Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic ...

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Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer1 Tsvetelina Pentcheva and Michael Edidin2 Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected. The Journal of Immunology, 2001, 166: 6625– 6632.

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ajor histocompatibility complex class I molecules present intracellular peptides to CD8⫹ CTLs. Each molecule comprises a polymorphic transmembrane heavy chain, noncovalently associated with soluble ␤2-microglobulin and a peptide of 8 –10 aa, usually generated in the cytosol by proteasomes. Biochemical studies have established a model for the assembly of human MHC (HLA) class I molecules in the endoplasmic reticulum (ER)3 (reviewed in Refs. 1 and 2). The model describes the associations of nascent class I molecules with ERresident chaperones and with TAP, which enable them to acquire antigenic peptides and enter the secretory pathway. However, it provides little information about the spatial organization of the nascent proteins within the ER or about the way in which they exit the ER. The 4:1 stoichiometry of MHC to TAP1/TAP2 heterodimer (3) suggests that class I molecules could be clustered at the TAP complex. Alternatively, fully folded, peptide-loaded MHC class I molecules could be clustered after their dissociation from TAP as part of a mechanism for ER export, analogous to that for soluble and GPI-anchored proteins (4, 5). Studying MHC class I spatial organization in the ER requires imaging on a scale beyond the resolution limit of the light microscope. Recently, we and others (6 –10) have developed a quantitative technique, fluorescence resonance energy transfer (FRET) microscopy, which can detect clusters of proteins carrying appro-

Department of Biology, Johns Hopkins University, Baltimore, MD 21218 Received for publication November 13, 2000. Accepted for publication March 20, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by National Institutes of Health Grant AI14584 (to M.E.).

2

Address correspondence and reprint requests to Dr. Michael Edidin, Department of Biology, Johns Hopkins University, Baltimore, MD 21218. E-mail address: [email protected] 3 Abbreviations used in this paper: ER, endoplasmic reticulum; CFP, cyan fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COPII, coat protein complex; D, diffusion coefficient; Endo H, endoglycosidase H; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; ROI, region of interest; YFP, yellow fluorescent protein.

Copyright © 2001 by The American Association of Immunologists

priate fluorophores. In FRET, nonradiative energy transfer occurs between two fluorophores, an energy donor and an energy acceptor. Because the efficiency of FRET decays as the sixth power of the donor-to-acceptor distance, the maximum separation allowing detectable FRET for typical donor-acceptor pairs is ⬃100 Å. Hence, significant energy transfer reports molecular proximity. Because FRET causes the quenching of donor fluorescence (energy is transferred to the acceptor instead of being emitted as a photon), it can be measured by imaging the increase in donor fluorescence after acceptor photobleaching (6, 7). A recent analysis of the theory for FRET on membranes has pointed the way to distinguishing between FRET due to clustering of donors and acceptors and FRET due to high concentrations of donors and acceptors randomly distributed in the membrane (7). If all donor and acceptor fluorophores are clustered, FRET is independent of acceptor concentration, whereas if they are randomly distributed, FRET increases with increasing acceptor surface density, and is independent of the ratio of donor-to-acceptor fluorophores. If a labeled population is a mixture of clustered and randomly distributed molecules, FRET increases with increasing acceptor concentration, but for a given acceptor surface density, it also depends on the ratio of donor-to-acceptor fluorophores (Fig. 1) (7). Because TAP was expected to mediate some class I clustering (3), we measured FRET between HLA-A2 or HLA-A2T134K, a mutant that does not associate with TAP (11, 12). Wild-type and mutant HLA-A2 molecules were tagged with either cyan fluorescent protein (CFP), or with yellow fluorescent protein (YFP), at their C termini (13). As a positive control for FRET, we tagged HLA-A2 with both CFP and YFP, separated by a 25-aa linker (14). At steady state, we could detect clusters of HLA-A2 molecules and clusters of A2-T134K molecules. However, there was no evidence for clusters containing both HLA-A2 and A2-T134K. Surprisingly, no HLA-A2 clusters could be detected by FRET after cells were treated with the proteasome inhibitor lactacystin (15), indicating that, if multiple molecules are simultaneously bound to TAP, the distance between them is beyond the FRET limit. In 0022-1767/01/$02.00

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CLUSTERING OF PEPTIDE-LOADED MHC CLASS I REVEALED BY FRET cataway, NJ). It recognizes a determinant in the ␣2 domain and is specific for conformed HLA-A2 molecules. Cy3-conjugated F(ab⬘)2 goat antimouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-TAP1 antiserum was purchased from StressGen Biotechnologies (Victoria, BC, Canada). All oligonucleotides were synthesized and purified by Integrated DNA Technologies (Coralville, IA). The Tax peptide (LLFGYPVYV) is derived from the human T cell lymphotropic virus HTLV-1 (aa 11–19) and binds with high affinity to HLA-A2 (21, 22). It was synthesized and purified to 97% purity by New England Peptide (Fitchberg, MA). It was dissolved in 5% DMSO to make a 2.5 mM stock and used at 250 ␮M.

