Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520 8002
Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5-8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5-5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.
The ER maintains an efficient machinery for protein
translation, folding, oligomeric assembly, and quality control (Gething and Sambrook, 1992 The chaperone-assisted folding and the formation of
disulfide bonds begin already while growing nascent chains
enter the lumenal compartment (Bergman and Kuehl, 1979 During folding, polypeptides with N-linked oligosaccharides interact transiently and specifically with two homologous lectin-like chaperones that are unique to the ER, the
membrane-bound calnexin and the soluble calreticulin.
A transmembrane protein of 64 kD, calnexin interacts
transiently with a large number of different glycoproteins
during their folding and maturation (Ou et al., 1993 Despite the increasing understanding of the processes
involved in protein maturation and quality control, it still
remains unclear how the ER works as a functional entity
and how calnexin and calreticulin carry out their functions.
In this paper, we have focussed on calnexin. To characterize the properties of individual calnexin molecules and
complexes between calnexin and its substrates, we analyzed detergent solubilized cell lysates by sucrose velocity gradient centrifugation and chemical cross-linking. To determine the nature of weaker contacts in situ, cross-linking
in live cells was employed. Our results indicated that the
unoccupied chaperones and chaperone complexes are
small entities, but part of an extended network of molecules that form larger structures in the ER.
Cells, Viruses, and Chemicals
CHO15B cells and the Lec 23 cells used in this study are mutant cells that
lack the medial-Golgi enzyme N-acetyl glucosamine transferase and glucosidase I, respectively (Gottlieb et al., 1975 Antibodies and Immunoprecipitations
The rabbit anti-influenza virus serum, the antiserum against the NH2-terminal 1-12 amino acid sequence of X31 influenza hemagglutinin (HA) referred to as NHA1, and the rabbit anti-calnexin COOH-terminal peptide
antibodies have been described (Doms et al., 1985 Pulse-chase Analysis of HA Folding and
Metabolic Labeling
The folding of HA was followed by the method described by Braakman
et al. (1991) Velocity Sedimentation on Sucrose Gradients
Monomeric and trimeric forms of HA were resolved on 5-25% continuous sucrose gradients in 20 mM MES, 100 mM NaCl, 30 mM Tris HCl, pH
8.6 (MNT buffer) with 0.1% Triton X-100 (Copeland et al., 1986
Cross-linking of Proteins in Cells,
Cell Lysates, or in Gradient Fractions Using
Homobifunctional Cross-linkers
Cross-linking was performed in three different ways: in intact cells, in cell
lysates, and in isolated gradient fractions. In intact cells, cross-linking was
performed as follows. After pulse and chase, the cells were washed and
left in phosphate buffer saline (PBS) with 20 mM NEM for 10 min on ice
and then washed once with cold PBS. Cells were scraped gently in 225 µl
of PBS (for ~106 cells) and 20 mM stock of DSP (freshly made for each
experiment) was added to give a final concentration of 2 mM. The stock
solution of DSP (20 mM) was prepared in dimethyl sulfoxide (DMSO). In
control samples an equivalent amount of DMSO without DSP was added.
The cell suspension was incubated on ice for 30 min (with intermittent shaking) after which the excess cross-linker was quenched with 50 mM
glycine. Cells were lysed by adding an equal volume of 4% CHAPS in
PBS. Lysates were spun at 1,500 g to obtain a postnuclear supernatant
(PNS) which was used for immunoprecipitations. Cross-linking with DSG
and DSS was performed in a similar way.
Cross-linking in cell lysates was performed by including 2 mM final
concentration of DSP in the lysis buffer (HBS) containing 2% CHAPS.
Cells were incubated in the presence of the lysis buffer for 30 min on ice
and cross-linker was quenched with 20 mM Glycine. An equal amount of
DMSO without DSP was added to uncross-linked controls.
For cross-linking of proteins in the fractions from the sucrose gradients,
DSP at a concentration of 2 mM was added to individual fractions and the
fractions were incubated on ice for 30 min. Excess DSP was quenched using 20 mM glycine and the fractions were processed further for immunoprecipitation.
Substrate-free Calnexin
Substrate-free calnexin was generated in CHO cells in
three different ways. First, prolonged incubation with cycloheximide was used to inhibit the synthesis of proteins
thus depleting the calnexin of its substrates (Fig. 1 A). Previous pulse-chase studies have shown that most substrates
bind to calnexin for not more than ~60 min (Hammond
et al., 1994
To incorporate radioactive label into calnexin, cells were
labeled overnight with [35S]methionine and cysteine. They
were then treated with inhibitors as described above for 5 h
(cycloheximide) or 1 h (castanospermine), whereafter they
were flooded with NEM, an alkylating agent that prevents
further disulfide oxidation, and lysed with CHAPS. Postnuclear supernatants were subjected to sucrose gradient
velocity centrifugation in the presence of the detergent.
The gradient fractions were immunoprecipitated with the
anti-calnexin antiserum, and analyzed by SDS-PAGE and
fluorography. The bands were quantified by densitometry.
