorption of MHC class I proteins from detergent lysates of TAP-positive cells (1, 2). We determined that over 40% of K^b synthesized into RMA microsomes in the absence of added peptide were recovered by coimmunoadsorption with an anti-TAP1 antiserum. When either OVA8 or SEV9 peptides were added cotranslationally, TAP association was increased. The relative increase in TAP association with the addition of peptide cotranslationally paralleled the increase in peptide-specific folding detected by the K^b-specific, ₂m- and peptide-sensitive conformational dependent Abs, Y-3 and K-10-56.

These results conflict with previous studies using permeabilized cells, in which a decrease in TAP association followed addition of high affinity peptide (1, 2). Differences in the timing when peptide was added relative to the synthesis of the MHC class I heavy chain between the two studies could explain these disparate results. In the permeabilized cell studies the peptide was added 20 min after heavy chain synthesis. Consequently, in this model system, peptide encounters class I molecules that have had a chance to interact with resident ER proteins that govern class I assembly. In our studies using isolated microsomes the peptide was added cotranslationally, before the complete synthesis of the MHC class I heavy chain. Consequently, peptide was available in the microsome at the earliest possible time for interactions with the nascent class I heavy chain. Additionally, the MHC class I heavy chain first encounters TAP while it is associated with high affinity peptide. Because the availability of high affinity peptide has been shown to be rate limiting in class I assembly, saturation of TAP with high affinity ligand is an anomalous condition.

To evaluate the importance of the timing of peptide additions to the assembling class I molecule, K^b assembled cotranslationally and posttranslationally with peptide was compared. We found that posttranslational addition of peptide to K^b molecules synthesized in TAP/TAPasin-positive microsomes resulted in heat-stable K^b₂m complexes that dissociated from TAP. The characteristics are similar to those observed for assembling class I molecules in permeabilized cells. The antigenic character of the class I molecules assembled posttranslationally with peptide is indistinguishable from those formed upon addition of peptide cotranslationally, an indication that peptide is bound similarly by both sets of class I-peptide complexes. These results indicated that K^b₂m complexes associated with TAP already associated with high affinity peptide influenced the conformational maturation of the K^b₂m-peptide complex.

We have previously shown that the TAP complex, in the absence of added high affinity peptide, influences the structure of nascent heavy chain, restraining the molecules from folding into a conformation detectable with K^b-specific mAb (20). The normal folding pathway for class I assembly appears to be association of class I-₂m complexes with TAPasin and calreticulin-associated TAP, followed by binding and subsequent transport of high affinity peptide by the TAP component of the chaperoning complex. Therefore, the TAP/TAPasin/calreticulin complex provides a docking site for newly synthesized class I molecules until high affinity binding peptides become available.

When nascent MHC class I heavy chain-₂m complexes bind to TAP molecules that are already associated with high affinity peptide the MHC molecule is able to bind this peptide. However, the resulting MHC-peptide complex maintains association with TAP and remains thermolabile upon solubilization of the microsomes. We hypothesize that an additional cofactor other than TAP is responsible for maintenance of the thermal-stabile phenotype while K^b₂m-peptide complexes reside within unsolubilized microsomes. TAP association is lost at 37°C, but the MHC-peptide complexes within the microsome remain stable. Likewise, in the absence of a functional TAP complex, K^b₂m molecules associate with an ER-derived component that results in the heavy chain acquiring conformational epitopes that are generally acquired by mature class I molecules that are ₂m associated and that have bound peptide. Recent results (15, 31, 32) suggest that TAPasin may be the ER-derived factor that is responsible for generating the thermal unstable K^b₂m-peptide complexes that result after cotranslational acquisition of peptide. Because calreticulin association with class I parallels TAP/TAPasin association, it remains a possibility that calreticulin is the cofactor required for maintaining the thermal stable complex within unsolubilized microsomes at 37°C. It is also possible that maintenance of thermal stability requires a combination of two or more chaperoning proteins.

The finding that K^b₂m-peptide complexes assembled on TAP saturated with high affinity peptide remain thermolabile upon solubilization of the microsomes raises the possibility that this may be a step along the normal pathway for mature class I assembly. Under what conditions within the cell would TAP be presented with an abundance of high affinity peptide substrate? Acute viral infection or increased intracellular degradation of proteins such as that found in the heat shock response would result in such a situation. As so elegantly demonstrated recently, TAP quickly becomes associated with peptides supplied from the cytosol that have accumulated due to inhibition of protein synthesis or viral infection (33, 34). The ordered assembly of class I molecules with peptide could serve as a counter balance to such a flood of peptides, preventing the overexpression of a small number of peptides that could be detrimental to the development of a high affinity response. However, we find that TAP complexes saturated with high affinity peptide may not interact with class I or cochaperones as efficiently, resulting in class I-₂m-peptide complexes that do not associate with peptide in a stable manner. The finding that the lateral mobility of peptide-associated-TAP is altered within the ER membrane suggests that the conformation of TAP bound to peptide differs substantially from peptide-free TAP supports this hypothesis (34). Within an intact cell, class I-₂m-peptide complexes formed under such conditions would have multiple opportunities to dissociate as they were transported from the ER to the Golgi and beyond under the constant surveillance by the general chaperone pathway.

