Reticulocalbin, a novel endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence.

A novel Ca(2+)-binding protein, tentatively designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence staining. The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca(2+)-binding motif, the EF-hand. Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+. The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This is similar to the Lys-Asp-Glu-Leu sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into medium in transient expression assays.

plasm) contains a number of soluble proteins including immunoglobulin heavy chain-binding protein (GRP78) (Munro and Pelham, 1986;Bole et al., 1986), protein disulfide-isomerase (Freedman, 1989;Edman et al., 1985), and GRP94 (endoplasmin, ERp99) (Mazzarella and Green, 1987;Sorger and Pelham, 1987). Many of these proteins are involved in the initial steps of the maturation of newly synthesized secretory proteins such as folding of nascent polypeptide chains and formation of the correct disulfide bonds. Retention of these resident proteins in the ER is dependent on a carboxylterminal signal, which in animal cells is usually Lys-Asp-Glu-Leu (KDEL). The KDEL sequence is recognized by a membrane-bound receptor that continually retrieves the proteins from a later compartment (&-Golgi cisternae) of the secretory pathway and returns them to the ER (Pelham, 1989(Pelham, , 1990. Although the above-mentioned proteins (immunoglobulin heavy chain-binding protein, protein disulfide-isomerase, and GRP94) reportedly bind Ca2+ (Macer and Koch, 1988), none of them have the EF-hand motifs.
Calreticulin, a well-characterized Ca2+-binding protein of the ER and the sarcoplasmic reticulum, binds Ca2+ with high affinity, but does not have an EF-hand motif (Smith and Koch, 1989;Fliegel et al., 1989). Calreticulin may be a nonmuscle functional analogue of calsequestrin, a major Ca2+binding (storage) protein of the skeletal muscle sarcoplasmic reticulum membrane (Milner et al., 1991). So far, there have been no reports of ER resident proteins having EF-hand motifs.
We previously isolated several independent cDNA clones from Xgtll libraries of mouse teratocarcinoma OTT6050 (Ozawa et al., 1988;Furukawa et al., 1990). These clones were isolated by screening the libraries with antibodies against Dolichos biflorus agglutinin-binding glycoproteins. The lectin D. biflorw agglutinin is known to bind specifically with the nonreducing terminal N-acetylgalactosamine of carbohydrate chains (Etzler, 1972). Although the majority of the clones showed developmentally regulated expression, others did not, suggesting that the latter are products of housekeeping genes. Sequencing a group of cDNA clones that belong to the latter category and characterizing the protein encoded by these clones revealed that the protein is an ER resident Ca2+binding protein with multiple EF-hand motifs for which we propose the nomenclature reticulocalbin.

MATERIALS AND METHODS
cDNA Cloning-cDNA clones 01, 09, and 032 were isolated from a Xgtll cDNA library of mouse teratocarcinoma OTT6050 (Ozawa et al., 1988) by screening with antibodies against D. biflorus agglutininbinding glycoproteins as previously described (Ozawa et al., 1988). Clones M10 and M22 were obtained from a primer extension cDNA library in X g t l O and subcloned into pUC18 and Bluescript KS(+) 699 (Stratagene) vectors, and the DNA was sequenced from both strands as described (Ozawa et al., 1988).