Gene construction

FIGURE 1. Theoretical dependence of FRET on acceptor concentration for different fluorophore distributions. a, If all donor and acceptor fluorophores are clustered, FRET is independent of acceptor concentration. b, If all fluorophores are randomly distributed, FRET increases with increasing acceptor concentration and is independent of the ratio of donor:acceptor. c, If a fluorophore population is a mixture of clustered and randomly distributed molecules, FRET increases with increasing acceptor concentration, but, for a given acceptor surface density, it also increases with higher ratios of acceptor:donor fluorophores.

contrast, addition of exogenous peptides that reached the ER independently of TAP (16, 17) resulted in some coclustering of wildtype and mutant HLA-A2 molecules. These data strongly suggest that HLA-A2 molecules cluster after peptide loading, perhaps as part of the process of their export from the ER.

Materials and Methods Cells, Abs, and reagents HeLa Tet-On cells (Clontech, Palo Alto, CA) were maintained in DMEM (Mediatech, Herndon, VA), supplemented with 10% tetracycline-free FBS (Clontech), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 100 ␮g/ml G418 (Life Technologies), and antibiotic/antimycotic (100 U/ml penicillin G (sodium salt), 100 ␮g/ml streptomycin sulfate, and 25 ␮g/ml amphotericin B) (Life Technologies). To produce stable clones, cells were transfected using LipofectAMINE (Life Technologies) with the pBI constructs described below mixed in a 20:1 ratio with vector pTK-Hyg (Clontech). They were selected with 200 ␮g/ml hygromycin B (Roche Molecular Biochemicals, Indianapolis, IN), sorted for high expression by flow cytometry, and cloned by limiting dilution. Positive clones were maintained in HeLa Tet-On medium, supplemented with 200 ␮g/ml hygromycin B. Expression of the transfected molecules was induced with 2 ␮g/ml doxycycline HCl (Sigma, St. Louis, MO) 48 h before each experiment. For FRET experiments, 105 cells were plated onto a sterile coverslip in 2 ml of medium. The second construct was transfected transiently 24 h later using FuGENE (Roche Molecular Biochemicals), and the cells were imaged after 48 h. The B-lymphoblast cell line LCL-721.45.1 (18, 19) was maintained in RPMI 1640 medium (Mediatech) containing 15% heat-inactivated FBS (Intergen, Purchase, NY). Cell line HMy2.C1R expressing HLA-A2T134K, a gift from Dr. J. Frelinger (University of North Carolina, Chapel Hill, NC) (11), was maintained in RPMI 1640, supplemented with 10% heat-inactivated FBS and 300 ␮g/ml G418. The mAb BB7.2 (ATCC HB-84) (20) was purified from hybridoma supernatants using GammaBindPlus Sepharose (Pharmacia Biotech, Pis-

YFP was generated from pEGFP-N3 (Clontech) by site-directed mutagenesis, which introduced the following amino acid substitutions: L64F, T65G, V68L, S72A, and T203Y. CFP was generated from pECFP (Clontech) by site-directed mutagenesis, which introduced the N212K substitution. The final PCR products were ligated into vector pGEM-T (Promega, Madison, WI) and sequenced. The EGFP was excised from the pEGFP-N3 with BamHI and BsrG I (New England Biolabs, Beverly, MA) and replaced with the PCR-generated products, cut with the same two enzymes, thus creating pYFP-N3 or pCFP-N3. The cDNA coding for HLA-A2 and HLA-A2-T134K were obtained by RT-PCR from LCL-721.45.1 and HMy2.C1R.T134K cells, respectively. RNA was purified from 107 cells using TRIzol (Life Technologies). It was converted to cDNA with random hexamer primers from the Advantage RT-PCR kit (Clontech). The two cDNA were amplified with specific primers and sequenced. They were excised with SalI (New England Biolabs) and BamHI and cloned into pYFP-N3 and pCFP-N3, digested with the same two enzymes. The constructs were excised out of the N3 vectors with SalI and XbaI (New England Biolabs) and cloned into pBI (Clontech), cut with the same two enzymes. To generate the untagged HLA-A2 and HLA-A2-T134K constructs (containing a STOP codon), their cDNA were PCR amplified and sequenced. They were excised with SalI and NheI and ligated into pBI, cut with SalI and XbaI. To generate the positive control for energy transfer, YFP and CFP were physically linked to the C terminus of HLA-A2. The linker SSMTGGQQMGGDLYDDDDGDPPAGS (based on Ref. 14) was created by PCR. The YFP STOP codon was deleted by PCR, and the linker was fused at the YFP C terminus. The product was sequenced, digested with BglII (New England Biolabs) and BamHI, and introduced into pA2-CFP-N3, cut with BamHI, and treated with calf intestinal phosphatase (New England Biolabs). The entire construct was moved into pBI, as described above for the tagged HLAs.

