(Received for publication, April 3, 1995; and in revised form, June 20, 1995)
From the
A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane-anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with other calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations.
Maturation of newly synthesized proteins in the ER ()is constantly monitored by the quality control apparatus,
which is thought to operate to prevent externalization of immature
proteins along the secretory pathway (Rose and Doms, 1988; Hurtley and
Helenius, 1989). Although the molecular mechanism of such a machinery
is unknown, the processing of N-linked oligosaccharides,
particularly glucose trimming, has been known to play a role in this
process (Fitting and Kabat, 1982; Gross et al., 1983;
Peyrieras et al., 1983; Lodish and Kong, 1984; Rizzolo and
Kornfeld, 1988; Rose and Doms, 1988). In 1989, Suh et al. found that the majority of misfolded G proteins of a vesicular
stomatitis virus mutant (ts045) retained in the ER possessed
mono-glucosylated oligosaccharides and that glucose was added
post-translationally on the deglucosylated N-linked
oligosaccharides. On the basis of these findings, they proposed a
hypothesis that the ER has a mechanism that selectively transfers a
single glucose onto the deglucosylated oligosaccharides of aberrant
molecules, resulting in their retention in the ER (Suh et al.,
1989). Although Parodi's group (Parodi et al., 1984;
Trombetta et al., 1989; Ganan et al., 1991; Sousa et al., 1992; Trombetta and Parodi, 1992), conclusively showed
that the majority of newly synthesized proteins are
post-translationally glucosylated by the enzyme
UDP-glucose:glycoprotein glucosyltransferase, which recognizes only
denatured polypeptides, the molecular machinery responsible for their
ER retention remained unknown. In 1994, Hammond et al. reported that calnexin, an ER membrane chaperone, formed a stable
complex (lasting at least 1 h) with misfolded G protein of a vesicular
stomatitis virus mutant (tsO45) at the permissive temperature and that
treatment of the cells with the glucosidase inhibitors, CAS or
1-deoxynojirimycin, abolished the association. They therefore proposed
that calnexin is a putative lectin molecule with a specificity for
mono-glucosylated oligosaccharides (Hammond et al., 1994).
Calnexin, which was originally identified as a major calcium-binding
protein of the ER membrane (Wada et al., 1991), has been known
to function as a molecular chaperone involved in the normal folding
process of nascent proteins (Bergeron et al., 1994). Its
chaperone function was initially discovered by analysis of cross-linked
products of nascent class I major histocompatibility antigen complex by
Degen and Williams(1991). Later, several membrane and soluble proteins,
most of which were N-glycosylated, were reported to
transiently interact with calnexin during their early stages of
maturation (Ahluwalia et al., 1992; Ou et al., 1993;
Anderson and Cresswell, 1994; Hammond et al., 1994; Le et
al., 1994; Lenter and Vestweber, 1994; Loo and Clarke, 1994; Pind et al., 1994; Wada et al., 1994; Kim and Arvan,
1995). In addition, nascent gp80, the major secretory protein in
Madin-Darby canine kidney cells, was highly susceptible to proteinase K
digestion when associated with calnexin, as compared to the nascent
dissociated molecules (Wada et al., 1994). Furthermore,
treatment of cultured cells with chemicals that disrupt protein folding
resulted in the formation of relatively stable complexes with calnexin
(Ou et al., 1993; Wada et al., 1994; Kim and Arvan,
1995). At the cellular level, unassembled multi-subunit proteins
(Jackson et al., 1994; Rajagopalan et al., 1994) as
well as the naturally occurring mutants, Phe
,
cystic fibrosis transmembrane conductance regulator (Pind et
al., 1994), or null
variant of
1-antitrypsin (Le et al., 1994), were also shown to be
retained in the ER by calnexin. Thus, the dissociation process is
highly correlated to the progress of proper protein folding. However,
the precise role of calnexin in the folding process is not clear.
Hammond and Helenius(1994) recently proposed that calnexin may be the
major chaperone for the folding of vesicular stomatitis virus G
protein, since CAS inhibited G protein maturation as well as calnexin
association. While this study demonstrated the importance of a
CAS-sensitive chaperone in the folding process, the involvement of
other chaperones that require glucose trimming for the association
cannot be ruled out.