Construction of Expression Vectors-To express reticulocalbin in animal cells, the cDNA was cloned into a mammalian expression vector, pCAGGS, which contains an enhancer derived from cytomegalovirus and the &actin promotor (Niwa et al., 1991). The Bluescript KS(+) vector containing the 1052-bp 5"fragment of reticulocalbin cDNA in the EcoRI-PstI site was restricted with SmaI and HincII. The fragment was isolated and cloned into pCAGGS, which had been digested with EcoRI and filled in with T4 DNA polymerase. The orientation of the cDNA in the vector was confirmed by restriction enzyme digestion. We constructed an expression vector encoding a mutant reticulocalbin lacking the carboxyl-terminal HDEL peptide by polymerase chain reaction (PCR). Oligonucleotides GACATCGA-CAAGAACGG and CCTGCAGTCAATTTTTGGTCAGGTCTTCC were synthesized and used as primers. The former corresponds to the sequence of the cDNA from positions 656 to 672. The latter contains a complementary sequence of the cDNA at positions 979-997 and the TGA termination codon as well as a PstI recognition sequence at the 5'-end. The template was the 1052-bp reticulocalbin cDNA in the Bluescript vector, which was linearized by digestion with PuuII. PCR was performed according to the manufacturer's instructions using the GeneAmp PCR reagent kit (Perkin-Elmer Cetus Instruments). The reaction mixture was subjected to 25 cycles of denaturation (93 "C, 1 rnin), annealing (50 "C, 2 rnin), and extension (72 "C, 3 rnin). The PCR product was purified on agarose gel, digested with PstI, and cloned into the SmaI-PstI site of Bluescript KS(+). After confirming the sequence of the 3'-BamHI-PstI fragment, the 5"region was replaced with the authentic fragment and cloned into the pCAGGS vector as described above.
To express recombinant reticulocalbin in Escherichia coli, the cDNA encoding the mature protein was cloned into the maltosebinding protein fusion vector (pMAL-c) (New England BioLabs, Inc.) as follows. The cDNA region coding for the signal sequence was eliminated by PCR. We synthesized oligonucleotide CAGCTGCGG-GCCAAGCCCACG for use as a primer. It corresponds to the nucleotide sequence encoding the first 6 amino acid residues of the mature protein with the 5"extension of CAG, which, together with CTG, constitutes a recognition sequence for PuuII. The second primer was oligonucleotide CAGGAAACAGCTATGAC, which was purchased commercially as a primer for dideoxy sequencing (the reverse primer; Takara, Kyoto, Japan). The template, the PCR program, and other conditions were the same as described above. The PCR product was cloned into the SmaI-PstI site of Bluescript KS(+) after the fill-in reaction and PstI digestion, and the sequence was confirmed. A 959bp Puu!!-PstI fragment that encodes the entire mature protein was cloned into the StuI-PstI site of the pMAL-c vector. The plasmid DNA was introduced into TB1 cells.
Cells-The mouse parietal endodermal cell line PYS-2, L cells, and COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a 10% CO, atmosphere at 37 "C. Genes were transfected by the DEAE-dextran method. Ten micrograms of DNA was mixed with DEAE-dextran (400 pg/ml) and added to COS cells (3 X lo5) that were plated 1 day in advance. After 4 h, the cells were washed and incubated with 100 p~ chloroquine for 3 h. Cells were cultured for 2 days and analyzed.
Biochemical Analysis-Immunoblot analysis was carried out as described before (Ozawa et al., 1989). Calcium blotting was performed as previously described (Maruyama et al., 1984). For immunoprecipitation, 5 x lo5 cells were preincubated for 30 min in Dulbecco's modified Eagle's medium without methionine and 10% dialyzed fetal calf serum and subsequently labeled with [36S]methionine (1000 Ci/ mmol; Du Pont-New England Nuclear) at 100 pCi/ml for 30 min. Cells were washed with normal Dulbecco's modified Eagle's medium with 10% fetal calf serum and 2 mM cold methionine and incubated in this medium for 2 h. Cells were washed with PBS and lyzed with PBS containing 1% Nonidet P-40, 1 mM CaCl,, and phenylmethylsulfonyl fluoride. After centrifugation, cell lysates or media were incubated with anti-reticulocalbin antibodies, and immunocomplexes were collected by protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology Inc.) as previously described (Ozawa et d., 1989). Lectinagarose fractionation proceeded as follows. Cell lysates were applied to columns (0.4-ml bed volume) containing concanavalin A (ConA)-Sepharose (Pharmacia) or Ricinus communis agglutinin-agarose (EY Laboratories) equilibrated with PBS containing 0.1% Nonidet P-40, 1 mM CaCl,, and 1 mM phenylmethylsulfonyl fluoride. After washing with 10 ml of this buffer, materials bound to the resin were eluted with the same buffer containing 0.2 M a-methylmannoside (for ConA-Sepharose) or 0.1 M lactose (R. communis agglutinin-agarose). The bound and unbound materials were analyzed by SDS-PAGE and immunoblot analysis. For subcellular fractionation, cells were homogenized in 20 mM Tris-HC1 (pH 7.4) containing 0.25 M sucrose. The suspension was centrifuged sequentially at 900 X g for 10 min, 5000 X g for 10 min, and 100,000 X g for 60 min. The precipitates from each centrifugation were designated nuclear, mitochondrial, and microsomal fractions, respectively. The supernatant after the third centrifugation was retained as the soluble fraction. The microsome fraction was further solubilized with Triton X-100 (1%) or sonicated, and the supernatants after centrifugation at 140,000 X g for 1 h were analyzed. Phase separation of reticulocalbin in Triton X-114 was performed as described (Bordier, 1981).
