Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.
ABSTRACT Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.
- SourceAvailable from: Sebastian Temme[Show abstract] [Hide abstract]
ABSTRACT: HLA class II α and β chains form receptors for antigen presentation to CD4+ T cells. Numerous pairings of class II α and β subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αβ heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study, we investigated assembly of isotype-mixed HLA class II α and βheterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and β subunits was dependent on the groove-binding section of the invariant chain (Ii). By mutation of polymorphic DPβ sequences, we identified two motifs, K69 and GGPM84-87, which are engaged in Ii-dependent assembly of DPβ with DRα. We identified five members of a family of DPβ chains containing K69 and GGPM 84-87, which assemble with DRα. The K/GGPM motif is present in the DPβ sequence of the Neandertal genome and this ancient sequence is related to the human allele DPB1*0401. By site- directed mutagenesis we inspected Neandertal amino acid residues which differ from the DPB1*0401 allele and whether matched heterodimers are formed by assembly of DPβ mutants with DRα. Since the *0401 allele is rare in the Sub-Saharan population but frequent in the European population it may have arisen in modern humans by admixture with Neandertals in Europe.Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mlg-associated Igα/Igβ heterodimers. The relative role of Igα and Igβ subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Igα and Igβ chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Igα targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Igβ targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.Immunity 09/1995; 3(3):335-347. · 19.80 Impact Factor
Defective Intracellular Transport as a
Common Mechanism Limiting Expression of
Inappropriately Paired Class II Major
Histocompatibility Complex ol/B Chains
By Andrea J. Sant7 Laura R. Hendrix,* John E. Coligan,$
W. Lee Maloy) and Ronald N. Germain*
From the *Lymphocyte Biology Section, Laboratory of Immunology, and
*Biological Resources Branch, National Institute of AllergIt and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892
Distinct combinations of class II major histocompatibility complex (MHC) oe and 3 chains show
widely varying efficiencies of cell surface expression in transfected cells. Previous studies have
analyzed the regions of the class II chains that are critically involved in this phenomenon of
variable expression and have shown a predominant effect of the NHrterminal domains
comprising the peptide-binding site. The present experiments attempt to identify the post-
translational defects responsible for this variation in surface class II molecule expression for both
interisotypic od3 combinations failing to give rise to any detectable cell membrane molecules
(e.g., EoeA3 k) and intraisotypic pairs with inefficient surface expression (e.g., AoedA3~). The
results of metabolic labeling and immunoprecipitation experiments using L cell transfectants
demonstrate that in both of these cases, the ol and 3 chains form substantial amounts of stable
intraceUular dimers. However, the isotype- and allele-mismatched combinations do not show
the typical post-translational increases in molecular weight that accompany maturation of the
N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no
or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These
data are consistent with active or passive retention of relatively stable and long-lived mismatched
dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticuhm itself. This
retention accounts for the absent or poor surface expression of these oe/3 combinations, and
suggests that conformational effects of the mismatching in the NH2-terminal domain results
in a failure of class II molecules to undergo efficient intracelhlar transport.
T cell receptor recognition of either self or foreign MHC
products is a cell surface event that depends on both the
qualitative and quantitative nature of MHC molecules on the
presenting cell membrane (1). For MHC class I, each locus
encoding a heavy chain can contribute only one new species
to a cell's MHC molecule display, albeit with a spectrum of
bound peptides. For class II, the situation is more complex,
as there is significant polymorphism in both the ot and B
chains comprising these heterodimeric molecules (2). Because
a minimum requirement for class II molecule cell surface ex-
pression is assembly of individual ol and 3 chains (2-7), if
free intracelhlar mixing were to occur, the number of pos-
sible class II MHC molecules would be the product of the
number of allelically and isotypically distinct oe and 3 chains
cosynthesized by the cell. However, biochemical analyses of
normal cells and transformed cell lines, as well as studies using
transfected cells, have shown that not all possible class II o~/3
pairs are detectable in or expressed on the surface of cells pos-
sessing a diversity of oe and 3 chains (2, 3, 7, 8).
Studies carried out over the past several years in this and
other laboratories have provided some insight into the basis
for these limitations in class II molecule expression. Two
different post-translational restrictions have been identified.
First, transfection experiments using cells not expressing any
endogenous class II gene products have shown that there is
a great deal of variability in the potential of distinct oe and
3 chain combinations to reach the surface membrane (9-15).
In mice, haplotype-matched (cis-encoded) Aol and Aft chains
are efficiently expressed on the cell surface, whereas haplotype-
mismatched combinations show variable surface expression
efficiency, ranging from almost that of haplotype-matched
combinations to no detectable expression at all (9, 10, 13).
Interisotypic combinations (e.g., EoeA3) can be expressed on
the membrane, but the restrictions on this process are even
799 The Journal of Experimental Medicine - Volume 174 October 1991 799-808
more stringent than for isotypically matched combinations
(11, 12, 15). Analyses using mutant and recombinant class
II genes have established that the sites of incompatibility
resulting in poor or absent surface expression in these cases
lie predominantly in the region of the class II peptide-binding
domain predicted to constitute the interacting pair of fl-pleated
sheets contributed by the ol and ~ polypeptides, with sec-
ondary regions of importance at the ends of the putative
binding domain helices (10, 12-14, 16).
A second mechanism limiting class II molecule expression,
which operates in cells synthesizing more than one class II
c~ and ~ combination, involves competition for precursor
chains among distinct c~/3 combinations. Thus, class II het-
erodimers that are moderately well expressed when only one
ol and one B chain are present in a cell become undetectable
if certain preferred partner ol and/or ~ chains are cosynthe-
sized (8, 17, 18). This feature of class II molecule assembly
and expression limits functional surface molecule diversity
to a smaller set of cff3 combinations than would be expected
based on the observed expression of individual ~/3 pairs by
Although these two classes of constraints on class II mole-
cule expression have been identified, less is known about the
precise biochemical and cell biological mechanisms under-
lying these expression phenotypes. Class II MHC dimers are
formed rapidly (within a few minutes) of initial chain syn-
thesis (19), and, in normal cells, are coassembled during this
time with a third, non-MHC-encoded, nonpolymorphic chain
termed the invariant chain (Ii) (7, 20, 21). This complex is
transported out of the rough endoplasmic reticulum (PER) 1
and through the Golgi stacks, undergoing post-translational
glycosylation during this passage. The class II-Ii complexes
then appear to move to an endosome-related compartment,
where low pH and acidic proteases contribute to the removal
of Ii and the loading of processed peptide antigen into the
class II molecule binding site (7, 22-25). The peptide-bearing
dimer then transits to the cell surface. Thus, limited or ab-
sent membrane expression could reflect either ineffective for-
mation of stable c~/3 dimers in the REK, impaired egress
from the PER or a subsequent intracellular organelle, degra-
dation before attainment of surface expression, or even rapid
loss from the cell surface. In the present experiments, we have
used biochemical analysis of L cell class II gene transfectants
to determine the site(s) of the intracellular defect(s) under-
lying the poor surface membrane display of several distinct
class II MHC o~/~ combinations.
