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*
Committee on Immunology and
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimers.
Although the membrane-proximal region of the Ii luminal domain is
structurally disordered, the C-terminal segment of the luminal domain
is largely -helical and contains a major interaction site for the Ii
trimer. In this study, we show that the Ii transmembrane domain plays
an important role in the formation of Ii trimers. The Ii transmembrane
domain contains an unusual patch of hydrophilic residues near the
luminal interface. Substitution of these polar residues with nonpolar
amino acids resulted in a decrease in the efficiency of Ii
trimerization and subsequent class II association. Moreover, N-terminal
fragments of Ii were found to trimerize independently of the luminal
-helical domain. Progressive C-terminal truncations mapped a
homotypic association site to the first 80 aa of Ii. Together, these
results implicate the Ii transmembrane domain as a site of trimer
interaction that can play an important role in the initiation of trimer
formation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical
structure (8). Ii trimerization is thought to precede class II assembly. First, Ii trimerization is rapid and monomeric intermediates have not been detected (2, 9). Second, Ii trimerization appears to precede class II association kinetically, and intermediates in assembly that contain one or two class II heterodimers associated with an Ii trimer have been detected (2). Thus, the final assembled class II-Ii complex is a nonamer, consisting of a single Ii trimer associated with three class II heterodimers (2, 10). The major class II-Ii interaction site maps to the CLIP region (aa 82–110) that binds to the class II peptide binding site (3, 11, 12, 13, 14, 15, 16, 17, 18). Occupancy of the class II peptide binding site by Ii plays an important role in class II assembly (19), preventing the aggregation of class II chains (20), and promoting the transport of class II out of the endoplasmic reticulum (ER) and through the Golgi (21, 22). Consistent with the importance of Ii binding within the class II antigenic peptide groove, addition of antigenic peptide or CLIP peptide alone suffices to enhance the folding and transport of class II heterodimers (23). In addition to the core class II-binding sequence, the flanking segments 81–89 and 101–110 of CLIP appear to interact with class II and may regulate Ii association to different class II alleles (16, 17, 24, 25). Finally, class II association sites outside of the CLIP region also might contribute to the Ii-class II complex (13, 26, 27).
After transit through the Golgi, the class II-Ii complex is diverted from the constitutive transport route from the trans-Golgi network (TGN) to the plasma membrane. Instead, most of the class II-Ii complexes are sorted directly from the TGN to the endosomal pathway (28). TGN sorting and endosomal targeting require two dileucine-like signals within the cytosolic tail of Ii (29, 30, 31, 32). Interestingly, trimerization of Ii plays an important role in TGN sorting, because class II-Ii complexes containing only a single intact Ii cytosolic tail are not sorted in the TGN and appear at the plasma membrane (33).
In addition to the dileucine signals, the Ii transmembrane domain (TMD) has been implicated in lysosomal targeting following internalization from the plasma membrane (34, 35). This raised the possibility that the Ii TMD functions in concert with the dileucine signals as an endosomal targeting signal at the TGN. However, studies of Ii localization in polarized cells suggest that sorting within the endosomal pathway may be distinct from the sorting at the TGN (36, 37). In this study, we have addressed the role of the Ii TMD in class II-Ii complex assembly and transport within the secretory pathway. Examination of the Ii TMD sequence and hydrophobicity profile reveals a patch of embedded polar amino acids. Surprisingly, mutation of these residues interferes with efficient Ii trimerization and Ii-class II assembly. These and other results implicate a critical role for the TMD in the initiation of Ii trimerization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pcEXV-3 expression vector (38), the p41 Ii cDNA
clone (39), and the 
31 Ii construct (32)
have been described previously. Two rounds of overlapping PCR were used
to generate TMmut Ii. First, the T49I, T50A
mutations were engineered into the TMD of wild-type p41 Ii, and the
Q47A substitution then was added to T49I, T50A p41 Ii. For in vitro
transcription/translation reactions, p41 Ii and
TMmut p41 Ii were subcloned into the pBluescript
expression vector (Stratagene, La Jolla, CA).
