The endoplasmic reticulum and calcium storage.

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.

The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I] concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combination of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.

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.

Perturbation of cellular calcium induces secretion of luminal ER proteins.

The endoplasmic reticulum (ER) contains a family of luminal proteins (reticuloplasmins) that are normally excluded from the secretory pathway. However, reticuloplasmins are efficiently secreted when murine fibroblasts are treated with calcium ionophores. The secreted and cellular forms of endoplasmin are clearly distinguishable on the basis of gel mobility and endoglycosidase H sensitivity. Reticuloplasmin secretion leads to the depletion of the proteins from the ER and their accumulation in the Golgi apparatus. The stress response to calcium ionophore induces reaccumulation of reticuloplasmins in the ER and suppresses their secretion. Secretion is also associated with changes in the structure and distribution of the ER. These observations show that perturbation of cellular calcium levels leads to the breakdown of the mechanism for ER retention of reticuloplasmins and suggest a role for calcium ions in their sorting from secretory proteins.

An association between actin and nucleocapsid polypeptides in isolated murine retroviral particles.

Mammalian cells infected with retroviruses frequently display virus particles budding at the tip of cellular projections resembling microvilli and filopodia. In normal and infected cells these cellular projections contain actin microfilaments and in specialized retrovirus-tipped projections from the P815 cell, a direct association between actin filaments and the apical virus particle could be demonstrated (Mortara and Koch, 1986). Here we confirm and extend these observations using a murine macrophage cell line chronically infected with a C-type retrovirus. Immunochemical and biochemical methods were used to identify actin-associated and actin-binding components among the retroviral polypeptides. The results show that Pr65gag and its p15 N-terminal domain can bind to actin in vitro and may be major binding sites for actin filaments on the retroviral nucleocapsid.

Dissociation and re-assembly of the endoplasmic reticulum in live cells.

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cisternae and lamellae which can be observed by immunofluorescence with anti-endoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dissociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.

Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum.

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich 'shells' followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.

Endoplasmin is a reticuloplasmin.

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

Visualization of the intact endoplasmic reticulum by immunofluorescence with antibodies to the major ER glycoprotein, endoplasmin.

Antibodies to endoplasmin were used to examine the morphology of the endoplasmic reticulum (ER) by immunofluorescence on permeabilized plasmacytoma and fibroblastoid cells. In unfixed cells, permeabilization led to a pronounced vesiculation of the ER. Therefore cells were first fixed lightly prior to permeabilization with detergent. Fibroblastoid cells gave a characteristic reticular pattern surrounding the nucleus with clear staining of the nuclear membrane. Plasmacytoma cells, in the conventional fluorescence microscope, gave a cisternae-like pattern. Optical sectioning with a confocal scanning laser microscope gave a distinct pattern of concentric cisternae similar to those obtained with transmission electron microscopy on cell sections. The overall morphology of the ER in such cells could be revealed by serial optical sectioning. Evidence was obtained that the ER does not undergo extensive vesiculation during mitosis in plasmacytoma cells. Using anti-endoplasmin immunofluorescence monitoring, conditions were developed for the retention of ER morphology in unfixed, permeabilized cells. These studies illustrate the value of endoplasmin as a general marker for the analysis of ER morphology in different types of cells by immunofluorescence microscopy.

Isolation and identification of partial cDNA clones for endoplasmin, the major glycoprotein of mammalian endoplasmic reticulum.

The amino acid sequences of peptides isolated from murine endoplasmin showed significant homology (approximately 50%) with sequences in the heat-shock proteins 90 and 83 of yeast and Drosophila, respectively, indicating that they are related proteins. Mixed oligonucleotide probes, deduced from the peptide sequences, were used to isolate cDNAs from a murine liver cDNA library. DNA sequencing confirmed the presence of a coding sequence for one of the endoplasmin peptides, formally establishing the authenticity of the cDNA. The identity of the murine and hamster endoplasmin sequences suggests a level of sequence conservation associated with proteins that perform a structural role in cells.

Differential expression of murine macrophage surface glycoprotein antigens in intracellular membranes.

A monoclonal antibody recognizing a novel murine macrophage glycoprotein antigen (MAG) was prepared by the hybridoma technique. Using live and permeabilized macrophages to estimate surface and total MAG, respectively, it was found that at least 50% of the total antigen was intracellular. In contrast, another macrophage surface glycoprotein antigen, Mac-1, was undetectable in the intracellular pool. Immunofluorescence studies confirmed the existence of a substantial intracellular pool of MAG antigen. Similar results were obtained with a panel of cultured tumour cell lines. In one such cell line, it was shown that surface MAG existed in a distinct punctate pattern indicative of microdomains, whereas surface Mac-1 antigen gave a uniform distribution. The possible role of the surface microdomains in the differential expression of the two surface glycoproteins in intracellular membranes is discussed.

Analysis of DNA repair in XP-HeLa hybrids; lack of correlation between excision repair of u.v. damage and adenovirus reactivation in an XP(D)-like cell line.

