Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.     

Perforin lytic activity is controlled by calreticulin

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.     

Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells

In the present study we have demonstrated the presence of calreticulin, a major Ca(2+)-sequestering protein of nonmuscle cells, in a variety of cell types in tissue culture. The protein localizes to the endoplasmic reticulum in most cell types and also to the nuclear envelope or nucleoli-like structures in some cell types. Calreticulin is enriched in the rough endoplasmic reticulum, suggesting a possible involvement in protein synthesis. Calreticulin terminates with the KDEL-COOH sequence, which is likely responsible for its endoplasmic reticulum localization. Unlike some other KDEL proteins, calreticulin expression is neither heat-shock nor Ca(2+)-shock dependent. Using a variety of metabolic inhibitors, we have shown that the pool of calreticulin in L6 cells has a relatively slow turnover and a stable intracellular distribution. In proliferating muscle cells in culture (both L6 and human skeletal muscle) calreticulin is present in the endoplasmic reticulum, and additional intranuclear staining is observed. When fusion of the L6 cells is inhibited with either a high serum concentration or TGF-beta or TPA, the nucleolar staining by anticalreticulin antibodies is diminished, although the presence of calreticulin in the endoplasmic reticulum remains unchanged. In contrast, in differentiated (i.e., fused) muscle cells neither intranuclear nor intracellular staining for calreticulin is present. We conclude, therefore, that calreticulin is abundant in the endoplasmic reticulum in proliferating myoblasts, while it is present in only small amounts in sarcoplasmic reticulum membranes in terminally differentiated myotubes. We propose a model for the domain structure of calreticulin that may explain the differential subcellular distribution of this protein. Because of its widespread distribution in nonmuscle tissues, we postulate that calreticulin is a multifunctional protein that plays an important role in Ca(2+) sequestering and thus that it is the nonmuscle analog of calsequestrin.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.     

The localization of the ER retrieval sequence for the calcium pump SERCA1

Abstract

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L     

A polypeptide binding conformation of calreticulin is induced by heat shock, calcium depletion, or by deletion of the C-terminal acidic region

It is widely believed that the chaperone activity of calreticulin is mediated by its ability to bind glycoproteins containing monoglucosylated oligosaccharides. However, calreticulin is also a polypeptide binding protein. Here we show that heat shock, calcium depletion, or deletion of the C-terminal acidic domain enhance binding of purified calreticulin to polypeptide substrates and enhance calreticulin's chaperone activity. These conditions also enhance calreticulin oligomerization, but oligomerization per se is not required for enhanced polypeptide binding. In cells, calreticulin oligomerization intermediates accumulate in response to conditions that induce protein misfolding (heat shock and tunicamycin treatments), and upon calcium depletion. Additionally, in cells, calreticulin binds to deglycosylated major histocompatibility complex class I heavy chains when significant levels of calreticulin oligomerization intermediates are induced. Thus, cell stress conditions that generate nonnative substrates of calreticulin also affect the conformational properties of calreticulin itself, and enhance its binding to substrates, independent of substrate glucosylation.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

Calreticulin: from Ca2+ binding to control of gene expression

Calreticulin is a highly conserved Ca(2+)-binding/storage protein of the endoplasmic reticulum (ER). Recently, it has been shown to play a role in the control of gene expression by interacting with the DNA-binding domain of various steroid receptors. How does this ER protein gain access to the nuclear steroid receptors? We propose that calreticulin undergoes unique intracellular trafficking that allows it to colocalize with and bind to steroid receptors.     

Determinant for endoplasmic reticulum retention in the luminal domain of the human cytomegalovirus US3 glycoprotein

US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum.     

ERp27, a new non-catalytic endoplasmic reticulum-located human protein disulfide isomerase family member, interacts with ERp57

Protein folding and quality control in the endoplasmic reticulum are critical processes for which our current understanding is far from complete. Here we describe the functional characterization of a new human 27.7-kDa protein (ERp27). We show that ERp27 is a two-domain protein located in the endoplasmic reticulum that is homologous to the non-catalytic b and b' domains of protein disulfide isomerase. ERp27 was shown to bind Delta-somatostatin, the standard test peptide for protein disulfide isomerase-substrate binding, and this ability was localized to the second domain of ERp27. An alignment of human ERp27 and human protein disulfide isomerase allowed for the putative identification of the peptide binding site of ERp27 indicating conservation of the location of the primary substrate binding site within the protein disulfide isomerase family. NMR studies revealed a significant conformational change in the b'-like domain of ERp27 upon substrate binding, which was not just localized to the substrate binding site. In addition, we report that ERp27 is bound by ERp57 both in vitro and in vivo by a similar mechanism by which ERp57 binds calreticulin.     

The glucose-regulated protein grp94 is related to heat shock protein hsp90

We report the sequence of a cDNA clone that encodes the C-terminal half of the hamster 94 X 10(3) Mr glucose-regulated protein, grp94. The amino acid sequence of this protein is about 50% homologous to Drosophila hsp83 and yeast hsp90, suggesting that grp94 and hsp90 have similar functional properties. Unlike hsp90, grp94 is associated with the endoplasmic reticulum. It has the same C-terminal tetrapeptide as two other luminal endoplasmic reticulum proteins, grp78 and protein disulphide isomerase. We suggest that this sequence forms part of a signal for retention of proteins in the lumen of the endoplasmic reticulum.     

Calreticulin: not just another calcium-binding protein

In this paper we review some of the rapidly expanding information about calreticulin, a Ca²⁺-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca²⁺-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca²⁺ storage protein in the endoplasmic reticulum.Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum     

Functional Conservation of Calreticulin in Euglena gracilis

Calreticulin is the major high capacity, low affinity Ca²⁺ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca²⁺ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca²⁺ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca²⁺ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phytogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca²⁺ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore iii that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca²⁺ binding function of calreticulin from Euglena traces the functional history of Ca²⁺ second messenger signal transduction pathways deep into eukaryotic evolution.     

The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment

In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (<100 micrometer), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca(2+) concentration up to 500 micrometer or 1 mm resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH(2)-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH(2)-terminal amino acid sequence analysis. Ca(2+)-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca(2+) concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn(2+) resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg(2+)-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.     

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.     

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein.

The complete amino acid sequence of CRP55, the major 55 kd calcium binding protein of the ER lumen, was deduced from the murine cDNA nucleotide sequence. This was completed using a novel application of PCR amplification. The mature 399 residue protein encoded is preceded by a 17 amino acid leader sequence and ends in the ER signal sequence, KDEL. The protein contains no calcium binding motifs of the EF hand type or of the form seen in calelectrin-related proteins. The major region of potential low affinity calcium binding sites is a polyacidic stretch towards the C terminus. The primary structure of the protein is markedly zonal. The N-terminal region, of approximately neutral net charge and hydrophobicity, is followed by a central proline-rich zone with repeat sequences separated from the polyacidic C-terminal stretch by a short hydrophobic sequence. The general shape suggested is a globular domain attached to an extended tail. Immunofluorescence studies show that the protein is present in skeletal muscle and indicate that it is a sarcoplasmic reticulum protein. We propose that the protein be named calreticulin to reflect its calcium binding activity and location in the ER and SR.