Materials and Methods
Transfected Cells Synthesizing Various Class II c~ and ~ Chains.
Drug-resistant, stable gene transfectants of the L cell subline DAP.3
were prepared as previously described using the calcium phosphate
coprecipitation method (26). The specific transfectants used in the
present experiments and the respective introduced class II genes
are as follows: AT5.2 (Ec~/A3 ~ ), AT7.1G9 (EeL/ABl~E3 ),
1Abbreviations used in this paper: CT, COOH terminal; PAS, protein
A-Sepharose; KEg, rough endoplasmic reticulum.
RT10.3 (EcffEB d 111]), KTa.17H3A5 (Ac~a/AB k ), and
KT7.3HB4.5 (Ac~k/Ac~ k ). For those a/B combinations yield-
ing cell surface molecules detected by mAbs, populations of cells
expressing levels of surface class II dimers similar to routine B cell
lymphomas and hybridomas were selected by preparative flow
microfluorimetry (9) and/or magnetic bead sorting. For those com-
binations not giving surface-expressed class II molecules, clones
of drag-resistant colonies were screened and those with the highest
levels of specific class II mRNA were chosen for study. All trans-
fected cells were maintained in culture in DMEM (high glucose)
with 10% heat-inactivated FCS in the presence of the appropriate
Monoclonal and Anti-COOH-terminal Peptide Antibodies. mAbs
were used as culture supernatants, both for immunofluorescent
staining and for immunoprecipitation. For class II molecules con-
taining the B1 domain of A8 ~, the antibody 10-2.16 was used
(28); for cells synthesizing Eot, the antibody 14-4-4S was used (29).
Polyclonal rabbit antisera reactive with each murine class II chain
were generated by immunization with peptides corresponding to
the COOH-terminal 13-15 residues of each chain together with
an NHrterminal cysteine. These peptides were conjugated to
KLH using m-maleimidebenzoic acid N-hydroxysuccinimide ester
(MBS). The sequences of the peptides used were: A~/, CQK-
GPKGPPPAGLLQ; E/~, CNQKGQSGLQFIGLLS; Ace, CRSG-
GTSKHPGPL; and E~, CKGIKKR.NVVEILR.QGAL. The spec-
ificity of each of the antisera pools used in this study is documented
in Fig. 1.
Flow Cytometry. Cells were analyzed for cell surface expression
of class II dimers as previously described (12), using mAbs as a
first-step reagent, followed by FITC-conjugated goat anti-mouse
Ig (Cappel Laboratories, West Chester, PA). Stained cells were ana-
lyzed using a FACScan | flow cytometer (Becton Dickinson & Co.,
Mountain View, CA).
Metabolic Labeling, lmmunoprecipitation, and SDS-PAGE. SDS-
PAGE analysis of metabolically labeled and immunoprecipitated pro-
teins from transfected cells was carried out by slight modifications
of previously described techniques (30, 31). Briefly, transfected cells
were harvested using trypsin-EDTA, washed three times in PBS
with 2.5% dialyzed FCS, and precultured for 1-2 h at 37~ in
1LPMI or DMEM lacking leucine, but supplemented with 5% dia-
lyzed FCS, 2 mM glutamine, 10 mM Hepes, and gentamicin. These
precultured cells were pelleted and resuspended in the same medium
containing 200-300 #Ci/ml [3H]leucine as [3H]leucine in water,
with 1/10 volume of 10x HBSS added to maintain isotonicity.
After incubation for the indicated time, cells were either washed
into label-free complete medium and reincubated for the chase times
indicated, or directly pelleted and lysed in 4 mM CHAPS/0.05 M
Tris/0.15 M NaC1 for 45 min at 4~
were removed by centrifugation, and the supernatant was used for
immunoprecipitation. Lysates were pre-cleared using normal rabbit
serum and Staphylococcus aureus, Cowan strain I (PanSorbin; Gibco
Laboratories, Grand Island, NY), followed by protein A-Sepharose
(PAS). Pre-cleared lysates were then mixed with antibody overnight,
and antigen-antibody complexes were isolated with PAS. The iso-
lated immunoprecipitates were either directly analyzed on SDS-
10% polyacrylamide gels under reducing conditions or dissociated
in SDS sample buffer and reprecipitated before gel electrophoresis.
For dissociation and repredpitation, immune complexes adsorbed
to PAS were suspended in 0.0625 M Tris, pH 6.8, containing 1%
SDS, incubated at room temperature for 15 min, boiled for 3 rain,
and centrifuged to remove PAS. Supernatants were adjusted to 0.2%
SDS by dilution in cell lysis buffer containing 0.5% NP-40. Samples
were then incubated for 3-4 h at 4~ and any free, renatured anti-
Nuclei and insoluble debris
800 Defective Intracellular Transport Limits Expression of a/B Chains
body was removed by incubation with PAS. Supematants were then
incubated overnight at 4~ with rabbit antisera, immune com-
plexes isolated with PAS, and the eluted complexes run on
All SDS gels were treated with EnBHance (New England Nu-
clear, Boston, MA), dried, and autoradiographs prepared at -70~
Endoglycosidase-H Treatment. The N-linked glycans of immu-
noprecipitated proteins were analyzed for sensitivity to endoglycosi-
dase H (Endo H) (Boehringer Mannheim Biochemicals, Indianapolis,
IN) digestion by suspending immune complexes adsorbed to PAS
in 30/zl of 50 mM sodium citrate, pH 5.5, containing 0.1% SDS
and 500 mlU/ml ofEndo H (32). Mock or enzyme-treated samples
were incubated at 37~ overnight under toluene vapor. 2 x con-
centrated Laemmli sample buffer was added to adjust samples to
2% SDS, 0.0625 M "Iris, pH 6.8, with 10% glycerol. The samples
were boiled for 3 rain and the dissociated proteins analyzed by
Stable cell3 Dimers Form in Transfectants Showing No
Surface Class II MHC Molecule Expression
We first asked whether combinations of el and 3 chains
showing no detectable cell surface expression assemble into
stable heterodimers by performing immunochemical studies
on L cell transfectants cotransfected with plasmid DNA en-
coding the chains comprising such combinations. For these
experiments, it was desirable to use antibodies that are not
conformation sensitive. Many monoclonal anti-class II anti-
bodies do not bind free cr or 3 chains and are sensitive to
the altered conformation of dimers composed of atypical (e.g.,
allele-mismatched) combinations of oe and 3 (10). To increase
the probability of detecting all forms of the class II chains
within the L cells, rabbit antisera raised against synthetic pep-
tides corresponding to the COOH-terminal (CT) segments
of c~ or 3 were utilized for immunoprecipitation. These re-
agents react with free ot and 3 chains, and also with SDS-
denatured proteins (.I.E. Coligan, and W.L. Maloy, unpub-
lished observations), making it likely that they would detect
all conformational forms of the class II chains, regardless of
To ascertain the specificity of these reagents, their reac-
tivity with the class II MHC o~ and 3 polypeptides synthe-
sized by normal hematopoietic cells was tested. BALB/c spleen
cells were biosynthetically labeled and aliquots of the deter-
gent lysates of the labeled cells were reacted with anti-CT
A3 or anti-CT E3 antisera. Antigen-antibody complexes were
isolated and denatured in SDS. The SDS concentration was
reduced to 0.2% by dilution in an NP-40-containing buffer,
and aliquots of the immunoprecipitated material were tested
for reactivity with Ac~-, A3-, Ec~-, or EB-specific antisera.
