ENDOPLASMIC RETICULUM CONTAINS A COMMON, … · The endoplasmic reticulum (ER) consists of a complex system of intracellular membranes, which can account for as much as 90% of the

J. Cell Sci. 86, 217-232 (1986) 217Printed in Great Britain © The Company of Biologists Limited 1986

ENDOPLASMIC RETICULUM CONTAINS A COMMON,ABUNDANT CALCIUM-BINDING GLYCOPROTEIN,ENDOPLASMIN

G. KOCH1, M. SMITH1, D. MACER1, P. WEBSTER2

AND R. MORTARA1

^Medical Research Council Laboratory of Molecular Biology, Hills Road, CambridgeCB2 2QH, England2International Laboratory for Research on Animal Diseases, Nairobi, Kenya

SUMMARYThe most abundant protein in microsomal membrane preparations from mammalian cells has

been identified as a 100X 103Mr concanavalin A-binding glycoprotein. The glycosyl moiety of theglycoprotein is completely sensitive to endoglycosidase H, suggesting a predominantly endo-plasmic reticulum localization in the cell. Using a monospecific antibody it was shown by bindingand immunofluorescence studies that the glycoprotein is intracellular. Immunoelectron mi-croscopy showed that the glycoprotein was at least 100 times more concentrated in the endoplasmicreticulum than in any other cellular organelle. It was found to be substantially overexpressed incells and tissues rich in endoplasmic reticulum. Since it is the major common protein componentassociated with the endoplasmic reticulum we refer to it as endoplasmin. Calcium-binding studiesshow that endoplasmin is a major calcium-binding protein in cells, suggesting that at least one of itsroles might be in the calcium-storage function of the endoplasmic reticulum. The amino-terminalsequence of endoplasmin is identical to that of a 100x 103A/r stress-related protein.

INTRODUCTION

The endoplasmic reticulum (ER) consists of a complex system of intracellularmembranes, which can account for as much as 90% of the total membrane in somecells (Alberts et al. 1983; Palade, 1975). It contains at least two distinct regions, thesmooth and rough ER. It is generally accepted that the ER is one of the majorsynthetic organelles involved in the synthesis of secretory products, plasma mem-brane, lysosomes and secretory granules. In addition, the ER is the site of synthesisand assembly of the lipid bilayer that forms the basic unit of membrane structure.

Recently, another major function of the ER has gained prominence, i.e. its role asa calcium store that serves as a source of calcium for intracellular signalling during avariety of physiologically important processes (Berridge & Irvine, 1984; Streb et al.1983). Thus in the PI cycle, hormone-receptor interactions result in the release ofinositol triphosphate, which passes to the ER and causes release of calcium, whichacts on a variety of cellular processes. In a general sense, therefore, the ER appears tosimulate the sarcoplasmic reticulum, which mediates calcium signalling duringmuscle contraction (MacLennan & Holland, 1975).

Key words: GPjoo, glucose-regulated glycoprotein, secretory differentiation, endoplasmicreticulum, endoplasmin.

218 G. Koch and others

In spite of extensive investigations, the molecular basis of many ER functionsremains obscure. One reason for this is that, unlike other membrane organelles suchas mitochondria, ER cannot be isolated without considerable disruption and dis-organization. Thus the definition of ER structure and function in molecular termsremains a major challenge in cell biology.

In this study we approached the analysis of ER structure and function by using acultured plasmacytoma cell as the starting material for endoplasmic reticulum.Plasma cells are almost completely dedicated to the secretion of immunoglobulin andtherefore contain large amounts of rough ER to effect this function (Alberts et al.1983).

Using ER-rich membranes we have demonstrated, for the first time, that ER con-tains at least one common glycoprotein, which is actually one of the most abundantcellular proteins. By analogy with other organelle-associated common abundantproteins, such as nucleoplasmin, we refer to this ER-associated glycoprotein asendoplasmin.

MATERIALS AND METHODS

Cells and cell cultureAll cells were grown in RPM1 1640 medium supplemented with 10 % foetal calf serum, 4 ITIM-L-

glutamine and 100 units ml"1 of penicillin and streptomycin. Cells were washed with phosphate-buffered saline (PBS) before use.