In each case, a major peak of calnexin was observed
with an S-value of 3-4 S20,W. When samples from the main
peak were cross-linked using a bifunctional chemical
cross-linker DSP, no higher molecular weight bands were
seen by SDS-PAGE suggesting that the peak corresponded to calnexin monomers (not shown). No evidence
of additional labeled subunits was found. Notably, the proteins associated with the so-called SSR complex (SSR- Calnexin-Substrate Complexes
When the experiments in CHO cells described above were
repeated without cycloheximide or castanospermine treatment, the endogenous calnexin-substrate complexes could
be analyzed. The distribution of calnexin in the gradients
was now clearly bimodal (Fig. 1 C). In addition to the peak
at 3-4 S20,W which cosedimented with the unoccupied calnexin (A and B), a more prominent rapidly sedimenting population of calnexin molecules was apparent. Calnexin
was distributed as heterogenously sized complexes over
the 3-8 S20,W range.
The proteins complexed to calnexin could be visualized
when the cells were pulse-labeled for 5 min before solubilization (Fig. 2). As expected (Ou et al., 1993
Analyzing Calnexin-Hemagglutinin Complexes
by Cross-linking
To further analyze the properties of calnexin substrate
complexes, we studied a single well-defined substrate molecule, influenza HA. This 84-kD glycoprotein associates
with calnexin cotranslationally and remains associated for
~4 min after chain completion until all the intrachain disulfides are formed (Hammond et al., 1994 Using sucrose gradients, we have previously shown that
calnexin-HA complexes sediment at 5-5.5 S20,W, calnexinfree HA subunits at 4.5-5 S20,W, and mature HA trimers at
8.9 S20,W (Tatu et al., 1995
To characterize the oligomeric structure of the detergent solubilized complexes, influenza-infected cells were
pulsed for 5 min, lysed, and fractionated on similar sucrose
gradients. Fractions 5-7, which contained the calnexin and
calreticulin complexes with HA, were pooled and aliquots
were subjected to chemical cross-linking using the cleavable cross-linker DSP. Together with uncross-linked controls, the samples were then immunoprecipitated with antiHA, anti-calnexin, and anti-calreticulin, respectively, and
analyzed in nonreduced form by SDS-PAGE and fluorography.
As shown in Fig. 3 C, anti-HA precipitates of uncrosslinked samples contained IT1, IT2, and NT (lane 1). The
cross-linked sample showed the presence of three crosslinked species, C1, C2, and C3 (lane 4). The most prominent was C2 which had an approximate molecular mass of
140-160 kD. Since this band was also precipitated by anticalnexin (lane 5) it corresponded to the 1:1 HA-calnexin complex. Less intense cross-linked bands were seen at
120 kD (C1) and just below 200 kD (C3). Of these, C1
could be precipitated with anti-calreticulin (lane 6) suggesting that it corresponded to 1:1 HA-calreticulin complexes. The composition of C3 remains unclear. It was,
however, precipitable both with anti-HA and anti-calnexin suggesting either a higher order complexes of these two
polypeptides or a ternary complex containing an additional component. In some experiments, the C3 band was
also precipitated with anti-calreticulin raising the possibility that it corresponded to ternary HA-calnexin-calreticulin complexes. Presence of such ternary complexes has also
been observed when HA is expressed in microsomes (Hebert,
D., and A. Helenius, manuscript in preparation). Occasionally we also saw higher molecular mass cross-links (>200
kD) in anti-calnexin and anti-calreticulin immunoprecipitates. The composition of these complexes is unclear.
Taken together, the results indicated that when cells
were solubilized with nondenaturing detergent and subjected to centrifugation on sucrose gradients, the majority
of calnexin- and calreticulin-bound HA was contained in
discrete calnexin and calreticulin complexes. Some higher
molecular weight complexes were also present.
Transient Association of HA with Larger Complexes
Next, we used cross-linking in situ to determine whether
additional associations existed between the newly synthesized HA and resident ER proteins. We pulse-labeled influenza-infected cells for 3 min and chased for various
times. The cells were then incubated with the cross-linker
DSP (which is membrane permeable), lysed with CHAPS,
immunoprecipitated with different antibodies, and analyzed by SDS-PAGE and fluorography. In the 5% gels
used in this experiment, the separation of IT1, IT2, NT,
and G (G is the Golgi form of HA) was improved, and
large cross-linked complexes were more completely resolved than above.
The results shown in Fig. 4 A, demonstrated that the
newly synthesized HA could be cross-linked inside the ER
of the cell. The products turned out to be quite different
than those seen after solubilization. When DSP was added
within the first 2 min of chase, cross-linking was very efficient as shown by the virtually complete disappearance of
the monomeric HA species (lanes 6 and 7, Nonreduced).
That the HA was present in the material on top of the
stacking gel was shown by boiling the samples in the presence of DTT, thus breaking the DSP links (lanes 6 and 7,
Reduced).
The result indicated that early folding forms of HA were
efficiently cross-linked to covalent complexes most of
which were so large that they did not enter the resolving
gel. The same large HA-containing complexes could also
be precipitated by anti-calnexin (Fig. 4 B, lanes 6 and 7),
suggesting that the 1:1 calnexin-HA complexes observed
by velocity centrifugation in CHAPS lysates (Fig. 3 C)
were part of much larger cross-linkable complexes in situ.
