(Received for publication, September 5, 1996, and in revised form, November 7, 1996)
From the Institut für Biochemie I der Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
Oligomeric proteins usually have to assemble into
their final quartenary structure to be secreted. However, most
immunoglobulin (Ig) light (L) chains can be exported as free chains,
whereas only a few Ig L chains, here referred to as export-incompetent, have to assemble with Ig heavy (H) chains into antibody molecules to be
secreted. In the absence of Ig H chain expression, these export-incompetent Ig L chains remain bound to BiP as partially folded
monomers with only one of the two internal disulfide bonds being
formed. To understand the apparent discrepancy in Ig L chain export, we
performed assembly studies with chimeric Ig chains and found that the
variable (V) domain of the export-incompetent NS1 chain cannot
mediate homodimer formation. Conversely, the V domain of the
export-competent J558L
1 chain supports homodimer formation and, concordantly, these Ig L chains are secreted as noncovalently or covalently linked homodimers. We show that the export-incompetent mutant
1 FS62 chain forms disulfide
bonds in both domains only upon pairing with Ig H chain and is secreted as part of an antibody. Therefore, Ig L chain assembly seems to be a
prerequisite for complete folding, indicating that Ig L chain secretion
generally depends on either homo- or heterodimer formation. We discuss
a mechanism that controls oligomerization by monitoring the
conformation of individual subunits that cannot proceed in folding
prior to successful assembly.
In eukaryotic cells, various soluble and membrane-bound proteins
travel along the secretory pathway to reach their final destination. These molecules first enter the endoplasmic reticulum
(ER)1 as unfolded polypeptides, which are
modified there to reach their final three-dimensional structure.
Attributes such as conformation, structure of attached carbohydrates,
and oligomeric state not only control the functional properties of a
protein but are also critical for intracellular transport. Newly
synthesized polypeptides are hampered in exiting the ER as long as they
have not acquired a so-called export-competent conformation (for
reviews, see Refs. 1-3). Oligomeric proteins such as influenza
hemagglutinin or vesicular stomatitis virus glycoprotein are
homo-oligomers that must be trimerized prior to exit from the ER
(4-6). In other cases, export is not dependent on complete assembly.
While the heptameric T-cell receptor complex only reaches the cell
surface when completely assembled, in the absence of chain
homodimer association, pentamers leave the ER and are subsequently
transported to lysosomes instead. Other assembly intermediates or
isolated subunits are retained in the ER (7). The export of major
histocompatibility complex class I antigens usually depends on the
noncovalent assembly of a transmembrane heavy chain, the soluble
2-microglobulin, and peptide (8), but soluble
2-microglobulin can be secreted as free chains (9).
Similarly, free immunoglobulin (Ig) heavy (H) chains are not exported
(10, 11) unless assembled with Ig light (L) chains into antibody
molecules (12), while most Ig L chains can also be secreted as free
molecules (13, 14). Astonishingly, some Ig L chains (here referred to
as export-incompetent) do not follow this rule but depend on Ig H chain
association to be secreted (14-16). To date, there is no valid model
to explain this apparent discrepancy.
Using immunoglobulins as a model to study the requirements of ER export, Sitia and colleagues have uncovered a quality control mechanism that acts on polypeptides to expose unpaired cysteine residues (17-20). Immunoglobulin L chains possess five cysteine residues, four of which are involved in two disulfide bonds that stabilize the variable (V) and constant (C) domain, respectively, and a carboxyl-terminal cysteine that is responsible for the intermolecular disulfide bond to Ig H chain. The thiol group of the carboxyl-terminal cysteine of unassembled Ig L chains causes a delay in secretion because it forms reversible disulfide bonds with the protein matrix of the ER (21). However, secreted Ig L chains no longer possess unpaired cysteines since the two internal disulfide bonds have already formed (22) and the carboxyl-terminal cysteine is either covalently linked to a second Ig L chain (or to Ig H chain) or paired with a free cysteine (21, 23).