Preparation of tissue extractsOrgans from Balb/c mice were dissected out by standard procedures. Dissection of pancreas was

kindly carried out by Dr N. Kamada. The tissues were weighed, coarsely minced with scissors andsuspended in PBS with 1 % Nonidet P40 (NP40) and the protease inhibitors (PI) describedpreviously (Koch et al. 1985). The suspension was homogenized in a Polytron homogenizer andthe debris removed by centrifugation at 10000#for 30min. The protein content of each extract wasmeasured (see below) and all extracts adjusted to lmgrnl"1 protein for gel electrophoresis andimmunoblotting.

Purification of endoplasmin for immunization and sequencingThe preparation of concanavalin A (ConA)-Sepharose, and its use in the purification of

glycoproteins was as described (Koch & Smith, 1982). When these procedures are applied to theMOPC-315 line endoplasmin is the major protein in the purified preparation (Fig. 1). Thereforeeluates from the ConA-Sepharose were used directly for immunization (see below). For se-quencing studies endoplasmin was purified further by preparative polyacrylamide gel electro-phoresis. The ConA-Sepharose eluate was electrophoresed on a 10% polyacrylamide gel, theprotein located by lightly staining with PAGE Blue 83, the band cut out and the protein eluted byhomogenization in 10 mM-Tris • HC1, pH 7-5. The eluate was dialysed extensively against deionizedwater and lyophilized. Sodium dodecyl sulphate (SDS)-polyacrylamide gel analysis showed thatthe preparation was >99% pure endoplasmin.

Preparation of monospecific antibody to endoplasminRabbits were immunized with the enriched endoplasmin preparation from MOPC-315 cells

prepared as described above. For the first injection 1 mg of protein in PBS was homogenized withFreund's complete adjuvant, 1:1 (v/v), in a total volume of 3 ml and injected subcutaneously intoDutch white rabbits. After 1 month the rabbits were re-injected subcutaneously with 1 mg of

Major endoplasmic reticulum glycoprotein 219

protein in 3 ml PBS without adjuvant. After a further 2 weeks all animals treated showed antibodiesto endoplasmin as measured by immunoblotting analysis.

The antibodies were rendered monospecific for endoplasmin by affinity purification. Apreparative gel was prepared as described above and electroblotted onto nitrocellulose paper (seebelow). The endoplasmin was localized by staining with Ponceau Red and the relevant strip cut outand blocked with neat foetal calf serum for 5 h. The strip was rinsed with undiluted antiserum on arolling mixer for at least 2 days at 4°C. The strip was then washed and eluted with 0-2 M-glycine-HCl buffer, pH2-8, followed by neutralization to pH7-0 with 1 M-Tris.

ImmunoblottingThe standard procedure described by Towbin et al. (1979) was used throughout these studies.

l25I-labelled protein A (purchased from Amersham, UK) was used to develop the antibody boundto the nitrocellulose. Unless otherwise stated, the antibody to endoplasmin was the affinity-purifiedreagent described above. For quantitative comparisons of endoplasmin expression in cells andtissues, a standard curve was prepared using various amounts of MOPC-315 endoplasmin run on astandard gel and immunoblotted by the standard procedure. The resultant curve was linear up to5 fig endoplasmin per sample. Therefore all the analyses were carried out at endoplasmin levelsbelow this.

ImmunofluorescenceSamples were treated with affinity-purified antibody to endoplasmin (see above) for 15min at

room temperature, washed with PBS/10% foetal calf serum (washing buffer) and developed withfluorescein conjugated (fl-) goat anti-rabbit immunoglobulin (Garig, Miles-Yeda) for IS min. Afterwashing in washing buffer the samples were mounted in 50% glycerol in PBS and examined forepifluorescence on a Zeiss microscope. Live cells (usually MOPC-315) were stained and examinedas above. 3T3 fibroblasts in culture medium were allowed to settle onto polylysine-coated glasscoverslips and incubated for varying times at 37°C to enable spreading to proceed. Cells wererapidly fixed by immersing the coverslips in 3'5 % formaldehyde in PBS (pH adjusted to 7-0 withNaOH) for 30 min at 37°C. The coverslips were washed and the cells permeabilized by immersionin 0-1 % saponin in PBS for 15 min. Excess aldehyde was neutralized with neat foetal calf serumand staining carried out as described above. When cells were analysed for both endoplasmin andtubulin, they were first stained for endoplasmin, treated with neat rabbit serum for 30 min toneutralize any excess fl-Garig and treated with monoclonal rat antibody to tubulin (YOLl/2;Kilmartin et al. 1982) followed by rhodamine conjugated (rh) rabbit anti-rat immunoglobulin(Rartig, Miles-Yeda). Photographs were taken on Kodak Tri-X film.