Gel analyses of the immunoprecipitated material (Fig. 1) dem-
onstrate, first, that each of the antisera reacts only with the
chain corresponding to the immunizing peptide, and second,
that only isotype-matched c~ chains are recovered after initial
precipitation with the B-specific antibodies (e.g., Ac~ with
Aft and Ec~ with Eft).
Using these antisera, we examined L cells synthesizing an
isotype-matched, well-expressed c~/fl combination (Ec~Efl)
Figure 1. Reactivity of anti-CT antisera with murine class II polypep-
tides. BALB/c spleen cells were labeled with [BH]leucine for 6 h, then
solubilized in 0.5% NP-40/0.05 M Tris/0.15 M NaC1. Precleared lysates
were divided in half and incubated with either anti-CT A3 (lanes I-5)
or anti-CT E3 (lanes 6-10) antisera. Antigen-antibody complexes were
isolated with PAS, then eluted by boiling in 1% SDS, as described in
Materials and Methods. SDS was reduced to 0.2% by dilution, and ali-
quots of the samples were incubated with antisera raised against synthetic
peptides corresponding to CT AB (lanes 2 and 7), Ao~ (lanes 3 and 8),
EB (lanes 4 and 9), or Eel (lanes 5 and I0), as indicated. Immunoprecipi-
tates were analyzed by SDS-10% PAGE. 5% of the AB and EB primary
immunoprecipitates were analyzed directly, and are shown in lanes 1 and
6, respectively. The bands are identified to the left of the gel.
and others producing non-cell surface-expressed c~/3 com-
binations (Ec~ plus A3 k or Eoe plus a hybrid B chain con-
taining the 31 NH2-terminal domain from AB k and the B2,
transmembrane, and cytoplasmic segments of E/~ [A31kE/~]
). Typical FACS | profiles of these transfizctants after indirect
immunofluorescence staining using monoclonal anti-class II
primary antibodies are shown in Fig. 2. They demonstrate
the dramatic effect of the NH2-terminal domain on cell sur-
face coexpression of c~ and ~, as previously reported (10, 12).
Precleared lysates of biosynthetically labeled cells were in-
cubated with antisera reactive with either the CT of the c~
or the 3 chain expressed by the transfectant. SDS-PAGE anal-
ysis of these immunoprecipitates (Fig. 3 A) indicated that
in each case, incubation of detergent lysates with Ec~-specific
reagents led to coisolation of the 3 chain, regardless of whether
or not an ol/3 dimer would ultimately be expressed at the
cell surface (lanes 4, 5, and 6). The E~/A3 k transfectant
showed a lower amount of coprecipitated 3 chain, consistent
with anti-3 precipitations showing a substantially lower level
of total 3 chain in these cells (data not shown). Screening
of additional clones from this and other transfectants did not
identify a cell with an amount orb chain similar to the other
two cells used in this analysis. Therefore, the remainder of
the experiments used the E~/A31kE3 transfectant as a rep-
resentative mixed isotype combination failing to show cell
surface expression. Fig. 3 B shows the reciprocal coprecipita-
tion from lysates of this latter cell of oe and B chains by each
anti-CT antibody. Fig. 3 C demonstrates that oe chain
coprecipitation is observed using the A31k-specific mAb 10-
2.16 (10) (lane 2). The capacity of the 10-2.16 mAb to precipi-
801 Sant et al.
(~ EQE~ d
lO 100 1000
Mean Fluorescent Intensity
(~ Ec~(~ k
Q EcxA~,lk El ~
l ,mwa~q j |||urn i ! i w|l|m I | wlwlww i i imlluq i IW|WlW I
I ||WlWl i
lo 100 1000 lO 100
Mean Fluorescent Intensity Mean Fluorescent Intensity
Figure 2. Cell surface expression of isotype-matched and isotype-mismatched r~//3 combinations. L cells transfected with plasmid DNA encoding
the isotype-matched E,v/EB a combination (A), the isotype-mismatched Ec~/A~' combination (B), or the isotype-mismatched combination EcffA/31kE b
(C), all of which express high levels of o~ and/3 mRNA, were stained for cell surface expression of class II, using the E~-speciilc antibody 14-4-4s
(A, solid line) or the A/3-specific antibody 10-2.16 (B and C). Also shown is background staining obtained with the FITC-goat anti-mouse Ig (GAM)
developing reagent (-4, dashed line). In B and C, all staining curves, including the background control, were identical.
tate an cr
also establishes that the failure to detect surface class II dimers
on these cells with this antibody (see Fig. 2) is not a result
of using the inappropriate reagent, but is a true reflection
of the inability of such c~/3 dimers to reach the plasma
dimer from the Ecc/A/31kE/3 cotransfected cells
Poorly or Nonexpressed Dimers Show Defective
Nonexpressed ol//3 Combinations. Pulse-chase studies were
performed to determine the intracellular fate and post-
translational modifications of various assembled c~//3 directs.