In the search for another chaperone whose function is coupled with glucose trimming, we focused on an abundant ER luminal protein, calreticulin (Michalak et al., 1992), which has a sequence similarity (33% identity in human) to calnexin including two unique sets of internal repeats (Wada et al., 1991; Hawn et al., 1993; Tjoelker et al., 1994). This protein is thought to be mostly responsible for calcium storage in the ER (MacLennan et al., 1972; Michalak et al., 1991; Baksh et al., 1992; Treves et al., 1992) and is one of the major ER luminal proteins, collectively called reticuloplasmins (Koch, 1987; Smith and Koch, 1989). Since nascent proteins would first come in contact with membrane proteins prior to luminal proteins, we designed a system to evaluate the maximum chaperone function of calreticulin. We constructed a membrane-anchored calreticulin by fusing a truncated calreticulin (residues 1-340) to the COOH-terminal portion of calnexin. The amino-terminal luminal domain of calnexin was confirmed to be essential for its chaperone function. We then studied the interaction of the calreticulin chimera with nascent secretory proteins in HepG2 cells. Finally, we tested if calreticulin indeed functions as a chaperone at its physiological location by expressing an internally tagged soluble calreticulin in HepG2 cells. Here, we demonstrate that calreticulin is another novel CAS-sensitive chaperone in the ER.
We intended to determine if calreticulin could function as a
chaperone when expressed in the same environment as calnexin (i.e. anchored to the luminal surface of the ER membrane). To test this,
we made constructs consisting of calreticulin residues 1-340
fused to residues 459-573 of the carboxyl terminus of calnexin.
This construct includes the membrane-spanning domain of calnexin as
well as the cytosolic tail. To distinguish the chimeric proteins from
cellular calnexin, we inserted an epitope tag YPYDVPDYA (influenza
virus hemagglutinin Tyr-Ala
) flanked
by an arginine residue near the carboxyl terminus of calnexin (Fig. 1, top), since the carboxyl-terminal six residues
of calnexin are reportedly required for its ER retention (Rajagopalan et al., 1994). The tagged calnexin was initially tested on 293
cells to confirm that the tag did not impair its chaperone function.
Cells were labeled with [
S]methionine and lysed
with the buffer containing Triton X-100. Immunoprecipitation with
anti-calnexin antibodies revealed that the endogenous calnexin
transiently interacted with nascent cellular proteins, including a
major 61-kDa polypeptide (Fig. 2, lanes1 and 2). When the tagged calnexin, CN(HA), was expressed in 293
cells, the HA tag monoclonal antibody 12CA5 precipitated the same set
of nascent cellular proteins including the 61-kDa protein. This protein
dissociated from CN(HA) after 60 min of chase (lanes3 and 4). These transiently associated proteins were not
observed in the immunoprecipitates of pRep8-transfected cells using
12CA5 antibody (lane7). Thus, these experiments
confirmed that the internal tag at the cytoplasmic tail was recognized
as a distinctive epitope by 12CA5 antibody and that the tag insertion
did not impair the apparent chaperone function of calnexin.
Figure 1:
Schematic illustration
of constructs. CN(HA), the amino acids of canine calnexin are numbered from the initiator methionine. HA epitope tag flanked
with Arg was inserted at the COOH terminus of Ser. TM, transmembrane domain; CN, calnexin.
PL/CN
(HA), prolactin (residues -30-198) was
connected to the carboxyl-terminal region of the tagged calnexin.
Ligation of the two cDNA fragments resulted in generation of two
additional amino acid residues, Lys and Leu at the joint. PL,
prolactin. CR/CN
(HA), calreticulin (residues
-17-340) was fused to the tagged calnexin lacking the
luminal domain. L was inserted at the joint as a result of ligation of
the two cDNA fragments. CR, calreticulin. CR(HA), HA epitope
tag flanked by a few amino acids as indicated was inserted at the COOH
terminus of Glu
. The arrows indicate the sites
of cleavage of signal sequences.
Figure 2:
The luminal domain is essential for the
chaperone function of calnexin in 293 cells. Control vectors (pRep8) (lanes1, 2, and 7), pCN(HA) (lanes3 and 4), or pPL/CN(HA) (lanes5 and 6) were transfected into 293
cells. The cells were labeled for 30 min with
[
S]methionine and chased for 0 min (lanes1, 3, 5. and 7) or 60 min (lanes2, 4, and 6). Cell extracts
were processed for immunoprecipitation using anti-calnexin antibodies
plus 12CA5 antibody (lanes1 and 2) or 12CA5
alone (lanes3-7). Efficiency of the
immunoprecipitation for the tagged molecule using 12CA5 was
90-100%. The immunoprecipitates were analyzed on a 9% SDS-gel.
