This information is current as
of June 16, 2017.
Cutting Edge: Tapasin Is Retained in the
Endoplasmic Reticulum by Dynamic
Clustering and Exclusion from Endoplasmic
Reticulum Exit Sites
Tsvetelina Pentcheva, Elias T. Spiliotis and Michael Edidin
J Immunol 2002; 168:1538-1541; ;
doi: 10.4049/jimmunol.168.4.1538
http://www.jimmunol.org/content/168/4/1538
Subscription
Permissions
Email Alerts
This article cites 38 articles, 22 of which you can access for free at:
http://www.jimmunol.org/content/168/4/1538.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
References
●
Cutting Edge: Tapasin Is Retained in the
Endoplasmic Reticulum by Dynamic Clustering and
Exclusion from Endoplasmic Reticulum Exit Sites1
Tsvetelina Pentcheva, Elias T. Spiliotis, and Michael Edidin2
he MHC class I heterotrimer, heavy chain, ␤2-microglobulin
and peptide, is assembled in the endoplasmic reticulum
(ER)3 lumen via interactions with ER-resident chaperones
and TAP and is subsequently transported to the plasma membrane (1).
One of the chaperones, tapasin, increases local peptide concentration
by tethering empty heavy chain/␤2-microglobulin dimers to the TAP
complex (2, 3), stimulates peptide transport by stabilization of the
TAP heterodimer (4, 5), stabilizes empty MHC dimers in a peptidereceptive conformation (3, 6), retains empty class I molecules until
peptide loading (7–9), and optimizes the affinity of class I-bound peptides by acting as a peptide editor (8, 10, 11).
Understanding the multiple roles of tapasin in MHC assembly requires understanding the mechanism of its retention in the ER. Tapasin bears a C-terminal dilysine (KKKXX) motif that could mediate its
T
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Received for publication November 2, 2001. Accepted for publication December
14, 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, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD
21218. E-mail address: [email protected]
3
Abbreviations used in this paper: ER, endoplasmic reticulum; CFP, cyan fluorescent
protein; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COPI/II, coat protein
I/II; endo H, endoglycosidase H; ERGIC, ER-to-Golgi intermediate compartment;
FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance
energy transfer.
Copyright © 2002 by The American Association of Immunologists
●
retrieval from post-ER compartments via associations with the coat
protein I (COPI) vesicle coat (13–18). However, KKAA retains a
CD4 chimera in the ER in the absence of COPI; therefore, the motif
could also effect direct ER retention (19). Several molecular mechanisms have been proposed to account for such retention, including
tight binding to microtubules (20), formation of large oligomers that
cannot be included into transport vesicles (21), or interactions with a
matrix of ER-resident proteins (22–25).
To determine the mechanism of tapasin-mediated class I retention, we investigated the intracellular organization of tapasin,
tagged with yellow fluorescent protein (YFP) or cyan fluorescent
protein (CFP) at the N terminus, by deconvolution fluorescence
microscopy, fluorescence recovery after photobleaching (FRAP),
and fluorescence resonance energy transfer (FRET). Tapasin did
not appear to reach the medial Golgi or to cycle rapidly between
the ER and the ER-to-Golgi-intermediate compartment (ERGIC);
it was excluded from ER exit sites under conditions favoring cargo
accumulation in those regions of the ER. Furthermore, whereas FRET
showed that all tapasin molecules were clustered, FRAP showed that
these oligomers were freely diffusing and highly mobile, making unlikely the possibility that tapasin is retained by association with a
static protein matrix. The data indicate that tapasin, and possibly class
I molecules bound to tapasin, form diffusing oligomers that are not
recognized by the ER export machinery.
Materials and Methods
Cells, Abs, and reagents
HeLa Tet-On cells (Clontech Laboratories, Palo Alto, CA) were maintained and transfected as described previously (26). The 721.220.B8 (.220)
cell line, a gift from Dr. P. Cresswell (Yale University, New Haven, CT),
was maintained as described (4). It was transfected with YFP-tapasin using
electroporation, selected, and sorted three times. mAbs HC10 (27), BB7.2
(28), and W6/32 (29) were purified as described (26). mAb G1/93, recognizing human ERGIC-53 (30), was a gift of Dr. H.-P. Hauri, University of
Basel (Basel, Switzerland). Other Abs were purchased from commercial
suppliers. Oligonucleotides were synthesized and purified by Integrated
DNA Technologies (Coralville, IA).