Purification of Recombinant Reticulocalbin-Bacterial cells containing the fusion plasmid were cultured, and the fusion protein was induced and collected according to the manufacturer's instructions (New England BioLabs, Inc.). The cells were disrupted by sonication in PBS containing 1 mM CaC1, and 1 mM phenylmethylsulfonyl fluoride and then centrifuged. The supernatant was applied to a column of amylose resin and washed with 5-10 column volumes of the following buffers: PBS containing 0.1% Tween 20, PBS containing 0.5 M NaCl and 0.1% Tween 20, and PBS. The fusion protein was eluted with PBS containing 10 mM maltose.
Antibodies-Antibodies were raised against D. biflorus agglutininbinding glycoproteins from teratocarcinoma OTT6050 as described (Ozawa et al., 1982). Monospecific antibodies against reticulocalbin were prepared as follows. The recombinant reticulocalbin of the MBP fusion described above was electrophoresed to remove any contaminating bacterial proteins. The corresponding band was excised from the gels after Coomassie Blue staining and used for immunization in rabbits. The antibodies were affinity-purified by coupling MBP or the fusion protein (1 mg each) to 1 ml of CNBr-activated Sepharose. Antibodies bound to the resin were eluted with 0.1 M glycine HC1 (pH 2.5) and immediately neutralized with Tris-HC1 (pH 8.8). In some experiments, antibodies eluted from MBP were used as a control.
Immunofluorescence-Cells on coverslips were washed with PBS containing 0.2 mM CaC12 and 0.2 mM MgCl, and fixed with 3% formaldehyde in PBS for 15 min at room temperature. After washing with PBS and incubation with 50 mM NH&l in PBS, cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 5 min. Cells were preincubated with a mixture (1:l) of PBS and Dulbecco's modified Eagle's medium with 10% fetal calf serum for 15 min and incubated sequentially with anti-reticulocalbin antibodies and goat anti-rabbit antibodies conjugated with fluorescein isothiocyanate (Jackson Laboratories) in the same solution for 30 min. For double staining, biotinylated ConA and rhodamine-labeled avidin (EY Laboratories) were included in the solutions containing anti-reticulocalbin antibodies or anti-rabbit antibodies, respectively. Stained cells were photographed with a Nikon microscope using Fujichrome 100 film. Images from the same slides were generated by a confocal scanning laser microscope (MRC500, Bio-Rad) on a video monitor and photographed using TriX film. Clones- Fig. 1 shows the scheme of isolated reticulocalbin cDNA clones. Clones 01, 09, and 032 were isolated from a cDNA library in Xgtll constructed from mouse teratocarcinoma OTT6050 cDNA by screening with antibodies against D. biflorus agglutinin-binding glycoproteins of the cells. These clones provide the 3'sequence to the internal EcoRI site at nucleotide 476, but they did not extend further because EcoRI sites were not methylated during construction of the library. Clone 032-8 was obtained by screening a Xgtll library constructed with a cDNA, whose internal EcoRI sites were protected with EcoRI methylase, by plaque hybridization using the EcoRI-BamHI fragment of clone 032 as a probe. To isolate clones M10 and M22, we used an oligonucleotide (30-mer) corresponding to nucleotides 625-655 of reticulocalbin cDNA as a primer for construction of another cDNA library. This library in X g t l O was screened by plaque hybridization using the 144-bp EcoRI-NdeI fragment of clone 032 as a probe. Northern hybridization of RNAs from teratocarcinoma OTT6050, embryonal carcinoma cell line F9, and parietal endodermal cell line PYS-2 revealed that the cDNA clones hybridized a major mRNA species of 2.3 kilobase pairs and a minor species of 2.0 kilobase pairs (data not shown).