31-TMmut Ii was generated by replacing the
SacI/PvuII fragment of 31 Ii with this
fragment from TMmut p41 Ii. For pcEXV-3 1–108
Ii, a stop codon and an EcoRI site were introduced after
position L108 using PCR. The PCR product was digested with
EcoRI and cloned into pcEXV-3. After subcloning p41 Ii into
pCF1E, 1–80 Ii was generated by making an internal
HindIII/SpeI deletion. pcEXV-3 1–131 Ii
(40) was provided by Dr. R. Germain (National Institute of
Allergy and Infectious Diseases (NIAID), Bethesda, MD). All constructs
generated through PCR techniques were confirmed by automated
sequencing.
Cell lines and transfections
Ltk- transfectants expressing wild-type
p41 Ii have been described previously, and stable transfectants
expressing TMmut p41 Ii or 31 Ii were
generated in the same manner (32, 39). Cells were
maintained in DMEM supplemented with 10% FCS, 10 mM HEPES, 2 mM
glutamine, 0.1 mM nonessential amino acids, and 10 µg/ml gentamicin.
To maintain selection of the transfected genes, G418 (200 µg/ml), MXH
(6 µg/ml mycophenolic acid, 250 µg/ml xanthine, 15 µg/ml
hypoxanthine), or blasticidin (10 µg/ml) was added as
appropriate.
For transient transfections, 1 x 106 Ltk- cells were plated per 60-mm tissue culture dish with DMEM and 10% FCS 24 h before transfection. Cells were washed twice in DMEM, 2 mM glutamine, 10 mM HEPES (DMEM-HEPES) before incubation in 2 ml DMEM-HEPES containing 500 µg DEAE-dextran, 50 µM chloroquine, and 2–10 µg Ii DNA. After 3 h at 37°C, the cells were treated with 10% DMSO in DMEM-HEPES for 1–2 min at room temperature, then incubated at 37°C for 48 h in DMEM and 10% FCS before immunoprecipitation experiments.
Radiolabeling and immunoprecipitation
Cells were incubated with cell culture media lacking leucine or methionine for at least 1 h before pulsing with media containing either 300 µCi/ml [3H]leucine or 200 µCi/ml [35S]methionine for the appropriate time periods. Immunoprecipitation and gel electrophoresis were conducted as previously described (33). Abs used were 10.2.16, specific for I-Ak (41); P4H5, specific for the luminal domain of murine Ii (42); and In-1, specific for the cytosolic domain of Ii (43).
Chemical cross-linking
Transfectants were labeled as above, but lysed in buffer containing 0.5% Nonidet P-40, 20 mM bicine, pH 8.2, and 0.13 M NaCl with or without 200 µg/ml dithiobis/(succinimidyl)/propionate (DSP; Pierce, Rockford, IL). After 30 min, the reaction was quenched by the addition of glycine to 100 mM. Immunoprecipitates were eluted in 2% SDS buffer without 2-ME. One half of each sample was boiled for 2 min and separated by 7% SDS-PAGE, while the second half was adjusted to 2% 2-ME before being boiled and separated by 10% SDS-PAGE.
In vitro transcription and translation
Transcription reactions were driven off the T7 promoter using the Ribomax Large Scale mRNA kit (Promega, Madison, WI). Translation reactions were performed using rabbit reticulocyte lysates and canine microsomal membranes (Promega) in the presence of 1 mCi/ml [35S]methionine for 1 h at 30°C. Microsomal membranes were isolated by centrifugation for 5 min at 14,000 rpm at room temperature and resuspended in bicine lysis buffer in the presence or absence of 25 µg/ml DSP. Immunoprecipitation and electrophoresis were performed as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, the Ii TMD has been predicted to extend from
a cytosolic anchor at Arg29 to a luminal
termination near Gln56–58 (Fig. 1
A) (44, 45).