The bands corresponding to endogenous calnexin, CN(HA), and
PL/CN
(HA) are indicated by arrows. Arrowhead indicates a 61-kDa polypeptide, the major calnexin ligand in 293
cells. The numbers to the left indicate the positions
of the molecular mass markers for
-galactosidase, bovine serum
albumin- and ovalbumin. Note that PL/CN
(HA) (lanes5 and 6) gave a fuzzy band of 47 kDa due to N-glycosylation.
We next
confirmed that the luminal portion of calnexin was essential for its
interaction with nascent proteins. Prolactin, a well-characterized
secretory protein, was fused to the truncated and tagged calnexin
lacking the luminal portion (Fig. 1, PR/CN(HA)).
Sheep prolactin used in the experiments contains one N-linked
oligosaccharide (Li, 1976). When the prolactin chimera was
immunoprecipitated from 293 cells transfected with
pPL/CN
(HA), a fuzzy band of 47 kDa corresponding to the
glycosylated chimera was observed (Fig. 2, lanes5 and 6). Although the calculated molecular weight is
22,562, the prolactin chimera migrated abnormally on SDS-PAGE,
presumably because of the extremely acidic charge of the calnexin
cytoplasmic tail (Wada et al., 1991). However, no interaction
of the chimera with nascent polypeptides, including the major 61-kDa
calnexin-associated protein, was observed (lane5 of Fig. 2). Localization of PL/CN
(HA) as well as
CN(HA) or wild type CN to the peripheral network structure around the
nucleus was confirmed by indirect immunofluorescence with 12CA5
antibody (data not shown). Therefore, we confirmed that the truncated
calnexin lacking the luminal domain has no apparent chaperone function.
For subsequent experiments, we used HepG2 cells, since these
hepatoma cells have a well developed secretion apparatus (Bouma et
al., 1989) and since the chaperone function of calnexin has been
well defined in these cells (Ou et al., 1993). We intended to
confirm that the calnexin association with HepG2 secretory proteins is
coupled to their glucose trimming. We pulse labeled HepG2 cells in the
absence (Fig. 3, lanes1 and 3) or
presence of 1 mM CAS (lanes2 and 4), followed by immunoprecipitation with anti-calnexin
antibody (Fig. 3, lanes1 and 2) or
anti-transferrin antibody (lanes3 and 4). Lane1 shows a set of nascent HepG2 polypeptides,
which were coimmunoprecipitated with calnexin. As reported by Ou et
al.(1993), the major bands of 80, 66, and 54 kDa corresponded to
transferrin, -fetoprotein, and
1-antitrypsin, respectively,
as confirmed by sequential immunoprecipitations (data not shown). The
band corresponding to
1-antichymotrypsin, which comigrated with
1-antitrypsin in the previous study (Ou et al., 1993)
gave a slightly faster migration than the
1-antitrypsin band in
our SDS-PAGE system, which used a 9% gel. Among the four major
associated polypeptides, the identity of the 62-kDa polypeptide is not
known. Association of these nascent polypeptides with calnexin was
markedly reduced by CAS treatment of the cells (Fig. 3, lane2). The CAS treatment did not affect synthesis or core
glycosylation of nascent transferrin but did produce a slightly slower
migrating band, presumably reflecting inhibition of glucose trimming.
Figure 3:
Effect of CAS on the association of newly
synthesized proteins with calnexin in HepG2 cells. HepG2 cells were
preincubated for 30 min with (lane2) or without (lane1) 1 mM CAS. The cells were
subsequently labeled for 30 min with
[S]methionine in the presence (lanes2 and 4) or absence (lanes1 and 3) of 1 mM CAS and then lysed with a buffer
containing 1% Triton X-100. Endogenous calnexin was immunoprecipitated
with anti-calnexin antibodies (lanes1 and 2). Cell extracts were also immunoprecipitated with
anti-transferrin antibodies (lanes3 and 4).
The immune complexes were resolved on a 9% SDS-gel and visualized by
BAS 2000 equipped with Pictrography. The arrows indicate the
positions of calnexin (CN, lanes1 and 2) and transferrin (TF, lanes3 and 4). Arrowheads, from top to bottom of gel, point to the major bands corresponding to transferrin,
-fetoprotein, a 62-kDa unidentified protein, and
1-antitrypsin, respectively. Numbers to the left show the positions of the molecular mass
markers.