Isolation of Reticulocalbin cDNA
The composite nucleotide sequence of the cDNA clones and the deduced amino acid sequence of reticulocalbin are presented in Fig. 2. The nucleotide sequence contains 34 bp of the 5"untranslated region and a 3"untranslated region of 1016 bp. The coding region specifies a protein of 325 residues with an M, of 38,112. The amino acid sequence of the first 20 amino acids of reticulocalbin has the typical features of a secretory leader peptide. There is a positively charged aminoterminal region and a hydrophobic central section followed by two small nonpolar residues with a single residue between them. This latter feature is thought to determine the cleavage point of the signal peptidase, which in this case could be between Ala-20 and Leu-21. Cleavage of this peptide would yield a mature protein of 305 residues with a predicted M, of 36,200. The sequence of reticulocalbin does not contain a hydrophobic transmembrane segment, which suggests transfer of the entire protein into the lumen of the ER. There is one consensus site for N-linked glycosylation and a number of serine-or threonine-rich regions that could be O-glycosylated.
Immunoblots of PYS-2 cells with antibodies against reticulocalbin revealed a band of 44 kDa and a faint band of 46 kDa (Fig. 3). The same bands were also detected in mouse fibroblast L cells (data not shown). The 46-kDa species binds to concanavalin A-Sepharose, whereas the 44-kDa species does not (Fig. 3). Neither of the two proteins bound to R. communis agglutinin-agarose, which recognizes terminal or sialylated Galpl-4GlcNAc sequences (Baenzinger and Fiete, 1979). Therefore, the 46-kDa species may be produced by Nglycosylation (high-mannose type) of the 44-kDa species at residue 47. To confirm that the cDNA codes the 44-kDa protein, the cDNA was cloned into an expression vector (pCAGGSneo) and introduced into COS cells. Upon immunoblot analysis, COS cells transfected with the cDNA in a sense orientation showed a 44-kDa band and a faint 46-kDa band, whereas COS cells transfected with the cDNA in an antisense orientation gave faint bands in the region (Fig. 4). We consider that the faint bands (46 and 44 kDa) represent endogenous reticulocalbin in COS cells. Even though the 44-kDa band is present in COS cells transfected with antisense cDNA, the intensity of the band greatly increased in COS cells transfected with sense cDNA. Therefore, we conclude  (lanes 2 and 3 ) or R. communis agglutinin (RCA)-agarose (lanes 4 and 5). Bound materials specifically eluted with the respective haptenic sugars (lanes  2 and 4 ) , and unbound materials (lanes 3 and 5) were immunoblotted with anti-reticulocalbin antibodies. that the cDNA clone has the entire sequence encoding the 44-kDa protein.
Reticulocalbin Is a Ca2+-binding Protein with EF-hand Motifs-Analysis of the cDNA sequence shows that the predicted protein contains six repeats of -30 amino acids (Fig. 5). Each repeat has the general feature of a high affinity Ca2+-binding EF-hand domain according to Kretsinger's rule (Kretsinger, 1980). Comparing the amino acid sequence of these domains with those required for a perfect EF-hand Ca2+-binding site, there is a varying degree of divergence. The 5 oxygen-containing residues important for the coordination of Ca2+ are present in all the predicted sites. The central glycine is conserved in sites I, IV, and V, whereas in sites 11,111, and VI, it is replaced either by glutamic acid or leucine. The replacements appear not to be cloning artifacts because the independent cDNA clones have the same sequence in the region. Furthermore, the sequence of the corresponding region of genomic DNA clones also has the same sequence.' The secondary structure * M. Ozawa, unpublished results. The protein sequence (Fig. 2) was analyzed for homologies to the test sequence for a Ca2+-binding EF-hand motif. The amino acid sequence is shown in one-letter code. In the test sequence, L represents hydrophobic residues, and 0 represents oxygen-containing residues.