Assignment of Tyr55 to the luminal interface
would be consistent with the finding that tyrosine residues are
preferentially located at the lipid/aqueous interface
(46). However, examination of the amino acid sequence in
conjunction with the hydrophobicity profile suggested an unusual
feature at the luminal end of this segment. The major region of
hydrophobicity within the TMD begins at Leu32 and
ends only 15 aa away. This stretch is followed by a hydrophilic spike
(attributed to the amino acids Gln47,
Thr49, and Thr50) and a
second hydrophobic patch (Fig. 1A). The presence of
Gln47 is especially striking within the TMD. In a
study of single pass type I transmembrane proteins, glutamine was
underrepresented 30-fold within transmembrane-spanning segments
(46).
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To examine the role of this hydrophilic spike within the Ii TMD, we
generated an Ii molecule, termed TMmut Ii, with
Glu47 substituted by Ala,
Thr49 substituted by Ile, and
Thr50 substituted by Ala. Substitution with
nonpolar amino acids effectively eliminates the patch of hydrophilicity
and results in a strongly hydrophobic stretch of 24 aa (Fig. 1
A). We chose to express TMmut Ii in
the context of the alternatively spliced gene product, p41, because p41
is resolved from the class II -chain in SDS-PAGE, whereas p31 and
the -chain comigrate. p31 and p41 Ii are indistinguishable in
trimerization, class II association, and intracellular trafficking, so
any effect on these events could be generalized to both isoforms.
Ii containing TM mutations fails to assemble with MHC class II
We first evaluated the effect of the TMD mutations on Ii assembly
with MHC class II. Stable Ltk- cell
transfectants coexpressing class II with either wild-type p41 Ii or
TMmut p41 Ii were continuously labeled for 2
h in the presence of [35S]methionine. After
lysis in Nonidet P-40, immunoprecipitations were conducted with mAbs
against either Ii or MHC class II. Wild-type Ii runs as a triplet on
SDS-PAGE (Fig. 2B). The two
faster migrating bands are the result of alternative glycosylation of
the four possible carbohydrate addition sites in p41 Ii. The broader,
upper band represents maturation of these sugars as they transit the
Golgi. Immunoprecipitation with anti-class II Ab demonstrates the
association of both mature and immature forms of wild-type Ii with MHC
class II (Fig. 2A). Although the addition of the core
N-linked glycans indicates ER insertion,
TMmut Ii fails to undergo significant maturation,
suggesting a defect in ER to Golgi transport (Fig. 2B).
Furthermore, very little TMmut Ii can be detected
in association with MHC class II (Fig. 2A). Pulse/chase
analysis confirmed that TMmut Ii largely is
retained within the ER (data not shown). These results suggest that
mutations within the Ii TM exert an effect very early in the Ii/class
II biosynthetic pathway before passage through the Golgi.
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Ii trimerization is thought to precede assembly with MHC class II
(2). The failure of TMmut Ii to
associate with MHC class II raised the possibility that the primary
defect in Ii-class II association could be a failure of Ii
trimerization. To assess the oligomeric state of
TMmut Ii, stable transfectants were radiolabeled
and then lysed in the absence or presence of the reducible cross-linker
DSP (2, 10). After immunoprecipitation with anti-Ii
mAb, samples were eluted and separated by SDS-PAGE under either
reducing or nonreducing conditions. In the absence of cross-linker,
wild-type Ii migrates at m.w. corresponding to monomeric and dimeric Ii
(Fig. 3A). This Ii dimer is
produced primarily by postlysis oxidation of the single cysteine
residue within the Ii cytosolic tail generating a disulfide-linked
homodimer (49). The addition of DSP during lysis results
in the majority of Ii migrating as a trimer with little detectable
monomer or dimer. Additional higher m.w. complexes may indicate Ii-MHC
class II nonamers or Ii-calnexin complexes (2, 50). In
contrast to wild-type Ii, very little disulfide-linked dimer of
TMmut Ii can be detected, suggesting that
TMmut Ii fails to self-assemble (Fig. 3A). In addition, no discernible trimer can be detected in
the presence of cross-linker.