Export-incompetent Ig L chains, which are not secreted in the absence of Ig H chains may also covalently interact with ER matrix proteins. However, the major cause of retention is probably the noncovalent binding to BiP, an ER-resident molecular chaperone (24, 25). It seems that only one of the domains can fold in vivo because such Ig L chains occur as partially oxidized BiP-bound monomers until they are degraded (22, 26). Complete oxidation of the Ig L chain reflecting disulfide bond formation in the second domain is only observed upon in vitro dissociation from BiP (22). As protein export is thought to be restricted to completely folded molecules, we reasoned that oligomerization might be a prerequisite for Ig L chain folding because Ig H chain association is sufficient to restore export competence to otherwise partially folded molecules.
The experiments reported here were designed to better understand the molecular mechanism underlying Ig L chain maturation and export. We have established an experimental system that allows us to investigate the capacity of particular V domains to pair with each other and found that not all support Ig chain assembly. Furthermore, we analyzed the in vivo folding state of unassembled and assembled Ig L chains. We will discuss our results with respect to the possible folding pathway of Ig L chains as well as to the general mechanism of quality control in the ER.
Expression vector p3 (named
B1-8.VHC
3 in Ref. 27) is derived from pEC
3 (28) and contains the
gene segments encoding the mouse
3 Ig H chain constant (C) region
and the rearranged Ig H chain B1.8 variable (V) region. pSVE2neo
1
(22) encodes wild type Balb/c
1 Ig L chain. pEVHC
neo
carries the rearranged Ig H chain B1.8 V region gene segment (27)
together with the mouse
chain C region gene segment. To construct
pEV
C
3, the rearranged V region gene segment of the
chain-expressing myeloma MOPC21 was isolated from the plasmid
pB1-14 (29) as a 2.3-kilobase SacI fragment,
inserted into the SacI site of pBluescript KS+
(Stratagene, La Jolla, CA), re-excised as
EcoRI/SalI fragment, and recloned into the
expression vector pEC
3, which had been digested similarly. To
construct pEV
C
3, the rearranged V region gene segment of the
1 chain-expressing myeloma A8-6 (identical to myeloma
A8-8; Ref. 30) was taken as a 2.6-kilobase
EcoRI/BamHI fragment from pSVE2neo
1 (22),
subcloned into pBluescript KS+, re-excised as an
EcoRI/SalI fragment, and ligated into pEC
3, which had been similarly digested.
Mouse myeloma X63Ag8.653 and
hybridoma H62 express no Ig chains (31, 32). J558L is a Ig H chain loss
variant of the mouse plasmacytoma J558 (,
1; Ref.
16). NS1 is a mouse plasmacytoma synthesizing but not secreting
chains (15). NSFS62 is a stable transfectant of NS1 that lost
endogenous
chain and expresses the export-incompetent Ig L chain
1 FS62 (22). Cell line E7.
1 was obtained
by transfecting E7 (a stable transfectant of H62 that expresses
3 Ig
H chains (33) bearing the B1.8 VH domain (34)) with
pSVE2neo
1. J558L.
3 co-expresses endogenous
1 chain and the same
3 chain as E7 (a generous gift of T. Simon).
Transfection of NSFS62 with p
3 gave rise to NSFS62.
3
(
1 FS62 Ig L and
3 Ig H chains). NS1.
3 is a stable
transfectant of NS1 and expresses endogenous
Ig L and the
p
3-encoded Ig H chain. The transfectant NS1.V
C
3 co-expresses
the endogenous
Ig L chain and the vector-encoded chimeric
V
C
3 Ig H chain. J558L.V
C
3 derives
from J558L and co-expresses endogenous
1 Ig L chain and
the chimeric V
C
3 Ig H chain. II4c.MEV
(co-expressing chimeric VHC
Ig L and V
C
3 Ig H chains) was made by
co-transfection of H62 with pEVHC
.neo and pEV
C
3. All cells
were maintained as described (24). Transfection of cells by
electroporation and selection of stable transfectants were performed in
principle as described by Allen et al. (28). A list of cell
lines, corresponding transfectants, and respective Ig chains expressed
is given in Table I.