L cell transfectants synthesizing od/3 combinations giving
either high or undetectable levels of surface molecules were
pulse labeled and then chased in nonradioactive media for
90 rain or 3 h. Immunopredpitates were prepared from these
cells and analyzed by SDS-PAGE. The resuhs of one such
experiment, shown in Fig. 4, suggest that for the non-cell
surface-expressed combination of Ec~ with A/31kE/3, al-
though the c~ and 13 chains assemble into a dimer, they do
not undergo the same types of post-translational modifica-
tions as do the dimers of the well-expressed, wild-type Eo~E/3
combination. Thus, in contrast to the chains of the latter
dimer, which undergo a time-dependent increase in both mo-
lecular weight and microheterogeneity (lanes 2, 4, and 6),
both the ol and the/3 chain of the Eo~ABlkE/3 dimer remain
homogeneous in molecular weight (lanes 1, 3, and 5). In-
terestingly, with time, the ol and/3 chains of the dimers that
are not transported to the cell surface undergo a slight de-
crease in apparent molecular weight (compare lane 1 to lanes
3 and 5). This analysis indicates that the nonexpressed but
assembled dimers persist for relatively long periods of time
within the cell, in a modified form that is biochemically dis-
tinct from o~//3 dimers that are uhimately expressed at the
Poorly Expressed c~//3 Combinations. Class II o~ and/3 chains
that are isotype and allele matched (e.g., AolkA/3 k) are ex-
pressed at the cell surface with high efficiency. In contrast,
some isotype-matched (e.g., AolAB) but allele-mismatched
(e.g., AccaA/3 k) combinations are very poorly expressed at
the cell surface (9, 10, 14, 16). To identify the intracellular
events that lead to these differences in cell surface expression,
and compare them to the intraceUular handling of the nonex-
pressed interisotypic dimers described above, we examined
Figure 3. Stable assembly of cx and/3 chains in transfected L cells. L
cells transfected with plasmids encoding the class II chains indicated in
each panel were biosynthetically labeled with [3H]leucine for 3-4 h, solu-
bilized in 6 mM CHAPS, 0.05 M Tris, 0.15 M NaC1, and pretreated with
NRS and PAS. Aliquots of the pretreated detergent lysates were incubated
with the antisera or mAb listed above each lane. Antigen-antibody com-
plexes were isolated with PAS and immunoprecipitates were analyzed by
SDS-10% PAGE. Shown at the left of each panel is the migration position
of the c~ and/3 chains.
802 Defective Intracellular Transport Limits Expression of ~/f3 Chains
Figure 4. Intracellular fate of efficiently expressed isotype- and allele-
matched or~~3 dimers vs. ineffaciently expressed isotype-mismatched or//3
dimers. L cells synthesizing the surface-nonexpressed interisotypic cx//3
combination EotA31kE3 (A) or the effidently expressed intraisotypic com-
bination EoeE/3 d (B) were biosynthetically labeled with [3H]leucine for 20
min (0), or pulsed and then chased in nonradioactive medium for 90 or
180 min (90 or 180, respectively). Detergent lysates were prepared from
the labeled cells and immunoprecipitated with antisera specific for Ecz.
Immunoprecipitates were analyzed by SDS-10% PAGE. The position of
each of the class II chains and of Ii is indicated on the left of the figure.
L cell transfectants that synthesized the same A/3 chain
(A/3 k) but that differed in the allelic origin of the c~ chain.
A/3 k is well expressed with its normal cis-encoded partner
chain (Ac~k), but is poorly expressed with the d aUelic
variant of Ao~ (9). L cells producing A/3 k and either Aol k or
Aot d were pulse labeled for 30 min and chased in nonra-
dioactive medium for 3 or 8 h. Class II chains were isolated
using Aot or AB CT-speciflc antisera and the immunoprecip-
itated material analyzed by SDS-PAGE.
As can be seen in Fig. 5, copredpitation ofc~ with/3-spedfic
antibodies in the pulse-labeled samples (lanes 1 and 4) demon-
strates that the assembly of the ot and/3 chains is similar in
both cell lines. This establishes that, as for the interisotypic
dimers, the defect in cell surface expression is not ol//3 coas-
sembly. Rather, as was seen in the L cells producing the surface-
nonexpressed interisotypic Ec~A/31kE/3 dimer, the subse-
quent intracellular processing of the interallelic AotaA/3 k
dimer appears to be defective. In the cell line generating the
efficiently expressed AolkA/3 k dimer, the individual c~ and/3
chains undergo an increase in molecular weight and micro-
heterogeneity between the pulse and 3-h chase period (lanes
1 and 2), and these forms persist at 8 h (lane 3). In contrast,
in the cell line producing the aUele-mismatched AoldA/3 k
combination, there are minimal detectable increases in mo-
lecular weight in the c~ or/3 chains at the 3-h chase period
(compared lanes 4 and 5). In fact, at 3 h, there is a significant
decrease in the amount of labeled class II MHC molecules
recovered in the immunoprecipitates, and a significant frac-
tion of the o~ chains that are precipitated display a lower ap-
parent molecular weight compared with the o~ chains from
pulse-labeled cells, as was also seen with the isotype-
mismatched EotA/31kE/3 combination.
803 Sant et al.
Figure 5. Comparison of the post-translational processing of allele-
matched and allele-mismatched intraisotypic od3 chain combinations. L
cell transfectants producing the dficiently expressed allele-matched chains
AotkA/3 k or the inefficiently expressed aUele-mismatched chains AoedA/3 k
were biosynthetically labded with [3H]leucine for 30 min and either har-
vested immediately from culture (T = 0), or chased for 3 or 8 h in non-
radioactive medium. Detergent lysates were immunoprecipitated with an-
tisera specific for Aot, and immunoprecipitates were analyzed by SDS-10%
PAGE. The positions of the or,/3, and Ii chains are indicated on the left
of the figure.
Biochemical Evidence for RER or Early Cis-Golgi
Retention of ot/fl Dimers Showing Defective Cell
The preceding analysis of nonexpressed and poorly expressed
class II MHC o~ and/3 combinations indicates a major defect
in post-dimerization transport that is reflected in an absence
of the usual post-translational modifications revealed by SDS-
PAGE. To determine where inside the cell the processing events
were interrupted, immunoprecipitated proteins were subjected
to degradation by Endo H, which primarily cleaves imma-
ture, high-mannose N-linked oligosaccharides that are char-
acteristic of glycoproteins that have not yet entered the medial-
Golgi (33). In the experiment shown (Fig. 6), three types
of class II dimers were examined; the isotype-matched pair
E~E/3 d, which is efficiently expressed at the cell surface, the
isotype-mismatched pair Ec~A/31kE/3, which is not detect-
ably expressed at the cell surface, and the haplotype-
mismatched pair AotdA/3 k, which is inefficiently expressed
at the cell surface. Ceils were pulse labeled for 45 min (P)
and either harvested from culture or chased for an additional
4 h (C). Immunoprecipitates, prepared using anti-oe antibodies,
were denatured in SDS, and divided into two aliquots. One
aliquot was treated with Endo H (E/q), and the other was
mock digested (UT). After such treatment, samples were in-
cubated with either anti-o~ or anti-/3 CT antibodies, and in-
dividual chains were reprecipitated, then analyzed by SDS-
PAGE (Fig. 6).