As HA maturation proceeded during the chase, the pattern of cross-linked products began to change. After 5-20
min of chase, the HA appeared in the form of three discrete bands (Fig. 4 A, lanes 8-10). Unlike the previous
large complexes, these were not coprecipitated with anticalnexin. One of them corresponded to the noncrosslinked monomeric species, the two others to HA homotrimers and homodimers familiar from previous studies
(Doms and Helenius, 1986 Similar large cross-linked complexes were obtained using another cross-linker DSG, indicating that the results
were not cross-linker dependent (not shown). Moreover,
when DSP was included in the lysis buffer, similar large
cross-linked complexes were observed (not shown). Thus,
the interactions between the HA and its cross-linking partners were apparently stable for some time after solubilization and dilution. Together with experiments described
below (Fig. 7 B), this result indicated that the extensive
cross-linking of early folding forms of HA was not just due
to a high local protein concentration in the ER.
To arrive at a size estimate for the cross-linked complexes containing HA, velocity sedimentation on sucrose
gradients was carried out following cross-linking. After
precipitating with anti-HA antibodies, the fractions were
analyzed on SDS-PAGE in Nonreduced form (Fig. 5). Sedimentation standards (urease, 18.9 S20,W and BSA, 4.5 S20,W)
were analyzed on identical gradients. While the uncrosslinked HA was found in fractions 2, 3, 4, and 5, the crosslinked complexes containing HA were present throughout the gradient indicating heterogeneous size. The cross-linked
complexes ranged in size from ~8 to more than 40 S20,W.
Taken together, the results showed that early folding
forms of HA (IT1, IT2, and NT) are cross-linked to large,
heterogeneous calnexin-containing complexes. Evidently,
the HA-calnexin and HA-calreticulin complexes present
at this stage of maturation were part of extensive structures that could be stabilized by cross-linking in situ and in
lysates immediately after solubilization. As folding proceeded, HA reached its trimeric form and was no longer
directly associated with this structure. Instead, cross-linking now occurred between the subunits of the mature HA
homotrimer.
The Effects of Brefeldin A
That the HA trimers escaped cross-linking after trimer
formation could mean that they were still in the ER but
dissociated from the chaperone complexes, or it could be
simply explained by their export out of the ER. To test
whether HA trimers retained in the ER would cross-link
to the large complexes, we employed BFA, a drug that
prevents transport of HA and other proteins from the ER
to the Golgi complex (Klausner et al., 1992 The result indicated that HA trimers, even though
present in the ER, did not get cross-linked to large complexes of ER proteins. The interaction of early folding
forms with constituent ER proteins was thus qualitatively
different than that of assembled trimers. It was concluded
that dissociation of HA from the chaperone machinery proceeded or coincided with trimer assembly, and that this
led to a situation where cross-linking no longer occurred.
We have previously shown that HA dissociates from calnexin just before trimer formation takes place (Tatu et al.,
1995 Composition of In Situ Cross-linked Complexes
To determine which lumenal ER proteins were part of the
HA-containing, cross-linked complexes formed in situ, long
term (36 h) labeling of CHO cells with [35S]methionine
and cysteine was performed so that the resident proteins would be labeled. The cells were then infected with influenza virus, and 5 h postinfection the HA synthesized was
labeled using a brief 5-min pulse. Half of the cells were
cross-linked with DSP, and the other half used as an
uncross-linked control. The lysates were immunoprecipitated with antibodies against five different antigens; HA,
calnexin, calreticulin, BiP, and GRP94. A background control received protein A beads without antibody ("PA
control"). The immunoprecipitates were analyzed in nonreduced form to determine whether cross-linking had occurred (NR), and in reduced form (R) to determine the
identity of the cross-linked components.
Judging by the amount of cross-linked HA precipitated
with anti-calnexin and anti-calreticulin compared to antiHA, almost all the HA was covalently cross-linked to either one or both of these chaperones (Fig. 7, Reduced,
lanes 7-9). A small amount of BiP was also present in the
complexes precipitated with anti-HA (bottom right panel,
lane 7). We have previously shown that ~5-10% of HA
misfolds and binds to BiP (Hurtley et al., 1989 The converse experiment using anti-HA did not bring
down detectable amounts of labeled cellular proteins except a small amount of BiP (bottom right panel, lane 7).
Considering the excess of chaperones present in the ER
over HA, this was not unexpected since only a small fraction of each would bind at any given time to the newly synthesized HA molecules. From these results, we tentatively
concluded that the large cross-linkable complexes containing HA also contained calnexin and calreticulin and possibly some BiP. Whether other ER proteins such as PDI
were present, remains to be determined.