|
As detailed elsewhere (24, 33), starved cells were labeled with [35S]methionine (>37 TBq/mmol; Amersham Corp.) and washed in phosphate-buffered saline prior to solubilization in NET lysis buffer (150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM Tris/HCl, pH 7.4). When indicated, wash and lysis buffer contained 20 mM NEM to prevent oxidation of free sulfhydryl groups. Immunprecipitation of Ig chains was performed with subunit-specific antibodies and/or protein A-Sepharose. Size fractionation of proteins was achieved by using a column (diameter, 1 cm; length, 40 cm) containing Sephacryl S-200HR (Pharmacia LKB, Freiburg, Germany). Proteins were separated by native or SDS-PAGE, transferred onto nitrocellulose filters by semidry blotting for Western blotting or visualized by autoradiograpy (24).
Native Gel ElectrophoresisPost-nuclear supernatant (10 µl; 4 × 107 cells/ml of NET lysis buffer) was mixed
with sample buffer (30 µl; 0.1 M Tris/HCl, pH 6.8, 8.7%
glycerol, 103% bromphenol blue) and applied onto a 6%
nondenaturing slab gel. The separating gel was prepared by diluting 6 ml of a mixture containing 30% acrylamide and 0.44% bisacrylamide in
24 ml of 0.1 M glycine/NaOH, pH 9.8. The stacking gel
contained 1 ml of the same acrylamide mixture, 2.5 ml of 0.5 M Tris/HCl, pH 6.8, and 6.3 ml of H2O.
Electrophoresis in 0.1 M glycine/NaOH, pH 9.8, at 4 °C
was done at 10 mA for 30 min until the blue dye reached the separating
gel and thereafter at 20 mA for 7 h. Proteins were transferred
onto nitrocellulose sheets and the Western blots developed as described
below.
All antisera were purchased from
Southern Biotechnology Associates (Birmingham, AL). Affinity-purified
goat anti-mouse antibody subunit-specific antisera bound to protein
A-Sepharose or protein A-Sepharose alone were used for
immunoprecipitations. Antibodies bearing the B1.8 idiotype react with
the hapten NP (4-hydroxy-3-nitro-phenyl)acetyl (34) and could be
isolated with NP-caproate coupled to Sepharose (NP-Sepharose). Western
blots were developed with biotinylated affinity-purified goat
anti-mouse 1 or
3 chain antisera, followed by a
staining reaction catalyzed by streptavidin-alkaline phosphatase. Alternatively, streptavidin-horseradish conjugate was used for detection with the BM chemiluminiscence blotting substrate (Boehringer, Mannheim, Germany).
The chain expressed by NS1 cells is a well known example of an
export-incompetent Ig L chain. Free NS1
chain is disulfide-bonded in one domain only and bound to BiP as a monomer (22), whereas the same
IgL chain is secreted when associated with Ig H chain (15). Therefore,
we reasoned that oligomerization might be the prerequisite for
secretion of Ig L chains. This model would predict that, in the absence
of Ig H chain expression, export-competent Ig L chains differ from
export-incompetent Ig L chains in their capability to form
homodimers.
As both export-competent and -incompetent Ig L chains may bear the same C domain (24, 35), the export competence of a free Ig L chain must be determined by its V region. That is, the V domain of the Ig L chain should determine whether or not homodimer formation is possible. To investigate this prediction, we used chimeric Ig chains in which the V regions of Ig H and L chains have been swapped. Such chimeric chains are assembled into antibodies as well as wild type chains (27). As the mode of Ig chain pairing does not seem to depend on the intramolecular V-C conjunction of the individual subunits, we can study the impact of specific V domains on chain pairing by investigating antibody formation in cells that expressed chimeric Ig chains.