Hybrids formed between HeLa cells and fibroblasts from xeroderma pigmentosum group D show either HeLa sensitivity or XPD-like hypersensitivity to u.v. radiation and corresponding high or low excision repair capability. Hybrids with low repair are presumed to have lost, via chromosome segregation, the HeLa wild type D alleles. In this paper we analyse the u.v. sensitivity and excision repair capability of another hybrid, HD1A, derived spontaneously from the normally sensitive hybrid HD1. While HD1A closely resembles the XPD phenotype in terms of u.v. sensitivity and excision repair it differs from XPD because of its ability to reactivate u.v.-irradiated adenovirus 2 to an extent similar to that of its HeLa parent. This capacity functionally dissociates excision repair of chromatin-based damage from damage in a viral environment. Moreover, on the basis of complementation studies the excision repair of genomic damage by HD1A is subtly different from that of a true XPD-like hybrid, HD2. The data are discussed in terms of a second change in the defective D allele of the HD1A cell.

Regulation of parasite-specific antibody responses in resistant (C57BL/6) and susceptible (C3H/HE) mice infected with Trypanosoma (trypanozoon) brucei brucei.

After infection with 10(3) T. brucei GUTat 3.1, C57BL/6 mice produced antibody responses and controlled the first parasitaemic wave whereas C3H/He mice did not. The inability of C3H/He mice to control parasitaemia resulted from an impaired ability of parasite-induced antibody-containing cells to secrete immunoglobulin. Antibody-containing cells in infected C3H/He mice regained the ability to secrete antibody within 24 h after trypanosome elimination by treatment with Berenil, suggesting that the block in antibody secretion was maintained by living parasites or short-lived components of degenerating parasites. Infected C3H/He mice also had an impaired ability to produce a rabbit erythrocyte-specific antibody response on challenge with rabbit erythrocytes and this response recovered when parasites were eliminated from the blood 24 h before analysis. It was not possible to inhibit secretion of antibody by rabbit erythrocyte-induced plasma cells either by incubating them with serum from infected C3H/He mice or by injecting large numbers of living trypanosomes into C3H/He mice already responding to rabbit erythrocytes. The process leading to failure of parasite and rabbit erythrocyte-induced antibody-containing cells to become high rate antibody-secreting cells was not identified but did not appear to correlate with any obvious change in the intra-cellular morphology of the antibody-containing cells.

Specificity of antibodies to the purified Con A acceptor glycoproteins of cultured tumour cells.

Con A acceptor glycoproteins from the human Molt 4 (T cell leukaemia) and HeLa (endocervical adenocarcinoma) cell lines were purified by affinity chromatography and used for the preparation of rat antisera. Cross-absorption analysis showed that each antiserum contained antibodies which recognised cell surface antigens preferentially expressed by the donor cell line. Molt 4-associated antigens were fully expressed on T cell tumour lines and normal thymocytes, but not on non T cell tumour lines, peripheral blood lymphocytes or other blood cells. Immunofluorescence studies showed that the antigens were preferentially expressed on a sub-population of immature thymocytes. HeLa-associated antigens were only fully expressed on one other epithelial tumour cell in a panel of 17 cell lines. Immunofluorescence studies showed that the HeLa-associated antigens were expressed on normal endocervical adenoepithelium but not on ectocervical, endometrial or intestinal epithelia. Thus purified Con A acceptor glycoproteins of cultured tumour cell lines are potent immunogens for the generation of antibodies recognising lineage-associated differentiation antigens. These antigens should be useful in tumour classification and in the study of normal differentiation.

Analysis of pseudopodial structure and assembly with viral projections.

The mechanisms by which cells extend motile pseudopodial projections are still poorly understood. Several fundamental mechanisms have been proposed on the basis of hydrostatic pressure, membrane addition and microfilament reorganization. A common focus of all such mechanisms is the growing tip of a pseudopodium. Yet some basic questions about the nature of the tip in natural pseudopodia remain obscure. However, one class of structure, the virus-tipped projections, often contains a well-defined particle, both morphologically and biochemically, and therefore provides a useful model system for the examination of the tips of cellular projections. In P815 cells the virus-tipped projections are long, thin structures closely resembling filopodia in other cells. The apical virus particle is a retrovirus particle produced by the chronic infection existing in this cell line. In demembranated filopodia, the virus particle retains a tight association with a single actin microfilament. Biochemical analyses indicate that the major retroviral structural polypeptide Pr65 is an actin-binding protein that could provide the anchorage site for the actin filament. The existence of a solid virus particle tethered by an actin filament to the cytoskeleton makes it very unlikely that these projections grow by membrane addition at the tip. The major positive implication is that the apex of a projection does not relinquish its interaction with the submembranous cytoskeleton during growth. Such an arrangement would be compatible with either a hydrostatic-pressure-driven or a cytoskeleton-driven mechanism of filopodial growth.

Xeroderma pigmentosum D-HeLa hybrids with low and high ultraviolet sensitivity associated with normal and diminished DNA repair ability, respectively.

Fusion between HeLa and fibroblasts from complementation group D xeroderma pigmentosum (XPD) followed by challenge with small doses of ultraviolet light (u.v.) results in the production of hybrid cells expressing either HeLa (HD1) or XPD-like (HD2) sensitivity to u.v. and related repair capacity. Assays used included unscheduled DNA synthesis (UDS), DNA break accumulation in the presence of inhibitors of DNA repair synthesis and host cell reactivation of irradiated adenovirus. Complementation assay in heterokaryons reveals limited ability of HD2 to restore UDS in XPD nuclei. We believe this complementation is more apparent than real since proliferating hybrids of HD2 and XPD parentage are without exception u.v.-sensitive and express limited excision repair. On the other hand hybrids between HD2 and XPC, XPE or XPF fibroblasts show true complementation resulting in a return to normal u.v. sensitivity and elevated repair ability.