In the L cells synthesizing the effciently expressed E~E/3 a
dimer, the c~ chains (compare Fig. 6 A, lanes 1 vs. 3) and
/3 chains (Fig. 6 A, lanes 5 vs. 7) are sensitive (S) to Endo
Figure 6. Analysis of intracellular processing of efficiently expressed, inef~ciently expressed, and nonexpressed class II MHC dimers. L cell transfec-
tants synthesizing the indicated class II chains were pulse labeled with [3H]leucine for 15 min and either harvested directly from culture (Pulse, P)
or chased in nonradioactive medium for 90 min (Chase, C). Detergent lysates were made from the labeled cells and ~x/fl immunoprecipitates were
prepared using anti-CT c~-specific rabbit antisera. Immunoprecipitates were either digested with Endoglycosidase H (EH) or mock digested (UT),
as described in Materials and Methods. Samples were then denatured in SDS to separate c, and fl chains and renatured with NP-40 buffer, cx and fl
chains were reisolated using CT-~x or -fl specific antisera as indicated. Immunoprecipitates were analyzed by SDS-10% PAGE. R and S to the left of
the figure refer to the positions of Endo H-resistant and Endo H-sensitive class II chains, respectively.
H after pulse labeling, as expected for glycoproteins that ex-
press high mannose oligosaccharides, but by 4 h, a significant
fraction of the o~ chains (Fig. 6 A, lanes 3 vs. 4) and fl chains
(Fig. 6 A, lanes 7 vs. 8) have Endo H-resistant (R) N-linked
glycans. This resistance presumably reflects the transport of
the dimer from the R.ER to the Golgi complex, where pro-
cessing of the N-linked oligosaccharides occurs (34). In con-
trast, in the cell lines that assemble dimers that are poorly
expressed at the cell surface, the cz and fl chains remain Endo
H-sensitive (S) throughout the chase (Fig. 6, B and C, lanes
3 vs. 4, and 7 vs. 8, respectively). This implies that they have
not yet entered the subcellular compartment in which the
glycosyltransferases responsible for conversion of oligosaccha-
rides from the high mannose to complex type reside. It thus
appears likely that the poorly expressed ol/fl dimers remain
in the PER or the c/s-Golgi, where they are degraded or from
which they directly exit to a degradative compartment. In-
terestingly, the time-dependent change in apparent molec-
ular weight of the transport-defective o~ and fl chains is no
longer apparent in the endo H-treated samples (compare Fig.
6, B and C, lanes I vs. 2 to lanes 3 vs. 4, and lanes 5 vs.
6 to lanes 7 vs. 8), indicating that the decrease in apparent
molecular weight of such claims seen in Figs. 4 and 5 is due
to modifications of the N-linked oligosaccharides.
A large body of prior work has documented that in cells
cosynthesizing a variety of allelic and isotypic forms of class
II MHC ol and 3 chains, only a limited subset of all possible
cz/fl dimer combinations is assembled and transported to the
surface membrane (2, 35). In part, this limitation reflects
preferences in stable ~/fl pairing during initial dimer assembly
so that favored combinations outcompete less-favored pairs
for available free chains (17, 18). However, even in the ab-
sence of competing chains, some combinations of cz and
give little or no detectable cell surface expression (9-15). In
the experiments described in this report, we have analyzed
the intracellular fate of combinations of oe and fl chains
showing poor or absent cell surface expression under non-
competitive conditions, which maximizes our ability to
visualize the formation and post-translational processing of
any dimers these combinations might form.
The fate of cosynthesized oe and fl chains has been exam-
ined biochemically in L cell transfectants representing three
distinct expression phenotypes: (a) two different cell lines
producing isotype- and/or allele-matched oe/fl combinations
showing efficient cell surface expression (Ecz/Efl a, Aolk/
Aflk); (b) a cell line making an inefficiently expressed allele-
mismatched combination (Aoed/Aflk); and (c) a cell line
producing an isotype-mismatched cz/fl combination showing
no detectable surface expression (Eoe/AfllkEfl). Biosynthetic
labeling studies revealed that despite very marked differences
in the efficiency with which each pair gave rise to surface
molecules, these o~/fl combinations showed similar initial levels
of assembled heterodimers stable to detergent solubilization
and immunoprecipitation. These results establish that the de-
fect in surface expression in the latter two cell lines is not
primarily due to deficient assembly per se, although our
experimental system does not permit us to exclude some
contribution of less efficient assembly to the overall pheno-
type. After initial assembly, however, the post-translational
processing of the various dimers appears to be distinct. The
allele- and/or isotype-matched combinations undergo readily
detectable post-translational modification of their N-linked
carbohydrate chains, whereas the ex/fl pairs showing poor
cell membrane expression do not. These data indicate that
both locus-specific and aUelically polymorphic residues in the
NH2-terminal domains of class II molecules control confor-
mational features of the assembled dimers that regulate post-
translational processing and intracellular transport.
Our results differ from those recently reported by Karp
et al. (15), who did not observe stable intracellular dimers
of human class II cz and fl combinations that failed to give
804 Defective Intracellular Transport Limits Expression of ol/fl Chains
detectable surface molecules. There are several possible ex-
planations for the differences seen in these two experimental
systems. The first is that the control of expression of human
and mouse class II gene products is distinct in the two spe-
cies. It is conceivable that the greater number ofdass II gene
products produced in individual human cells (2) has led to
a need for stricter regulation of assembly, so that competi-
tion for component chains does not deplete intraceUular chain
levels required for adequate expression of each isotype. The
importance of such intracellular competition in determining
patterns of cell surface class II molecule expression has been
demonstrated in both our transfection model (18) and more
recently in A3 transgenic mice (36). A second possibility is
that there may be substantially more diversity in the capacity
of various ot/B combinations to stably assemble than revealed
in the set of molecules we have examined in this report. The
pair tested by Karp et al. (15) might then represent one of
the more unfavored combinations. It is also likely that detec-
tion of assembled dimers depends on the experimental con-
ditions used to immunoprecipitate the chains, with instability
of weakly associated dimers in certain buffer/detergent com-
binations. The importance of the particular conditions of cell
lysis and immunoprecipitation in preservation of TCR-CD3
associations is well known and perhaps relevant in this re-
gard (37). This same explanation may underlie the failure
to coprecipitate A~ f with Eo~ from cells of A.TFR5 mice
(38), which show weak but significant expression ofEc~ epi-
topes on the cell surface, as our own studies indicate that
such expression requires dimer formation and is not due to
Ec~ transport to the membrane alone (A.J. Sant, C. Layet,
and R.N. Germain, unpublished observations).