We next attempted to see if calreticulin could function as a
chaperone when anchored to the ER membrane, where newly synthesized
proteins are highly enriched. Since the carboxyl terminus region of
calreticulin is composed of an ER retention signal (residues
396-399) and an acidic cluster(342-395), both of which are
also found in the cytoplasmic tail of calnexin, we reasoned that
calreticulin residues 1-341 might be sufficient to act as a
chaperone. We therefore constructed a chimeric molecule (Fig. 1,
CR/CNtc(HA)) by fusing calreticulin (residues -17-340) to
the membrane/cytoplasmic domain of calnexin. We then expressed the
chimera in HepG2 cells and lysed under denaturing conditions. The
membrane-anchored calreticulin gave a major band of 67 kDa and a faint
band of 75 kDa (Fig. 4A, lane3). The
calculated molecular weight is 54,076, again showing an anomalous
mobility on the SDS-PAGE gel. Both bands were also detected on
immunoblots of cellular membranes that had been pre-washed with 0.1 M sodium carbonate, pH 11 (Morimoto et al., 1983;
Wada et al., 1992), and probed with antibodies against
calreticulin or calnexin (data not shown). The identity of the 75-kDa
band, which was not clearly seen upon a shorter pulse (e.g.Fig. 4B, lanes1-3), is
not known. A similar doublet was also observed for the intact
calreticulin (Dupuis et al., 1993). ()Although rat
calreticulin has one putative N-glycosylation site at
Asn
(Murthy et al., 1990), tunicamycin treatment
did not change the mobility of the two bands, suggesting that they were
not N-glycosylated. When HepG2 cells transfected with
pCR/CN
(HA) were lysed under non-denaturing conditions
using Triton X-100, 12CA5 monoclonal antibody precipitated several
nascent polypeptides (lane4 of Fig. 4A) including the four major bands. Essentially
the same pattern was obtained when CN(HA) was analyzed (lane2 of Fig. 4A). The identity of the
coprecipitates with CR/CN
(HA) was confirmed by a series of
sequential immunoprecipitations with 12CA5 antibody followed by
individual antibodies against the secretory proteins,
1-antitrypsin (lane7),
-fetoprotein (lane8), and transferrin (lane9).
Interaction of CR/CN
(HA) with cellular calnexin or
albumin, which is the major secretory protein of HepG2 cells, was not
observed (data not shown).
Figure 4:
Membrane-anchored calreticulin transiently
interacts with nascent proteins. A, pCN(HA) (lanes1 and 2), pCR/CN(HA) (lanes3 and 4), or the control vector (pRep8) (lanes5 and 6) was transfected into HepG2
cells. The cells were lysed with a buffer containing SDS (lanes1, 3, and 5) or Triton X-100 (lanes2, 4, and 6) and immunoprecipitated
with 12CA5 antibody. Four major bands coimmunoprecipitating with the
tagged molecules are indicated by arrowheads. Arrows show the bands corresponding to CN(HA) (lanes1 and 2) or CR/CN
(HA) (lanes3 and 4). For sequential immunoprecipitation,
CR/CN
(HA) was expressed in HepG2 cells cultured in a 35-mm
dish. The cells were labeled for 30 min with
[
S]methionine, and the cell extract was prepared
using 1% Triton X-100, followed by immunoprecipitation with 12CA5
antibody. The proteins that coprecipitated with CR/CN
(HA)
were extracted by incubating the immune complexes in 1% SDS at 65
°C for 10 min. This extract was then divided into three aliquots
and re-immunoprecipitated with antibodies against
1-antitrypsin (lane7),
-fetoprotein (lane8), or transferrin (lane9). Asterisks indicate unknown polypeptides that were observed in
the immunoprecipitates of 12CA5. B, HepG2 cells expressing
CR/CN
(HA) (lanes1-3) or the
control vector (lanes4-7) were pulse labeled
with [
S]methionine for 15 min and chased for the
indicated periods of time. The cell extracts were prepared using buffer
containing 1% Triton X-100 and then immunoprecipitated with 12CA5
antibody (lanes1-4) or anti-calnexin
antibodies (lanes5-7). Immune complexes were
analyzed on a 9% gel. Arrowheads indicate the positions of the
four major coprecipitates as described above. Asterisks indicate unknown polypeptides that were observed in the
immunoprecipitates of 12CA5. C,
CR/CN
(HA)-transfected 293 cells were labeled for 30 min
followed by immunoprecipitation with 12CA5 anti-tag antibody. Arrowhead indicates the 61-kDa major cellular protein
associating with the calreticulin chimera.