To rule out the possibility that the cross-linking of newly
synthesized HA into large complexes containing ER chaperones in situ was merely a result of the high protein concentration, we carried out a cross-linking experiment in
castanospermine-treated cells. Castanospermine inhibits HA
association with calnexin and calreticulin by preventing
trimming of glucose residues from the N-linked core oligosaccharides (Elbein, 1991 Constitutive Association of Chaperone Proteins
in the ER
Interestingly, the precipitations shown in Fig. 7 revealed
extensive cross-linking of calreticulin with BiP and GRP94,
and of GRP94 with BiP (see lanes 3 and 4). To examine
whether these associations were dependent on the presence of newly synthesized substrate proteins, the cells
were prelabeled as described. Then cycloheximide was
added for 5 h before cross-linking in order to clear the ER
of substrate proteins.
It was evident from the results (Fig. 8, A and B) that,
under conditions where no newly synthesized proteins
were present, the chaperones were associated with each
other in a variety of cross-linkable combinations. The anticalnexin immunoprecipitates after reduction of the large
cross-linked complexes formed (Fig. 8, lane 2) showed,
for example, bands corresponding to calnexin, calreticulin,
BiP, and GRP94. In addition to BiP, anti-BiP precipitates showed GRP94 and some calreticulin. Also, in Lec 23 cells where calnexin and calreticulin do not bind to substrates (Ora and Helenius, 1995
The results indicated that calnexin and possibly calreticulin formed stable, discrete 1:1 complexes with substrate
glycoproteins in the ER. These complexes were resistant
to detergent solubilization, gradient centrifugation, and immunoprecipitation. In the ER of intact cells, calnexin and
calreticulin were, however, part of a larger network of interacting proteins which included other ER chaperones.
While this network was stabilized by weaker contacts that
dissociated upon detergent solubilization it could be analyzed using chemical cross-linkers in situ.
In CHAPS solubilized samples, substrate-free calnexin
molecules were found to sediment at 3-4 S20,W. No evidence for the presence of additional, tightly bound subunits was observed although a shoulder of faster sedimenting forms were occasionally seen. This indicated that
calnexin is a monomer when it is not associated with a substrate molecule. Judging by the sedimentation, we estimated that no more than about a third of the calnexin in
CHO cells was normally in an unoccupied state. The rest
was associated with a variety of different substrate proteins (see also Ou et al., 1993 The sedimentation rate (4-8 S20,W) in sucrose gradients
after solubilization with CHAPS, indicated that complexes
between calnexin and its substrate glycoproteins were only
somewhat larger than calnexin alone. The sedimentation
profiles suggested that most of them contained no more
than a single substrate molecule. The molecular weight of
the substrate proteins determined how much faster the
complex sedimented than calnexin itself. Very few of the
complexes were in the 12 S20,W range previously reported by Ou et al. (1993) The 1:1 stoichiometry was found to apply to the majority
of stable calnexin-HA and calreticulin-HA complexes extracted from virus-infected cells. While monomeric HA
has an S-value of ~4.5 S20,W (Doms and Helenius, 1986 The observation that calnexin and calreticulin interact
with their substrates as monomers renders them somewhat
unusual because most lectins are homooligomeric (Weis
and Drickamer, 1996 While calnexin and calreticulin seem to bind to their
substrates as monomers, the in situ cross-linking experiments indicated that they share weaker interactions with
larger chaperone entities. Particularly obvious were crosslinks between calreticulin and BiP, calreticulin and GRP94,
calreticulin and calnexin, as well as BiP and GRP94. Similar contacts have been previously reported in pancreatic
acinar microsomes (Baksh et al., 1995 The existence of a network of resident ER proteins has
been postulated for some time (Kreibich et al., 1978 Pulse-chase experiments combined with in situ crosslinking showed that early full-length folding intermediates
of HA were connected to the network. Once the HA reached
its mature trimeric conformation, it was no longer crosslinked. While BiP and GRP94 were part of larger crosslinked structures, there was no evidence that they interacted directly with HA (Hurtley et al., 1989 The ER matrix is likely to provide a structural framework for the resident chaperones and associated substrates, and functional structure necessary for efficient
quality control. Thus, it may serve as a complex, mixedbed, "affinity chromatography matrix" that transiently adsorbs incompletely folded and assembled proteins, and
prevents their nonspecific aggregation with each other.
The substrate proteins undergo controlled on- and -off cycles with selected chaperone components of the matrix.
Glycoproteins will, for example, associate with calnexin and
calreticulin, with glucosidase II and UDP-glucose:glycoprotein glucosyl transferase driving the on- and -off cycle
(Hammond and Helenius, 1993; Helenius
et al., 1992b
). The lumen provides an exclusive, highly specialized environment for the controlled folding and maturation of membrane proteins and soluble proteins most of
which are destined for export to other organelles or for secretion. The redox environment and the ionic milieu are
carefully maintained and concentration of molecular chaperones and folding enzymes such as protein disulfide isomerase (PDI),1 GRP 94, calnexin, calreticulin, and ERp72
is very high. At nearly millimolar concentrations, the most
abundant of them greatly outnumber the substrate proteins.
;
Chen et al., 1995
). It continues posttranslationally often
with the formation of additional disulfide bonds and in
many cases with the assembly of oligomers (Hurtley and
Helenius, 1989
; Braakman et al., 1991
; Gething and Sambrook, 1992
). Proteins that fail to fold or oligomerize
properly are, as a rule, prevented from export, and get degraded (Hurtley and Helenius, 1989
; Klausner, 1989
;
Hammond and Helenius, 1995
).