Immunoelectron microscopyThe methods used were essentially those described by Webster et al. (1985), based on the work

of Griffiths et al. (1984a,b) and Tokuyasu (1973, 1980); the only modifications being that the cellswere fixed in 1 % glutaraldehyde in PBS for 15 min, and that after gelatin embedding, prior tofreezing, the cells were infused with 2M-sucrose in lOOmM-phosphate buffer containing 8%formaldehyde (from paraformaldehyde).

Gold particles (5 nm) made according to Slot & Geuze (1985). Staphylococcal protein A(Pharmacia Fine Chemicals AB., Uppsala, Sweden) was absorbed onto the gold particles and thereagents purified by centrifugation (Geuze et al. 1981).

Gel electrophoresisOne-dimensional SDS-polyacrylamide gel electrophoresis was carried out according to the

method of Laemmli (1970) using 10% polyacrylamide gels or in the microgradient gel system ofMatsudaira & Burgess (1978). Gels were stained with PAGE Blue 83.

Preparation of microsomal membranesThe plasmacytoma line MOPC-315 was used. Cells were suspended in 20 vol. of phosphate-

buffered saline (PBS) and saponin added to 0'01 %. After all cells had been permeabilized, as


evident from light microscopy (15min), the soluble components were separated from the 'ghosts'by centrifugation at 1000 jf for lOmin. The ghosts were then disrupted by syringing through an 18gauge hypodermic needle into a cushion of 45% sucrose/PBS at lOOOOOX^ for 30min. Alloperations were carried out in the presence of a protease inhibitor mix (Koch et al. 1985).

Calcium binding assaysCalcium-binding proteins were detected after SDS—polyacrylamide gel electrophoresis and

electroblotting into nitrocellulose by overlay with 45Ca (Koch et al. 1986). For quantitative testsendoplasmin was purifed from the MOPC-315 cell line by affinity with ConA-agarose as describedabove. The preparation was >90% pure and endoplasmin was the only calcium-binding proteindetectable (see Fig. 1C). Samples (10/ig) were spotted onto nitrocellulose paper (Schleicher andSchiill, BA 85/20) in triplicate and incubated with varying concentrations of 45CaCl2 (4xl06ctsmin~ 'ml" 1 , adjusted to the appropriate concentration with cold CaC^) in 25 /ZM-Hepes, pH 7-2,100/iM-KCl and 10^M-MgCl2. After 15 min incubation at room temperature, the spots were rinsedin deionized water for 5 s, dried out on a vacuum filter and subjected to scintillation counting. Theabsorption of protein onto filter was monitored by staining the spots with Ponceau Red, followed bydensitometry in the reflectance mode. Background controls were carried out in exact parallel, usingidentical sample spots (10 fig each) of cytochrome c which shows no calcium binding.

RESULTS

Composition of membranes from a murine plasmacytoma line

Fig. 1 shows the protein composition of the soluble and membrane fractions fromthe MOPC 315 cell line. Most of the major proteins are absent from the membranefraction with the exception of an abundant 100xl03Mr protein and a less-abundant75 X10 MT species, which are selectively retained by the membranes. Similar resultsare obtained with other cell lines (data unpublished). These studies revealed theexistence of a previously undescribed abundant protein in microsomal membranes.

The major membrane-associated WOxlO3 Mrprotein is a glycoprotein

Fig. 2 shows that the 100 K (100xl03Mr) protein, selectively retained in micro-somal membrane preparations, binds to ConA and is eluted with or-methylman-noside. The binding to ConA is eliminated by glycosidase treatment (see below) andthe protein can be metabolically labelled with radioactive sugars such as glucosamineand mannose (data not shown). Thus it was concluded that the 100 K protein is aglycoprotein (GPIQO), which is the same as the major mammalian cell glycoproteindescribed previously (Koche/ al. 1985).