FIG. 6. Calcium blots of recombinant reticulocalbin.
Maltose-binding protein (2 pg) (lanes I ) or a recombinant reticulocalbin of the maltose-binding protein fusion (2 pg) (lanes 2 ) purified on amylose resin was electrophoresed and transferred to a nitrocellulose membrane. After incubation with 4sCaz+, the membrane was exposed to x-ray film. The same membrane was stained with Amido Black to detect proteins. The apparent molecular mass (88 kDa) of the fusion protein is in good agreement with the expected size (86 kDa).
prediction (Chou and Fasman, 1978) shows that the replacement of glycine with glutamic acid in sites I1 and VI causes the formation of an a-helix at the sites instead of a loop structure. Therefore, sites I1 and VI of reticulocalbin probably no longer bind Ca2+. Although the glycine is replaced by leucine, site I11 appears capable of forming the loop structure. The replacement of the normally conserved glycine with lysine has been reported in the EF-hands of the fibrinogen ychain (Dang et al., 1985) and secreted protein acidic and rich in cysteine (BM-40, osteonectin) (Engel et al., 1987). Therefore, site I11 seems to bind Ca2+.
To test whether reticulocalbin actually binds Ca", recombinant reticulocalbin was analyzed by 45Ca2+ blotting. The cDNA coding mature reticulocalbin was cloned into the MBP fusion vector (pMAL-c), and the recombinant protein was expressed in E. coli as a fusion protein with MBP. After purification by affinity chromatography on a column containing amylose resin, the fusion protein was electrophoresed on a gel, transferred to a nitrocellulose membrane, and incubated with Ca2+. MBP alone did not bind Ca2+; however, the fusion protein did (Fig. 6).
Reticulocalbin Is a Luminal ER Protein-After homogenization of PYS-2 cells, reticulocalbin remained in the low speed (5000 x g) supernatant, but sedimented together with crude membranes during high speed (100,000 X g) centrifugation (Fig. 7A). It was solubilized from the membrane by Triton X-100 or by sonication in the absence of detergents (Fig. 7A). Reticulocalbin partitioned into the aqueous phase when cells fractions and immunoblotted. Reticulocalbin in the microsomal fraction was solubilized with Triton X-100 ( l a n e 5) or released by sonication (Sonic; lane 6). B, cells were extracted with Triton X-114, and the extract ( l a n e I ) was analyzed by phase separation. Reticulocalbin was found in the aqueous (uque.) phase (lune 2), and not in the detergent (deter.) phase ( l a n e 3).
T o localize reticulocalbin, PYS-2 cells were stained with anti-reticulocalbin antibodies by indirect immunofluorescence. When the cells were fixed with formaldehyde and permeabilized with Triton X-100, the intracellular ER regions (but not the cell surface) were stained (Fig. 8A). Fig. 8A shows that reticulocalbin is localized in the perinuclear system of membranes corresponding to that of the ER of PYS-2 cells. Since the ER seemed not to be well developed in PYS-2 cells, we stained transfected COS cells expressing reticulocalbin.
Untransfected cells were not detectably stained with the antibody (data not shown). All cells expressing reticulocalbin exhibited prominent staining of the perinuclear region as well as a lattice of fine tubular structures (Fig. 8C), but there was no obvious staining of the Golgi apparatus. This pattern is typical of that obtained for proteins that are retained in the ER (Munro and Pelham, 1987). Furthermore, the structure that contained reticulocalbin also stained with concanavalin A (Fig. 8 0 ) , which predominantly stains E R structures (Tartakoff and Vassalli, 1983), confirming that they represented the ER. Because of the bright staining in the perinuclear region, however, it was difficult to get a clearly visible pattern of the tubule network. We overcame this issue using a confocal scanning laser microscope. Fig. 8E shows the distribution of reticulocalbin in the transfected COS cells. The image confirms the results obtained using conventional microscopy and allows detailed examination of the distribution of the protein.
There is an intense reticular staining of the ER.
