(Received for publication, August 2, 1995; and in revised form, October 21, 1995)
From the
The ubiquitous eukaryotic protein calreticulin has been detected in a wide variety of different cell types. Recently, calreticulin was found to bind in vitro to a number of proteins isolated from the endoplasmic reticulum. In addition, calreticulin has sequence similarities with the molecular chaperone calnexin. These data suggest that calreticulin might also act as a chaperone. We found that calreticulin associated transiently with a large number of newly synthesized cellular proteins. In cells expressing recombinant human immunodeficiency virus (HIV) envelope glycoprotein, gp160 bound transiently to calreticulin with a peak at 10 min after its synthesis. Binding of gp120 to calreticulin was not detected because proteolytic cleavage of gp160 occurs in the trans-Golgi. Nonglycosylated HIV envelope protein was not associated with calreticulin, suggesting a requirement for N-linked oligosaccharides on newly synthesized proteins as has been reported for calnexin. The in vivo binding kinetics of calnexin and calreticulin to gp160 were very similar. Sequential immunoprecipitations provided evidence for the existence of ternary complexes of gp160, calreticulin, and calnexin. The data suggested that most of the gp160 associated with calreticulin was also bound to calnexin but that only a portion of the gp160 associated with calnexin was also bound to calreticulin.
Highly conserved cDNAs encoding calreticulin, an abundant
protein that has been localized to the lumen of the endoplasmic
reticulum (ER), ()have been isolated from human, mouse,
rabbit, rat, Xenopus, Aplysia, Drosophila, Onchocerca, Caenorhabditis, and Schistosoma libraries (for a review see (1) ). There is over 90% amino
acid sequence identity between rabbit, mouse, human, and rat
calreticulins(2) . Calreticulin is considered to be the major
Ca
-binding protein in the nonmuscle ER(2) .
Other Ca
-binding proteins in the lumen of this
membrane system include endoplasmin (GRP94), immunoglobulin-binding
protein (BiP or GRP78), and protein disulfide isomerase. As with
calreticulin, these three proteins contain the C-terminal ER retention
sequence KDEL preceded by clusters of acidic residues. A role for
Ca
in the interaction of KDEL proteins with the KDEL
receptor has been proposed(3, 4) . There is evidence
that the four ER proteins might have a similar transcriptional
regulation(5) . Because BiP, GRP94, and protein disulfide
isomerase are believed to be involved in protein transport, folding, or
assembly(6) , a similar role has been considered for
calreticulin. Calreticulin can be induced by amino acid deprivation (7) and heat shock(8) , suggesting that calreticulin,
like many chaperones, is a stress protein. In addition, several groups
have reported that calreticulin binds to a number of proteins isolated
from the ER(9, 10, 11) . Also, calreticulin
has sequence similarities with calnexin, a known molecular chaperone.
In accordance with these data, a recent study provided evidence that
calreticulin serves as a chaperone in the biosynthesis of
myeloperoxidase(12) . Here, we show that calreticulin and
calnexin bind transiently to glycosylated forms of the integral
membrane protein encoded by the human immunodeficiency virus type 1
(HIV-1). Sequential immunoprecipitations suggest the existence of
ternary complexes containing calreticulin, calnexin, and the HIV-1
envelope protein.
Figure 1:
Association of newly synthesized
cellular proteins with calreticulin. BS-C-1 cells were metabolically
labeled with [S]methionine for 5 min and chased
for the times indicated at the top of the figure. Then
extracts of the cells were incubated with calreticulin-directed
antiserum and protein A-Sepharose beads. The washed beads were treated
with SDS and analyzed by SDS-PAGE and autoradiography. The arrow indicates calreticulin.
Figure 2:
The calreticulin-directed serum showed no
cross-reactivity for calnexin. BS-C-1 cells were labeled with
[S]methionine starting 1 h after infection.
After approximately 16 h, the cells were lysed, and the extracts were
incubated with calnexin-directed serum (lane 1) or with
calreticulin-directed serum (lane 2). In lanes 3 and 4, the beads were incubated with SDS to release bound
proteins, diluted tenfold in Triton X-containing lysis buffer and used
for a second round of immunoprecipitations with anti-calnexin or
anti-calreticulin antibodies, respectively.
Figure 3:
Calreticulin complexes with gp160 but
not with gp120. BS-C-1 cells infected with vPE16 were harvested at 16 h
after infection and lysed. One portion was immunoprecipitated by a
gp160-directed serum (lane 1), and one portion by
calreticulin-directed serum (lane 2). After separation of the
proteins by SDS-PAGE, they were blotted onto nitrocellulose and
detected by incubation with both sera followed by incubation with
protein A-Sepharose labeled with I.