). Calreticulin (46 kD), a soluble protein interacts with an overlapping but not identical group of proteins (Nauseef et al.,
1995
; Peterson et al., 1995
; Wada et al., 1995
). Both bind specifically to partially trimmed, monoglucosylated forms
of the N-linked core glycans present on folding intermediates (Hammond et al., 1994
; Hebert et al., 1995
; Peterson
et al., 1995
; Ware et al., 1995
; Spiro et al., 1996
). They promote proper folding, prevent premature oligomerization,
inhibit degradation, and mediate quality control for a variety of glycoproteins (David et al., 1993
; Ou et al., 1993
;
Hammond and Helenius, 1994
; Jackson et al., 1994
; Kearse
et al., 1994
; Le et al., 1994
; Loo and Clarke, 1994
; Pind et al.,
1994
; Tector and Salter, 1995
; Hebert et al., 1996
; Vassilakos et al., 1996
).
Materials and Methods
; Ray et al., 1991
). They were
grown as described (Balch et al., 1986
; Ora and Helenius, 1995
). The X31/
A/Aichi/1968 strain of Influenza virus was used to infect CHO15B cells
(Braakman et al., 1991
). Infected cells were used 4-6 h after infection for the pulse-chase experiments. Media and reagents for cell culture were obtained from Gibco BRL (Grand Island, NY). Brefeldin A (BFA) was purchased from Epicenter Technologies (Madison, WI). It was used at a final
concentration of 5 µg/ml, from a stock of 1 mg/ml in ethanol. CHAPS and
the chemical cross-linking reagents [Dithiobis(succinimidylpropionate)] (DSP), Disuccinimidyl Glutarate (DSG), and Disuccinimidyl Suberate (DSS) were purchased form Pierce (Rockford, IL). Castanospermine (CST), Cycloheximide (CHX), N-ethylmaleimide (NEM), and Triton X-100
were purchased from Sigma Chem. Co. (St. Louis, MO). [35S]-Promix containing cysteine and methionine was purchased from Amersham Corp.
(Arlington Heights, IL).
; Hammond et al., 1994
;
Peterson et al., 1995
). The polyclonal rabbit anti-rat-calreticulin antisera
were a gift from Dr. Hans Dieter Söling (Göttingen, Germany). The latter
was affinity purified against rat calreticulin for use in immunoprecipitation (Peterson et al., 1995
). The rabbit anti-calreticulin polyclonal antiserum and the rat anti-GRP94 monoclonal antiserum used in the cross-linking experiments in intact cells was purchased from Affinity Bioreagents
(Neshanic Station, NJ). The mouse anti-BiP monoclonals were purchased
from StressGen (Victoria, Canada).
. For studying calnexin binding the cells were lysed with 2%
CHAPS in 0.2 M NaCl, 50 mM Hepes, pH 7.5 (HBS buffer, Hammond et
al., 1994
). For overnight labeling, the cells were starved for 15 min for methionine and cysteine and then 4 ml of complete
-MEM containing 0.3 mCi of radiolabeled methionine and cysteine (Amersham Promix) was
added to each 60-mm dish.
). In experiments where the gradient fractions were to be used for cross-linking,
the sucrose gradients were made in HBS buffer containing 0.1% Triton
X-100. The cell lysates were layered on top of 5-ml gradients and centrifuged in an SW 55 rotor at 42,000 rpm for 15 h at 4°C (Beckman Instrs.,
Fullerton, CA). After centrifugation the gradients were fractionated manually from top to bottom. To characterize the size of the large cross-linked complex (Fig. 6), the cross-linked cell lysates were sedimented on a 5-25%
sucrose gradient in TLS 55 rotor tubes. The tubes were sedimented at
160,000 g (50,000 rpm) for 3 h at 4°C in a Beckman table top ultracentrifuge. The gradients were fractionated into 12 fractions and precipitated
with anti-HA antibodies.
Fig. 6.
Effects of Brefeldin A. Ten confluent dishes of CHO15B
cells were infected with Influenza virus. At 6 h postinfection five dishes were pretreated with Brefeldin A as described in Materials and Methods. All the dishes were pulse labeled and chased as indicated in the figure in the presence (lanes 6-10) or absence
(lanes 1-5) of BFA and the chases were stopped with ice cold
PBS with NEM. Cross-linking was performed before lysis as in
Fig. 4 and the cell lysates were precipitated with anti-NHA1 antiserum. The immunoprecipitates were analyzed in the nonreduced
form by 5% SDS-PAGE and fluorography. Top panels show uncross-linked samples and the bottom panels show cross-linked
samples precipitated with anti-NHA1 antiserum.
[View Larger Version of this Image (47K GIF file)]
Fig. 4.
