Direct peptide-regulatable interactions between
MHC class I molecules and tapasin
Syed Monem Rizvi and Malini Raghavan†
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620
Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved October 10, 2006 (received for review June 21, 2006)
Tapasin (Tpn) has been implicated in multiple steps of the MHC
class I assembly pathway, but the mechanisms of function remain
incompletely understood. Using purified proteins, we could dem-
onstrate direct binding of Tpn to peptide-deficient forms of MHC
class I molecules at physiological temperatures. Tpn also bound to
M10.5, a pheromone receptor-associated MHC molecule that has
an open and empty groove and that shares significant sequence
identity with class I sequences. Two types of MHC class I–Tpn
complexes were detectable in vitro depending on the input pro-
teins; those depleted in ?2m, and those containing ?2m. Both were
competent for subsequent assembly with peptides, but the latter
complexes assembled more rapidly. Thus, the assembly rate of
Tpn-associated class I was determined by the conditions under
class I inhibited Tpn–class I-binding interactions, and peptide-
depletion enhanced binding. In combination with ?2m, certain
peptides induced efficient dissociation of preformed Tpn–class I
complexes. Together, these studies demonstrate direct Tpn–MHC
class I interactions and preferential binding of empty MHC class I
by Tpn, and that the Tpn–class I interaction is regulated by both
?2m and peptide. In cells, Tpn is likely to be a direct mediator of
peptide-regulated binding and release of MHC class I from the TAP
antigen presentation ? HLA ? TAP transporter
(?2m)] within the endoplasmic reticulum (ER) of cells and are
then transported to the cell surface, where they are available for
immune surveillance by cytotoxic T lymphocytes (1). The trans-
porter associated with antigen processing (TAP) is a peptide
transporter that translocates cytosolic peptides into the ER
lumen, for assembly with MHC class I HC and ?2m. Other ER
resident proteins that assist in MHC class I (class I) assembly
include the chaperones calnexin and calreticulin, the thiol-
disulfide isomerase ERp57, and the MHC-encoded transmem-
brane protein tapasin (Tpn). Individually or in combination,
these components ensure quality control of class I-peptide
For many human and mouse class I allotypes, Tpn increases
cell surface class I expression. How Tpn mediates this increase
has been a matter of considerable debate, and many functions
have been proposed for Tpn. Tpn stabilizes TAP, and increases
steady-state levels of TAP, thereby allowing more peptides to be
translocated into the ER (2, 3). Tpn also allows a physical link
between class I and the TAP (4), and by doing so, may increase
I binding. Tpn may also be important for preventing ER exit of
peptide-deficient class I (5–7), and may thereby indirectly pro-
mote the accumulation of more stable class I at the cell surface.
Soluble Tpn (sTpn) that does not stably bind TAP, enhance TAP
expression levels, or mediate the TAP–class I interaction, is able
that at least some of Tpn’s functions are independent of its
effects upon TAP. There are also indications from cell-based
experiments, that Tpn edits/optimizes the class I peptide reper-
eptide products of foreign and self-proteins bind to MHC
class I heavy chains (HC) and light chains [?2-microglubulin
toire (8), or directly facilitates peptide loading of class I mole-
The absence of a direct binding assay between Tpn and class
I has hampered a better understanding of the mechanisms of
Tpn’s function. Although cell-based experiments have demon-
strated class I-Tpn binding in the absence of other components
of the class I pathway (5, 12), there are no reports thus far of
direct Tpn–class I-binding interactions outside the context of a
cell. Here, we reconstituted the Tpn–class I interaction by using
purified proteins and investigated the effects of Tpn upon the
class I–peptide interaction, and the effects of peptide upon the
class I–Tpn interaction.
Tpn Can Bind to Peptide-Deficient Class I Molecules at Physiological
from CHO cells (Fig. 6, which is published as supporting
information on the PNAS web site), and soluble peptide-
deficient class I heterodimers were purified from insect cells (13,
14). Fluorescent peptides and native-PAGE-based assembly
assays (13) were used to ask whether sTpn binding influenced
peptide loading of soluble peptide-deficient HLA-A2 (A2) or
HLA-B*3501 (B35). Under the conditions that were examined,
no significant differences in peptide binding were observed in
the presence or absence of sTpn (Fig. 7 A–D, which is published
as supporting information on the PNAS web site).
Absence of a significant effect of sTpn on peptide loading
could arise because of absent or inefficient sTpn–class I-binding
interactions under these in vitro conditions, or because of
enhanced kinetics of class I peptide loading compared with sTpn
binding. Therefore, it was important to investigate binding
interactions between sTpn and A2. Equimolar amounts of A2
heterodimers and sTpn monomers were incubated for 2 h at 4°C
or 37°C. Samples were then immunoprecipitated with the anti-
FLAG antibody, proteins separated by SDS/PAGE, and visual-
ized by silver-staining to observe sTpn and any A2 that bound to
(IPs) after protein incubations at 37°C (Fig. 1A, lane 6), but not
4°C (Fig. 1A, lane 3).