To determine
the time course of gp160 binding by calreticulin, pulse-chase
experiments were performed. BS-C-1 cells expressing gp160 were labeled
with [S]methionine for 5 min and chased for
times between 0 and 120 min. Immunoprecipitation of extracts from these
cells by calreticulin-directed antibodies followed by SDS-PAGE showed
that newly synthesized gp160 coprecipitated with calreticulin (Fig. 4A). Quantitation of the radioactivity in the
bands revealed that the highest amount of gp160 was bound to
calreticulin about 10 min after synthesis (Fig. 4B).
After a chase of 25 min, the labeled gp160 bound to calreticulin was
reduced by 50%, and by 90 min only trace amounts of gp160 were still
associated with calreticulin. In the extracts of cells lysed directly
after the 5 min pulse, the amounts of gp160 precipitated by either the
calreticulin- or gp160-directed antibodies were less than that after a
chase of at least 5 min. The delay could reflect continued
incorporation of labeled methionine as well as the time needed for
glycosylation(18) . Because vaccinia virus infection stops host
cell protein synthesis(19) , metabolically labeled calreticulin
was not detected in infected cells (data not shown). Nevertheless, in
Western blot of those cells (Fig. 3) or in
radioimmunoprecipitation assays of uninfected cells (Fig. 1),
calreticulin was easily detectable.
Figure 4:
Transient binding of calreticulin to
gp160. A, BS-C-1 cells at 16 h after infection with vPE16 were
metabolically labeled with [S]methionine for 5
min and chased for the times indicated in min at the top of
the figure. Then extracts of the cells were immunoprecipitated by
calreticulin-directed or gp160-directed serum, respectively. The
resulting immunoprecipitates were analyzed by SDS-PAGE and
autoradiography. B, the amount of radioactivity of each band
in A was quantitated with a
PhosphorImager.
Figure 5:
Calreticulin binds to truncated HIV-1
gp160. BS-C-1 cells were infected with vaccinia viruses expressing the
wild type gp160 (lane 1) and six C-terminally truncated
gp160-proteins (lanes 2-7). The cells were metabolically
labeled with [S]methionine for 15 min and chased
for an additional 10 min. Then extracts of the cells were
immunoprecipitated by calreticulin-directed or gp160-directed serum,
respectively. Resulting immunoprecipitates were analyzed by SDS-PAGE
and autoradiography.
Figure 6:
Effects of glycosylation inhibitors on
calreticulin binding. The inhibitor tunicamycin (TM, 5
µg/ml), castanospermine (CST, 1 mM),
1-deoxynojirimycin (dNM, 2 mM), bromoconduritol (Br, 1 mM), and 1-deoxymannojirimycin (dMAN,
1 mM) were added to BS-C-1 cells expressing gp160 from the
beginning of their depletion for methionine. The cells were labeled
with [S]methionine for 10 min and chased for an
additional 10 min. Then extracts of the cells were immunoprecipitated
by calreticulin-, calnexin-, or gp160-directed serum, respectively. The
resulting immunoprecipitates were separated by SDS-PAGE and
autoradiography.
Tunicamycin-treatment of
uninfected BS-C-1 cells and immunoprecipitation with
calreticulin-directed serum of their
[S]methionine-labeled extracts led to similar
results: tunicamycin prevented newly synthesized cellular proteins from
binding to calreticulin (not shown).
Figure 7:
Comparison of the binding kinetics of
gp160 to calnexin and calreticulin. A, BS-C-1 cells were
metabolically labeled with [S]methionine for 5
min and chased for the times indicated in min at the top of
the figure. Then extracts of the cells were immunoprecipitated by
calreticulin-directed or calnexin-directed serum, respectively. The
resulting immunoprecipitates were analyzed by SDS-PAGE and
autoradiography. B, the amounts of radioactivity associated
with the gp160 bands were quantitated with a PhosphorImager.
,
gp160 precipitated by calreticulin-directed serum;
, gp160
precipitated by calnexin-directed serum.
To find
out whether calnexin and calreticulin bind sequentially or
simultaneously to gp160, successive immunoprecipitations were
performed. Again, BS-C-1 cells expressing gp160 were pulse-labeled with
[S]methionine for 5 min and chased for times
between 0 and 60 min. Half of each extract from these cells was
depleted of calreticulin-bound gp160 by two successive
immunoprecipitations with calreticulin-directed serum prebound to
protein A-Sepharose; the other half of each extract was depleted of
calnexin-bound gp160 in a similar way. This procedure was followed by
incubation of the supernatants with a surplus of protein A-Sepharose to
remove remaining antibodies left from the first two rounds of
immunoprecipitations. Finally, the samples depleted for
calreticulin-bound gp160 were used for an immunoprecipitation by
calnexin-directed serum, and the samples depleted for calnexin-bound
gp160 were used for an immunoprecipitation by calreticulin-directed
serum. We found that extracts depleted of calreticulin-bound gp160
still contained significant amounts of calnexin-bound gp160 (Fig. 8A). However, calnexin-directed serum effectively
depleted calreticulin-bound gp160 because only trace amounts of gp160
were still precipitable by anti-calreticulin (Fig. 8A).
