Ca2+ Regulation of Interactions between
Endoplasmic Reticulum Chaperones*
Elaine F.
Corbett
,
Kim
Oikawa§,
Patrice
Francois¶,
Daniel
C.
Tessier
,
Cyril
Kay§,
John J. M.
Bergeron**,
David Y.
Thomas
,
Karl-Heinz
Krause¶, and
Marek
Michalak

From the
Medical Research Council of Canada (MRC)
Group in Molecular Biology of Membranes, the § MRC Group in
Protein Structure and Function, Protein Engineering Network of Centers
of Excellence and the Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada, the ¶ Division of
Infectious Diseases, University Hospital, CH-1216 Geneva, Switzerland,
the
Genetics Group, Biotechnology Research Institute, National
Research Council of Canada, Montreal H4P 2R2, and the ** Department of
Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A
2B2, Canada
 |
ABSTRACT |
Casade Blue (CB), a fluorescent dye, was
used to investigate the dynamics of interactions between endoplasmic
reticulum (ER) lumenal chaperones including calreticulin, protein
disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with
CB, and subsequently, we show that the fluorescence intensity of the
CB-conjugated proteins changes upon exposure to microenvironments
of a different polarity. CD analysis of the purified proteins revealed
that changes in the fluorescence intensity of CB-ERp57 and CB-PDI
correspond to conformational changes in the proteins. Using this
technique we demonstrate that PDI interacts with calreticulin at low
Ca2+ concentration (below 100 µM),
whereas the protein complex dissociates at >400 µM
Ca2+. These are the Ca2+ concentrations
reminiscent of Ca2+ levels found in empty or full ER
Ca2+ stores. The N-domain of calreticulin interacts with
PDI, but Ca2+ binding to the C-domain of the protein is
responsible for Ca2+ sensitivity of the interaction. ERp57
also interacts with calreticulin through the N-domain of the protein.
Initial interaction between these proteins is
Ca2+-independent, but it is modulated by Ca2+
binding to the C-domain of calreticulin. We conclude that changes in ER
lumenal Ca2+ concentration may be responsible for the
regulation of protein-protein interactions. Calreticulin may play a
role of Ca2+ "sensor" for ER chaperones via regulation
of Ca2+-dependent formation and maintenance of
structural and functional complexes between different proteins involved
in a variety of steps during protein synthesis, folding, and
post-translational modification.
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INTRODUCTION |
Calreticulin is a ubiquitous and highly conserved
Ca2+-binding, resident protein of the endoplasmic reticulum
(ER)1 membranes (1). The
protein has been implicated to play a role in a variety of cellular
functions including Ca2+ storage and signaling, regulation
of gene expression, cell adhesion, and autoimmunity while also serving
as a lectin-like chaperone (2-10). Calreticulin can be divided into
three regions: a highly conserved N-domain, the proline rich P-domain,
containing a high affinity Ca2+-binding site and
lectin-like function, and the C-domain that contains the high capacity,
low affinity Ca2+-binding (storage) site (11-14). The
P-domain of calreticulin shares amino acid sequence identity with
calnexin, an integral ER membrane chaperone (15). Calnexin and
calreticulin are both lectins, which recognize and bind to
N-glycans in the form of
GlcNAc2Man9Glc3 (9, 14). Helenius
and co-workers (9) proposed that the lectin-like activity of
calreticulin and calnexin plays a critical role in quality control
process during protein synthesis and folding.
In our earlier studies (1, 16) we have identified calreticulin as one
of the major Ca2+-binding, multifunctional proteins of the
ER membrane, and we documented that the protein interacts with PDI. PDI
is an abundant, ER lumenal protein that catalyzes a variety of
thiol/disulfide exchange reactions (17, 18). ERp57 is another ER
chaperone, a homologue of PDI, with thiol-dependent
reductase (19) and cysteine-dependent protease activities
(20). Recently, High and co-workers (21, 22) reported that ERp57 can be
cross-linked to monoglucosylated glycoproteins that are substrates for
calnexin and calreticulin suggesting that ERp57 may also be a
lectin-like chaperone. Zapun et al. (23) identified
functional complexes between ERp57, calreticulin, and calnexin and
showed that disulfide isomerase activity of ERp57 is much greater in
the presence of calreticulin or calnexin suggesting a functional
association between these proteins. Direct interaction between
calreticulin and ERp57 has not yet been reported.
In addition to their chaperone function, the majority of ER resident
proteins, including calreticulin and PDI, bind Ca2+ and
Zn2+ and contribute to the Ca2+ storage
capacity of the ER and cellular Ca2+ homeostasis (10, 13,
16, 24-33). Therefore, changes in the lumenal Ca2+
concentration due to Ca2+ release via inositol
trisphosphate receptor/ryanodine receptor and Ca2+ uptake
via Ca2+-ATPase (SERCA) (34) are expected to play an
important role in the control of chaperoning and other functions of
these proteins (13, 32). How chaperones interact to facilitate protein
folding and what is a role of the ER lumenal ions in these processes is not known.