The class II molecules that are retained within the trans-
fectants we have studied do not have any detectable confor-
mational features that distinguish them from efficiently trans-
ported dimers. We have tested their reactivity with a panel
of mAbs, and have not observed the absence of any serolog-
ical epitopes predicted to be present from studies on conven-
tional dimers containing these chains (A.J. Sant, L.R. Hen-
drix, and R.N. Germain, unpublished observations). This
contrasts with results obtained studying expression-defective
mutants of various viral envelope proteins (39, 40), but per-
haps is more related to the limited number of epitopic sites
seen by murine anti-MHC mAbs than to an absence of con-
formational differences among the well- and poorly expressed
class II MHC ol/~8 dimers.
In addition, both well- and poorly or non-expressed dimers
appear to coassemble with invariant chain (see Fig. 4, lane
I). Although we do not know whether the sites or affinity
of interaction are equivalent in the two cases, recent work
in this laboratory has revealed that the cell surface expression
that is observed for such complexes as AotaA~ k is due in
large measure to the ability of the invariant chain to "rescue"
these dimers from retention in a late ER/early Golgi com-
partment (41). These recent results showing that Ii can con-
tribute in a positive sense to cell surface expression of other-
wise poorly expressed dimer combinations make it unlikely
that inappropriate ol/3 dimer interaction with invariant chain
is responsible for the transport defect(s) we have observed.
Because it is not required for and does not markedly aug-
ment the transport and surface expression of haplotype- and
isotype-matched dimers (41-43), it remains possible, how-
ever, that a limitation in availability of Ii in the L cells con-
tributes disproportionately to the poor expression observed
for allele- and isotype-mismatched ot/B combinations in these
The structural features of proteins that regulate their in-
tracellular movement are poorly defined at the present time.
One major area of uncertainty is whether successful trans-
port between organelles is a consequence of expression of an
appropriate positive signal, which allows recognition by a
receptor protein responsible for transport, or if successful trans-
port is due to the lack of expression of retention signals. Evi-
dence in favor of each of these mechanisms exists in different
model systems (44-49). A variety of reports provide strong
support for the view that some proteins are retained in the
endoplasmic reticulum as a result of their association with
other proteins containing specific retrieval signals that mediate
their recycling from an intermediate compartment interposed
between the endoplasmic reticulum and c/s-Golgi compart-
ment (50-52). One such retrieval (retention) protein is BiP,
one of the KDEI.-containing lumenal endoplasmic reticulum
proteins believed to play an important role in polypeptide
folding (53). Rothman (54) has speculated that BiP and related
polypeptide chain binding proteins interact with varying
affinity with discrete peptide patches on proteins during their
import into the endoplasmic reticulum (54). When appro-
priate folding occurs, these sites become unavailable for con-
tinued BiP binding, and coretrieval of the protein with BiP
from the salvage pathway ceases. This allows egress to the
later Golgi and secretory/transport compartments. Inappropri-
ately folded molecttles would not lose these interaction patches
and be subject to continuous retrieval by association with
a KDEL-containing molecule.
This model of transport blockade may be applicable to both
our present results and the earlier work of Griffith et al. (55),
who studied B lymphomas with defects in surface class II
molecule expression. An identical mutation at a conserved
residue in the A~ d or E~ d chains led to assembly of im-
munoprecipitable dimers without full transport to the mem-
brane. For the AotaAB a molecule, fully modified N-linked
glycans were observed, suggesting arrest in a post-medial Golgi
compartment, whereas for Ec~aEB a, the dimers exhibited
only core glycosylation, consistent with the results reported
here (56). These dimers again were serologically indistinguish-
able from wild-type, despite their arrested intracellular trans-
port. It would thus appear that rather subtle structural vari-
ation from fully wild-type molecules is sufficient to interfere
with normal dass II molecule movement within the cell.
We have not yet precisely localized the site of retention
of the aberrantly assembled class II molecules by ultrastruc-
tural methods. The time-dependent loss in molecular weight
that the nonexpressed dimers undergo appears to be due to
the activity of glycosidases rather than proteases, based on
the finding that the difference in molecular weight disappears
805 Sant et al.
when the N-linked oligosaccharides are removed by Endo
H. Trimming of N-linked oligosaccharides chains occurs in
both the ILEK and cis-Golgi, through the activity of glucosi-
dases and mannosidases (34). Based on the magnitude of de-
crease in molecular weight that a and ~3 undergo (1,500),
it is likely that they have been completely trimmed, suggesting
that they have reached the cis-Golgi compartment. Glyco-
proteins bearing such trimmed carbohydrate groups remain
sensitive to Endo H. Addition of N-acetyl glucosamine,
through the activity of GlcNAc transferase I and II, which
occurs in the medial-Gol~ compartments, renders molecules
resistant to Endo H (33). Given the complete sensitivity of
the surface nonexpressed but assembled dimers to Endo H,
our data suggest that the block in intraceUular transport is
before arrival in the raediabGol~ compartment. The newly
described intermediate or salvage compartment between the
RER and Golgi compartment (52) may actually constitute
the site of carbohydrate trimming referred to above, consis-
tent with a retention mechanism involving retrograde trans-
port back to the REK in association with BiP-like molecules.
However, prdiminary studies examining the relative associa-
tion of the transportable vs. retained dimers with such en-
doplasmic reticulum-resident polypeptides have not shown
any striking differences. Additional studies of the transport-
defective molecules reported here will be necessary to define
the mechanism of this block, and should provide useful in-
formation about intracellular protein trafficking in general.
We thank Drs. Eric Long and David Margulies for their thoughtful reviews of this manuscript, and Dr.
Jim Miller for helpful discussions of these studies.
This work was supported in part by a National Institutes of Health postdoctoral fellowship (A. J. Sant)
and by an Arthritis Foundation Investigator Award (A. J. Sant).
Address correspondence to Andrea J. Sant, Department of Pathology, University of Chicago, 5841 South
Maryland Avenue, Box 414, Chicago, IL 60637. W. Lee Maloy's present address is Magainin Sciences
Inc., Plymouth Meeting, PA 19462.
Received for publication 9 November 1990 and in revised form 12 June 1991.