Gene construction
To generate N-terminal fusions of tapasin to YFP and CFP, the signal
sequence of the tapasin cDNA (3), a gift of Dr. P. Cresswell, was deleted
by PCR. The YFP linker fragment from the positive control for FRET (26)
was excised and ligated into the tapasin construct above, which had been
cloned into vector pEGFP-N3 (Clontech) lacking the green fluorescent protein (GFP). CFP and YFP were fused to the folate receptor signal sequence
and the myc tag from a GFP-GPI construct, a gift of Dr. S. Lacey (University of Texas Southwestern Medical Center, Dallas, TX), using PCR.
These fusions were then ligated into the leaderless tapasin construct above
lacking the YFP tag. The inserts were also introduced in the pBI vector
(Clontech).
0022-1767/02/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Tapasin retains empty or suboptimally loaded MHC class I
molecules in the endoplasmic reticulum (ER). However, the
molecular mechanism of this process and how tapasin itself is
retained in the ER are unknown. These questions were addressed by tagging tapasin with the cyan fluorescent protein or
yellow fluorescent protein (YFP) and probing the distribution
and mobility of the tagged proteins. YFP-tapasin molecules
were functional and could be isolated in association with TAP,
as reported for native tapasin. YFP-tapasin was excluded from
ER exit sites even after accumulation of secretory cargo due to
disrupted anterograde traffic. Almost all tapasin molecules
were clustered, and these clusters diffused freely in the ER.
Tapasin oligomers appear to be retained by the failure of the
export machinery to recognize them as cargo. The Journal of
Immunology, 2002, 168: 1538 –1541.
The Journal of Immunology
Pulse-chase, immunoprecipitations, and Western blotting
Cells were incubated in cysteine- and methionine-free medium containing
10% dialyzed FBS for 30 min, labeled with 500 ␮Ci/ml Tran 35S-label
(ICN Pharmaceuticals, Costa Mesa, CA) for 90 min and chased in complete medium for 16 h as described (26). They were lysed in 0.5% Triton
X-100 (Sigma, St. Louis, MO) and precleared, and tapasin was immunoprecipitated with anti-tapasin serum (StressGen Biotechnologies, Victoria,
Canada) and treated with endoglycosidase H (endo H), as described (26).
For sequential immunoprecipitations with anti-TAP1 and anti-tapasin
antisera, 3 million cells were treated with 400 U/ml human IFN-␥ for 48 h
before labeling. Cells were starved in methionine- and cysteine-free medium containing 5% dialyzed FBS for 60 min, pulsed with 1.25 mCi/ml
Tran 35S-label for 5 min, and chased for 75 min in medium containing 3
mM methionine and cysteine. Solubilization, preclearing, and immunoprecipitation were all performed as previously described (4, 31).
For Western blotting, cells were lysed in buffer containing 1% 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). All
immunoprecipitations and protein transfers were performed as described
(32). Membranes were incubated with anti-tapasin serum and HRP-conjugated anti-rabbit Ab and visualized by ECL (Amersham Pharmacia Biotech, Piscataway, NJ).
Deconvolution fluorescence microscopy
tapasin function. Expression of the chimeric molecule in tapasindeficient .220 cells restored MHC class I surface expression (Fig.
1A) and association of MHC class I heavy chains with TAP (Fig.
1B). The level of MHC class I expression was proportional to the
level of YFP-tagged tapasin over a 10- to 20-fold range of tapasin
expression (data not shown). In digitonin lysates of HeLa cells,
most molecules of YFP-tapasin and endogenous tapasin were
bound to TAP (Fig. 2A), similarly to endogenous tapasin in nontransfected cells (31). There was a small population of TAP-free
molecules; this was also observed for native tapasin in overexposed gels (Fig. 2A, inset). In addition to TAP, YFP-tapasin also
associated with calnexin, calreticulin (Fig. 2B) and MHC class I
heavy chains (Fig. 2C).