We next tested whether the association of
CR/CN(HA) with nascent proteins was transient. Pulse-chase
experiments (Fig. 4B, lanes1-3) revealed that the majority of the polypeptides
bound to the calreticulin chimera dissociated as the cells were chased.
When quantitated by BAS2000, 55% of
-fetoprotein and 31% of
transferrin dissociated from the membrane-anchored calreticulin at 15
min of chase and 80 and 62%, respectively, at 45 min. Comparable
results were obtained for cellular calnexin (Fig. 4B (lanes5-7);
-fetoprotein, 48% at 15
min and 78% at 45 min; transferrin, 22% at 15 min and 65% at 45 min) as
well as for CN(HA) (data not shown).
The transient interaction of
calreticulin (residues 1-340) with nascent chains on the ER
membrane was also confirmed in 293 cells. Immunoprecipitation from 293
cells transfected with pCR/CN(HA) using 12CA5 antibody
showed that the 61-kDa polypeptide observed in Fig. 2was also
the major protein in the immunoprecipitates and dissociated after 1 h
of chase (Fig. 4C).
Nigam et al.(1994)
reported that calreticulin as well as immunoglobulin heavy chain
binding protein (BiP), calnexin, ERp72, Grp94, and protein-disulfide
isomerase bound to urea-denatured secretory proteins immobilized on
Sepharose beads and that all of them with the exception of calnexin
were eluted from the beads by incubation with 1 mM ATP.
Therefore, we tested if ATP could disrupt the nascent
chain/membrane-anchored calreticulin complex. We isolated the immune
complex by incubating 12CA5 monoclonal antibody with lysate from HepG2
cells expressing pCR/CNtc(HA), which had been pre-labeled with
[S]methionine for 30 min. The complex was then
incubated with 1 mM ATP on ice for 1 h or at 16 °C for 15
min. However, ATP treatment did not release the nascent chains from the
complex (data not shown).
Next, we examined whether the chaperone
function of calreticulin (residues 1-340) was dependent on
glucose trimming. We treated the HepG2 cells expressing
CR/CN(HA) with 1 mM CAS and then isolated the
immune complex with 12CA5 antibody. As shown in Fig. 5, the set
of polypeptides coisolated from non-treated cells including
transferrin,
-fetoprotein, and
1-antitrypsin was not
recovered in the immune complex of CR/CN
(HA) isolated from
the CAS-treated cells.
Figure 5:
CAS abolishes the interaction of the
membrane-anchored calreticulin with nascent proteins.
CR/CN(HA)-transfected HepG2 cells were incubated for 30
min with (lane2) or without (lane1) 1 mM CAS and then labeled for 30 min with
[
S]methionine in the presence (lane2) or absence (lane1) of 1 mM CAS. The cell lysates were processed for immunoprecipitation with
12CA5 antibody. Arrowheads indicate the four major
coprecipitates. Asterisks indicate unknown polypeptides that
were observed in the immunoprecipitates of
12CA5.
Finally, we tested if the apparent chaperone
function of calreticulin (residues 1-340) above described is
expressed at its physiological location, i.e. the lumen of the
ER. Since calreticulin contains an ER retention signal
(KDEL
) at the COOH terminus, which can
function as an independent domain (Munro and Pelham, 1987), we placed a
HA tag internally near the COOH terminus of calreticulin (Fig. 1, bottom) and expressed it in HepG2 cells. The
expressed molecules were extractable from the cells by 0.1 M sodium carbonate, pH 11, confirmed by an immunoblot, and were
shown to be confined to the perinuclear structure of HepG2 cells when
tested by indirect immunofluorescence (data not shown).