In this paper we studied interactions between calreticulin and two
related ER membrane chaperones, PDI and ERp57, and we investigated the
role of Ca2+ in these protein-protein interactions. We show
that calreticulin interacts with PDI in a
Ca2+-dependent manner reminiscent of emptying
and refilling of the ER Ca2+ stores. Formation of the
ERp57-calreticulin complex was initiated by a
Ca2+-independent conformational change in ERp57 followed by
additional Ca2+-dependent conformational
changes in the complex. Ca2+ sensitivity of the
calreticulin-PDI and calreticulin-ERp57 complexes was confined to the
high capacity Ca2+ binding, C-domain of calreticulin. We
conclude that calreticulin interacts with ER lumenal chaperones and
that the protein may play a role of Ca2+ "sensor" for
these interactions.
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EXPERIMENTAL PROCEDURES |
Materials--
The FluroTag FITC Conjugation Kit, Mops, Pipes,
DTPA, RNase, and EGTA were obtained from Sigma. Dithiothreitol was
purchased from ICN Biomedicals, Inc. Cascade Blue acetyl azide (catalog number C-2284) was from Molecular Probes, Inc. QuixSep microdialyzer was obtained from Membrane Filtration Products, Inc. Sephadex G-25M was
from Amersham Pharmacia Biotech. Fresh canine pancreas was obtained
from the Surgical Medical Research Institute at the University of
Alberta. All chemicals were of the highest grade available.
Isolation of Proteins--
Canine pancreatic calreticulin was
purified by ammonium sulfate precipitation procedures as described
previously (35, 36). The recombinant domains of calreticulin and
recombinant glutathione S-transferase were expressed in
Escherichia coli and purified (11, 36). Protein disulfide
isomerase (PDI) was isolated from bovine liver by a procedure of
Lambert and Freedman (37) and modified as described by Baksh et
al. (16). Human ERp57 was expressed in E. coli and
purified (23).
Fluorescence Labeling--
Proteins were labeled with Cascade
Blue (CB) acetyl azide by adaptation to a FluroTag FITC Conjugation Kit
procedure as recommended by the manufacturer. Briefly, CB (1.11 mg/ml)
was dissolved in 100 mM carbonate/bicarbonate buffer, pH
9.0. Six hundred µg of purified PDI or ERp57 (2.6 mg/ml) in a 100 mM sodium carbonate/bicarbonate buffer, pH 9.0, was used
directly for labeling. The dye was added dropwise to the protein
mixture with constant stirring. The reaction vial was incubated in the
dark for 2 h at room temperature with gentle stirring. Labeled
proteins were separated from the free dye on a Sephadex G-25M column
(3.5 ml, bed height 2.6 cm) previously equilibrated with PBS. The
reaction mixture was applied, and fractions (0.25 ml each) were eluted
with PBS. The fluorescence of each fraction was determined at the
excitation wavelength 385 nm and the emission wavelength 430 nm using a
Perkin-Elmer spectrophotometer. Fractions containing labeled protein
were combined and used directly for protein-protein interaction
studies. Determination of the stoichiometry of the labeling revealed
that there were 4 and 5 molecules of CB conjugated per each molecule of
ERp57 and PDI, respectively, indicating that over 90% of the protein
was labeled with the dye.
Fluorescence Measurements--
Fluorescence measurements were
performed at an excitation wavelength 385 nm (slit, 15 nm) and an
emission wavelength 430 nm (slit, 15 nm) at room temperature using a
Luminescence Spectrometer LS50B (Perkin-Elmer). Fluorescence
intensities were measured with constant stirring in 1.5 ml of a binding
buffer containing 10 mM Mops, pH 7.0, 100 mM
KCl, 2 mM MgCl2, 0.5 mM EGTA.
Appropriate proteins and/or ions were added to the reaction mixture,
and the corresponding changes in fluorescence intensity were monitored. Quantum yields of CB or CB-labeled proteins were calculated from the
emission spectra (420-460 nm) obtained at the
excitation of 385 nm. Initial rates of the
protein-protein interaction and time constants were calculated using an
exponential decay function using Origin version 4.1 software.
Circular Dichroism Measurements--
For circular dichroism (CD)
analysis, proteins were dialyzed for 16 h against a buffer
containing of 25 mM Pipes, pH 6.8, 100 mM NaCl,
1 mM EGTA, 1 mM dithiothreitol. Dialysis was
performed using Spectra/Por dialysis tubing (cut-off 12-14 kDa) in a
QuixSep Microdialyzer. CD measurements were carried out on a Jasco
J-720 spectropolarimeter (Jasco Inc., Easton, MD), interfaced to an Epson Equity 386/25 and controlled by Jasco software. The thermostable cell holder was maintained at 25 °C with a Lauda RMS circulatory water bath (Lauda, Westbury, NY). The instrument was routinely calibrated with ammonium D-(+)-10-camphor sulfonate at
290.5 and 192 nm. Each sample was scanned 10 times, and noise reduction was applied to remove the high frequency before calculating molar ellipticities. The voltage to multiplier was kept below 500 V to
prevent distortion of the CD spectrum. The cell path length used was
0.02 cm, and the protein concentrations were 0.6 mg/ml in the far
ultraviolet. Concentrations were determined on a Cary 3 UV-visible
spectrophotometer and were corrected for light scattering. Molar
extinction coefficients were calculated from tyrosine and tryptophan
compositional values and were 43,780, 45,040, and 81,480 for ERp57,
PDI, and calreticulin, respectively. Molar ellipticities were
calculated from the following equation: [
] =
obs/10 × l × c, where
obs is in millidegrees, l is the pathlength
in centimeters, and c is the concentration in
moles/liter × number of amino acids in the sequence. The unit for
molar ellipticity is degree centimeter squared per pmol. The CD spectra
were analyzed for secondary structure elements by the Contin ridge
regression analysis program of Provencher and Glöckner (38).