	Acknowledgments

 
We thank Scott Kuhns for critical review of this manuscript, Dr. Peter Cresswell for the RMA cell line, Dr. Ted Hansen for anti-TAP Ab, and Dr. Kristin Hogquist for supplying the 100.3 mAb. Rudy Hanson, Kathleen Allen, and Mike Hansen provided expert technical assistance, and Terri Felmlee provided secretarial assistance in preparation of this manuscript.

	Footnotes

 
¹ This study was partially supported by National Institutes of Health Award AI28230.

² Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Guggenheim 3, Mayo Clinic/Foundation, 200 First Street Southwest, Rochester, MN 55905.

³ Abbreviations used in this paper: ER, endoplasmic reticulum; NP40, Nonidet P-40; ₂m, ₂-microglobulin; PK, proteinase K.

Received for publication February 25, 2000. Accepted for publication November 14, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ortmann, B., M. J. Androlewicz, P. Cresswell. 1994. MHC class I/₂-microglobulin complexes associate with TAP transporters before peptide binding. Nature 368:864.[Medline]
2. Suh, W. K., M. F. Cohen-Doyle, K. Fruh, K. Wang, P. A. Peterson, D. B. Williams. 1994. Interaction of MHC class I molecules with the transporter associated with antigen processing. Science 264:1322.[Abstract/Free Full Text]
3. Sadasivan, B., P. J. Lehner, B. Ortmann, T. Spies, P. Cresswell. 1996. Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity 5:103.[Medline]
4. Sheperd, J. C., T. N. M. Schumacher, P. G. Ashton-Rickardt, S. Imaeda, H. L. Ploegh, C. A. Janeway, S. Tonegawa. 1993. TAP-1-dependent peptide translocation in vitro is ATP dependent and peptide selective. Cell 74:577.[Medline]
5. Heemels, M.-T., H. L. Ploegh. 1995. Generation, translocation and presentation of MHC class I-restricted peptides. Annu. Rev. Biochem. 64:463.[Medline]
6. Hsu, V. W., L. C. Yauan, J. G. Nuchtern, J. Lippincott-Schwartz, G. J. Hammerling, R. D. Klausner. 1991. A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules. Nature 352:441.[Medline]
7. Hayes, B. K., F. Esquivel, J. R. Bennink, J. W. Yewdell, A. Varki. 1995. Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. J. Immunol. 155:3780.[Abstract]
8. Raposo, G., H. S. van Santen, R. Leijenkekker, H. J. Geuze, H. L. Ploegh. 1995. Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment. J. Cell Biol. 131:1403.[Abstract/Free Full Text]
9. Day, P. M., F. Esquivel, J. Lukszo, J. R. Bennink, J. W. Yewdell. 1995. Effect of TAP on the generation and intracellular trafficking of peptide-receptive major histocompatibility complex class I molecules. Immunity 2:137.[Medline]
10. Solheim, J. C., B. M. Carreno, T. H. Hansen. 1997. Are transporters associated with antigen processing (TAP) and tapasin class I MHC chaperones?. J. Immunol. 158:541.[Abstract]
11. Suh, W.-K., E. K. Mitchell, Y. Yang, P. A. Peterson, G. L. Waneck, D. B. Williams. 1996. MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains. J. Exp. Med. 184:337.[Abstract/Free Full Text]
12. Peace-Brewer, A. L., L. G. Tussey, M. Matsui, G. Li, D. G. Quinn, J. A. Frelinger. 1996. A point mutation in HLA-A*0201 results in failure to bind the TAP complex and to present virus-derived peptides to CTL. Immunity 4:505.[Medline]
13. Suh, W.-K., M. A. Derby, M. F. Cohen-Doyle, G. J. Schoenhals, K. Fruh, J. A. Bersofsky, D. B. Williams. 1999. Interaction of murine MHC class I molecules with TAPasin and TAP enhances peptide loading and involves the heavy chain 3 domain. J. Immunol. 162:1530.[Abstract/Free Full Text]
14. Yu, Y. Y. L., H. R. Turnquist, N. B. Myers, G. K. Balendiran, T. H. Hansen, J. C. Solheim. 1999. An extensive region of an MHC class I 2 domain loop influences interaction with the assembly complex. J. Immunol. 163:4427.[Abstract/Free Full Text]
15. Li, S., K. M. Paulsson, H.-V. Sjogren, P. Wang. 1999. Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing. J. Biol. Chem. 274:8649.[Abstract/Free Full Text]