Flow cytometry Cells were harvested in PBS containing trypsin, chicken serum, collagenase, and EDTA (Worthington Biochemical, Lakewood, NJ). They were washed once with 1% BSA in PBS and either analyzed directly or stained with 40 ␮g/ml BB7.2 mAb for 1 h at 4°C, then washed in PBS, and incubated with 5 ␮g/ml Cy3-conjugated F(ab⬘)2 goat anti-mouse IgG for 30 min at 4°C. After washing, cells were resuspended in PBS-1% BSA and analyzed on an EPICS 752 flow cytometer (Coulter, Miami, FL).

Pulse chase and immunoprecipitation Cells were incubated in cysteine- and methionine-free medium for 30 min, then labeled with 260 ␮Ci/ml Tran35S-label (ICN Biochemicals, Costa Mesa, CA) for 20 min. The cells were washed in PBS and chased in complete medium, supplemented with 2 mM cysteine and 2 mM methionine for the indicated time intervals. They were washed with cold PBS and lysed in buffer containing 0.5% Triton X-100. Postnuclear supernatants were precleared overnight with protein A-Sepharose (Sigma), then incubated with 25 ␮g/ml BB7.2 mAb, and the HLA-Ab complexes were recovered with protein A-Sepharose. They were washed in buffer containing 0.1% Triton X-100 and eluted. The eluates were digested overnight with endoglycosidase H (Endo H; Roche Molecular Biochemicals) in buffer G5 (New England Biolabs). The samples were analyzed by 10% SDS-PAGE and autoradiography. To detect MHC class I interactions with TAP, cells were radiolabeled and washed as above, then lysed in buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Sigma). Precleared lysates were incubated with anti-TAP1 antiserum at 1/100 dilution. The Ag-Ab complexes were recovered with protein A-Sepharose, washed

The Journal of Immunology in buffer containing 0.1% CHAPS, and eluted. The eluates were treated with Endo H and analyzed by 10% SDS-PAGE and autoradiography, as above. Bands were quantified using Scion Image (Scion, Frederick, MD). For experiments in the presence of peptides or lactacystin, cells were incubated for 1.5 h in medium supplemented with 250 ␮M Tax peptide or 100 ␮M lactacystin (Kamiya Biomedical, Seattle, WA) before their lysis.

Fluorescence recovery after photobleaching (FRAP) measurements Cells were grown on coverslips for 2 days prior to the experiments. Coverslips were washed twice in HBSS (Life Technologies), supplemented with 1% FBS and 10 mM HEPES (pH 7.3), mounted on slides in the same solution, and sealed with nail polish. The peptide and lactacystin treatments were the same as in the TAP coimmunoprecipitation experiments. Lateral diffusion of A2-YFP or A2-T134K-YFP was measured as described previously (17). Data were collected with custom software. From each curve, the percentage of recovery of fluorescence (the mobile fraction) and the half-time for recovery were obtained. The diffusion coefficient (D) was calculated from the half-time value, assuming one-dimensional diffusion.

6627 range 1500 –2500 fluorescence units, because in this range all plots of FRET vs YFP concentration reached a plateau for all YFP:CFP ratios.

Results Tagging with CFP or YFP did not perturb the folding and surface expression of the class I molecules. Both the chimeric and the untagged proteins were detected on the surface of transfected cells with mAb BB7.2 specific for native HLA-A2. The plasma membrane levels of the mutant A2-T134K were lower than the levels of the wild-type molecules, as expected (12) (data not shown).