Miscellaneous--
All recombinant techniques were conducted
according to standard protocols (39). Refolding of RNase B in the
presence of CB-labeled proteins was carried out as described previously
(23). Free Ca2+ concentrations were calculated using Max
Chelator, Winmaxc version 1.70.
 |
RESULTS |
Changes in Fluorescent Intensity of Cascade Blue (CB) Are
Indicative of Conformational Changes in PDI and ERp57--
CB acetyl
azide reacts with aliphatic amines in proteins to yield stable
carboxamides, is highly fluorescent, and resists quenching upon protein
conjugation (40). One unique feature of CB is that it has a different
fluorescence intensity depending on the polarity of the solvent used
(Fig. 1 and Table
I). For example, CB alone had relatively
high fluorescence intensity in PBS and water but significantly lower
fluorescence in nonpolar solvents such as n-propyl alcohol
and n-butanol (Fig. 1 and Table I). This observation
suggested to us that the dye may also be sensitive to exposure to the
hydrophilic or hydrophobic environments in a protein. To test these ER
lumenal chaperones, PDI and ERp57 were labeled with CB to generate
CB-PDI and CB-ERp57, respectively. Labeled proteins displayed emission
and excitation spectrum similar to the unconjugated fluorophore.
Similar to CB alone, fluorescence intensity of the protein-conjugated
CB (CB-ERp57 and CB-PDI) had different relative quantum yields in
various solvents (Table I) suggesting that the protein-conjugated dye
had a different fluorescence intensity depending on whether it was
exposed to a polar or a nonpolar microenvironment.

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Fig. 1.
Emission spectra of Cascade Blue in different
solvents at excitation of 380 nm. Emission spectra analysis of CB was carried out in PBS,
methanol, n-propyl alcohol (n-propanol), and
n-butyl alcohol (n-butanol).
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Table I
Relative quantum yield of Cascade Blue acetyl azide (CB) and CB
coupled to ERp57 (CB-ERp57) or PDI (CB-PDI) in different solvents
of various polarity
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Native PDI and ERp57 catalyze a variety of thiol/disulfide exchange
reactions (17-19, 23), and their chaperone activity can be estimated
by their ability to refold RNase B (23). Furthermore, ERp57 disulfide
isomerase activity is increased in the presence of calreticulin or
calnexin (23). It was, therefore, important to demonstrate that CB
labeling of PDI and ERp57 does not interfere with the function of these
chaperones. We examined disulfide isomerase activity of CB-PDI and
CB-ERp57 on the refolding of RNase B the presence and absence of
calreticulin and calnexin (Fig. 2). Fig. 2 shows that similar to native PDI and
ERp57 (23), CB-PDI and CB-ERp57 catalyzed refolding of RNase B
indicating that labeled proteins retained their chaperone activity.
Refolding of RNase B catalyzed by CB-PDI or CB-ERp57 was not influenced
by changes of Ca2+ or Zn2+ concentration.

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Fig. 2.
Refolding of RNase B catalyzed by CB-PDI and
CB-ERp57. Refolding of reduced RNase B was carried out in the
presence of CB-ERp57 (ERp57), CB-PDI (PDI), or
without the catalyst (CTL) and in the presence of calnexin
(CNX) or calreticulin (CRT) by the method
described previously (23). The reaction was terminated after indicated
times, and the RNase B conformation was examined by nondenaturing
polyacrylamide gel electrophoresis. Unfolded RNase B has the slowest
mobility (U), and the native form (N) has the
greatest mobility.
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Next we tested effects of ions (Ca2+ and Zn2+)
and purified calreticulin on CB alone and on CB labeled proteins.
Fluorescence intensity of CB alone did not change in the presence of
Ca2+, Zn2+ or purified calreticulin (Fig.
3A). However, the amplitude of fluorescence of the labeled proteins was sensitive to ion-induced conformational changes. Fig. 3, B and C show that
addition of Zn2+ to CB-PDI or CB-ERp57 resulted in
significant decrease in fluorescence intensity of the labeled proteins.