Reticulocalbin has a putative amino-terminal signal peptide, but no hydrophobic transmembrane segment, which is consistent with transfer of the entire protein into the lumen of the ER. To verify that the amino-terminal stretch of hydrophobic amino acids functions as a signal sequence, we carried out the experiments described below. The rationale is as follows. Luminal ER proteins in animal cells are prevented from being secreted by a sorting system that recognizes the carboxyl-terminal sequence KDEL (Munro and Pelham, 1987). Instead of KDEL at the carboxyl terminus, reticulocalbin has the closely related sequence HDEL. Although the HDEL sequence has been reported to be inefficient as an ER retention signal in animal cells because a lysozyme fusion protein with the HDEL sequence at the carboxyl terminus was efficiently secreted into the medium (Pelham et al., 1988), were double-labeled with anti-reticulocalbin antibody and biotinylated ConA followed by fluorescein-labeled goat anti-rabbit antibody and rhodamine-labeled avidin, and distribution of reticulocalbin (C) and ConA (D) was visualized. Arrows in D shows cells negative for reticulocalbin staining ( C ) . A-D, cells were photographed using an immunofluorescence microscope at the same magnification. Bur, 20 pm. E , an image generated by an MRC500 confocal scanning laser microscope on a video monitor. Bur, 25 pm. The microscope was focused on the lower part of the cell. recent experiments on liver carboxylesterases have demonstrated that the sequence is functional at least in this case (Robbi and Beaufay, 1991). Therefore, the removal of the carboxyl-terminal HDEL sequence could allow the mutant protein to escape from the retrieval machinery and to be secreted into the medium if the amino-terminal sequence is the signal sequence. For this, a mutant protein specifically lacking the HDEL sequence was constructed. The synthesis and secretion of the wild-type and mutant reticulocalbins were evaluated by transient expression in COS cells (Fig. 9).
After labeling with ["S]methionine for 30 min and a chase (3 h) with excess unlabeled methionine, proteins were immunoprecipitated from the cell lysates and from media and then analyzed by SDS-PAGE. Under these conditions, wild-type reticulocalbin was secreted very slowly from the COS cells.
Less than 10% of the pulse-labeled protein was found in the medium. Reticulocalbin lacking the HDEL sequence, however, was secreted rapidly from the COS cells (Fig. 9). Densitometry of the fluorographs showed that -80% of the mutant protein was recovered from the medium. No reticulocalbin was secreted from PYS-2 cells under the same conditions (data not shown). These results show that the amino-terminal hydrophobic amino acids of reticulocalbin function as a signal for the transfer of the entire protein into the lumen of the ER.

DISCUSSION
We identified and characterized a novel ER resident Ca2+binding protein called reticulocalbin by cDNA cloning, sequence analysis, and biochemical as well as cell biological studies. The major structural features of reticulocalbin, as deduced from the cDNA sequence, are outlined in Fig. 10. The protein consists of 325 amino acids with a single hydrophobic sequence at the amino terminus that constitutes a leader sequence. At the amino-terminal region of the mature protein, there is a potential N-glycosylation site, which was indeed partially glycosylated. The carboxyl terminus contains a version of the KDEL ER retention signal, HDEL. The rest of the protein consists of the six domains of the EF-hand motif of high affinity Ca2+-binding proteins.
The most interesting feature of the sequence of reticulocalbin is the presence of the domains of the EF-hand motif. Therefore, reticulocalbin can be classified into the EF-hand calcium-binding protein superfamily, which includes calmodulin, troponin C, and myosin light chain. All members of this diverse protein family share multiple conserved sequence domains based on a distinct helix-loop-helix structure, the EF-hand (Kretsinger, 1980). The loop constitutes the Ca2+binding site. The proteins of the superfamily identified to date contain from two to eight EF-hands or variants thereof. In some of them, the EF-hands have been duplicated or lost; and in others, the calcium binding properties have been altered or lost entirely (Heizman and Hunziker, 1991). Although the reticulocalbin sequence has no significant homology to any other proteins except for the EF-hand motifs, the overall structure of reticulocalbin is similar to that of calbindin D28 and calretinin (Rogers, 1989) in that both proteins have six EF-hand motif domains. Calbindin D28 and calretinin have been found at high concentrations in the central and peripheral nervous systems of many species, but their function is presently unknown. Calbindin D28 binds only four Ca2+ atoms/mol of protein, and the second and sixth domains may have lost their Ca2+ binding capability because some oxygen-containing amino acids in the loop are missing (Hunziker, 1986). Similarly, the second and sixth domains of reticulocalbin seem to have lost their Ca2+ binding ability. Reticulocalbin, however, has a long amino-terminal extension as well as a short carboxyl-terminal extension. The latter has an HDEL sequence that could serve as part of the retention signal for the protein in the ER. The amino-terminal extension is composed of a leader sequence, which directs the protein to be translocated into the lumen of the ER, and the amino-terminal region of the mature protein of -50 amino acids, where a single N-glycosylation site resides.