Deconvolution microscopy showed that YFP-tapasin localized
exclusively to the ER (Fig. 3A). In pulse-chase experiments, the
YFP-tapasin carbohydrates remained fully sensitive to endo H digestion, indicating that they were never processed by medial Golgi
enzymes (Fig. 3B, upper gel). At steady state, endogenous and
YFP-tagged tapasin molecules were also fully sensitive to endo H
digestion (Fig. 3B, lower gel). To see whether tapasin localization
to the ER was due to retrieval from earlier post-ER compartments
(i.e., ERGIC, cis-Golgi), the transfected cells were incubated at
15°C or treated with nocodazole. Incubation at 15°C leads to the
formation of a nonphysiological hybrid compartment resulting
from the fusion of ER exit sites with the ERGIC (33). Nocodazole
Flow cytometry, FRAP measurements, and FRET microscopy
Cells were stained with mAb W6/32 at 4°C and analyzed by flow cytometry,
as described (26). The lactacystin and peptide treatments, lateral diffusion measurements at 37°C by FRAP, and FRET measurements were as detailed in our
earlier papers (26, 32). FRET was calculated from five 10 ⫻ 10-pixel (680 ⫻
680 nm) nuclear envelope regions per cell as the percent increase in CFP
fluorescence after YFP photobleaching, as described (26).
Results and Discussion
Because tapasin contains a C-terminal KKKXX motif (3, 12), we
fused YFP to its N terminus. The YFP tag did not interfere with
FIGURE 1. YFP-tapasin (tpn, TPN) restores class I surface expression
and TAP association in .220 cells. A, Tapasin-deficient .220 cells (gray
line) or .220 cells transfected with YFP-tagged tapasin (black line) were
stained with mAb W6/32 and Cy3-conjugated F(ab⬘)2 goat anti-mouse IgG.
B, Tapasin-deficient .220 or .220 cells transfected with YFP-tapasin were lysed
in 1% CHAPS and immunoprecipitated with anti (␣)-TAP1 antiserum or with
mAb W6/32. The samples were transferred to nitrocellulose filters and probed
with anti-TAP1 or mAb HC10 (anti-HLA free heavy chains).
FIGURE 2. YFP-tapasin (tpn, TPN) interacts with the components of
the peptide-loading complex. A, HeLa cells were metabolically labeled
with [35S]methionine for 5 min and chased for 75 min. After solubilization
in buffer containing 1% digitonin, TAP-bound tapasin molecules were precipitated with anti (␣)-TAP1 antiserum, and TAP-free molecules were sequentially precipitated with anti-tapasin antiserum. Inset, Bands of endogenous tapasin in overexposed autoradiographs. B, HeLa cells expressing
YFP-tapasin (lane b) were lysed in 1% CHAPS and immunoprecipitated
with anti-calnexin (clnx), anti-calreticulin (clrt), and anti-tapasin (tpn) antisera. Untransfected HeLa cells were used as a negative control (lane a).
The samples were blotted with anti-tapasin antiserum. C, Untransfected
and YFP-tapasin-expressing HeLa cells were lysed in 1% digitonin and
treated with anti-GFP antiserum. Immunoprecipitates were blotted for
HLA heavy chains with mAb HC10. As a positive control for HLA heavy
chains, a fraction of HeLa cell lysate was run next to the precipitates.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Cells fixed in 4% paraformaldehyde in PBS were washed with 0.25%
NH4Cl in PBS and permeabilized with 0.2% saponin in PBS-1% BSA.
Coverslips were mounted in SlowFade Light (Molecular Probes, Eugene,
OR) and imaged on a DeltaVision deconvolution system (Applied Precision), using a 1.4 numerical aperture 100⫻ Zeiss Plan-apochromat objective. Images were collected with a 12-bit CH300-cooled charge-coupled
device (Roper Scientific, Trenton, NJ) and out-of-focus light was subtracted using the softWoRx software (Applied Precision, Issaquah, WA).