To test whether changes in fluorescence intensity induced by
Zn2+ (Fig. 3, B and C) reflected
conformational alterations in CB-PDI and CB-ERp57 we carried out CD
analysis of the purified proteins. Fig. 4
reveals that the CD spectra of ERp57 and PDI are very similar in shape,
having minima at 219 and 210 nm with molar ellipticity values of
10330o and
10180o at 219 nm and
9490°
and
9670° at 210 nm, respectively. The Contin version program for
calculating secondary structural elements (Table
II) indicates the
-helical content for
both proteins was 25-30%, and the combined
-sheet and
-turn was
~50%. The values for PDI are similar to those reported in previous
studies by Wetterau et al. (41, 42). Both proteins underwent
a conformational change upon addition of Zn2+ with a loss
in the amount of
-helix and a concomitant increase in
-sheet-
-turn. Similarly, pancreatic calreticulin showed no change upon addition of Ca2+ but underwent a reduction in
-helix and an increase in combined
-sheet-
-turn upon addition
of 2 mM Zn2+, a finding also reported earlier
by Khanna et al. (26). The apoprotein has ~10%
-helix
and 50%
-sheet-
-turn, upon analysis (Table II). We concluded
that Zn2+-dependent changes in the fluorescence
intensity of CB-ERp57 and CB-PDI (Fig. 3) may be due to conformational
changes in the proteins (Fig. 4) resulting in exposure of the
conjugated CB to protein microenvironments of a different polarity
(Fig. 1).

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Fig. 3.
Zn2+-induced conformational
changes in Cascade Blue-labeled PDI and ERp57. CB labeling
of PDI and ERp57 was carried out as described under "Experimental
Procedures." Changes in the fluorescence intensities of CB
(A) and CB-PDI (B) and ERp57 (C) were
monitored in the absence or presence of 500 µM
Zn2+ (A and B), and 1 mM
Zn2+ (C). A, the effects of
Zn2+, 1 mM Ca2+, and purified
pancreatic calreticulin on fluorescence intensity of CB alone was
examined. B and C, effects of 500 µM Zn2+ on CB-PDI and CB-ERp57.
Bars represent the duration of incubation with ions or
purified proteins. Arrowheads depict the time of
addition.
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Fig. 4.
CD analysis of ER lumenal proteins. CD
spectra of purified calreticulin, PDI, and recombinant ERp57 were
carried out as described under "Experimental Procedures." The data
are plotted as molar ellipticity versus wavelength for the
proteins in the absence ( ) and presence of Zn2+
(- - -).
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Table II
Provencher-Glöcker secondary analysis of ERp57, calreticulin,
PDI, and complexes of calreticulin-ERp57 and calreticulin-PDI
Measurements were carried out in the presence of a buffer containing 25 mM Pipes, pH 6.8, 100 mM NaCl, 1 mM
dithiothreitol, 1 mM EGTA. The Zn2+ and
Ca2+ concentrations given are the free amount in the solution.
Complexes are 1:1 molar ratio, and calculated complexes are weight
percentage of each component. The delta values are the difference
between observed and calculated apo
spectra.
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Interactions between Calreticulin and PDI Are Regulated by
Ca2+--
Can the observed conformational changes in the
CB-PDI and CB-ERp57 be applied to study protein-protein interactions?
We first investigated the interaction between CB-PDI and calreticulin
(Fig. 5). CB-PDI was incubated with
Zn2+ to induce conformational changes in the protein
followed by addition of the purified pancreatic calreticulin (Fig.
5A). Fig. 5A shows that calreticulin induced a
very rapid increase in fluorescence intensity of CB-PDI indicative of
protein-protein interaction-induced conformational changes in the
protein. The calculated initial rate of calreticulin and PDI
interaction was 21.4 ± 0.6 units/s (mean ± S.E.,
n = 3), whereas the time constant of the process was
0.16 ± 0.01 s (mean ± S.E., n = 3).
Fig. 5B illustrates that the
effect was saturable with respect to calreticulin and did not require
the presence of Zn2+. Identical results were obtained with
E. coli- and Pichia-expressed recombinant
proteins (data not shown). Fig. 5C reveals that, in agreement with our earlier observations (16), calreticulin did not
interact with PDI in the presence of 1 mM Ca2+.
Hormone-stimulated Ca2+ depletion results in lowering of
the ER lumenal free Ca2+ concentration below 100 µM (32, 43, 44). We have tested, therefore, if
calreticulin and CB-PDI will interact under the conditions of
Ca2+ store depletion. Addition of EGTA to lower free
Ca2+ concentration to 50 µM restored
interaction between calreticulin and CB-PDI as revealed by changes in
the fluorescence intensity of the CB-labeled protein (Fig.
5C). Re-addition of Ca2+ (to 1 mM)
rapidly reduced the fluorescence to the original level indicative of
dissociation of the protein complex (Fig. 5C).
Ca2+ titration experiments revealed that the
EC50 for Ca2+ for calreticulin-PDI dissociation
was approximately 110 ± 15 µM (mean ± S.E.,
n = 3). Thus, there is a saturable, rapidly reversible and Ca2+-dependent (under physiologically
relevant Ca2+ concentrations) interaction between
calreticulin and PDI.

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Fig. 5.
Interaction between calreticulin and
CB-PDI. CB-PDI was generated as described under "Experimental
Procedures." A, effects of 500 µM
Zn2+ and purified calreticulin on the fluorescence
intensity of CB-PDI; B, effects of increased levels of
calreticulin on fluorescence of CB-PDI; C, Ca2+
dependence of interaction between calreticulin and CB-PDI. Levels of 50 µM or 1 mM free Ca2+
concentration were maintained by the addition of 1 mM EGTA.