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A. This loss of material raised the possibility that the
TMD substitutions could be inducing nonspecific aggregation and then
large aggregates could have been lost during preclearing steps before
immunoprecipitation. Aggregation of TMmut Ii
seems unlikely because TMmut Ii sediments as a
monomer in sucrose gradients (data not shown) and because
overexpression in a transient transfection system drives
TMmut Ii trimerization (Fig. 4). In addition, significant loss of
TMmut Ii is not detected when we expressed either
wild-type Ii or TMmut Ii using an in vitro
transcription and translation assay (Fig. 3B). In this
assay, wild-type Ii readily forms dimers and trimers, as previously
noted (3). Consistent with the in vivo results,
TMmut Ii does not form dimers efficiently (Fig. 3B). Moreover, upon cross-linking,
TMmut Ii remains largely as a monomer with little
detectable trimer. Aggregation is not a concern in this assay because
loss of material in the presence of cross-linker was equivalent between
wild-type Ii and TMmut Ii. Together, these
results suggest that TMmut Ii is defective in its
ability to efficiently self-assemble into homotypic trimers.
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31 Ii (Fig. 1B). 31 Ii shares with p41 Ii the
luminal epitope for the mAb P4H5, but lacks the cytosolic tail In-1 mAb
epitope. Because p31 and p41 Ii randomly associate into mixed trimers
(33, 51), any 31 Ii appearing in an In-1
immunoprecipitate must be assembling with full-length p41 Ii.
Cells expressing wild-type p41 Ii were supertransfected either with
31 Ii or 31-TMmut Ii. Stable subclones were
matched for equivalent levels of 31 Ii expression. To assess Ii
oligomerization, cells were continuously labeled for 1 h with
[35S]methionine, postnuclear lysates were
divided in half, and immunoprecipitations were performed with either
P4H5 or In-1. P4H5 reacts with all Ii species equivalently and
illustrates the equivalent levels of expression of each of the Ii
molecules (Fig. 5A). The
efficient coprecipitation of 31 Ii with p41 Ii in the In-1
precipitate demonstrates the formation of mixed complexes containing
wild-type TMD. In contrast, In-1 does not coprecipitate
31-TMmut Ii with p41 Ii, consistent with an
inability of TMmut Ii to trimerize. To further
support these findings, reciprocal experiments were performed by
coexpressing TMmut p41 Ii with 31 Ii or
31-TMmut Ii. In this case, neither 31 Ii
nor 31-TMmut Ii can be coprecipitated with
TMmut p41 Ii (Fig. 5B). Therefore, in
agreement with the cross-linking data, the coprecipitation experiments
confirm that the mutations within the TMD inhibit Ii trimerization.
Moreover, the lack of detectable trimers upon cross-linking is not the
result of a conformational change within an intact trimer that
prohibits the cross-linking reaction. Together, these data show that
substitution of polar residues within the Ii TMD with small hydrophobic
residues results in monomeric Ii molecules that do not efficiently
assemble into trimers.
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Carboxyl-terminal Ii truncations expressed in vivo or in vitro
have not been observed to form trimers (3, 4, 5), yet
analogous Ii molecules generated through in vivo proteolytic processing
remain trimeric in MHC class II-associated nonamers (6, 7). Our observation that sequences within the TMD play an
important role in the trimerization of full-length Ii led us to
reexamine the ability of N-terminal fragments of Ii to trimerize. To
test whether the TMD could mediate oligomerization independently of the
luminal trimerization domain, we generated a C-terminal truncation
mutant of Ii, 1–108 Ii, which corresponds approximately to the
intracellular p12 proteolytic fragment that remains trimerized (Fig. 1B). Transient transfection of 1–108 Ii into
Ltk- cells was conducted in the absence of class
II, and, 2 days posttransfection, radiolabeling and cross-linking were
performed as above. Both disulfide-linked dimers and cross-linked
trimers of 1–108 Ii could be detected (Fig. 6B). Nonreducing/reducing
two-dimensional gel electrophoresis confirmed that the bands identified
as dimeric and trimeric Ii were in fact derived from 1–108 Ii (data
not shown). This observation is consistent with the ability of
truncated Ii molecules to trimerize independently of the luminal
-helical domain.