Fluorescence microscopy and FRET measurements Cells on coverslips were washed in PBS and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. They were washed five times in PBS, and then incubated for 10 min in equilibration buffer from the SlowFade Light antifade kit (Molecular Probes, Eugene, OR). They were mounted on slides in antifade from the same kit and sealed with nail polish. The peptide and lactacystin treatments were the same as in the TAP coimmunoprecipitation experiments and the FRAP measurements. Cells were imaged on a Zeiss Axiovert 135 TV microscope (Zeiss, Thornwood, NY) using a 1.4 NA ⫻100 Zeiss Plan-apochromat objective. Fluorescence was excited with a 75-W arc lamp. CFP and YFP were detected with XF114 (excitation 440DF20, dichroic 455DRLP, emission 480DF30) and XF30 (excitation 510DF23, dichroic 540DRLP, emission OG550) filter sets, respectively (Omega Optical, Brattleboro, VT). Digital images were collected with a 12-bit Series 300 cooled CCD (Roper Scientific, Trenton, NJ), operated by the IC300 digital imaging system (Inovision, Raleigh, NC). FRET was measured as the percentage increase in CFP fluorescence after the bleaching of YFP (adapted for CFP and YFP from Ref. 7). Four images were acquired in a FRET experiment: 1) an image of the CFP fluorescence (in the presence of YFP), 2) an image of the YFP fluorescence, 3) an image of the YFP fluorescence after 30-s-long continuous excitation leading to its destruction, and 4) an image of the CFP fluorescence after the YFP bleach. Data were collected from more than five fields per coverslip, and the results from more than four independent experiments were pooled together, because they were comparable. To control for CFP bleaching and noise at low CFP levels, cells expressing only CFP-tagged molecules were taken through all four steps of a FRET experiment. The two CFP images were registered using ISee (Inovision) to account for any x-y drift of the slide during the bleach. Custom software output the mean values for CFP and YFP fluorescence before and after the bleach, after a dark current correction, from five 5 ⫻ 5-pixel (340 nm ⫻ 340 nm) regions of interest (ROI) per cell. The ROI were placed on the nuclear envelope. For each ROI, FRET was calculated as (CFPpostbleach ⫺ CFPprebleach)/CFPpostbleach, and the ratio of acceptor to donor was calculated as YFPprebleach/CFPpostbleach. To convert the fluorescence ratios to approximate protein ratios, cells were transiently transfected with chimeric molecules in known cDNA ratios. Because the noise of the experiment (⫾ 5% FRET) increased at very low CFP levels, data with CFP below 150 arbitrary units were excluded from the graphs. Our data could not be fit by theoretical curves (23, 24). This is because the dependence of FRET on YFP surface density is not linear, and because a number of the variables, needed for a fit, are unknown. These variables include the fraction of clustered molecules (which in turn may depend on YFP concentration), and the FRET efficiency within a cluster, which may not be the same for all clusters. Furthermore, in a mixture of clustered and randomly distributed molecules, FRET may occur both within and between clusters. No single theoretical model accounts for all these parameters, and any fit that tries to take them into account has too many free parameters to yield any useful information. However, the relative extent of clustering can be evaluated in terms of the dependence of FRET on donor-to-acceptor ratio for a given surface concentration of acceptor, and can be expressed as the difference in the mean FRET, over a range of YFP concentration, for different YFP:CFP ratios. This was done for YFP surface density in the

FIGURE 2. Tagging with YFP or CFP does not significantly interfere with the intracellular processing or with the interactions of the class I molecules with the TAP complex. Cells expressing A2-YFP (a), untagged HLA-A2 (b), A2-T134K-CFP (c), or untagged A2-T134K (d) were metabolically labeled with a mixture of [35S]methionine and cysteine and chased in nonradioactive medium for the indicated intervals. The cells were lysed in 0.5% Triton X-100 and immunoprecipitated with mAb BB7.2. The samples were digested with Endo H. Acquisition of resistance to Endo H indicates that the sugar moieties of the labeled molecules have been processed by enzymes residing in the medial Golgi. R and S refer to the Endo H-resistant and the Endo H-sensitive forms of the proteins, respectively. e, Cells expressing A2-YFP or A2-T134K-CFP were metabolically labeled as before and lysed in 1% CHAPS. They were immunoprecipitated with either anti-TAP1 antiserum or BB7.2 mAb and treated with Endo H. R and S refer to the Endo H-resistant and the Endo H-sensitive forms of the proteins, respectively. f, Cells expressing A2-YFP were treated with 100 ␮M lactacystin or 250 ␮M Tax peptide for 1.5 h, metabolically labeled, and lysed in 1% CHAPS. Equal amount of lysates was immunoprecipitated with anti-TAP1 antiserum or BB7.2 mAb (as a positive control). Untransfected cells were used as a negative control. Mix refers to the immunoprecipitate from a 1:1 lysate mixture of radiolabeled untreated cell and nonradioactive cells treated with 250 ␮M Tax peptide for 1.5 h as a control for peptide-induced A2-YFP dissociation from TAP in vitro.