Bars represent the duration of incubation with ions or
purified proteins. Arrowheads depict the time of
addition.
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Fig. 6.
Interaction between calreticulin and
CB-ERp57. ERp57 was labeled with CB as described under
"Experimental Procedures." A, effects of calreticulin, 1 mM Ca2+, and 1 mM Zn2+
on fluorescence intensity of CB-ERp57. DTPA was added to chelate
Zn2+. B, effects of increasing levels of
calreticulin and Ca2+ on fluorescence intensity of
CB-ERp57. Bars represent the duration of incubation with
ions or purified proteins. Arrowheads depict the time of
addition.
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Interactions between Calreticulin and ERp57--
High and
co-workers (21, 22) have shown that ERp57, similar to calreticulin and
calnexin, is involved in chaperoning of glycoproteins. Furthermore,
Zapun et al. (23) reported functional interactions between
calreticulin and ERp57. However, direct interaction between
calreticulin and ERp57 has not yet been documented. We utilized
CB-ERp57 to investigate binding of calreticulin to ERp57 and the role
of Ca2+ in these protein-protein interactions. Fig.
6A shows that addition of calreticulin to CB-ERp57 either in
the presence or absence of Ca2+ had no effect on
fluorescence intensity of CB-ERp57, suggesting that under these
conditions the proteins may have not interacted. Since Zn2+
induced conformational changes in ERp57 (Fig. 3), we tested if the
presence of Zn2+ had any effect on the protein interaction
with calreticulin. Zn2+ induced a decrease in
fluorescence intensity of CB-ERp57 alone (Fig. 3). In contrast,
addition of calreticulin to CB-ERp57 in the presence of
Zn2+ resulted in an increase in the fluorescence
intensity (Fig. 6A) indicating that conformational changes
in ERp57 were required to initiate its interaction with calreticulin.
Chelating of Zn2+ with DTPA resulted in a decrease in the
intensity of fluorescence, indicative of dissociation of the protein
complex (Fig. 6A). The initial rate of this interaction was
23.4 ± 0.4 units/s (mean ± S.E., n = 3),
and it was similar to that observed for calreticulin and PDI
interaction (21.4 ± 0.6 units/s; mean ± S.E.,
n = 3). The time constant of interaction between ERp57
and calreticulin (1.4 ± 0.1 s; mean ± S.E.,
n = 3) was approximately 9-fold higher than between PDI
and calreticulin. Fig. 6B illustrates that changes in the
fluorescence intensity of CB-ERp57 were saturable with respect to
calreticulin. Initial interactions between ERp57 and calreticulin were
not affected by Ca2+ (Fig. 6A). Surprisingly,
Ca2+ had an effect on already formed calreticulin-ERp57
protein complex. Fluorescence intensity of CB-ERp57 and calreticulin
complex was increased by addition of 1 mM Ca2+
(Fig. 6B). Titration of calreticulin binding to ERp57 in the presence or absence of Ca2+ revealed that Ca2+
had no effect on the calreticulin affinity to bind ERp57 but only on
the fluorescence intensity of the complex. We concluded that
Ca2+ induced new conformational change in the
calreticulin-ERp57 complex without recruiting additional molecules of
calreticulin. The Ca2+-dependent enhancement of
fluorescence was reversed by addition of EGTA (Fig. 6B).
Analysis of the Ca2+ dependence of this process revealed
that the EC50 for Ca2+ was approximately
400 ± 30 µM (mean ± S.E., n = 3). Thus, interactions between calreticulin and ERp57 initially
required a Zn2+-dependent but
Ca2+-independent conformational change in the ERp57. The
properties of the complex were further influenced by increased
Ca2+ concentration.
Interaction between Calreticulin and PDI and ERp57 Induces
Conformational Changes in Protein Complexes--
We used CD analysis
to further investigate interaction between calreticulin and ERp57 and
PDI. Fig. 7 represents the observed and
calculated CD spectra of complexes between calreticulin and PDI and
between calreticulin and ERp57. Their secondary structural analysis is
indicated in Table II. In the absence of Ca2+ and
Zn2+ the ERp57 complex with calreticulin showed comparable
observed and calculated spectra, the latter estimated by weight
percentage of each component of the complex. This would imply that
under these conditions there was an interaction with a minimal
attendant conformational change. In agreement with our fluorescence
measurements, addition of Zn2+ to the mixture resulted in a
larger change in the observed versus calculated spectra
(Fig. 7 and Table II). The PDI complex with calreticulin produced small
changes for the observed apo and Ca2+ conditions relative
to the calculated values, whereas Zn2+ once again produced
the greatest change. Ca2+ resulted in a slight increase in
-helix. The apo and Zn2+ conditions showed a drop in
-helix upon Contin analysis.

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Fig. 7.
CD analysis of protein complexes.
Representative CD spectra of calreticulin, PDI, and ERp57 in the
absence ( ) and presence of Zn2+ (- - -). Also included
are the calculated spectra for the apo state of the complexes
(···).