To examine the specificity of the A2–sTpn interaction, we
related to class I. HFE (the protein mutated in hereditary
hemochromatosis) HC folds into a structure similar to class I
HC, and associates with ?2m, but does not bind peptides, and has
a closed peptide-binding groove (15). By IP assays similar to
Author contributions: S.M.R. and M.R. designed research; S.M.R. performed research;
S.M.R. and M.R. analyzed data; and S.M.R. and M.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: HC, heavy chain; ER, endoplasmic reticulum; TAP, transporter associated
with antigen processing; ?2m, ?2-microglubulin; class I, MHC class I; Tpn, tapasin; sTpn,
soluble Tpn; A2, HLA-A2; B35, HLA-B*3501; IP, immunoprecipitation; M10, M10.5.
†To whom correspondence should be addressed at: Department of Microbiology and
Arbor, MI 48109-0620. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
November 28, 2006 ?
vol. 103 ?
complexes were isolated could determine the assembly kinetics
of Tpn-associated MHC class I molecules.
In summary, our studies demonstrate that Tpn binds to
peptide-deficient class I, that physiological temperatures pro-
mote binding, and that conditions favorable for class I assembly
dissociate Tpn–class I complexes. In cells, Tpn interaction with
network comprising Tpn, ERp57, and the lectin chaperones,
which together, may create an environment that favors peptide
loading and assembly over degradation.
Proteins and Peptides. Soluble A2/?2m, B35/?2m, and full-length
Tpn expression in insect cells has been previously described (13,
14, 31). Expression and purification of soluble tapasin and ?2m
and detection of class I/Tpn complexes in insect cells are
described in Supporting Materials and Methods, which is pub-
lished as supporting information on the PNAS web site. Fluo-
rescently labeled versions of class I-binding peptides were ob-
tained by cysteine substitutions at positions 4 or 5 of the parent
sequences, and labeling with iodoacetamidofluorescein, as pre-
viously described (13, 14). Purified human HFE (15), mouse
M10.5 (16), and a baculovirus encoding mouse M10.5/human
?2m (16) were obtained from the laboratory of P. J. Bjorkman
(California Institute of Technology, Pasadena, CA).
Detection of Tpn–Class I Complexes Using Purified Proteins. Soluble
class I, HFE, or M10 (2.5–5 ?M each) was incubated with sTpn
(2.5–5 ?M) in 50 mM Tris, 150 mM NaCl, pH 7.5, at indicated
temperatures for indicated time. After incubations, the samples
were centrifuged, diluted in the same buffer containing 1% Triton-
immunoprecipitation) overnight at 4°C. The supernatants were
further centrifuged and incubated for 2–4 h at 4°C with protein G
beads. The beads were washed three times with buffer containing
0.25% gelatin (10 mM Tris/10 mM phosphate buffer/130 mM
NaCl/1% Triton X-100, pH 7.5) and once with buffer without
gelatin. The beads were boiled in SDS/PAGE buffer, samples were
resolved by SDS/PAGE, and immunoprecipitated proteins were
visualized by silver staining. For ?2m detection, the anti-FLAG
immunoprecipitated samples or direct proteins loads were trans-
ferred to PVDF membrane, immunoblotted with anti-?2m antisera
(Roche, Nutley, NJ), HRP-conjugated secondary antibody, and
developed by ECL plus kit (Amersham Biosciences, Piscataway,
Isolation of Tpn–Class I Complexes with Anti-FLAG Beads. sTpn–class
I complexes were formed by incubation of proteins (5 ?M) in the
presence or absence of 50 ?M ?2m in a total volume of 40 ?l at
37°C for 2–4 h. Proteins were then diluted to 1 ml buffer (50 mM
Tris/150 mM NaCl/1% Triton X-100), incubated overnight at
4°C with 100-?l FLAG-beads, loaded onto an empty column,
and washed with 5–15 ml buffer to remove free class I. For some
analyses, beads were eluted with FLAG peptide (100 ?M in 100
?l). For other analyses, beads were then incubated with 100 ?l
of buffer (50 mM Tris/150 mM NaCl) containing 50 ?M ?2m, 50
?M ?2m plus 500 ?M specific or nonspecific peptides, or buffer
alone at room temperature for 4 h. Specific peptide used for A2
was FLPSDDFPSV and for B35 was LPSSADVEF and nonspe-
cific peptide for both was RRYQKSTEL. After incubations,
supernatants were collected and analyzed by SDS/PAGE and
immunoblotting analyses with anti-His (for A2) or HC10 (for
We thank Chris Perria for conducting the experiment shown in Fig. 1D;
Dr. Peter Cresswell (Yale University School of Medicine, New Haven,
of Technology, Pasadena, CA) for M10 and HFE constructs; the
University of Michigan Biomedical Research Core Facilities for DNA
sequencing and peptide syntheses and purification; the Hybridoma Core
for ascites production; and the Rheumatic Diseases Core Center and the
Michigan Diabetes Research and Training Centers for financial support.
This work was supported by National Institutes of Health Grant AI-
44115 and a Cancer Research Institute Investigator Award (to M.R.).
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