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Tagging with CFP or YFP had some effect on the intracellular processing of the HLA molecules, without affecting their associations with the TAP complex. In pulse-chase experiments, the intracellular processing rates of both A2-YFP and A2-T134K-CFP were lower than those of their untagged equivalents (Fig. 2, a– d), which is consistent with previous data (25). However, the associations of the tagged molecules with the TAP complex were the same as those of their untagged counterparts. A2-YFP coimmunoprecipitated with TAP, whereas A2-T134K-CFP did not coimmunoprecipitate with TAP, consistent with results in other cells (Fig. 2e) (11, 12). To confirm that inhibiting the proteasome results in a larger number of A2-YFP molecules associating with TAP, whereas adding exogenous peptides has the opposite effect, we coimmunoprecipitated A2-YFP and TAP after treatment with 100 ␮M lactacystin for 1.5 h or 250 ␮M Tax peptide for 1.5 h (Fig. 2f). Densitometric analysis showed that lactacystin increased the number of A2-YFP molecules coimmunoprecipitating with TAP by a factor of ⬃1.2, whereas the addition of Tax peptides decreased that number by a factor of ⬃1.2. To ensure that peptide-induced dissociation did not occur in vitro, we compared the number of A2YFP molecules coimmunoprecipitating with TAP from lysate of radiolabeled untreated cells and from the same lysate mixed in a 1:1 ratio with lysate of nonradioactive cells incubated with 250 ␮M Tax peptide for 1.5 h (Fig. 2f, “mix”). Densitometric analysis showed that the same number of molecules coimmunoprecipitated with TAP under both conditions. The lateral diffusion of green fluorescent protein (GFP)-tagged MHC class I molecules measured by FRAP reports their association with TAP in the ER (17). The data are summarized in Table I. At steady state, the diffusion coefficient (D) represents the average of two populations of MHC class I: TAP bound (D ⬃1 ⫻ 10⫺9cm2s⫺1) and TAP free (D ⬃4 ⫻ 10⫺9cm2s⫺1) (17). At steady state, A2-YFP diffusion was intermediate to that previously measured for free or TAP-bound molecules, D ⬃2.3 ⫻ 10⫺9 cm2s⫺1 ⫾ 2 ⫻ 10⫺10 cm2s⫺1, indicating that some fraction of the population was associated with TAP. D of A2-T134K-YFP was higher, D ⬃3.5 ⫻ 10⫺9 cm2s⫺1 ⫾ 3 ⫻ 10⫺10 cm2s⫺1, consistent with the biochemical evidence that A2-T134K does not bind TAP at all (11, 12) (Fig. 2e). Lactacystin treatment lowered the average A2-YFP diffusion to D ⬃1.7 ⫻ 10⫺9 cm2s⫺1 ⫾ 3 ⫻ 10⫺10 cm2s⫺1, indicating an increase in the fraction of A2-YFP bound to TAP, but it had no effect on the lateral diffusion of A2-T134KYFP. Exogenously added peptides did not significantly affect the diffusion of either A2-YFP or A2-T134K-YFP; however, when added after lactacystin treatment, they restored A2-YFP diffusion to its original value, D ⬃2.5 ⫻ 10⫺9 cm2s⫺1 ⫾ 4 ⫻ 10⫺10 cm2s⫺1. This suggests that the peptides were bound by empty, TAP-associated HLA-A2 molecules that were then released from TAP. The fraction of mobile A2-YFP molecules was slightly, but significantly, lower than that of A2-T134K-YFP (70 ⫾ 4% vs 80 ⫾ 3%) and was unaffected by the treatments.

FRET was measured in terms of the dequenching of donor (CFP) fluorescence after acceptor (YFP) bleaching (Fig. 3). After acquiring images of the initial donor (CFPpre, Fig. 3a) and acceptor (YFPpre, Fig. 3b) fluorescence, the acceptor is destroyed by continuous excitation (YFPpost, Fig. 3d). The increase in donor fluorescence (CFPpost, Fig. 3c) after acceptor bleaching is proportional to the amount of FRET occurring before the destruction of the acceptor: FRET (%) ⫽ 100 ⫻ (CFPpost ⫺ CFPpre)/CFPpost. Our positive control for FRET was YFP and CFP connected with a 25-aa linker and fused to the C terminus of HLA-A2. In cells expressing this construct, FRET was insensitive to YFP surface density, indicating that all donor and acceptor fluorophores were clustered. Neither lactacystin nor the addition of Tax peptides altered this distribution of FRET values (Fig. 4, compare with the theoretical predictions in Fig. 1a). FRET between labeled HLA-A2 molecules depended on acceptor surface density, but for a given surface density of acceptor, FRET increased with increasing acceptor:donor ratios, indicating that a fraction of the molecules was clustered at steady state (Fig. 5a, compare with the theoretical predictions in Fig. 1c). The difference between the mean FRET over a range of YFP concentrations (1500 and 2500 arbitrary units) for YFP:CFP ratios of 1:1 and 2:1 was statistically significant (Fig. 5d). Because we assumed that the clusters reflected multiple HLA-A2 molecules bound simultaneously to TAP, it was surprising to observe that some A2T134K molecules were also clustered. FRET between labeled A2T134K molecules depended on acceptor surface density, but for a given acceptor concentration, FRET increased with increasing acceptor:donor ratios (Fig. 5, b and d). In contrast, FRET between A2-T134K-CFP and A2-YFP was insensitive to the acceptor:donor ratio, indicating that these molecules were randomly distributed relative to one another (Fig. 5, c and d, also compare with the theoretical predictions in Fig. 1b). After lactacystin treatment, which increased the fraction of TAP-bound HLA-A2 molecules (Fig. 2f), FRET among HLA-A2 molecules no longer depended on the donor-to-acceptor ratio (Fig. 6, a and d), whereas FRET among A2-T134K molecules did (Fig. 6, b and d). Thus, cutting off peptide supply reduced the fraction of clustered HLA-A2 molecules, without affecting A2-T134K clustering, confirming that the clusters we observed at steady state were not mediated by TAP. Lactacystin had no effect on FRET in the mixture of HLA-A2 and A2T134K (Fig. 6, c and d). When Tax peptides were added to otherwise untreated cells, a fraction of the molecules in the mixture of HLA-A2 and A2T134K was now clustered, because for a given surface concentration of acceptor, FRET between these molecules depended on the donor-to-acceptor ratio (Fig. 7, c and d). Peptide addition had no effect on FRET among HLA-A2 or among A2-T134K molecules (Fig. 7b– d).