Newly synthesized HA is a part of large complexes until it assembles into trimers. Influenza-infected CHO15B cells were labeled for 3 min and chased for different time intervals as indicated. The chase was stopped by ice cold PBS containing alkylating agent NEM. The cells in each dish were scraped and divided into two aliquots. One aliquot was cross-linked with DSP as described in Materials and Methods and the other was used as an uncross-linked control. After cross-linking the cells were lysed. The lysates from the crosslinked (lanes 6-10) and the control samples (lanes 1-5) were immunoprecipitated with anti-NHA1 (Fig. 4 A), anti-calnexin (CNX, Fig. 4
B, top panel), and the trimer specific monoclonal antibody (Fig. 4 B, bottom panel) as described in Materials and Methods. The anti-calnexin and anti-trimer immunoprecipitates were analyzed only in their nonreduced form (Fig. 4 B). The anti-NHA1 immunoprecipitates
were analyzed both in the nonreduced (Fig. 4 A, top panel) and the reduced forms (Fig. 4 A, bottom panel). The immunoprecipitates
were analyzed by 5% SDS-PAGE and fluorography.
[View Larger Versions of these Images (36 + 44K GIF file)]
Results
; Ou et al., 1993
). Castanospermine, an
-glucosidase inhibitor that prevents trimming of newly synthesized glycoproteins to the required monoglucosylated
form (Elbein, 1991
), was similarly used to prevent the formation of binding-competent substrates (Fig. 1 B) (Hammond et al., 1994
).
Fig. 1.
Sedimentation analysis of substrate-free calnexin and
calnexin-substrate complexes. Three dishes of CHO15B were labeled for 16 h and chased for 5 h in the absence (C) or presence
(A) of 500 µM cycloheximide. To one untreated dish of CHO15B
cells, castanospermine (B) was added at a concentration of 1 mM
in the last 1 h of chase. After the chase the cells were lysed in 2%
CHAPS in HBS. The cell lysates were subjected to velocity sedimentation on sucrose gradients at 42,000 rpm for 15 h at 4°C. After centrifugation the gradients were fractionated from top to
bottom and each fraction was precipitated with anti-calnexin antiserum. The precipitates were analyzed in Nonreduced form on
5% SDS-PAGE. The band corresponding to calnexin was quantified by scanning and plotted as the percentage of total for each
gradient.
[View Larger Version of this Image (11K GIF file)]
,
SSR-
), previously reported to copurify with calnexin from
MDCK cells (Wada et al., 1991
), were absent. We concluded that in its unoccupied form, calnexin is solubilized from the ER mainly as a monomer.
; Peterson et al.,
1995
), a large number of different cellular proteins were
found coprecipitating with calnexin. These were present as
a relatively discrete peak in the 4-8 S20,W range centered at
~5.5 S20,W. The substrate proteins that had a higher apparent molecular weight were present in the faster sedimenting complexes indicating that their size determined the increment in sedimentation rate over that of calnexin alone. Given the relatively narrow size distribution and the low
overall sedimentation rates, it seemed likely that most of
the CHAPS-solubilized complexes contained a single calnexin and a single substrate molecule.
Fig. 2.
Association of
newly synthesized CHO-substrate proteins with calnexin.
CHO15B cells were labeled
for 5 min and lysed with 2%
CHAPS containing buffer.
The cell lysate was layered
on a 5-25% sucrose gradient
and centrifuged in SW 55 rotor at 42,000 rpm for 15 h.
After centrifugation, the gradient was fractionated and
each fraction was precipitated with anti-calnexin antiserum. The immunoprecipitates were analyzed in the nonreduced form using 7.5% SDS-PAGE and fluorography. The positions of the
sedimentation markers are indicated; BSA, 4.5 S20,W, HA-trimers, 8.9 S20,W.
[View Larger Version of this Image (28K GIF file)]
; Chen et al.,
1995
; Tatu et al., 1995
). After dissociating from calnexin,
HA monomers proceed to assemble into noncovalent homotrimers that are transported to the Golgi complex and
eventually to the plasma membrane.
). Fig. 3 A shows the sedimentation of the folding intermediates (IT1, IT2), the fully oxidized NT, and the trimers of HA on sucrose gradients. As
shown in Fig. 3 B, anti-calnexin antiserum brought down
IT1, IT2, and some of the fully oxidized HA (NT) from the 5-5.5 S20,W region of the gradient. Calnexin itself was seen
as a weakly labeled band just above IT1 (Fig. 3 B).
Fig. 3.
Calnexin and calreticulin form
cross-linkable complexes with folding intermediates of HA. Influenza-infected
CHO15B cells were labeled for 20 or 5 min and lysed as described above. The lysates were analyzed by velocity centrifugation on 5-25% sucrose gradients. After centrifugation the gradient was fractionated from top into 15 fractions. For
the sample pulsed for 20 min, the fractions were immunoprecipitated with
anti-HA antiserum (A) and with anticalnexin antiserum (B), and analyzed in
nonreduced form by 7.5% SDS-PAGE.
For the 5-min pulsed samples, each fraction was divided into two parts. Crosslinker DSP was added to one aliquot and
cross-linking was performed as described in Materials and Methods. To
the other, an equal amount of DMSO
was added. The cross-linked (+ DSP,
lanes 4, 5, and 6) and the uncross-linked ( DSP, lanes 1, 2, and 3) samples were
further divided into two parts. These
were immunoprecipitated with anti-HA
(lanes 1 and 4), with anti-calnexin
(CNX) antisera (lanes 2 and 5), or with anti-calreticulin (CRT) antiserum (lanes
3 and 6). The immunoprecipitates were
analyzed in the nonreduced form by
7.5% SDS-PAGE and fluorography.