Using this system, we assayed the assembly of Ig H and L chains bearing
identical V domains derived from either an export-competent Ig L chain
(the 1 chain of J558L), or an export-incompetent Ig L
chain (the
chain of NS1). Hybrid genes
(V
C
3 and V
C
3) were constructed and stable
transfectants of J558L (expressing
1 chains) or NS1
(expressing NS1
chains) cells established (Fig. 1;
J558L.V
C
3 and NS1.V
C
3, respectively). In order to verify
that chimeric chains can, in principle, form antibodies, we made other
transfectants expressing Ig H and L chains with complementary V
domains. As summarized Fig. 1 (and Table I), these
include NS1.
3 (endogenous
and wild type
3 chains), II4c.MEV
(VHC
and V
C
3
chains with mutually exchanged V domains), and J558L.
3 (endogenous
1 and wild type
3 chains).
To analyze the assembly state of the Ig chains, cellular proteins were
biosynthetically labeled with [35S]methionine and
material containing Ig H chains was isolated with protein A-Sepharose
and analyzed by SDS-PAGE under non-reducing conditions (Fig.
2). In the control cell line, NS1.3 (NS1
chains and wild type
3 chains), most of the Ig H chains migrated as H2L2 molecules. In addition, all covalent
assembly intermediates as well as some free Ig H chains (H2
and H) were detected (Fig. 2a, lane 1). Only
40-50% of the labeled Ig L chains were associated with Ig H chains,
as quantified by densitometry of the fluorograph (data not shown) seen
in Fig. 2b (lanes 1 and 2),
i.e. Ig L chain seems to be in a molar excess over Ig H
chain. Ig chains expressed by II4c.MEV cells
(VHC
and V
C
3)
differ from those of the NS1.
3 cell line only by a mutual exchange
of the V
and VH domains. Although most
intracellular II4c.MEV Ig H chains migrated as monomers under
non-reducing conditions, Ig molecules were also isolated as
H2L2 tetramers, H2L, HL, and
H2 assembly intermediates (Fig. 2a, lane
2) in agreement with the data of Simon and Rajewsky (27). We
assume that the low levels of Ig L chains synthesized by the II4c.MEV
cells limited antibody formation, since all of the intracellular Ig L
chains coprecipitated with the Ig H chains (Fig. 2b, compare
lanes 3 and 4). In contrast, no covalently
associated Ig molecules were detected in NS1.V
C
3 cells, which
express Ig H and L chains each bearing the V domain of the NS1
chain (Fig. 2a, lane 3). In this cell line, Ig L chain is not limiting; high levels of endogenous
chain were produced (Fig. 2b, lane 5). Only traces of
labeled
chain (<0.5%, as determined by densitometry) were
co-isolated with Ig H chain (Fig. 2b, lane 6).
Thus, it seems clear that NS1
chains assemble with Ig H chains
bearing the VH domain but not the V
domain of NS1.
In contrast, export-competent J558L 1 chains assemble
well with Ig H chains bearing either the VH domain or the
V
domain. For example, most Ig H chains in both
transfectants assembled into H2L2 tetramers and
covalently linked H2L and HL intermediates (Fig.
2a, lanes 4 and 5, respectively). Only
part of the total intracellular Ig L chains of both J558L.
3 and
J558L.V
C
3 coprecipitated with the Ig H chains (Fig.
2b, compare lanes 7 and 9 with
lanes 8 and 10, respectively), i.e.
endogenous Ig L chains were in molar excess over the vector-encoded Ig
H chains.