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A Role of Calreticulin Domains in Protein-Protein
Interactions--
In order to establish the specific region of
calreticulin involved in the interaction with PDI and ERp57, we
expressed calreticulin domains in E. coli. The following
domains of calreticulin were used: the N-domain (the
NH2-terminal 182 amino acids of the protein), the P-domain
(residues 182-273), the N + P-domain (residues 1-273), and the
C-domain (residues 270-401) (11). Previously we showed by ligand
blotting, affinity chromatography, and the yeast two-hybrid system that
PDI interacts with the N-domain and P-domain of calreticulin in the
absence of Ca2+ (16). Fig. 8
shows that in the absence of Ca2+ the P- and
C-domains of calreticulin had no effect on the intensity of
fluorescence of CB-PDI, suggesting that under those conditions these
domains did not interact with calreticulin. Addition of the N-domain or
N + P-domain of calreticulin to CB-PDI in the presence of 50 µM Ca2+ induced changes in the fluorescence
intensity of the CB-PDI indicative of protein-protein interactions
similar to those observed in the presence of full-length calreticulin
(compare Fig. 8C and Fig. 5C). Increasing
Ca2+ concentration up to 1 mM had no effect on
fluorescence intensity of the CB-PDI and N-domain (Fig. 8D)
or CB-PDI and N + P-domain complexes (Fig. 8C). This is in
contrast to the Ca2+-dependent dissociation of
the full-length calreticulin and CB-PDI complex (Fig. 5). We concluded
that Ca2+ binding to the C-domain of calreticulin was
responsible for Ca2+-dependent dissociation of
the calreticulin-PDI complex. Thus, the N-domain of calreticulin is
required and sufficient for interaction between calreticulin and PDI.
The C-domain of the protein plays a role in dissociation of the protein
complex.

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Fig. 8.
Effects of calreticulin domains on
fluorescence intensity of CB-PDI and CB-ERp57. Calreticulin
domains were expressed in E. coli and purified as described
by Baksh and Michalak (11). CB labeling of PDI and ERp57 was carried
out as described under "Experimental Procedures." The effect of the
C-domain (A and E), P-domain (B and
F), N-domain (C and G), and N + P-domain (D and H) of calreticulin on
fluorescence intensity of CB-PDI (A-D) and ERp57
(E-H) was measured as described under "Experimental
Procedures." Zn2+ or Ca2+ were added where
indicated by the arrowheads. Bars represent the duration of
incubation with ions or purified proteins as indicated.
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Next we examined the role of calreticulin domains in interactions with
ERp57. Fig. 8, E and F, shows that the
P- and C-domain of calreticulin did not have any effect on
the fluorescence intensity of CB-ERp57 regardless of the conditions
used. However, addition of the N-domain or N + P-domain of the protein
resulted in an increase in the intensity of fluorescence of CB-ERp57
suggesting that the two proteins interacted (Fig. 8, G and
H). The effect of the N-domain or N + P-domain was identical
to that of the full-length calreticulin (compare Fig. 8, G
and H, and Fig. 5). Since the P-domain alone did not have
any effect on the CB-ERp57 fluorescence, we concluded that the N-domain
of calreticulin interacted with ERp57. Similar to the full-length
calreticulin, initial interaction between the N-domain of calreticulin
and CB-ERp57 was Ca2+-independent and required the presence
of Zn2+ (Fig. 8, G and H).
Importantly, Ca2+ had no effect on the N-domain and
CB-ERp57 complex (Fig. 8, G and H). This is in
contrast to full-length calreticulin (compare Fig. 8, G and
H, and Fig. 6). We concluded that the N-domain of calreticulin is essential for interaction between calreticulin and
ERp57. The P-domain does not participate in the interaction. Once again
these results suggest that the C-domain of the protein plays a role in
Ca2+-dependent augmentation of the
calreticulin-ERp57 complex.
 |
DISCUSSION |
In this report we utilized CB, a fluorescent dye, to investigate
the dynamics of the interactions between calreticulin and two related
ER membrane chaperones, PDI and ERp57. These interactions are modulated
by changes in protein conformation and by fluctuations in free
Ca2+ concentrations reminiscent of emptying and refilling
of the ER Ca2+ stores. Calreticulin interacts with PDI at
low Ca2+ concentration (<100 µM), but the
protein complex dissociates upon increased Ca2+
concentration (>500 µM). Formation of the
ERp57-calreticulin complex was initiated by a
Ca2+-independent conformational change in ERp57 followed by
a Ca2+-dependent conformational change in the
complex. Ca2+ binding to the C-domain of calreticulin is
responsible for the dissociation of the calreticulin-PDI complex and
for the modulation of the interaction between calreticulin and ERp57,
suggesting that this domain of the protein may play a role of a
Ca2+ sensor for ER membrane chaperones. These results
suggest changes in the free ER lumenal Ca2+ concentration
may be important for the regulation of these chaperone-chaperone interactions.
