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Department of Biology, Johns Hopkins University, Baltimore, MD 21218
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ER-resident 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, 7, 8, 9, 10) have developed a quantitative
technique, fluorescence resonance energy transfer (FRET) microscopy,
which can detect clusters of proteins carrying appropriate
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).
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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 contrast, addition of exogenous peptides that reached the ER independently of TAP (16, 17) resulted in some coclustering of wild-type 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg/ml G418 (Life Technologies), and antibiotic/antimycotic (100 U/ml penicillin G (sodium salt), 100 µg/ml streptomycin sulfate, and 25 µg/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 µg/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 µg/ml hygromycin B. Expression of the transfected molecules was induced with 2 µg/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-A2-T134K, 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 µg/ml G418.
The mAb BB7.2 (ATCC HB-84) (20) was purified from
hybridoma supernatants using GammaBindPlus Sepharose (Pharmacia
Biotech, Piscataway, NJ). It recognizes a determinant in the 
2
domain and is specific for conformed HLA-A2 molecules. Cy3-conjugated
F(ab')2 goat anti-mouse 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 µM.
Gene construction
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 µg/ml BB7.2 mAb for 1 h at 4°C, then washed in PBS, and incubated with 5 µg/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 µCi/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 µg/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 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 µM Tax peptide or 100 µM 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.
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 x100 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 x 5-pixel (340 nm x 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 range 1500–2500 fluorescence units, because in this range all plots of FRET vs YFP concentration reached a plateau for all YFP:CFP ratios.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µM lactacystin for 1.5 h
or 250 µM 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 A2-YFP 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 µM Tax peptide for 1.5 h (Fig. 2f,
"mix"). Densitometric analysis showed that the same number of
molecules coimmunoprecipitated with TAP under both conditions.
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. At steady state, the diffusion
coefficient (D) represents the average of two populations of
MHC class I: TAP bound (D 1 x
10-9cm2s-1)
and TAP free (D 4 x
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 x 10-9
cm2s-1 ± 2 x
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 x
10-9
cm2s-1 ± 3 x
10-10
cm2s-1, consistent with
the biochemical evidence that A2-T134K does not bind TAP at all
(11, 12) (Fig. 2
e). Lactacystin treatment
lowered the average A2-YFP diffusion to D 1.7 x
10-9
cm2s-1 ± 3 x
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-T134K-YFP. 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 x 10-9
cm2s-1 ± 4 x
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.
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). 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
occurringbefore the destruction of the acceptor: FRET (%) =
100 x(CFPpost -
CFPpre)/CFPpost.
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, compare with the
theoretical predictions in Fig. 1a).
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a, 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 A2-T134K molecules were also clustered.
FRET between labeled A2-T134K 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).
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f), 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 A2-T134K (Fig. 6, c and d).
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, c and d).
Peptide addition had no effect on FRET among HLA-A2 or among A2-T134K
molecules (Fig. 7b–d).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (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 A2-YFP 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 x
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 A2-T134K 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 A2-T134K-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).
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 A2-T134K 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 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 (HLA-A2) 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 di-acidic (DxE) motifs (32, 33, 34, 35, 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 peptide-loaded 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Edidin, Department of Biology, Johns Hopkins University, Baltimore, MD 21218. E-mail address: edidin{at}jhu.edu
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.
Received for publication November 13, 2000. Accepted for publication March 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation in cells. Science 283:2085.2 domain of HLA-A2.1 define a functionally relevant interaction with TAP. Curr. Biol. 6:873.[Medline]
Ray-induced loss of expression of HLA and glyoxalase I alleles in lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 77:4251.3): complex formation with other p24 family members. Mol. Biol. Cell 10:1939.This article has been cited by other articles:
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  A. Tuli, M. Sharma, M. M. McIlhaney, J. E. Talmadge, N. Naslavsky, S. Caplan, and J. C. Solheim Amyloid Precursor-Like Protein 2 Increases the Endocytosis, Instability, and Turnover of the H2-Kd MHC Class I Molecule J. Immunol., August 1, 2008; 181(3): 1978 - 1987. [Abstract] [Full Text] [PDF]
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 G. R. Chichili and W. Rodgers Clustering of Membrane Raft Proteins by the Actin Cytoskeleton J. Biol. Chem., December 14, 2007; 282(50): 36682 - 36691. [Abstract] [Full Text] [PDF]
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M. W. Everett and M. Edidin Tapasin Increases Efficiency of MHC I Assembly in the Endoplasmic Reticulum but Does Not Affect MHC I Stability at the Cell Surface J. Immunol., December 1, 2007; 179(11): 7646 - 7652. [Abstract] [Full Text] [PDF]
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J. J. Ladasky, S. Boyle, M. Seth, H. Li, T. Pentcheva, F. Abe, S. J. Steinberg, and M. Edidin Bap31 Enhances the Endoplasmic Reticulum Export and Quality Control of Human Class I MHC Molecules J. Immunol., November 1, 2006; 177(9): 6172 - 6181. [Abstract] [Full Text] [PDF]
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L. H. Boyle, A. K. Gillingham, S. Munro, and J. Trowsdale Selective Export of HLA-F by Its Cytoplasmic Tail. J. Immunol., June 1, 2006; 176(11): 6464 - 6472. [Abstract] [Full Text] [PDF]
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S. Bhatia, M. Edidin, S. C. Almo, and S. G. Nathenson Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling PNAS, October 25, 2005; 102(43): 15569 - 15574. [Abstract] [Full Text] [PDF]
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). This distribution did not change upon treatment
with 100 µM lactacystin for 1.5 h
(
) or 250 µM
Tax peptide for 1.5 h (•).
), A2-T134K-YFP and A2-T134K-CFP
(
), and A2-YFP and A2-T134K-CFP (
).