In summary, no association is observed when both Ig H and L chains
carry the V domain of the NS1 chain, indicating that the V domain
of the export-incompetent Ig L chain is not capable of forming the
homodomain dimers required to support Ig chain assembly. However, the
individual chains are, in principle, able to assemble into antibody
molecules because they assemble with partner chains that carry the V
domain of the Ig H chain. Covalent assembly of Ig H and L chains also
takes place when both Ig chains bear the V domain of the
export-competent J558L
1 chain, a result indicating that
these V domains are capable of homodomain pairing.
Our results
indicated that the V domain of the export-competent J558L
1 chains could support Ig L chain homodimer formation. Indeed, J558L
1 chains migrate as a single species when
total cellular proteins were separated under native conditions that ought to have preserved all native complexes (Fig. 3).
Together with the fact that a fraction of the J558L
1
chains migrate as covalently linked dimers in non-reducing SDS-gels
(24), the finding of a single band in the native gel suggests that most intracellular Ig L chains are in the same assembly state,
i.e. dimers.
To verify this assumption, we analyzed total cellular proteins as well
as proteins from the culture supernatant of J558L cells by gel
filtration (Fig. 4). Most of the intracellular
covalently linked Ig L chain dimers eluted at a position corresponding
to the expected molecular mass of 55 kDa (maximum at fraction 18 in
Fig. 4, left panel). Most of the non-disulfide linked Ig L chains also left the column in the dimer fraction, indicating that
these Ig L chains are noncovalently linked dimers. Only a minor
fraction eluted in the low molecular mass fractions from 10 to 25 kDa
(fractions 23-25). The asymmetric elution profile of the noncovalently
linked Ig L chains suggests that some dimers disintegrated during the
gel filtration procedure (36) and, as a consequence, eluted in the
lower molecular mass fractions. A similar co-elution profile of
covalently and noncovalently linked Ig L chain dimers was evident with
the culture supernatant (Fig. 4, right panel). The
overwhelming majority of J558L 1 chains form homodimers
and are secreted as such; some of are covalently linked and some are
not. In contrast, a similar analysis showed that the export-incompetent
NS1
chain is monomeric and complexed with BiP (22).
A Mutation in the
It was reported that the Phe to Ser
mutation at position 62 in a conserved sequence exposed on the surface
of the 1 chain V domain blocked the secretion of the Ig
L chain whether free or associated with µ Ig H chain (37). We
previously analyzed this particular Ig L chain,
1 FS62,
expressed in the absence of Ig H chains and showed that they are indeed
not transported but are degraded instead (22). Here, we report an
analysis of the export of the same Ig L chain in stably transfected
cells, NSFS.
3, that co-express
3 Ig H chain. In contrast to the
model used by Dul and Argon (37), our experiments involve (i) stable rather than transient expression and (ii) expression of
rather than µ chain. We observed that
1 FS62 was secreted as part
of assembled IgG. In fact, IgG secretion was as efficient as in the control cell line, E7.
1, which expresses
3 chain in
combination with a
1 chain that does not carry the Phe
to Ser substitution (Fig. 5). Since the mutation in the
V domain of the
1 chain does not lead to a general block
in Ig secretion, these results demonstrate that it does not destroy a
putative transport signal postulated by Dul and Argon (37).
Both Internal Disulfide Bonds Are Formed in Export-incompetent
Previous studies had shown that the export-incompetent
1 FS62 chain, like NS1
chain, is bound to BiP as a
partially oxidized molecule, whereas the export-competent J558L
1 chain exhibits a fully oxidized conformation (22). In
order to investigate the oxidation state of the
1 FS62
chain in cells co-expressing Ig H chain, lysates of NSFS62.