1539
1540
CUTTING EDGE: DYNAMIC CLUSTERS OF TAPASIN ESCAPE ER EXPORT
Table I. Diffusion coefficients (D) mobile fraction (R) and 95%
confidence limits (CL) for YFP-tapasin and A2-YFP
Molecule
Treatment
YFP-tapasin only
YFP-tapasin ⫹ untagged
HLA-A2
Lactacystin
Lactacystin
Tax peptide
A2-YFP
Lactacystin
causes the accumulation of secretory cargo and proteins that cycle
between the ER and the ERGIC at ER exit sites consequent to
microtubule depolymerization (33). YFP-tapasin was excluded
from the ER exit sites, even after incubation at 15°C (Fig. 3C) or
treatment with nocodazole (Fig. 3D), whereas the ERGIC marker
ERGIC-53 accumulated in the hybrid compartment after incubation at 15°C (Fig. 3C) or at discrete ER exit sites after nocodazole
treatment (Fig. 3D). These data are not consistent with ER retention of tapasin by tight binding to microtubules or by continuous
retrieval from a post-ER compartment. They also argue against
rapid tapasin cycling between the ER and the ERGIC as a possible
mechanism for retrieving suboptimally loaded class I molecules.
YFP-tapasin diffusion was high (3.2 ⫾ 0.5 ⫻ 10⫺9 cm2s⫺1),
comparable with the diffusion of A2-YFP (Table I). Because treatment with lactacystin decreased A2-YFP diffusion as a result of an
increased A2-YFP fraction bound to TAP (Table I; Ref. 26), its
effect on YFP-tapasin was also investigated. Lactacystin treatment
did not affect YFP-tapasin diffusion (Table I). Because FRAP of
lactacystin-treated cells detected TAP binding by a fraction of the
total A2-YFP, it should have detected an increased fraction of TAP
binding by YFP-tapasin in these cells. The lack of effect of lactacystin shows that at steady state, all tapasin-binding sites at TAP
are already occupied and that this association is not static but involves rapid cycles of association and dissociation. Besides associating with the peptide-loading complex (TAP1/2, MHC class I,
calreticulin, ERp57), tapasin molecules are also known to form
complexes with TAP alone and with TAP bound to calnexin and
ERp57 (31). Lactacystin would not change the diffusion coefficient
(D) observed for YFP-tapasin if most tapasin molecules are dynamically bound to subcomplexes of TAP that remain free of
3.2 ⫾ 0.5
3.4 ⫾ 0.4
2.8 ⫾ 0.2
88 ⫾ 4
87 ⫾ 3
82 ⫾ 3
3.1 ⫾ 0.3
3.1 ⫾ 0.3
3.0 ⫾ 0.3
2.1 ⫾ 0.3
80 ⫾ 3
85 ⫾ 4
67 ⫾ 4
78 ⫾ 6
MHC dimers, whereas only a small fraction forms a stable bridge
between TAP and MHC class I. Overall, the diffusion data indicate
that the mechanism of tapasin retention in the ER is dynamic and
does not appear to involve stable interactions with TAP or an ER
matrix. Similar observations have been made for the ER retention
of misfolded vesicular stomatitis virus G protein-GFP (25).