1. Schwartz, R.H. 1985. T-lymphocyte recognition of antigen
in association with gene products of the major histocompati-
bility complex. Annu. Rev. Imraunol. 3:237.
2. Kaufman, J.F., C. Auffray, A.J. Korman, D.A. Shackelford,
and J. Strominger. 1984. The class II molecules of the human
and murine major histocompatibility complex. Cell. 36:1.
3. Jones, P.P., D.B. Murphy, and H.O. McDevitt. 1981. Variable
synthesis and expression of Ec~ and Ae (EB) Ia polypeptide
chains in mice of different H-2 haplotypes. Imraunogenetics.
4. Rabourdin-Combe, C., and B. Mach. 1983. Expression of HLA-
DR antigens at the surface of mouse L cells co-transfected with
cloned human genes. Nature (Lond.). 303:670.
5. Malissen, B., M. Steinmetz, M. McMillan, M. Pierres, and
L. Hood. 1983. Expression of I-A k class II genes in mouse L
cells after DNA-mediated gene transfer. Nature (LoncL). 305:440.
6. Norcross, M.A., D.M. Bentley, D.H. Margulies, and R.N. Get-
main. 1984. Membrane Ia expression and antigen presenting
accessory function of L cells transfected with class II major
histocompatibility genes. J. Ex F Med. 160:1316.
7. McMillan, M., J.A. Frelinger, P.P. Jones, D.B. Murphy, H.O.
McDevitt, and L. Hood. 1981. Structure of murine Ia antigens.
Two dimensional electrophoretic analysis and high pressure
liquid chromatography tryptic Peptide maps of products of the
I-A and I-E subregions and of an associated invariant chain.
J. EXl~ Med. 153:936.
8. Schlauder, G.G., M.P. Bell, B.N. Beck, A. Nilson, and D.J.
McKean. 1985. The structure-function relationship of I-A mol-
ecules: a biochemical analysis of A polypeptides from mutant
antigen-presenting cells and evidence of preferential associa-
tion of allelic forms. J. Immunol. 135:1945.
9. Germain, R.N., D.M. Bentley, and H. Quill. 1985. Influence
of alhlic polymorphism on the assembly and cell surface ex-
pression of class II MHC (Ia) molecules. Cell. 43:233.
10. Brannstein, N.S., and R.N. Germain. 1987. AUele-specific con-
trol of Ia molecule surface expression and conformation: im-
plications for a general model of Ia structure-function relation-
ships. Pro~ Natl. Acad. Sci. USA. 84:2921.
11. Germain, R.N., and H. Quill. 1986. Unexpected expression
of a unique mixed-isotype class II MHC molecule by trans-
fected L cells. Nature (Lond.). 320:72.
12. Sant, A.J., N.S. Braunstein, and IL.N. Germain. 1987. Pre-
dominant role of amino-terminal sequences in dictating
e~dency of class II major histocompatibility complex a/3 dimer
expression. Pwc Natl. Acad. Sci. USA. 84:8065.
13. Lechler, R.I., A.J. Sant, N.S. Braunstein, R. Sekaly, E. Long,
and K.N. Germain. 1990. Cell surface expression of hybrid
murine/human MHC class II ~/a dimers. Key influence of
residues in the amino-terminal portion of the 81 domain. J.
14. Braunstein, N.S., R.N. Germain, K. Loney, and N. Berkowitz.
1990. Structurally interdependent and independent regions of
allelic polymorphism in class II MHC molecules: implications
for Ia function and evolution. J. Immunol. 145:1635.
806 Defective Intracellular Transport Limits Expression of c~/13 Chains
15. Karp, D.R., C.L. Tr
Coligan, and E.O. Long. 1990. Structural requirements for
pairing of ot and 3 chains in HLA-DR and HLA-DP mole-
cules, J. Extx Med. 171:615.
16. Buerstedde, J.M., L.R. Pease, A.E. Nilson, M.P. Bell, C. Chase,
G. Buerstedde, and D.J. McKean. 1988. Regulation of murine
MHC class II molecule expression. Identification of A3 residues
responsible for allele-slxxific cell surface expression..]. Extx Med.
17. Lottean, V., J. Sands, L. Teyton, P. Turmd, D. Charron, and
J.L. Strominger. 1989. Modulation of HLA class II antigen
expression by transfection of sense and antisense DR~ cDNA.
J. ExF Med. 169:351.
18. Sant, A.J., and K.N. Germain. 1989. Intracellular competi-
tion for component chains determines class II MHC cell sur-
face phenotype. Cell. 57:797.
19. Kvist, S., K. Wiman, L. Claesson, P.A. Peterson, and B. Dob-
berstein. 1982. Membrane insertion and oligomeric assembly
of HLA-DR histocompatibility antigens. Cell, 1:61.
20. Jones, P.P., D.B. Murphy, D. Hewgill, and H.O. McDevitt.
1979. Detection of a common polypeptide chain in I-A and
I-E sub-region immunoprecipitates. Mol. Immunol. 16:51.
21. Sung, E., and P.P. Jones. 1981. The invariant chain of murine
Ia antigens: its glycosylation, abundance, and subcellular lo-
calization. MoI. Immunol. 10:899.
22. Machamer, C.E., and P. Cresswell. 1984. Monensin prevents
terminal glycosylation of the N- and O-linked oligosaccharides
of the HLA-DR associated invariant chain and inhibits its dis-
sociation from the ot-~ chain complex. Prec. Natl. Acad. Sci.
23. Guagliardi, L.E., B. Koppelman, J.S. Blum, M.S. Marks, P.
Cresswell, and F.M. Brodsky. 1990. Co-localization of mole-
cules involved in antigen processing and presentation in an early
endocytic compartment. Nature (Lend.). 343:133.
24. Neefjes, J.J., V. Stollorz, P.J. Peters, H.J. Geuze, and H.L.
Ploegh. 1990. The biosynthetic pathway of MHC class II but
not class I molecules intersects the endocytic route. Cell. 61:171.
25. Harding, C.V., F. Leyva-Cobian, and E.R. Unanue. 1988.
Mechanisms of antigen processing. Immunol. Rev. 106:77.
26. Margulies, D.H., G.A. Evans, K. Ozato, R.D. Camerini-Otero,
K. Tanaha, E. Appella, and J.G. Seidman. 1983. Expression
of H-2D d and H-2L d mouse major histocompatibility antigen
genes in L cells after DNA-mediated gene transfer.J, Immunol.
27. Lechler, R.I., M.A. Norcross, and R.N. Germain. 1985.
Qualitative and quantitative studies of antigen-presenting cell
function by using I-A-expressing L cells../, lmmunol. 135:2914.