Table I. Diffusion coefficients (D), mobile fraction (R), and 95% confidence limits (C.L.) for HLA-A2 and A2-T134K Molecule

Treatment

A2-YFP Lactacystin Tax peptide Lactacystin ⫹ Tax peptide A2-T134K-YFP Lactacystin Tax peptide Lactacystin ⫹ Tax peptide

D (⫻10⫺9 cm2s⫺1) ⫾ 95% C.L.

R (%) ⫾ 95% C.L.

2.3 ⫾ 0.2 1.7 ⫾ 0.3 2.5 ⫾ 0.3 2.5 ⫾ 0.4 3.5 ⫾ 0.3 3.7 ⫾ 0.3 3.5 ⫾ 0.3 3.7 ⫾ 0.3

70 ⫾ 4 73 ⫾ 4 71 ⫾ 3 72 ⫾ 4 80 ⫾ 3 83 ⫾ 3 82 ⫾ 2 81 ⫾ 2

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FIGURE 3. FRET was measured by imaging the increase in CFP fluorescence after photobleaching YFP. Images are pseudocolored for fluorescence intensity, and the color scheme is shown in the bar on the lower right. a, Donor fluorescence before bleaching the acceptor (CFPpre); b, acceptor fluorescence before photobleaching (YFPpre); c, donor fluorescence after bleaching the acceptor (CFPpost); and d, acceptor fluorescence after the bleach (YFPpost).

Discussion Tagging with CFP or YFP did not significantly interfere with the folding, the intracellular processing, and the associations of the class I molecules with TAP (Fig. 2) (25). This allowed us to use FRET between CFP- and YFP-tagged class I molecules to monitor molecular clustering during their assembly in and export from the ER. Changes in the fraction of TAP-associated HLA-A2 molecules were measured biochemically and in terms of changes in lateral diffusion in the ER membrane using FRAP. D measured for A2YFP in HeLa cells was comparable with that reported for A2-GFP in Mel JuSo cells (26). However, whereas adding peptide increased D of the mouse MHC class I molecule, H2Ld-GFP, in L cells (17), it had no detectable effect on D of HLA-A2 in HeLa cells. This probably reflects the relatively low affinity, 6 ⫻ 107 M⫺1 (22, 27), of Tax peptide for HLA-A2 compared with the 30-fold higher affinity of mouse cytomegalovirus peptide amino acid sequence YPHFMPTNL (17) peptide for H2Ld. Exogenous peptides reached the ER of HeLa cells, because they decreased the number of A2-YFP molecules coimmunoprecipitating with TAP (Fig. 2f), and also reversed the effects of lactacystin on D. However, the fraction of TAP-bound HLA-A2 displaced by Tax was much smaller than reported for mouse cytomegalovirus peptide amino acid sequence YPHFMPTNL (17) peptide and H2Ld (17). D for A2-T134K was higher than D for wild-type HLA-A2 under all conditions. This is consistent with the inability of A2T134K to bind to TAP; however, because each of these constructs was expressed in a stable, clonal, cell line, it may be that the maximum value for D represents clonal variation in some aspect of the ER affecting lateral diffusion in its membrane. Clonal variation may also account for the slightly higher mobile fraction of A2T134K-YFP molecules relative to A2-YFP.