FRET between fluorescent tapasin molecules depended on the
YFP:CFP ratio and was insensitive to the YFP surface density both
at steady state (Fig. 4, A and C) and after treatment with lactacystin
(Fig. 4, B and C). These characteristics are hallmarks of a clustered
FIGURE 4. All tapasin molecules are clustered. In cells expressing
tagged tapasin, FRET was largely insensitive to YFP concentration but
depended on the ratio of YFP to CFP; YFP:CFP ⬃1:1 (䡺), YFP: CFP
⬃2:1 ( ), YFP: CFP ⬃3:1 (F) at steady state (A) and after treatment with
100 ␮M lactacystin (B). C, The mean FRET (%) for YFP concentration in
the range 1500 –2500 fluorescence units for YFP:CFP ratios of 2:1 and 3:1,
as well as the 98% confidence limits (bars) were plotted for untreated cells
(o; a) and for cells treated with lactacystin (p; b).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. YFP-tapasin (tpn) resides in the ER and does not appear to
cycle between the ER and the ERGIC/Golgi. HeLa cells were fixed, permeabilized, and stained with mAb G1/93 and Cy5-conjugated F(ab⬘)2 goat
anti-mouse IgG. The intracellular distribution of YFP-tapasin and human
ERGIC-53 was analyzed by fluorescence deconvolution microscopy at
steady state (A), after incubation at 15°C for 3 h (C), or after treatment with
5 ␮g/ml nocodazole for 2 h at 37°C (D). The images are overlays of
YFP-tapasin (green) and ERGIC-53 (red) fluorescence. Scale bar, 10 ␮m.
B, Upper gel, HeLa cells expressing YFP-tapasin were metabolically labeled with [35S]methionine and chased in nonradioactive medium for 16 h.
The cells were lysed in 0.5% Triton X-100, immunoprecipitated with antitapasin serum and digested with Endo H. R and S refer to the expected
endo H-resistant and the endo H-sensitive forms of the proteins, respectively. Lower gel, HeLa cells expressing YFP-tapasin were lysed in 0.5%
Triton X-100. Mock treated and endo H-treated lysates were run on SDSPAGE and blotted with anti-tapasin antiserum.
D (⫻ 10⫺9 cm2s⫺1) R (%) ⫾
⫾ 95% CL
95% CL
The Journal of Immunology
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Acknowledgments
We thank Drs. Peter Cresswell, Hans-Peter Hauri, and Stephen Lacey for
cells, Abs, and plasmids; and Taiyin Wei, Andrew Nechkin, and Dr. Gerry
Sexton of the Integrated Imaging Center, Department of Biology, The
Johns Hopkins University, for expert technical support.
27.
References
29.
1. Cresswell, P., N. Bangia, T. Dick, and G. Diedrich. 1999. The nature of the MHC
class I peptide loading complex. Immunol. Rev. 172:21.
2. Sadasivan, B., P. J. Lehner, B. Ortmann, T. Spies, and P. Cresswell. 1996. Roles
for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class
I molecules with TAP. Immunity 5:103.
3. Ortmann, B., J. Copeman, P. J. Lehner, B. Sadasivan, J. A. Herberg,
A. G. Grandea, S. R. Riddell, R. Tampe, T. Spies, J. Trowsdale, and P. Cresswell.
1997. A critical role for tapasin in the assembly and function of multimeric MHC
class I-TAP complexes. Science 277:1306.
4. Lehner, P. J., M. J. Surman, and P. Cresswell. 1998. Soluble tapasin restores
MHC class I expression and function in the tapasin-negative cell line .220. Immunity 8:221.
5. Bangia, N., P. J. Lehner, E. A. Hughes, M. Surman, and P. Cresswell. 1999. The
N-terminal region of tapasin is required to stabilize the MHC class I loading
complex. Eur. J. Immunol. 29:1858.
6. Grandea, A. G., 3rd, P. J. Lehner, P. Cresswell, and T. Spies. 1997. Regulation
of MHC class I heterodimer stability and interaction with TAP by tapasin. Immunogenetics 46:477.
7. Schoenhals, G. J., R. M. Krishna, A. G. Grandea, 3rd, T. Spies, P. A. Peterson,
Y. Yang, and 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.
8. Barnden, M. J., A. W. Purcell, J. J. Gorman, and J. McCluskey. 2000. Tapasinmediated retention and optimization of peptide ligands during the assembly of
class I molecules. J. Immunol. 165:322.
9. Grandea, A. G., 3rd, T. N. Golovina, S. E. Hamilton, V. Sriram, T. Spies,
R. R. Brutkiewicz, J. T. Harty, L. C. Eisenlohr, and L. Van Kaer. 2000. Impaired
assembly yet normal trafficking of MHC class I molecules in Tapasin mutant
mice. Immunity 13:213.