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B). These two constructs were transiently
transfected alone and in combination into class II-negative L cells,
and equivalent expression of both forms of Ii was confirmed by
immunoprecipitation with P4H5 (Fig. 7).
18–108 Ii was detected in In-1 precipitates only when it was
coexpressed with 1–108 Ii, confirming that N-terminal fragments of Ii
can associate in the absence of the luminal trimerization domain.
Coprecipitation of these N-terminal fragments was less efficient than
previously detected with Ii constructs containing the luminal
trimerization domain (see Fig. 5), possibly because interactions
mediated through the TMD are destabilized in the presence of
detergent.
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B). Upon
cross-linking, 1–80 Ii also can be demonstrated to migrate at a m.w.
corresponding to trimers (Fig. 6C). Therefore, these results
identify a site of homotypic interaction within the first 80 aa of Ii.
Because mutation of TMD polar residues can inhibit trimerization in the
context of either full-length (
Figs. 3–5) or truncated Ii (data not
shown), this N-terminal interaction site most likely maps to the
Ii TMD.
To attempt to reconcile our data with previous studies that did not
detect trimerization of N-terminal fragments of Ii (3, 4, 5),
we have explored several technical differences between these studies.
First, in earlier studies, the shortest molecule reported not to
trimerize was 1–127 Ii (4, 5), which contains the luminal
glycosylation sites and the initial portion of the -helical domain
not present in either 1–80 Ii or 1–108 Ii. To test whether the length
of these constructs might account for differences in trimerization seen
in ours and previous studies, we assayed the oligomeric state of 1–131
Ii, a construct analogous to 1–127 Ii that was used in the previous
studies (Fig. 1B). Consistent with our results for 1–80 Ii
and 1–108 Ii, 1–131 Ii was found to form trimers upon cross-linking
(Fig. 6A). Thus, the presence of the amino acid segment
between residues 108 and 131 and the N-linked carbohydrate
sites at residues 113 and 119 does not interfere with trimerization of
N-terminal fragments of Ii. Second, these truncated trimers may be
particularly sensitive to detergent-induced dissociation if their
assembly is mediated by the TMD. However, we have found that trimers of
1–80 Ii, 1–108 Ii, and 1–131 Ii can be detected in several different
detergents, including Nonidet P-40 and
C12E9 (data not shown).
Third, we have found equivalent trimerization of 1–131 Ii in
Ltk- cells and in COS cells (data not shown),
which were used in previous studies (4, 5). Finally,
although the N-terminal trimers spontaneously dissociate more rapidly
than intact trimers, trimers of both 1–108 Ii and 1–131 Ii remain
intact 60 min after cell lysis in either Nonidet P-40 or
C12E9 (data not shown).
Therefore, while it is not clear why oligomerization of C-terminal
truncation mutants of Ii has not been previously observed, the
data presented in this study clearly demonstrate that N-terminal Ii
fragments do trimerize and implicate the Ii TMD as the association
site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inability of TMmut p41 Ii to trimerize
efficiently is a striking result because the Ii luminal domain alone
can trimerize in solution (8, 52). This raised the
question of whether the mutations within the TMD could perturb
interactions between the luminal domains. However, the
membrane-proximal region of the Ii luminal domain is structurally
disordered (53), and it is unlikely that a conformational
change could be transmitted from the TMD that would affect the
independent folding of the -helical domain. It is possible that the
mutant TMD might alter the orientation of Ii with respect to the lipid
bilayer. Alterations in the length of a membrane-spanning region can
result in the deformation of the lipid bilayer or in the tilting of the
TMD to minimize a hydrophobic mismatch (54, 55, 56). This
alteration in orientation could adversely affect luminal domain
interactions. Alternatively, the ability of soluble, recombinant Ii to
trimerize could reflect differences between the in vitro conditions and
the ER environment. Sample concentrations of purified, soluble Ii used
in vitro ranged from 0.3 to 2 mM (8, 52, 53). These
concentrations are likely to be 100-1000-fold greater than the
concentration within the ER of moderately expressed proteins such as Ii
(57). In vivo, trimerization of Ii may depend on the
initial TMD-mediated associations to increase the relative
concentration of the luminal domain trimerization site.