16. Ortmann, B., J. Copeman, P. J. Lehner, B. Sadasivan, J. A. Herberg, A. G. Grandea, S. R. Riddell, R. Tampe, T. Spies, J. Trowdale, P. Cresswell. 1997. A critical role for TAPasin in the assembly and function of multimeric MHC class I-TAP complexes. Science 277:1306.[Abstract/Free Full Text]
17. Greenwood, R., Y. Shimizu, G. S. Sekhon, R. DeMars. 1994. Novel allele-specific, post-translational reduction in HLA class I surface expression in a mutant human B cell line. J. Immunol. 153:5525.[Abstract]
18. Grandea, I. A. G., M. J. Androlewicz, R. S. Athwal, D. E. Geraghty, T. Spies. 1995. Dependence of peptide binding by MHC class I molecules on their interaction with TAP. Science 270:105.[Abstract/Free Full Text]
19. Li, S., K. M. Paulsson, S. Chen, H. O. Sjogren, P. Wang. 2000. TAPasin is required for efficient peptide binding to transporter associated with antigen processing. J. Biol. Chem. 275:1581.[Abstract/Free Full Text]
20. Owen, B. A. L., L. R. Pease. 1999. TAP association influences the conformation of nascent MHC class I molecules. J. Immunol. 162:4677.[Abstract/Free Full Text]
21. DeSilva, A. D., A. Boesteanu, R. Song, N. Nagy, E. Harhaj, C. V. Harding, S. Joyce. 1999. Thermolabile H-2K^b molecules expressed by transporter associated with antigen processing-deficient RMA-S cells are occupied by low-affinity ligands. J. Immunol. 163:4413.[Abstract/Free Full Text]
22. Cresswell, P., N. Bangia, T. Dick, G. Diedrich. 1999. The nature of the MHC class I peptide loading complex. Immunol. Rev. 172:21.[Medline]
23. Solheim, J. C.. 1999. Class I MHC molecules: assembly and antigen presentation. Immunol. Rev. 172:11.[Medline]
24. Rohren, E. M., D. J. McCormick, L. R. Pease. 1994. Peptide-induced conformational changes in class I molecules. J. Immunol. 152:5337.[Abstract]
25. Hogquist, K. A., A. G. Grandea, M. J. Bevan. 1993. Peptide variants reveal how antibodies recognize major histocompatibility complex class I. Eur. J. Immunol. 23:3028.[Medline]
26. van Endert, P. M., R. Tampe, T. H. Meyer, R. Tisch, J.-F. Bach, H. O. McDevitt. 1994. A sequential model for peptide binding and transport by the transporters associated with antigen processing. Immunity 1:491.[Medline]
27. Russ, G., F. Esquivel, J. W. Yewdell, P. Cresswell, T. Spies, J. R. Bennink. 1995. Assembly, intracellular localization, and nucleotide binding properties of the human peptide transporters TAP1 and TAP2 expressed by recombinant vaccinia viruses. J. Biol. Chem. 270:21312.[Abstract/Free Full Text]
28. N. Craig. 1975. Regulation of translation in rabbit reticulocytes and mouse L-cells: comparison of the effects of temperature. J. Cell. Physiol. 87:157.[Medline]
29. Li, S., H. O. Sjogren, M. Hellman, R. F. Pettersson, P. Wang. 1997. Cloning and functional characterization of a subunit of the transporter associated with antigen processing. Proc. Natl. Acad. Sci. 94:8708.[Abstract/Free Full Text]
30. Owen, B. A. L., L. R. Pease. 1998. Early association of MHC class I with TAP and peptide determine MHC class I conformational stability. FASEB J. 12:A589.
31. Schoenhals, G. J., R. M. Krishna, A. G. Grandea, T. Spies, P. A. Peterson, Y. Yang, K. Fruh. 1999. Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells. EMBO J. 18:743.[Medline]
32. Bangia, N., P. J. Lehner, E. A. Hughes, M. Surman, P. Cresswell. 1999. The N-terminal region of tapasin is required to stabilize the MHC class I loading complex. Eur. J. Immunol. 29:1858.[Medline]
33. Schuber, U., L. C. Anton, J. Gibbs, D. C. Norbury, J. W. Yewdell, J. R. Bennink. 2000. Rapid degradation of a large fraction of newly synthesized proteins by proteosomes. Nature 404:770.[Medline]
34. Reits, A. J., J. C. Vos, M. Gromme, J. Neefjes. 2000. The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404:774.[Medline]

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