10. Lewis, J. W., and T. Elliott. 1998. Evidence for successive peptide binding and
quality control stages during MHC class I assembly. Curr. Biol. 8:717.
11. Purcell, A. W., J. J. Gorman, M. Garcia-Peydro, A. Paradela, S. R. Burrows,
G. H. Talbo, N. Laham, C. A. Peh, E. C. Reynolds, J. A. Lopez De Castro, and
28.
30.
31.
32.
33.
34.
35.
36.
37.
38.
J. McCluskey. 2001. Quantitative and qualitative influences of tapasin on the
class I peptide repertoire. J. Immunol. 166:1016.
Li, S., H. O. Sjogren, U. Hellman, R. F. Pettersson, and P. Wang. 1997. Cloning
and functional characterization of a subunit of the transporter associated with
antigen processing. Proc. Natl. Acad. Sci. USA 94:8708.
Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. Retrieval of transmembrane
proteins to the endoplasmic reticulum. J. Cell Biol. 121:317.
Gaynor, E. C., S. te Heesen, T. R. Graham, M. Aebi, and S. D. Emr. 1994.
Signal-mediated retrieval of a membrane protein from the Golgi to the ER in
yeast. J. Cell Biol. 127:653.
Cosson, P., and F. Letourneur. 1994. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science 263:1629.
Letourneur, F., E. C. Gaynor, S. Hennecke, C. Demolliere, R. Duden, S. D. Emr,
H. Riezman, and P. Cosson. 1994. Coatomer is essential for retrieval of dilysinetagged proteins to the endoplasmic reticulum. Cell 79:1199.
Orci, L., B. S. Glick, and J. E. Rothman. 1986. A new type of coated vesicular
carrier that appears not to contain clathrin: its possible role in protein transport
within the Golgi stack. Cell 46:171.
Waters, M. G., T. Serafini, and J. E. Rothman. 1991. “Coatomer”: a cytosolic
protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349:248.
Andersson, H., F. Kappeler, and H. P. Hauri. 1999. Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to
retrieval. J. Biol. Chem. 274:15080.
Dahllof, B., M. Wallin, and S. Kvist. 1991. The endoplasmic reticulum retention
signal of the E3/19K protein of adenovirus-2 is microtubule binding. J. Biol.
Chem. 266:1804.
Weisz, O. A., A. M. Swift, and C. E. Machamer. 1993. Oligomerization of a
membrane protein correlates with its retention in the Golgi complex. J. Cell Biol.
122:1185.
Kreibich, G., B. L. Ulrich, and D. D. Sabatini. 1978. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I
and II, membrane proteins characteristics of rough microsomes. J. Cell Biol.
77:464.
Booth, C., and G. L. Koch. 1989. Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59:729.
Tatu, U., and A. Helenius. 1997. Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum. J. Cell Biol. 136:555.
Nehls, S., E. L. Snapp, N. B. Cole, K. J. Zaal, A. K. Kenworthy, T. H. Roberts,
J. Ellenberg, J. F. Presley, E. Siggia, and J. Lippincott-Schwartz. 2000. Dynamics
and retention of misfolded proteins in native ER membranes. Nat. Cell Biol.
2:288.
Pentcheva, T., and M. Edidin. 2001. Clustering of peptide-loaded MHC class I
molecules for endoplasmic reticulum export imaged by fluorescence resonance
energy transfer. J. Immunol. 166:6625.
Stam, N. J., H. Spits, and H. L. Ploegh. 1986. Monoclonal antibodies raised
against denatured HLA-B locus heavy chains permit biochemical characterization
of certain HLA-C locus products. J. Immunol. 137:2299.
Parham, P., and F. M. Brodsky. 1981. Partial purification and some properties of
BB7.2: a cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28. Hum. Immunol. 3:277.
Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein,
A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to
group A erythrocytes, HLA and other human cell surface antigens-new tools for
genetic analysis. Cell 14:9.
Schweizer, A., J. A. Fransen, T. Bachi, L. Ginsel, and H. P. Hauri. 1988. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulovesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol. 107:
1643.