The evidence presented here demonstrates that reticulocalbin is a luminal Ca2+-binding protein residing in the ER. The sequence of the cDNA clone showed that it has a secretory leader peptide that is not present on the mature protein. This leader sequence is functional in that it causes reticulocalbin to enter the ER, as shown by its glycosylation, and to be secreted into the medium since a mutant reticulocalbin lacking the carboxyl-terminal HDEL sequence was secreted in COS cells. Mature reticulocalbin does not contain a hydrophobic transmembrane sequence, and there is no evidence for its secretion from the cells; it thus appears likely that it accumulates as a soluble protein within the intracellular membrane system. Consistent with this is the finding that reticulocalbin cosediments with crude membranes (microsomes) and can be released from the membranes with detergents or by sonication. Upon phase separation in Triton X-114, reticulocalbin partitioned into the aqueous phase. Furthermore, anti-reticulocalbin antibody stained endogenous reticulocalbin in PYS-2 cells as well as transiently expressed reticulocalbin in COS cells with an ER-like pattern.
The carboxyl-terminal HDEL tetrapeptide, a variant of the KDEL ER retention signal, seems to be part of a signal that prevents it from being secreted from the cell since the mutant reticulocalbin lacking HDEL is secreted. After identifying the carboxyl-terminal KDEL sequence as an ER retention signal, a number of variants of this sequence have been reported (Pelham, 1990), including sequences KEEL for the protein ERp72 (Mazzarella e t al., 1990) and RDEL in a 55-kDa thyroid hormone-binding protein (Fliegel et al., 1990). The retrieval system in the yeast Saccharomyces cerevkiae recognizes the carboxyl-terminal HDEL sequence of ER resident proteins (Pelham et al., 1988). The HDEL sequence, however, is reportedly inefficient as an ER retention signal in animal cells because addition of the sequence to a lysozyme fusion protein did not cause its retention in the ER when the protein was expressed in COS cells (Pelham e t al., 1988). Despite this observation, HDEL can be used as the ER retention signal in some ER resident proteins such as reticulocalbin because the efficiency of retention can vary, depending on the proteins to be analyzed, as has been reported (Zagouras and Rose, 1989). Recently, HDEL in carboxylesterase has been shown to be functional in animal cells (Robbi and Beaufay, 1991). The other possibility, that the removal of HDEL caused altered protein folding, which in turn affected the interaction of reticulocalbin with other ER resident proteins, however, is not formally excluded.
Although our studies revealed valuable information about the structure and localization of reticulocalbin, the function of this protein remains unknown. Its localization in the lumen of the ER and its expression in different types of cells suggest a role in protein synthesis, modification, and intracellular transport. We speculate that reticulocalbin functions in the regulation of Ca2+-dependent activities in the lumen of the ER or post-ER compartment. One intriguing possibility is that reticulocalbin is involved in the retention mechanism of KDEL-terminated proteins in the ER, in which Ca2+ may be involved (Booth and Koch, 1989;Kelly, 1990). Reticulocalbin may associate with an as yet unidentified protein and regulate its activity by binding Ca2+. That reticulocalbin may perform multiple functions like calmodulin should be considered.
Finally, we believe that the name reticulocalbin is appropriate for the protein because it reflects its intracellular localization in the lumen of the ER (reticuloplasm), established Ca2+ binding properties, and the six calbindin D28-like domains of the EF-hand motifs.