CB has been used previously as a fluorescent indicator for labeling of
protein, including antibodies (40). However, to our knowledge, this is
the first time the dye has been utilized to study changes in protein
conformation due to either ion binding or protein-protein interactions.
This property of the dye is likely a result of different behaviors of
CB in solvents (environments) of various polarities. Indeed, we show
that the fluorescence intensity of CB changes with the exposure to
hydrophobic or hydrophilic environments suggesting that exposure of the
dye to different regions in proteins may also lead to changes in its
fluorescence intensity. For example, changes in fluorescence intensity
of CB-PDI and CB-ERp57 due to Zn2+ binding or interaction
with calreticulin correspond with changes in their CD spectra. It is
conceivable that the dye may be used to study other protein-protein
interactions involving conformational changes in proteins.
One of the most important observations in this study is that the
interactions between calreticulin, PDI, and ERp57 are regulated by
fluctuation of Ca2+ concentration. For example calreticulin
interacts with PDI only when the Ca2+ concentration is
below 100 µM, a Ca2+ concentration found upon
emptying of the Ca2+ stores (32, 43, 44). The complex
dissociates at increased Ca2+ concentration (>500
µM), a concentration common for refilled Ca2+
stores (32, 43, 44). Ca2+ is released from the ER via
inositol trisphosphate receptor/Ca2+ channel, and it is
taken up by Ca2+-ATPase (SERCA) (34). In stimulated cells
this is a rapid process resulting in continuous changes in the levels
of free ER lumenal Ca2+ concentrations (32). We show that
interaction between calreticulin and PDI and calreticulin and ERp57 are
also very rapid with time constants of 0.16 ± 0.01 and 1.4 ± 0.1 s, respectively. Therefore, as far as the kinetics of these
protein-protein interactions are concerned, they are physiologically
relevant and capable of responding to rapid fluctuations in the lumenal
Ca2+ concentrations.
Interactions between calreticulin and CB-PDI is restricted to the
NH2-terminal region (N-domain) of calreticulin, but
Ca2+ sensitivity of this interaction is confined to the
COOH-terminal, high capacity Ca2+-binding region (C-domain)
of the protein. We also established that ERp57 forms protein complexes
with calreticulin, but unlike the calreticulin-PDI complex, binding of
calreticulin to ERp57 is initiated by a Ca2+-independent,
conformational change in ERp57 followed by a
Ca2+-dependent modulation of the complex. Once
again the N-domain of calreticulin plays a key role in this
protein-protein interaction, and the C-domain is responsible for the
Ca2+-dependent enhancement of the complex. The
N-domain of calreticulin is the most conserved region in the protein
with over 70% amino acid similarity between human and higher plants
(1). One important function of the N-domain of calreticulin may be
formation of specific complexes between ER lumenal chaperones. The
C-domain of the protein may play a
Ca2+-dependent regulatory role in these
protein-protein interactions. The C-domain of calreticulin is the least
conserved region of the protein, and in some organisms it is even
missing (1). Although in all organisms the N-domain of calreticulin may
be responsible for formation of protein complexes between different ER
chaperones, not all of them will have a
Ca2+-dependent (C-domain-dependent)
regulation of these complexes.
What is the physiological relevance of these specific protein-protein
interactions? Fig. 9 shows a proposed
model for a role of calreticulin and Ca2+ in controlling
interactions between ER lumenal proteins and regulation of their
chaperone function. Under the conditions of empty Ca2+
stores (Fig. 9A), when free Ca2+ concentration
is below 100 µM (44), calreticulin will form tight
complexes with PDI. This protein-protein interaction may result in
inhibition of PDI activity (16). Under low Ca2+ conditions
unfolded proteins may be released from PDI to enable them to interact
with other chaperones to continue the process of folding and quality
control. This may allow a flux of proteins undergoing a folding
process, from one chaperone to the other. It is well established that
Ca2+ depletion of the ER Ca2+ stores by
thapsigargin leads to induction of the unfolded protein pathway
(45-50). Results of our work indicate that prolonged Ca2+
depletion of the ER may result in a massive release of unfolded proteins from PDI and calreticulin activating the unfolded protein pathway, without actually changing a total concentration of unfolded proteins.

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|
Fig. 9.
Proposed model for a role of calreticulin in
dynamics of the ER lumen. Interactions between ER lumenal
chaperones under low Ca2+ conditions (A,
Empty Ca2+ stores) and high
Ca2+ conditions (B, Refilled
Ca2+ stores). Under low Ca2+
conditions (A) calreticulin associates with both PDI and
ERp57 but not with carbohydrate of newly synthesized glycoproteins.
When Ca2+ is taken up by SERCA and ER lumenal concentration
is increased above 400 µM, Ca2+ binds to the
C-domain of calreticulin, and the protein dissociates from PDI.