[View Larger Version of this Image (36K GIF file)]
). All three bands were precipitable with trimer-specific monoclonal antibodies to HA
(Copeland et al., 1986
) (Fig. 4 B, anti-trimer). Moreover,
within each band, a slight shift occurred with time from a
slower to a somewhat faster migrating band. This shift is
caused by mannose trimming of N-linked glycans in the Golgi complex (Balch et al., 1986
). The shift revealed that
the trimeric HA had moved from pre-Golgi compartments
to the Golgi complex.
Fig. 7.
Composition of the folding complex. (A) CHO15B cells were prelabeled for 36 h and then infected with the Influenza virus. 6 h
after infection the cells were pulse labeled for 5 min and the pulse was stopped with ice cold PBS containing NEM. The cells were
scraped from the dish and divided into two aliquots. One aliquot was cross-linked with DSP as in Fig. 4. The cells were lysed with 2%
CHAPS in HBS after cross-linking, and the lysates from the cross-linked and the control samples were divided into aliquots. These were
precipitated with antibodies against HA (lanes 1 and 7), calnexin (CNX, lanes 2 and 8), calreticulin (CRT, lanes 3 and 9), BiP (lanes 4 and 10), GRP94 (lanes 5 and 11) and with Protein A beads without any antibody (lanes 6 and 12). The precipitates were analyzed in
nonreduced (top panels) and the reduced form (bottom panels) by 5% SDS-PAGE and fluorography. (B) Two confluent dishes of CHO
cells were infected with Influenza virus. 4 h after infection one dish was preincubated with 1 mM castanospermine for 1 h and then pulse
labeled for 5 min in the presence of 1 mM castanospermine. The other untreated dish was pulse labeled without castanospermine for 5 min. Cross-linking was performed as above and cells were lysed with 2% CHAPS in PBS. Lysates were divided into four aliquots and
immunoprecipitated as above. The immunoprecipitates were analyzed in the reduced form by 7.5% SDS-PAGE and fluorography.
[View Larger Versions of these Images (43 + 39K GIF file)]
Fig. 5.
Velocity gradient centrifugation to determine the size
of the cross-linked complex containing HA. CHO cells were prelabeled for 15 h with [35S]methionine and cysteine and then infected with Influenza virus. 6 h after infection cells were pulse labeled for 5 min and the pulse was stopped with ice cold PBS
containing 20 mM NEM. Cross-linking and cell lysis was carried
out as in Fig. 5 and as described in Materials and Methods. The
cross-linked and the control samples were layered on a 5-25%
sucrose gradient in a 2-ml tube and sedimented in TLS 55 rotor in
a table top ultracentrifuge as described under Materials and
Methods. The gradients were fractionated and the fractions were
precipitated with anti-HA antibodies. The immunoprecipitates were analyzed in the nonreduced form by 5% SDS-PAGE and
fluorography.
[View Larger Version of this Image (49K GIF file)]
). As shown in
Fig. 6 (lanes 6-10, +DSP), BFA had no effect on the crosslinking of HA either early or late in the chase. That the inhibitor was working, was demonstrated by the lack of conversion of HA to the mannose-trimmed form marked G in
the figure.
).
). No detectable HA-precipitation occurred with anti-GRP94, but precipitations with this antibody were not very efficient (bottom right panel, lane 11).
; Hammond et al., 1994
; Peterson et al., 1995
). If cross-linking would simply occur as a
result of high protein concentration, the lack of specific binding of HA to these chaperones should not prevent formation of large calnexin and calreticulin complexes containing HA. In castanospermine-treated cells, the newly
synthesized HA failed, however, to precipitate with anticalnexin, anti-calreticulin, or anti-BiP antibodies regardless of whether the cells had been treated with DSP or not
(Fig. 7 B, lanes 2 and 3 without DSP, lanes 6 and 7 after
DSP cross-linking and reduction). The result indicated
that to be cross-linked with a complex containing calnexin and calreticulin, HA had to be specifically associated with
these chaperones. It was not enough that they were
present in the same compartment.
) calreticulin could be
cross-linked to calnexin, BiP, and GRP94 (Fig. 8 A, lane 3).
Fig. 8.
Constitutive association of the chaperone proteins of
the ER. Lec 23 (A) and CHO (B) cells were prelabeled for 36 h
as described in Materials and Methods. CHO's were treated with
500 µM cycloheximide for 5 h and cross-linking was performed,
in both, as in Fig. 4. The cell lysates were divided into six different
aliquots and immunoprecipitated with five antibodies. Lane 1,
anti-NHA1; lane 2, anti-calnexin (CNX); lane 3, anti-calreticulin
(CRT), lane 4, anti-BiP; lane 5, anti-GRP94. One aliquot was
used for the protein A control without any antibody. The immunoprecipitates were analyzed in the reduced form by 5% SDSPAGE and fluorography. Only the cross-linked samples are
shown.