Consistent with this idea, we found that overexpression of Ii
TMmut Ii in cells can drive trimerization,
possibly by increasing the relative concentration of Ii and allowing
luminal domain interactions in the absence of efficient TMD-mediated
association.
Aberrant binding to TMmut Ii by proteins such as ER chaperones may physically block luminal domain interactions. The observation that TMmut Ii is retained within the ER suggests associations with ER chaperones. Calnexin is an ER chaperone that can interact both with the N-linked carbohydrate side chains and with the TMD (58, 59, 60). Both of these interactions have been implicated in calnexin-Ii association (9, 40, 50). Interestingly, three times as much TMmut Ii than wild-type Ii has been found associated with calnexin (data not shown), raising the possibility that mutations in the Ii TMD created an exceptionally strong calnexin binding site. The stoichiometry of calnexin association to wild-type Ii trimers has been reported to be one calnexin molecule for each Ii trimer (50). This 3-fold increase then could be reflective of a 1:1 ratio of calnexin to monomeric TMmut Ii. This raised the possibility that increased calnexin binding to TMmut Ii could prohibit trimerization, as has been shown for influenza hemagglutinin (61). However, treatment with either tunicamycin or castanospermine reduces the amount of calnexin-associated TMmut Ii to a proportion equivalent to that for wild-type Ii, but does not allow TMmut Ii to trimerize (data not shown). Thus, the carbohydrates appear to be the major site of calnexin association for both wild-type and mutant Ii, and the specific mutations within the Ii TMD do not appear to create a high affinity calnexin binding site. These results support a conclusion that increased association of calnexin is not actively inhibiting trimerization, but is instead a consequence of the retention of monomeric Ii within the ER. This result does not rule out the possibility that another protein(s) may be associating with TMmut Ii to block trimerization.
One possible scenario that could account for a role of the TMD in Ii
trimerization is that newly synthesized Ii monomers form transient
intramolecular interactions between the luminal domain and a
membrane-proximal segment. Initial interactions mediated by the polar
residues within the TMD of Ii monomers would induce the dissociation of
these cis interactions, freeing the -helical domain for
intermolecular trimerization. Under normal conditions, Ii assembly is a
rapid process and Ii monomers are typically not detected even with
short pulse times (2, 9). In the
TMmut Ii, loss of the polar residues within the
TMD would decrease the efficiency of TMD interaction and would
effectively stabilize this monomeric intermediate.
The importance of the polar residues in the TMD and the ability of
short N-terminal fragments of Ii to trimerize support a structure of
the Ii trimer that is maintained by interactions at both the luminal
trimerization domain and the TMD (62). The intervening
sequences between these two trimerization domains contain the major
class II interaction sites, yet display little secondary structure
(52, 53). Ii trimerization mediated through both
N-terminal and C-terminal interactions may impose some structural order
onto the CLIP region that could facilitate the assembly of class II
with Ii. In our studies, mutations of the TMD that interfere with
trimerization also interfere with class II binding. These results
suggest that trimerization of Ii, either by disrupting intramolecular
interactions or by adding structural order to the CLIP region, may play
an important role in class II-Ii assembly. Consistent with this idea,
when trimerization of TMmut Ii is driven by
overexpression (Fig. 4), the resulting trimers do assemble with class
II (data not shown).