This result suggests that under our conditions the majority of gp160
molecules that are bound to calreticulin are also bound to calnexin in
a transient ternary complex, whereas there are many gp160-calnexin
complexes without calreticulin. To rule out the trivial possibility
that the calreticulin-gp160 complex was unstable or lost during the
anti-calnexin steps, the immunoprecipitations were repeated with
unrelated antibodies in the first two rounds (Fig. 8B).
After two immunoprecipitations with VSV G protein-directed antibodies,
there were still high amounts of calnexin-bound gp160 as well as
calreticulin-bound gp160 (Fig. 8B), indicating that the
calreticulin-gp160 complexes were not lost during the procedure.
Figure 8:
Evidence for ternary complexes composed of
gp160, calnexin, and calreticulin. A, infected BS-C-1 cells
were metabolically labeled with [S]methionine
for 5 min and chased for the times indicated in min at the top of the figure. The cells were lysed, and each extract was divided
into two aliquots and bound by calreticulin-directed or
calnexin-directed serum that had been attached to protein A-Sepharose
beads (row 1). To completely deplete calnexin- or
calreticulin-bound gp160 from the cell extract, this procedure was
repeated with the supernatants using fresh beads coated with the same
respective antibodies (row 2). To remove any free calnexin- or
calreticulin-directed antibodies from the supernatants, an excess of
protein A-Sepharose was added to each supernatant for 1 h (row
3). Finally, the supernatants depleted of calnexin were used for
an immunoprecipitation by anti-calreticulin, and the supernatant
cleared from calreticulin was used for an immunoprecipitation by
anti-calnexin (row 4). The gp160 associated with the protein
A-Sepharose beads at each step was analyzed by SDS-PAGE and
autoradiography. The gp160 bands or equivalent positions on the gels
are shown. The amounts of gp160 co-precipitated with calnexin and
calreticulin were quantitated with a PhosphorImager. At the 10- and
15-min chase times, the gp160-calnexin/gp160-calreticulin was 0.043 for
the calnexin-gp160 depletion experiment (left panel), and the
gp160-calreticulin/gp160-calnexin was 0.17 (at 10 min) or 0.20 (at 15
min) for the calreticulin-gp160 depletion experiment (right
panel). B, BS-C-1 cells were labeled with
[
S]methionine for 10 min and chased for an
additional 10 min. The cells were lysed, and each extract was divided
into four aliquots and treated as described in A. Lane
I, anti-calreticulin followed by anti-calnexin; lane II,
anti-calnexin followed by anti-calreticulin; lane III,
anti-VSV G protein followed by anti-calnexin; lane IV,
anti-VSV G protein followed by anti-calreticulin. The rows correspond to the same steps as in A. The gp160
associated with protein A-Sepharose at each step was analyzed by
SDS-PAGE.
Calreticulin is a highly conserved ER membrane protein that shares several regions of 42-78% identity with calnexin(25) . The sequence similarity between calnexin and calreticulin suggests that these distinct ER proteins may have common functions. Calnexin associates transiently and selectively with newly synthesized glycoproteins, indicating that it may act as a chaperone(17, 20, 26, 27) . Moreover, calnexin binds specifically to molecules with monoglucosylated core glycans, consistent with a role in the synthesis and secretion of glycoproteins(22) . In addition to the structural similarities between calreticulin and calnexin, there have been several reports that calreticulin binds to a wide variety of proteins(9, 10, 11) , including a processing intermediate of myeloperoxidase(12) . To examine the transient association of calreticulin with a newly synthesized integral membrane glycoprotein, we took advantage of the slow processing rate of the well studied HIV envelope glycoprotein gp160(18) .
Initially, we
checked the binding of our antibodies to calreticulin and calnexin and
observed no cross-reactivity. Using coprecipitation assays, we then
demonstrated that calreticulin was associated with many newly
synthesized proteins. The association was detected after a 5-min pulse
with [S]methionine. This association was found
to be transient; almost all of these proteins dissociated from
calreticulin within the first 90 min of chase. When we performed
similar experiments with extracts of cells expressing gp160,
calreticulin coprecipitated with uncleaved gp160 but not with gp120.
Most likely, this is the result of different locations of these
proteins. Cleavage of gp160 to gp41 and gp120 is a late processing
event that occurs in the Golgi(18, 28) , and
calreticulin contains a KDEL sequence and is therefore ER residential.