28. Oi, V.T., P.P. Jones, J.W. Goding, L.A. Herzenberg, and L.A.
Herzenberg. 1978. Properties of monoclonal antibodies to
mouse Ig allotypes, H-2, and Ia antigens. Curt. Totx Microbiol.
29. Ozato, K., N. Mayer, and D.H. Sachs. 1980. Hybridoma cell
lines secreting monodonal antibodies to mouse H-2 and Ia an-
tigens. J. Immunol. 124:533.
30. Sant, A.J., S.E. Cullen, K.S. Giacoletto, and B.D. Schwartz,
1985. Invariant chain is the core protein of the h-associated
chondroitin sulfate proteoglycan. J. Exlx Med. 162:1916.
31. Sant, A.J., S.E. Cullen, and B.D. Schwartz. 1985. Biosynthetic
relationships of the chondroitin sulfate proteoglycan with Ia
and invariant chain glycoproteins. J. Immunol. 135:416.
32. Tarentino, A.L., T.H. Plummet, Jr., and F. Maley. 1974. The
release of intact oligosaccharide from specific glycoproteins by
D. Jaraquemada, W.L. Maloy, J.E.
endo-3-N-acetylglucosaminidase. J. Biol. Chem. 249:818.
33. Kobata, A. 1979. Use of endo- and exoglycosidases for struc-
tural studies of glycoconjugates. Anal. Biochem. 100:1.
34. Kornfeld, R., and S. Kornfeld. 1985. Assembly of asparagine-
linked oligosaccharides. Annu. Reg Biochem. 54:631.
35. Mengle-Gaw, L., and H.O. McDevitt. 1985. Genetics and ex-
pression of mouse Ia antigens. Annu. Rev. Immunol. 3:367.
36. Gilfillan, S., S. Aiso, S.A. Michie, and H.O. McDevitt. 1990.
The effect of excess ~-chain synthesis on cell-surface expres-
sion of allele-mismatched class II heterodimers in rive. Proc
Natl. Acad. Sci. USA. 87:7314.
37. Ashwell, J.D., and R.D. Klausner. 1990. Genetic and muta-
tional analysis of the T-cell antigen receptor. Annu. Rev. Im-
38. Begovich, A.B., and P.P. Jones. 1985. Free Ia Eot chain expres-
sion in the Eoe+:E/3-recombinant strain A.TFR5. Immune-
39. Gething, M.J., K. McCammon, and J. Sambrook. 1986. Ex-
pression of wild-type and mutant forms of influenza hemag-
glutinin: The role of folding in intracellular transport. Cell.
40. Copeland, C., K-P. Zimmer, K. Wagner, G. Healey, I. Mellman,
and A. Hdenius. 1988. Folding, trimerization and transport
are sequential events in the biogenesis of influenza virus hemag-
glutinin. Cell. 53:197.
41. Layer, C., and R.N. Germain. 1991. Invariant chain (Ii) pro-
motes egress of poorly expressed, haplotype-mismatched class
II major histocompatibility complex ActA[3 dimers from the
endoplasmic reticulum/c/s-golgi. Prec. Natl. Acad. Sci, USA.
42. Miller, J., and R.N. Germain. 1986. Efficient cell surface ex-
pression of class II MHC molecules in the absence of associated
invariant chain. J. Exl~ Med. 164:1478.
43. Sekaly, R.-P., C. Tonnelle, M. Strubin, B. Much, and E.O.
Long. 1986. Cell surface expression of class II histocompati-
bility antigen occurs in the absence of the invariant chain, jr.
Ex!a Med. 164:1490.
44. Colman, A., and C. Robinson. 1986. Protein import into or-
ganelles: Hierarchical targeting signals. Cell. 46:321.
45. Pfeffer, S.R., and J.E. Rothman. 1987. Biosynthetic protein
transport and sorting by the rough endoplasmic reticulum and
Golgi. Annu, R~. Biochem. 56:829.
46. Fitting, T., and D. Kabat. 1982. Evidence for a glycoprotein
"signal" involved in transport between subcellular organelles.
J. Biol. Chem. 257:14011.
47. Horwich, A.L., F. Kalousek, W.A. Fenton, R.A. Pollack, and
L.E. Rosenberg. 1986. Targeting ofpre-ornithine transcarbamy-
lase to mitochondria: definition of critical regions and residues
in the leader peptide. Cell. 44:451.
48. Hurt, E.C., B. Pesold-Hurt, and G. Schatz. 1984. The amino-
terminal region of an imported mitochondrial precursor poly-
peptide can direct cytoplasmic dihydrofolate reductase into the
mitochondrial matrix. EMBO (Eur. Mol. Biol. Organ.)J. 3:3149.
49. Wieland, F., M.G. Gleason, T. Serafini, andJ. Rothman. 1987.
The rate of bulk flow from the endoplasmic reticulum to the
cell surface. Cell. 50:289.
50. Bole, D.G., L.M. Hendershot, and J.F. Kearney. 1986. Post-
translational association of immunoglobulin heavy chain
binding protein with nascent heavy chains in non-secreting
and secreting hybridomas, j, Cell Biol. 102:1558.
51. Hurtley, S.M., D.G. Bole, H. Hoover-Litty, A. Helenius, and
C.S. Copeland. 1989. Interactions of misfolded virus hemag-
807 Sant et al.
glutinin with binding protein (BiP). J. Cell Biol. 108:2117.
52. Lippincott-Schwartz, J., J.G. Donaldson, A. Schweizer, E.G.
Berger, H.-P. Hauri, L.C. Yuan, and lk.D. Klausner. 1990.
Microtubule-dependent retrograde transport of proteins into
the ER in the presence of Brefeldin A suggests an ER recy-
cling pathway. Cell. 60:821.
53. Munro, S., and H.R.B. Pelham. 1987. A C-terminal signal pre-
vents secretion of luminal ER proteins. Cell. 48:899.
54. Rothman, J.E. 1989. Polypeptide chain binding proteins:
Catalysts of protein folding and related processes of cells. Cell.
55. Gri~th, I.J., N. Nabava, Z. Ghogawala, C.G. Chase, M. Rodri-
quez, D.J. McKean, and L.H. Glimcher. 1988. Structural mu-
tation affecting intracellular transport and cell surface expres-
sion of murine class II molecules. J. EXl~ IVied. 167:541.
56. Glimcher, L.G., D.J. McI~an, E. Choi, andJ.G. Seidman. 1985.
Complex regulation of class II gene expression: analysis with
class II mutant cell lines. J. Immunol. 135:3542.
808 Defective IntraceUular Transport Limits Expression of c~//~ Chains