If the initiation of Ii trimerization is dependent upon the
interactions among TMD, this TMD-mediated association should function
independently of the trimerization domain. We have mapped such an
N-terminal interaction site to the amino acid segment 18–80,
containing only part of the cytosolic tail, the TMD, and the
membrane-proximal luminal segment. TMD have been demonstrated to play
an important role in the assembly of a number of oligomeric complexes,
including glycophorin A (63, 64), Fc
RIII
(65), TCR (66, 67), MHC class II
(68), and influenza hemagglutinin (69, 70, 71).
Considering this diverse array of complexes, a mechanism must exist to
generate specificity of interactions among TMD. TMD are thought to fold
into -helical segments, and interactions between TMD have been
predicted to be similar to those of soluble coiled coils
(72). Specificity of helix-helix interactions should then
arise from the preference for close packing of the side chains at the
protein-protein interface compared with packing at the lipid-protein
interfaces (73). Interestingly, single buried polar
residues such as asparagine and glutamine have been shown to confer
specificity upon the oligomeric state and the orientation of soluble
coiled coils (74, 75). Indeed, there is a strong
preference for glutamine residues within coiled coil trimers (76, 77). Similarly, oligomerization and Golgi localization of the
transmembrane M protein of the avian coronavirus depend upon a single
glutamine within its TMD (78, 79). In addition, a recent
study has implicated a TMD-specific glutamine in the dimerization of
the bovine papilloma virus E5 protein and in its association with its
target platelet-derived growth factor ß receptor (80).
Finally, serine and threonine residues can affect the structure of
-helices by forming both inter- and intrahelical hydrogen bonds
(81). Seen in this context, the embedded polar residues
within the Ii TMD may play a significant role in promoting the specific
assembly of Ii trimers. In the absence of these residues, Ii
trimerization is inhibited and the Ii TMD may lose specificity and
interact with the TMD of other proteins within ER. This may account for
the loss of TMmut Ii observed after
cross-linking. In this context, the biochemical data presented in this
study support an important role for the TMD in the initiation of
homotypic interactions between Ii monomers that lead to their rapid
assembly into trimers.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jim Miller, Department of Molecular Genetics and Cell Biology, University of Chicago, 920 E. 58th Street, Chicago, IL 60637. E-mail address:
3 Abbreviations used in this paper: Ii, invariant chain; CLIP, class II-associated invariant chain peptide(s); DSP, dithiobis/(succinimidyl)/propionate; ER, endoplasmic reticulum; TGN, trans-Golgi network; TMD, transmembrane domain.
Received for publication April 27, 1999. Accepted for publication June 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßIi complexes. J. Exp. Med. 182:2027.-helical peptides and gramicidin A. Biochemistry 37:9333.[Medline]
-helices. J. Biol. Chem. 267:7683.-helices and its relevance to membrane-bound proteins. J. Mol. Biol. 175:75.[Medline]
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G. Rajagopalan, M. K. Smart, C. J. Krco, and C. S. David Expression and Function of Transgenic HLA-DQ Molecules and Lymphocyte Development in Mice Lacking Invariant Chain J. Immunol., August 15, 2002; 169(4): 1774 - 1783. [Abstract] [Full Text] [PDF]
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 D. Matza, F. Lantner, Y. Bogoch, L. Flaishon, R. Hershkoviz, and I. Shachar Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity PNAS, February 20, 2002; (2002) 52703299. [Abstract] [Full Text] [PDF]
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D. Matza, F. Lantner, Y. Bogoch, L. Flaishon, R. Hershkoviz, and I. Shachar Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity PNAS, March 5, 2002; 99(5): 3018 - 3023. [Abstract] [Full Text] [PDF]
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10-fold greater than expression levels
observed in stable cell lines, based on comparison of exposure times of
autoradiographs between experiments. This is likely to be an
underestimate of the level of overexpression in individual cells,
because only a subset of cells are expressing Ii after transient
transfection. Despite the increased amount of monomer recovered after
cross-linking compared with WT Ii, both dimers and trimers of
TMmut Ii can be detected.