FRET was measured by imaging the increase in CFP fluorescence after YFP bleaching (Fig. 3). An advantage of this method is that the experiments are internally controlled: the parameters needed to calculate FRET (donor fluorescence in the presence and the absence of the acceptor) can be obtained from the same cells, without having to correct for absolute donor and acceptor concentrations. Furthermore, in contrast to quantitative measurements of acceptor-sensitized emission due to FRET, this method does not require the experimental determination of spectral correlation factors (8).

FIGURE 4. Dependence of FRET on acceptor (YFP) concentration in cells expressing CFP and YFP physically linked to the cytoplasmic tail of HLA-A2. Each point represents the calculated FRET and the mean value of YFP fluorescence, from a 5 ⫻ 5-pixel square placed on the nuclear membrane of the cells. In untreated cells, FRET was independent of YFP concentration (䡺). This distribution did not change upon treatment with 100 ␮M lactacystin for 1.5 h ( ) or 250 ␮M Tax peptide for 1.5 h (F).

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CLUSTERING OF PEPTIDE-LOADED MHC CLASS I REVEALED BY FRET

FIGURE 5. Dependence of FRET on YFP concentration for different acceptor:donor ratios: YFP:CFP, ⬃1:2 (䡺); YFP:CFP, ⬃1:1 ( ); YFP:CFP, ⬃2:1 (F). a, In cells expressing A2-YFP and A2-CFP, FRET depended both on YFP concentration and the ratio of YFP:CFP. b, In cells expressing A2-T134K-YFP and A2-T134K-CFP, FRET depended both on YFP concentration and the ratio of YFP:CFP. c, In cells expressing A2-YFP and A2-T134K-CFP, FRET depended on YFP concentration, but was independent of the ratio of YFP:CFP. d, The mean FRET (%) for YFP concentration in the range 1500 –2500 fluorescence units for YFP:CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (o), A2-T134K-YFP and A2-T134K-CFP (p), and A2-YFP and A2-T134KCFP (s).

As expected, the positive control, in which CFP and YFP were physically linked to the C terminus of HLA-A2, reported that all fluorescent molecules were clustered (Fig. 4). This set an upper limit for our observations on FRET between mixtures of CFP- and YFP-labeled molecules. At steady state, these mixtures showed that some HLA-A2 molecules were clustered (Fig. 5, a and d) and some A2-T134K molecules were clustered (Fig. 5, b and d), but no clustering could be detected in the FRET profile of a mixture of HLA-A2 and A2-T134K (Fig. 5, c and d). Given the 4:1 stoichiometry of MHC class I association with TAP, we expected that lactacystin treatment would increase the fraction of clustered HLA-A2 molecules, because it increased the fraction bound to TAP (Fig. 2f). Surprisingly, FRET did not detect any clustered HLA-A2 molecules after lactacystin treatment (Fig. 6, a and d), although the A2-T134K clusters were not perturbed by the proteasome inhibitor (Fig. 6, b and d), consistent with the inability of A2-T134K to bind TAP (Fig. 2e) (11, 12). Because

HLA-A2 can bind peptides derived from proteasome-independent signal sequences, it is possible that some of the clustered A2T134K molecules are still peptide loaded. To be out of FRET range, the fluorophores of the multiple HLA-A2 molecules bound to TAP must be separated by ⬎80 Å, because R0, the distance for 50% FRET between CFP and YFP, is 60 Å. This is consistent with our earlier calculation that the TAP complex is large, ⬃600 –1000 Å in diameter (17). It is also consistent with our recent observation that FRET between mouse TAP1d-CFP and H2Ld-YFP was less than 10% even after lactacystin treatment (data not shown). FRET among HLA-A2 molecules or among A2-T134K molecules did not change after the addition of Tax peptides (Fig. 7, a, b, and d). It depended both on acceptor surface density and, for a given acceptor density, on the donor-to-acceptor ratio, indicating that each labeled population was a mixture of clustered and randomly distributed molecules. However, after peptide addition, FRET between A2-YFP and A2-T134K-CFP depended both on

FIGURE 6. Dependence of FRET on YFP concentration after treatment with 100 ␮M lactacystin for 1.5 h for different acceptor:donor ratios: YFP:CFP, ⬃1:2 (䡺); YFP:CFP, ⬃1:1 ( ); YFP:CFP, ⬃2:1 (F). a, In cells expressing A2-YFP and A2-CFP, FRET depended on YFP concentration but was independent of the YFP:CFP ratio. b, In cells expressing A2T134K-YFP and A2-T134K-CFP, FRET depended both on YFP concentration and the ratio of YFP:CFP. c, In cells expressing A2-YFP and A2-T134K-CFP, FRET depended on YFP concentration, but was independent of the YFP:CFP ratio. d, The mean FRET (%) for YFP concentration in the range 1500 –2500 fluorescence units for YFP:CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (o), A2T134K-YFP and A2-T134K-CFP (p), and A2-YFP and A2-T134K-CFP (s).