Immunoprecipitation with the tag antibody from the SDS-lysate of cells
transfected with pCR(HA) yielded a single 58-kDa band (Fig. 6A, lane2), a molecular mass
comparable to that previously reported (Michalak et al.,
1992). When the expressed molecule of 58 kDa was isolated under
non-denaturating conditions, two additional bands of 100 and 80 kDa
were reproducibly observed in the immunoprecipitate (Fig. 6A, lane3). The prominent
47-kDa band in lane3 could not be observed in other
repeated experiments because it comigrates with the band that was
usually observed in control immunoprecipitations. Sequential
immunoprecipitation (Fig. 6B, lane5)
identified the 80-kDa band as transferrin. The identity of the 100-kDa
band is not known. In contrast, three other major calnexin substrates (Fig. 6B, lane1) were recovered to a
limited extent in the CR(HA)-immune complex (Fig. 6B, lanes2-4). The interactions of CR(HA)-bound
proteins were also transient, and 20% of transferrin was released at 15
min of chase and 65% at 45 min (Fig. 6C, lanes2-4). As shown for the membrane-anchored
calreticulin, CAS treatment abolished the transient association of
nascent polypeptides with calreticulin (Fig. 6D, lane3), confirming that the apparent chaperone
property of calreticulin at its physiological location is also coupled
with glucose processing.
Figure 6:
Soluble calreticulin interacts with
nascent glycoproteins at varied efficiencies in a CAS-sensitive manner. A, HepG2 cells transfected with wild type vector (pRep8) or
pCR(HA) were labeled for 30 min with
[S]methionine and lysed with buffer containing
1% SDS (lane2) or 1% Triton X-100 (lanes1 and 3). Immunoprecipitation was carried out
with 12CA5 antibody. Asterisks indicate unknown polypeptides
directly bound to 12CA5. Two bands of 80 and 100 kDa in lane3 coisolated with CR(HA) are indicated by dots. B, HepG2 cells expressing CN(HA) (lane1) or
CR(HA) (lane2) were labeled for 30 min and lysed
with Triton X-100-containing buffer followed by immunoprecipitation
with 12CA5. For sequential immunoprecipitations, CR(HA) was expressed
in HepG2 cells plated in 35-mm dishes. Sequential immunoprecipitation
with anti-
1-antitrypsin (lane3) or
anti-
-fetoprotein (lane4) or anti-transferrin (lane5) antibodies was carried out as described in Fig. 4. The four major calnexin substrates are indicated by arrowheads. Asterisks and dots are as
indicated above for panelA. C, HepG2 cells
harboring pRep8 (lane1) or pCR(HA) (lanes2-4) were labeled for 15 min with
[
S]methionine and chased for 0 min (lanes1 and 2), 15 min (lane 3), and 45 min (lane4) by adding 5 mM methionine and 1
mM cysteine. The Triton X-100 extracts were processed for
immunoprecipitation with 12CA5. Asterisks and dots are as indicated above for panelA. D,
HepG2 cells harboring pRep8 (lane1) or pCR(HA) (lanes2 and 3) were labeled with
[
S]methionine for 30 min in the absence (lanes1 and 2) or presence (lane3) of 1 mM CAS after a 30-min preincubation
period. Immunoprecipitation was carried out using 12CA5
antibody.
This study demonstrates that calreticulin (residues 1-340) transiently interacts with secretory glycoproteins of HepG2 cells when expressed as a membrane-anchored chimera and that the association was sensitive to CAS treatment. Although calnexin interacts with a selected set of proteins synthesized in the ER with various efficiencies (Margolese et al., 1993; Ou et al., 1993; Kim and Arvan, 1995; Arunachalam and Cresswell, 1995), the spectrum of proteins that calreticulin (residues 1-340) recognized on the membrane was remarkably similar to what was observed for calnexin. Therefore, we conclude that calreticulin per se is another novel chaperone that requires glucose processing of N-linked oligosaccharides to interact with nascent proteins. We further confirmed that calreticulin, expressed at its physiological location, indeed transiently associated with a set of nascent proteins in a CAS-inhibitable manner. Different from the membrane-anchored form, the soluble calreticulin appeared to interact preferentially with transferrin and only weakly with other major calnexin ligands (Fig. 6B). However, it is apparent that calnexin and calreticulin share a basically similar ligand specificity. These results clearly suggest that the ER possesses two kinds of molecular chaperones to fulfill its quality control function. At present, it is difficult to assess the quantitative contributions of the two chaperones in the folding process of secretory proteins. However, it is reasonable to assume that calnexin should play the major role in the maturation process, since the membrane rather than the ER lumen would be primarily enriched with nascent proteins. Such nascent proteins may have differential folding kinetics, and some of them, like transferrin as observed in this experiment, might have access to the luminal calreticulin without being trapped by calnexin. In this context, the soluble calreticulin may represent a ``back-up'' mechanism for the kinetically diverse process of nascent chain maturation.