3 cells
were prepared in the presence of N-ethylmaleimide (to
prevent oxygen-mediated oxidation of thiol groups) and
immunoprecipitated Ig was analyzed by Western blotting under
non-reducing conditions (Fig. 6). Analysis of total Ig
chains revealed two Ig L chain bands (Fig. 6, lane 4)
corresponding to a partially and a completely oxidized form; both forms
migrated more rapidly than completely reduced Ig L chain (compare
lane 4 with lane 1). When Ig H chain-associated
material was analyzed (lane 5), the co-precipitated Ig L
chains migrated as fully oxidized molecules as did the J558L
1 chains (lane 2). In contrast, Ig L chains
remaining in the Ig H chain-depleted fraction were partially oxidized
(lane 6) as they were in the absence of Ig H chain
expression (lane 3). Thus, in the same cell, free
1 FS62 chain is partially oxidized, while Ig L chain
co-precipitated with Ig H chain is completely oxidized.
The finding that 1
FS62 chains have formed the second internal disulfide bond upon Ig H
chain association raises the question whether assembly precedes
oxidation of the second Ig L chain domain or is required for
stabilization of completely folded
1 FS62 chain. To
determine the sequence of events, we performed pulse-chase experiments
with NSFS62.
3 cells and analyzed the oxidation state of Ig H
chain-bound L chains (Fig. 7). Both fully and partially oxidized labeled Ig L chains were co-precipitated with Ig H chain isolated directly after the pulse (lane 2), indicating that
Ig L chains can assemble with Ig H chains as partially oxidized
molecules. The majority of labeled Ig H chains migrated as monomers
(lane 2) and resolved into at least four bands (clearly
visible in shorter exposed autoradiographs; data not shown), which
might not only reflect Ig H chain with different glycomoiety structure
but could also be due to Ig H chain with various numbers of internal
disulfide bonds, as the fully reduced Ig H chain exhibited a higher
apparent molecular weight and resolved into only two bands (lane
1).
After a chase period of 4 h, a different pattern was obtained. As compared to the situation present directly after the pulse, more noncovalently linked labeled Ig L chains were co-precipitated with Ig H chain after 4 h of chase indicating that unlabeled Ig H chains synthesized during the chase associated with pre-existing labeled Ig L chains (Fig. 7, lane 3). The majority of these Ig L chains migrated as fully oxidized molecules. More label is also found with covalently linked Ig H-L chain assembly intermediates as well as with complete H2L2 molecules. Conversely, a decreased amount of labeled molecules was present in the monomeric Ig H chain pool (lane 3). Interestingly, the amount of slower migrating Ig H chain monomers was more reduced as compared to the amount of the more rapidly migrating species. This could reflect that assembly with Ig L chain allowed Ig H chain to form internal disulfide bonds that increase Ig H chain motility (33).
Consecutive precipitation of total Ig chains remaining in the Ig H chain-depleted fraction revealed that the majority of labeled intracellular Ig L chains were unassembled and partially oxidized (Fig. 7, lane 4). The presence of Ig L chains migrating as fully oxidized molecules in this fraction could be due to disassembly of the molecules from Ig H chains during the first immunoprecipitation procedure because of weak binding. Decrease in the total amount of free partially oxidized Ig L chain during the chase period (compare lanes 4 and 5) might reflect assembly with Ig H chain, on the one hand, or the degradation of free Ig L chains as is the case with these Ig L chains when expressed in the absence of Ig H chains, on the other hand (22). BiP co-immunoprecipitated with Ig H chains as well as with Ig chains present in the Ig H chain-depleted samples (lanes 2-5).
In conclusion, these experiments demonstrate that partially oxidized
1 FS62 chain is able to bind Ig H chain and could
oxidize in the second domain only as the result of Ig H chain
co-expression. However, these experiments did not allow us to
unambiguously determine the fate of partially oxidized Ig L chains in
Ig H chain-expressing cells because the conversion of noncovalent Ig
L-H chain complexes into covalently linked complexes interfered with
the analysis of the Ig L chain oxidation state, which could only be
followed in Ig L chains noncovalently associated with Ig H chain.