Nauseef et al.(12) observed that the nonglycosylated form of the cellular enzyme myeloperoxidase synthesized in the presence of tunicamycin did not interact with calreticulin. We observed the same effect for gp160. It has been reported that the inhibitors of the glucosidases I and II, castanospermine and 1-deoxynojirimycin, block the association of calnexin to newly synthesized proteins(20) . Later, it was shown that calnexin is specific for monoglucosylated core glycans(22) . Castanospermine partially blocked the binding of gp160 to calreticulin and calnexin, whereas 1-deoxynojirimycin had little inhibitory effect. Castanospermine showed some inhibition, but even calnexin still bound detectable amounts of gp160 produced in the presence of this inhibitor. However, gp160 is very heavily glycosylated with almost half of the molecular weight of the mature envelope protein derived from the carbohydrate side chains. For this reason, the specific trimming events may not have all been blocked, allowing calnexin to bind. Although clearer data might be obtained with another protein with fewer glycosylation sites, our data suggest that removal of the terminal glucose residues is important for the association of calreticulin to processing intermediates. Otherwise castanospermine would not have reduced its association, as it also did for calnexin.
The binding of some chaperones, such as BiP, can be reversed by the addition of ATP(29) . Nigam et al.(11) found that after applying ER microsomes to columns coated with denaturated proteins, calreticulin but not calnexin was eluted by ATP. In our study we did not observe an ATP-dependent release of calreticulin from gp160 either when the complex was in solution or bound to protein A-Sepharose beads via antibodies. However, it remains possible that additional cofactors necessary for an ATP-induced release of calreticulin were not present in our assay or inactive under the conditions we have used.
Taken together, our data add to the suggestion that calreticulin may act as a molecular chaperone. Especially its transient binding to a variety of cellular proteins and gp160 early during their processing and its specific binding to glycosylated proteins confirm this assumption. This role for calreticulin may also explain a recent finding that long term sensitization training in aplysia lead to an increase of the expression of calreticulin. The increase of calreticulin was first detected when the increase in overall protein synthesis reached its peak and the formation of new synaptic terminals became apparent(30) . Because similar findings were published by the same group for BiP(31) , a chaperone function of calreticulin and BiP might serve to fold proteins necessary for the structural changes characteristic of long term memory.
Because of the striking
structural and functional similarities between calreticulin and
calnexin, we compared the kinetics of gp160 binding to calreticulin
with its binding to calnexin and observed similar patterns. About 10
min after synthesis, a maximal amount of gp160 was bound to calnexin
and calreticulin and about half remained bound after 25-35 min.
The long t of dissociation is consistent with
earlier reports that gp160 is processed rather slowly (18) . Ou
and co-workers (17) showed recently that maximal binding of
1-antitrypsin, complement 3, transferrin, and apolipoprotein B-100
to calnexin occurred 2-10 min after the pulse. This is probably
due to the time needed for the translation of nascent polypeptide
chains. Recently, it was shown that association of calnexin to newly
synthesized hemagglutinin starts when the chains are about half
translated(32) . The t
of dissociation
of the proteins investigated by Ou et al.(17) from
calnexin varied between 5 and 35 min.
Calnexin coprecipitated gp160 and small amounts of calreticulin (data not shown). This could be explained by the existence of either a ternary complex composed of gp160, calnexin, and calreticulin or of two binary complexes in which calnexin is bound to calreticulin or to gp160. However, the very similar binding kinetics of both chaperones to gp160 suggested the existence of a ternary complex. To prove that both chaperones bind to the same gp160-folding intermediates, extracts of cells were depleted of gp160 bound to calnexin by anti-calnexin antibodies, and the supernatants were assayed for calreticulin-bound gp160 and vice versa. Calnexin-directed serum depleted calreticulin-bound gp160, suggesting the existence of a transient ternary complex composed of gp160, calreticulin, and calnexin. However, calreticulin-directed serum did not efficiently deplete calnexin-bound gp160. We ruled out the possibility that this effect results from differences in the stability of complexes between gp160 and calnexin or calreticulin. We concluded that calreticulin is mostly bound to gp160 molecules that are also bound to calnexin, whereas calnexin-gp160 complexes lacking calreticulin exist. Whether calnexin is required for calreticulin binding to gp160 cannot be deduced from our data. However, because calnexin is a transmembrane protein, whereas calreticulin is lumenal, we consider a model in which calnexin holds the newly translated gp160 in the ER, whereas calreticulin associates with and dissociates from the protein. Alternatively, calnexin may fulfill its role on the parts of nascent proteins associated or close to the ER membrane, whereas the soluble calreticulin may manipulate the lumenal parts of those proteins.