Calreticulin was originally discovered as a ``high affinity calcium binding protein'' of skeletal muscle (MacLennan et al., 1972). Later, ER localization of this molecule was found in cells of non-muscular type from various species (see Michalak et al. (1992) and references therein). Several physiological functions have been ascribed to this abundant protein. At first, a role for calcium storage was proposed since calreticulin binds, per mole of protein, 20-50 mol of calcium at low affinity and 1 mol of calcium at high affinity. It was also known that the majority of calcium in the ER was found bound to proteins (MacLennan et al., 1972; Michalak et al., 1991; Baksh et al., 1992; Treves et al., 1992). At least three physiological phenomena have been reportedly correlated to this role. First, calreticulin expression was moderately induced upon activation of T lymphocytes (Burns et al., 1992). Second, calreticulin as well as calcium-ATPase sequestrated in vesicles were concentrated around the undigested particles when heat-killed yeasts were presented to neutrophils (Stendahl et al., 1994). This suggested that calreticulin may play a role in the regulation of calcium homeostasis upon phagocytosis. Finally, in cytolytic lymphocytes, calreticulin was found as the major constituent of lytic granules (Dupuis et al., 1993). In this case, calreticulin has been speculated to function as a calcium chelator in the granules, since an increase of free calcium led to perforin-mediated autolysis. Thus, it has been suggested that calreticulin plays a dynamic role in regulating calcium homeostasis. On the other hand, several studies revealed that calreticulin directly bound to polypeptides through the sequence KXFF(K/R)R, which is found in the DNA binding domain of nuclear hormone receptors as well as in the regulatory cytoplasmic domain of the integrin family (Burns et al.(1994), Dedhar et al.(1994), and reviewed by Dedhar(1994)). In fact, nuclear as well as cytoplasmic localization of calreticulin was also suggested (Rojiani, 1991; Michalak et al., 1992; Dedhar, 1994; Leung et al., 1994). Since calreticulin contains a cleavable signal sequence at the amino terminus, elucidation of a mechanism by which this protein escapes targeting by the signal recognition particle (Rapoport, 1992) to the ER would be of great interest. The chaperone function of calreticulin described in this study apparently differs from its association with polypeptides previously reported (Dedhar, 1994; Nigam et al., 1994) in that the association is limited to newly synthesized proteins and is inhibited by CAS (Fig. 5) or tunicamycin treatment (data not shown). Recently, Nauseef et al.(1995) reported that calreticulin bound to the lysosomal enzyme myeloperoxidase, being slowly dissociated over 2 h of chase. Since no lysosomal enzymes have so far been reported to interact with calnexin, it would be interesting to know if lysosomal enzymes preferentially interact with calreticulin and whether the interaction is also dependent on glucose trimming.
The luminal domain of calnexin was
demonstrated to have a weak but specific affinity for the structure of
GlcMan
GlcNAc
itself (Ware et
al., 1995). Remarkably, it was also found that a complex of
calnexin with nascent class I major histocompatibility antigen
molecules or
1-antitrypsin was resistant to endoglycosidase H
treatment. Therefore, it was proposed that the recognition of
Glc
Man
GlcNAc
triggers stable
association of the polypeptide chain with calnexin. At present, the
molecular basis for the proposed two-step mechanism is not clear. Given
that calreticulin appears to have the same chaperone function as
calnexin, it would be reasonable to assume that the two units of
sequence repeats, PXXIPDPXAXKPEDWDE and
PXXIPDPXAXKPEDWDE may be primarily
responsible for the unique molecular properties common to both calnexin
and calreticulin. These two sets of unique motifs are repeated four
times in calnexin and three times in calreticulin. Furthermore, it
should be pointed out that both molecules contain a short stretch of
hydrophobic residues (Phe
-Val
,
calnexin; Phe
-Ile
, calreticulin)
despite the fact that the amino acid sequences in this region are not
well conserved. Such a hydrophobic domain may play a role in binding to
the immature polypeptides, as the hydrophobic patch is thought to be
the common structure exposed on the surface of unfolded molecules. The
tagged expression system described in this study may be suitable for
mapping of the binding sites.