[View Larger Version of this Image (30K GIF file)]
Discussion
; Peterson et al., 1995
).
to be the average size of calnexin-substrate complexes after cholate solubilization (Ou et al., 1993
).
The majority of complexes were in the size range observed
for calnexin/
1-antitrypsin complexes (Le et al., 1994
) i.e.,
smaller than originally assumed.
),
the calnexin-HA complexes sedimented at ~5-5.5 S20,W.
When the gradient fractions were treated with the chemical cross-linker, the main cross-linked, HA-containing species had a MW of 140-160 kD, consistent with a 1:1 complex. Calreticulin-HA dimers somewhat smaller than the
calnexin-HA dimers could also be demonstrated. Only a
small amount of larger cross-linked species were observed,
possibly representing ternary complexes containing both
cross-linked calnexin, calreticulin, and HA. Previously we
have shown that on sucrose gradients calnexin associated
folding intermediates IT1 and IT2 sediment somewhat
faster than calnexin associated NT (Tatu et al., 1995
). Together with the observation that calreticulin preferentially
binds to IT1 and some IT2 (Hebert et al., 1996
), it seems
probable that the folding intermediates form ternary complexes of HA, calnexin, and calreticulin. That ternary complexes can occur, has been recently shown for HIV gp160, a protein which has 24 N-linked glycans and therefore a
large number of potential attachment sites for calnexin
and calreticulin (Otteken and Moss, 1996
). Others have
shown that calnexin can bind to oligomeric assembly intermediates such as assemblies of
,
, and invariant chains
of MHC class II antigens (Anderson and Cresswell, 1994
).
If present in lysates from CHO cells, such large oligomeric, calnexin-containing complexes were too few to be
detectable in our gradients.
). Even if lectins have extended binding sites that recognize more than just a single terminal
residue (as calnexin and calreticulin do [Ware et al., 1995
;
Spiro et al., 1996
]), the affinity for individual glycans generally reaches only the millimolar range which is not sufficient for stable association (Weis and Drickamer, 1996
). Most lectins have, therefore, either multiple lectin domains
in the same polypeptide chain or they are multimeric. For
example, other membrane-bound lectins in the secretory
pathway, the two mannose-6-P receptors and ERGIC 53, are oligomeric (Schweizer et al., 1988
; Zhongmin et al.,
1992
). The members of the calnexin family of lectins have,
however, conserved sequence repeats (Wada et al., 1995
)
which could provide as many as 3-4 glycan-binding domains per polypeptide.
) and in chondrocytes (Nakai et al., 1992
). Since inhibition of protein synthesis
had no effect on the cross-linking patterns, associations
are present whether substrate proteins are synthesized or
not. They probably represent authentic interactions in a
dynamic network throughout the rough ER. Inhibition of
substrate binding to calnexin and calreticulin by castanospermine inhibited cross-linking of HA to them further
supporting that the cross-links represent highly specific interactions and ruling out the possibility of nonspecific crosslinks simply due to high protein concentrations in the ER.
;
Hortsch et al., 1987
; Booth and Koch, 1990
; Sambrook,
1990
; Helenius et al., 1992a
; Hammond and Helenius,
1995
). At a concentration exceeding 100 mg/ml, the lumenal resident proteins, most of which are chaperones and
folding enzymes, easily outnumber the newly synthesized
substrate proteins present in the ER (Koch, 1987
; Marquardt et al., 1993
). Electronmicrographs of the ER reveal
a lace-like material spanning the lumenal space. The majority of resident proteins have COOH-terminal KDEL
sequences that provide a signal for retrieval from the Golgi complex (Pelham, 1991
), and most of them have long negatively charged sequences that constitute low affinity, high
capacity calcium-binding sites. Both of these sequence elements contribute to efficient localization of calreticulin in
the ER lumen (Sönnichsen et al., 1994
). While providing
structure to the lumen and the membrane of the rough
ER, the matrix is probably very dynamic and highly responsive to the fluctuations in free calcium and ATP concentrations (Sambrook, 1990
; Nigam et al., 1994
).
).
). These interactions may
promote folding, suppress irreversible aggregation, and at the same time limit the mobility of the newly synthesized
folding and assembly intermediates, preventing their premature exit from the ER.
Received for publication 4 September 1996 and in revised form 30 November 1996.
Please address all correspondence to A. Helenius, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06520-8002. Tel.: (203) 785-4301. Fax: (203) 7857226.The authors wish to thank Dr. Jani Simons for critically reading the manuscript and all the members of the Helenius-Mellman labs for helpful discussions and comments.
This work was supported by National Institutes of Health grants GM 38346 and CA 46128 to A. Helenius. U. Tatu was supported by a fellowship from the American Heart Association, Connecticut Affiliate, and a Human Frontier Science Program grant to A. Helenius.
BFA, Brefeldin A; DSP, dithiobis(succinimidylpropionate); DSG, disuccinimidyl glutarate; HA, hemagglutinin; NEM, N-ethylmaleimide; PDI, protein disulfide isomerase.