The Journal of Immunology

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FIGURE 7. Dependence of FRET on YFP concentration after addition of 250 ␮M Tax peptide for 1.5 h for different acceptor:donor ratios: YFP:CFP, ⬃1:2 (䡺); YFP: CFP, ⬃1:1 ( ); YFP:CFP, ⬃2:1 (F). In cells expressing A2-YFP and A2-CFP (a), A2-T134K-YFP and A2T134K-CFP (b), or A2-YFP and A2-T134K-CFP (c), FRET depended both on YFP concentration and the ratio of YFP:CFP. d, The mean FRET (%) for YFP concentration in the range 1500 –2500 fluorescence units for YFP: CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (o), A2-T134K-YFP and A2-T134K-CFP (p), and A2-YFP and A2-T134K-CFP (s).

acceptor surface density and, for a given acceptor density, on the donor-to-acceptor ratio, indicating that some of these molecules were also clustered (Fig. 7, c and d), even though they are loaded with peptide by different mechanisms and at different sites in the ER (see below). Taken together, the data indicate that MHC class I molecules are clustered after peptide loading, either in proximity to TAP (HLAA2) or elsewhere in the ER (A2-T134K). This strongly suggests that MHC class I molecules are clustered for export out of the ER. MHC class I exit from the ER may occur by either nonselective bulk flow (28) or by specific receptor-mediated export (29). The specific clustering of peptide-loaded MHC that we observed supports the second ER export mechanism. It is generally believed that transmembrane proteins are selectively recruited into ER exit sites by interactions of their cytoplasmic tails with the coat protein complex (COPII) coat (30, 31). HLA-A2 lacks either of the putative signals for ER export, the di-phenylalanine (FF), or the diacidic (D⫻E) motifs (32–36). Conceivably, it may contain another, yet unidentified, ER exit signal. However, because COPII proteins are concentrated in distinct punctate structures, known as ER exit sites (37, 38), and we randomly sampled uniformly fluorescent regions of the nuclear envelope, if COPII is responsible for the clusters that we observed, it must form them before the proteins reach the ER exit sites. Another possibility is that the clusters are created by class I interactions with cargo receptors for transmembrane proteins. Both of the known cargo receptors, ERGIC-53 and p24, are multimeric transmembrane complexes (for reviews, see Refs. 39 and 40). ERGIC-53 exists as dimers and hexamers (39), whereas p24 complexes are heterotetramers (41, 42). If the exit of MHC class I molecules involves interactions with specific cargo receptors, and if these have the properties of known cargo receptors, then HLA-A2 molecules should be clustered for export. The observation of clusters containing only HLA-A2 molecules or only A2-T134K molecules and the failure to observe steady state clustering in the mixture of HLA-A2 and A2-T134K probably reflects differences in their spatial distribution for export, i.e., their physical segregation in distinct ER subdomains. It is possible that HLA-A2 molecules are sequestered immediately after their TAP dissociation by factors selecting cargo for the vesicles leaving the ER and transported along the secretory pathway. In contrast,

A2-T134K does not bind to TAP and leaves the ER loaded with peptides of suboptimal affinity (43). It is likely that the peptideloaded A2-T134K molecules are also capable of interacting with the cargo-selecting factors; however, they will not bind them in proximity to TAP, but elsewhere in the ER. In this scenario, in the mixed population, there are separate clusters containing HLA-A2 or A2-T134K molecules, but few, if any, clusters containing both proteins. The addition of high affinity peptides that reach the ER independently of TAP may enable both wild-type and mutant molecules to bind peptide and complete their folding away from TAP. Thus, they would have an equal chance of binding to the factors selecting cargo for export. Clustering of peptide-loaded MHC class I molecules immediately after their dissociation from TAP, as our data indicate, may enhance host response against viral infections. It is likely that in cases in which viruses appropriate the host protein synthesis machinery for the dedicated production of their own proteins, the clusters would contain class I molecules loaded almost exclusively with a few dominant viral peptides. Assuming that the clusters persist throughout the secretory pathway until their delivery at the plasma membrane, they might simultaneously engage multiple TCRs, and as a result, may constitute better targets than single molecules. The question whether the cargo receptors responsible for their formation, assuming they exist, would be dedicated to MHC class I export or would be shared with other transmembrane proteins, as well as their actual identification, is a matter of future investigation.

Acknowledgments We thank Dr. J. Frelinger for providing the HMy2.C1R.T134K cells; T. Wei and A. Nechkin of the Integrated Imaging Center, Department of Biology, for expert technical support; and Dr. D. Marguet, Dr. A. Kenworthy, and E. Spiliotis for technical advice.

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