Therefore, these data could not prove that assembly is a prerequisite
for Ig L chain folding. Nevertheless, the fact that partially oxidized Ig L chains coprecipitated with Ig H chain together with the
time-dependent increase in the amount of fully oxidized Ig
H chain-associated L chains (that could have been recruited from the
partially oxidized labeled Ig L chain pool) are compatible with the
interpretation that newly synthesized partially oxidized Ig L chains
can pair with Ig H chains and may fully oxidize as Ig H chain-bound
molecules.
A major aim of this study was to understand the molecular basis
underlying Ig chain secretion. In particular, we were puzzled by the
phenomenon that some Ig L chains depend on Ig H chain association for
export, whereas others are also secreted as free chains even in the
absence of Ig H chain expression. The latter fact was interpreted as a
possible example of polypeptides that may escape ER quality control
(1). The model that led us to design the experiments was based on the
information that, on the one hand, export-incompetent Ig L chains are
monomeric, oxidized in one domain only, and turn into fully oxidized
molecules when experimentally released from BiP interaction (22, 26).
On the other hand, export-competent Ig L chains interact only
transiently with BiP and are secreted as completely oxidized molecules
(22, 24). The data presented here provide evidence for Ig L chain
monomers having to assemble with another Ig chain prior to export. This
means that export competence of an Ig L chain is determined by its
capability to form homodimers in case Ig H chains are not available for
assembly. Furthermore, the requirement of assembly for export could
reflect that Ig L chain proceed in folding only upon chain association as demonstrated for the export-incompetent 1 FS62 that
forms both internal disulfide bonds only when associated with an Ig H
chain.
When 1 FS62 chains are co-expressed with Ig H chains, Ig
H chain-bound L chains are completely oxidized whereas, in the same cell, free Ig L chains are still partially oxidized (Fig. 6). This
could reflect that the partially oxidized Ig L chain is a folding
intermediate used for Ig chain assembly, which implies that assembly
precedes disulfide bond formation in the second Ig L chain domain.
Alternatively, Ig H-L assembly could be required for stabilization of a
completely folded
1 FS62 chain. In favor of the first
hypothesis is the fact that we never detected completely oxidized Ig L
chains in the absence of Ig H chain expression. Furthermore, the
results of our pulse-chase experiments showed that Ig L chains are
principally able to bind to Ig H chains as partially oxidized
molecules. Our attempt to determine the folding pathway of Ig L chains
was impeded by the formation of covalently linked HL, H2L,
and H2L2 molecules. However, the existence of assembly-competent Ig L chain folding intermediates is independently supported by the finding that Ig chain assembly is also a prerequisite for prolyl isomerization (38). Furthermore, export-competent Ig L
chains accumulate in a partially oxidized conformation in COS cells
that transiently overexpress a mutant BiP defective in its ATPase
activity (39). From these results it seems that disulfide bond
formation in one of the two Ig L chain domains is independent of
BiP-ATPase function, whereas oxidation of the second domain is not.
Interestingly, export-incompetent Ig L chains are in the partially
oxidized form irrespective of whether the ATPase of BiP is functional
or not. In the light of our results, it is tempting to speculate that
oligomerization of Ig L chains is the initial step required for
ATP-dependent dissociation of the BiP·L chain
complex.
It seems clear that structural features intrinsic to the V domain must
determine whether Ig L chains are export-competent or not as both types
of Ig L chains with identical constant domains are found (24, 35).