Newly synthesized glycoproteins can now associate with
calreticulin, and formation of calreticulin-ERp57 complexes is enhanced
allowing accelerated chaperoning of glycoproteins. N, P, and C, depict
calreticulin domains. IP3, inositol
trisphosphate.
|
|
Under the low Ca2+ conditions calreticulin will also
interact with ERp57 (Fig. 9A). These complexes may not be
functional, as far as chaperoning is concerned, because at a low
Ca2+ concentration calreticulin displays a very weak, if
any, binding to monoglucosylated glycoproteins (14). Refilling of
Ca2+ stores via function of SERCA will result in
significant changes in the dynamics of lumenal ER proteins. Under these
conditions free Ca2+ concentration will rise above 400 µM (44) followed by a rapid dissociation of calreticulin
from PDI (Fig. 9B). Since, under these conditions, PDI does
not associate with calreticulin (or calnexin), it is likely that the
protein may play an important role in disulfide bond formation of newly
synthesized proteins that are not glycosylated. Importantly, elevations
in the free Ca2+ concentration in the lumen of the ER will
promote calreticulin (and calnexin) lectin-like activity and their
interaction with monoglucosylated glycoproteins (Fig. 9B).
Calreticulin will recruit ERp57 to provide an "attachment" site for
the protein to chaperone (disulfide bond formation) newly synthesized
glycoproteins. ERp57 chaperone activity is greatly increased when
complexed with calreticulin (23). Under these conditions other ER
lumenal chaperones may also be recruited to further assist in proper
folding of newly synthesized glycoproteins. One significant finding is
that the high capacity Ca2+-binding site (C-domain) of
calreticulin may play a role of a Ca2+ sensor for these
protein-protein interactions in response to continuous fluctuations of
ER lumenal Ca2+ concentrations. The C-domain of
calreticulin is expected to bind Ca2+ only in fully
re-filled Ca2+ stores but not under the ER Ca2+
depletion conditions (11). It is likely, therefore, that calreticulin, via its Ca2+ binding ability, may play multiple roles in
the lumen of the ER: regulation of free Ca2+ concentration
and Ca2+-dependent modulation of chaperone
function of other ER lumenal proteins such as PDI and ERp57. It has
previously been reported that the function of other chaperones may be
sensitive to fluctuations in the ER lumenal Ca2+. At low
Ca2+ concentrations in the lumen of the ER, the activity of
BiP, another ER lumenal chaperone, is also inhibited (51). Protein
synthesis, glycoprotein processing, and transport competence are also
blocked under the conditions of ER Ca2+ depletion (52-54).
Our work suggests that calreticulin may play a key role in the control
of these processes.
Calreticulin, from the lumen of the ER, may regulate Ca2+
levels in the lumen of ER via potential interactions with the ER
Ca2+-ATPase (SERCA) and/or the inositol trisphosphate
receptor (5, 10). Favre et al. (55) demonstrated a highly
supralinear feedback inhibition of Ca2+ uptake via SERCA
and postulated existence of an ER lumenal molecule(s) which may
regulate SERCA activity (10). Calreticulin may be a potential ER
lumenal candidate protein that modulates SERCA activity. Furthermore,
calreticulin, from the lumen of the ER, affects steroid-sensitive gene
expression (7), cell adhesiveness (6), and store-operated
Ca2+ influx (29, 30). Results of this work support our
earlier hypothesis (13) and suggest that fluctuation of the ER lumenal Ca2+ concentration will regulate the free concentration of
calreticulin in the lumen of the ER. For example, at high lumenal
Ca2+ concentration levels of free calreticulin, in the
lumen of the ER, will be significantly increased. Thus,
Ca2+-dependent changes in the free calreticulin
may play a role in modulation of a variety of
calreticulin-dependent signals, including gene expression
and Ca2+ homeostasis.
In summary, results of this work show that calreticulin appears to be
one of the key players of the chaperone network in association with
calnexin, PDI, and ERp57. Furthermore, present work indicates that
calreticulin may play a role of Ca2+ sensor for these
chaperones. The protein modulates
Ca2+-dependent formation and maintenance of the
structural and functional complexes between different ER lumenal
proteins involved in a variety of steps during protein synthesis,
folding, and post-translational modification. It is conceivable that
other chaperones may form similar functional complexes with
calreticulin and that these interactions may be controlled by
hormone-dependent fluctuations of ER lumenal free
Ca2+.
 |
ACKNOWLEDGEMENT |
The superb technical assistance of Monika
Dabrowska is greatly appreciated.
 |
FOOTNOTES |
*
This work was supported by grants from the Medical Research
Council of Canada (to M. M., J. J. M. B., and D. Y. T.), from the
Heart and Stroke Foundations of Alberta and Zyma Foundation (to
M. M.), from the Swiss National Foundation (to K. H. K.), and from
the Protein Engineering Network of Centers of Excellence (to
C. M. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

Medical Research Council of Canada Senior Scientist and Medical
Scientist of the Alberta Heritage Foundation for Medical Research. To
whom correspondence should be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-2256; Fax: 780-492-0886; E-mail:
marek.michalak{at}ualberta.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
PDI, protein disulfide isomerase;
CB, Cascade Blue;
PBS, phosphate-buffered saline;
Pipes, 1,4-piperazinediethanesulfonic acid;
Mops, 4-morpholinepropanesulfonic acid;
DTPA, diethylenetriaminepentaacetic acid;
SERCA, sarcoplasmic/endoplasmic
reticulum Ca2+-ATPase.
 |
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