Diedrich, G., N. Bangia, M. Pan, and P. Cresswell. 2001. A role for calnexin in
the assembly of the MHC class I loading complex in the endoplasmic reticulum.
J. Immunol. 166:1703.
Marguet, D., E. T. Spiliotis, T. Pentcheva, M. Lebowitz, J. Schneck, and
M. Edidin. 1999. Lateral diffusion of GFP-tagged H2Ld molecules and of GFPTAP1 reports on the assembly and retention of these molecules in the endoplasmic reticulum. Immunity 11:231.
Hammond, A. T., and B. S. Glick. 2000. Dynamics of transitional endoplasmic
reticulum sites in vertebrate cells. Mol. Biol. Cell 11:3013.
Kenworthy, A. K., and M. Edidin. 1998. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a
resolution of ⬍100 A using imaging fluorescence resonance energy transfer [published erratum appears in 1998 J. Cell Biol. 142:881]. J. Cell Biol. 142:69.
Stephens, D. J., and R. Pepperkok. 2001. Illuminating the secretory pathway:
when do we need vesicles? J. Cell. Sci. 114:1053.
Kappeler, F., D. R. Klopfenstein, M. Foguet, J. P. Paccaud, and H. P. Hauri.
1997. The recycling of ERGIC-53 in the early secretory pathway: ERGIC-53
carries a cytosolic endoplasmic reticulum-exit determinant interacting with
COPII. J. Biol. Chem. 272:31801.
Dominguez, M., K. Dejgaard, J. Fullekrug, S. Dahan, A. Fazel, J. P. Paccaud,
D. Y. Thomas, J. J. Bergeron, and T. Nilsson. 1998. gp25L/emp24/p24 protein
family members of the cis-Golgi network bind both COP I and II coatomer.
J. Cell Biol. 140:751.
Spiliotis, E. T., H. Manley, M. Osorio, M. Zuniga, and M. Edidin. 2000. Selective
export of MHC class I molecules from the ER after their dissociation from, TAP.
Immmunity 13:841.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
fluorophore population (26, 34). The lack of effect of lactacystin
suggests that at least some of the clusters are located at TAP.
In conclusion, tapasin retention in the ER does not involve continuous retrieval from post-ER compartments and is not mediated
by static associations with microtubules or with an ER protein
matrix. If tapasin is excluded from ER-budding vesicles by forming large clusters ⬎60 – 80 nm in diameter (the size of a small
coated vesicle; Ref. 35), then D should be significantly lower than
that of export-competent MHC class I molecules (see Ref. 32).
However, in HeLa cells, D of tapasin (3.2 ⫾ 0.5 ⫻ 10⫺9 cm2 s⫺1)
closely resembles D of peptide-loaded HLA-A2 molecules (2.5–
3.5 ⫻ 10⫺9 cm2 s⫺1; Ref. 26). Therefore, we believe that tapasin
forms dynamic oligomers that are not recognized by the ER export
machinery. This interpretation is consistent with lack of phenylalanine at positions ⫺1 and ⫺2 of the C⬘-terminal sequence
(KKKAE) of tapasin. Phenylalanine-dependent interactions with
COPII have been shown for ERGIC-53 and the p24 protein family
(36, 37); alanine substitution (KKAA) results in ER retention (19).
The potential coclustering of tapasin and empty or suboptimally
loaded MHC class I molecules might ensure class I retention in the
ER until loading with high affinity peptides. However, such complexes could not be directly demonstrated because FRET between
YFP-tapasin and N-terminally tagged A2-CFP or A2-T134-CFP
was below 10% even at the highest YFP:CFP ratio (data not
shown), possibly because direct binding involves the tapasin N
terminus (5) and might place the two fluorescent tags out of FRET
range. Because MHC class I molecules cluster after peptide loading (26), perhaps as part of the mechanism for their export from the
ER, the incorporation of tapasin in such clusters may reduce the
multivalency of potential interactions between COPII and the cytoplasmic tails of MHC class I molecules or of their cargo receptors (38).
1541