Indeed, mutations in the V domain of Ig L chains can alter their
secretory phenotype (37, 40). Our experiments with the chimeric chains
demonstrate that the competence of an Ig chain to assemble in
vivo with another chain is strongly influenced by the nature of
its V domain. For example, antibodies are formed when both chains carry
the V domain of the export-competent J558L 1 chain,
while the chains do not associate when both subunits bear the V domain
of the export-incompetent NS1
chain. It was shown that secretion of
NS1
chain is restored when the histidine in position 87 is replaced
by tyrosine or phenylalanine (35), suggesting that histidine 87 located
in the contact surface of paired V domains (41) prevented Ig L chain
homodimer formation. However, in the light of our results, the final
three-dimensional structure known from crystallographic data is only
achieved upon Ig chain pairing. As a consequence, amino acid residues
that are not embedded in the final contact area may also be involved in the process of V domain pairing. For instance, the
1
FS62 chain, which is found exclusively as unpaired monomer in NSFS62
cells, carries the mutation on the solvent accessible
outer surface of the folded structure.2
If complete Ig L chain folding is required for secretion and depends on
the capability of free Ig L chains to self-assemble, one should expect
export-competent Ig L chains to be secreted as dimers. We found that
the J558L 1 chains are fully oxidized (22) and are
secreted as covalently and noncovalently linked homodimers (Figs. 3 and
4). The monomeric Ig L chains reported in some cases (42) may
correspond to molecules with low affinity interaction that fall apart
easily when exported as noncovalently linked dimers. In fact, the
in vitro analysis of 17 human
Bence Jones proteins
showed that the ratio between Ig L chain monomers and noncovalently
linked dimers depended on the dimerization constant, which ranged from
<103 to >106 M
1
(36). In this context, it is interesting to note that the dimerization constant does not necessarily correlate with the ratio between covalently and noncovalently linked Ig L chain dimers (36).
It had been suggested that V domains of Ig L chains carry a transport
signal conferring export competence to the isolated Ig L chain as well
as to assembled antibody molecules (37). This notion was based on
experiments performed with the 1 chain mutant,
1 FS62, in which this putative signal was destroyed. The
1 FS62 mutation not only blocked secretion of the Ig L
chain itself but also led to the retention of assembled IgM in a
transient transfection model. Here, we have analyzed transport of the
same Ig L chain in stably transfected cells that co-express a
chain and found that assembled IgG molecules were secreted. Moreover, about
the same amount of IgG was secreted when the antibodies contained J558L
1 or
1 FS62 chains, i.e. the
mutation did not alter the efficiency of antibody export. The
conflicting results might be explained by the different experimental
systems used to study Ig secretion; for instance, transient
transfections might not ensure that sufficient amounts of both chains
are expressed in the same cell. Be that as it may, however, our results
exclude the possibility that a putative transport signal located on the Ig L chain had been destroyed by the FS62 mutation.
Our studies on Ig L chain assembly and secretion provide an example to
illustrate how the cellular machinery could control the oligomerization
state of multi-subunit proteins by monitoring the folding state of the
individual subunits. In our model, oligomeric proteins would become
export-competent only when the subunits proceed in folding as the
result of successful assembly. Retention in the ER of unassembled
subunits or of partial complexes would be due to ER chaperones binding
to incompletely folded structures as does BiP with unassembled,
partially oxidized Ig L chains (22). This view clearly differs from a
model in which the role of BiP is to cover the hydrophobic interface of
a non-assembled folded subunit (1). The observation that BiP binding
prevents complete oxidation of unassembled Ig L chains (22, 39)
strongly indicates that BiP interferes with the in vivo
folding pathway of Ig chains. A quality control mechanism that monitors
oligomerization at the level of subunit folding could not exclude the
formation of different oligomers when subunits fulfill their folding
requirements by assembling with various partner chains. For example, Ig
L chains pass the export checkpoint by forming either heterodimeric
antibody molecules or Ig L homodimers. The thiol-mediated retention
described by Sitia and co-workers (18) monitors the oligomerization
state of normally covalently linked subunits and may act as a second mechanism to retain folded molecules that still exhibit free thiol groups (17, 21). As 2-microglobulin also exhibits an Ig
fold structure and molecules can be exported without major
histocompatibility complex class I H chain association, our model
predicts that free
2-microglobulin might form homodimers
prior to exit from the ER.
We thank T. Simon for the generous gift of vectors, cells, and reagents and H.-M. Jäck, T. Neubert, C. Steinberg, H. Tschochner, and F. Wieland for discussions and critical reading of the manuscript.