Binding of ConA to the 100 Kglycoprotein is completely eliminated by digestion withendoglycosidase H

When the partially purified protein is treated with endoglycosidase H (endo H),there is a slight increase in mobility on SDS—polyacrylamide gels, similar to thatobserved with ovalbumin but not with a non-glycosylated protein such as serumalbumin (Fig. 3). Analysis of ConA binding shows a corresponding loss of binding tothe lectin. In the fully digested protein no residual ConA binding is detectable. Thusthe glycosyl moiety of the glycoprotein is completely sensitive to endoglycosidase Hdigestion. Since glycoproteins that have proceeded into the Golgi complex acquire

Major endoplasmic reticulunt glycoprotein H I

resistance to endo H, the sensitivity of the 100 K glycoprotein is reminiscent ofglycoproteins such as HMG Co A reductase (Liscum et al. 1983), cytochrome P4S0and glucosidase 1 (Brands et al. 1985), which are confined to the endoplasmic

Fig. 1. A major 100 K protein (endoplasmin) in microsomal membrane preparationsfrom a murine plasmacytoma cell. Microsomal membranes were prepared from theMOPC-315 cell line as described in Materials and Methods. The soluble (lane a) andmembrane fractions (lane b) were run on SDS-polyacrylamide gels and stained withCoomassie Blue; 1X 106 cell equivalents of each fraction were analysed. The two proteinsenriched in the membrane fraction (large arrows) and the molecular weight markers (95,65, 45, 30, 20 and 15 K; small arrows) are shown.

G. Koch and others

M 10 5 2 1 M

Fig. 2. Isolation of endoplasmin with ConA. A sample of MOPC-315 microsomalmembranes was solubilized in 1 % NP40 in PBS, mixed with ConA-Sepharose andeluted with cr-methyl mannoside (see Materials and Methods). The eluate (10, 5, 2 and1 ng protein, respectively) was analysed by SDS-polyacrylamide gel electrophoresis andstained with Coomassie Blue. The major 100 K protein species (see Fig. 1) is quan-titatively extracted by the ConA. M, protein marker standards (see Fig. 1).

reticulum, and suggests, but does not prove, that the 100 K glycoprotein may bepredominantly an ER glycoprotein.

Monospecific antibody to the 100Kglycoprotein

To examine the cellular localization of the glycoprotein, a monospecific affinity-purified antibody was prepared. In whole cell lysates the antibody recognizes the100 K glycoprotein only (Fig. 4). Binding studies with the antibody, to intactMOPC-315 cells, failed to reveal any antigen at the cell surface, although largeamounts were detected in permeabilized cells (unpublished observation) suggestingthat the glycoprotein is localized intracellularly. This was confirmed by immuno-fluorescence, using attached fibroblasts after permeabilization (Fig. 5). The antigenwas associated with large structures dispersed throughout the cytoplasm. Stainingwas not observed at the cell surface or within the nucleus, although the nuclearmembrane was often clearly stained. At early stages of cell spreading the glycoproteinwas associated with perinuclear structures that completely surrounded the nucleus,

EndoH(mg ml"1)

Major endoplasmic reticulum glycoprotein

0 0 25 0-5 1 0 0 0-25 0-5 1 0 0 0-25 0-5 1 0 0 0-25 0-5 1 0

100K

BSA

Ovalb.

Protein [125l]ConA

Fig. 3. Endoglycosidase H sensitivity of endoplasmin. Samples (5 jig) of ConA-purifiedglycoprotein were mixed with 5/ig BSA in 30/il 0-lM-sodium citrate, pH 5-5, andincubated at 37 °C with 0, 0-25, 0 5 and 1-0 fi units endoglycosidase H (Brands et al.1985). A parallel analysis was carried out with 5 fig chicken ovalbumin (Ovalb.; Sigma) aspositive control. The samples (ovalbumin set on the right half of the same gel) wereanalysed by SDS-polyacrylamide gel electrophoresis, stained for protein with CoomassieBlue (left panel) and the same gel stained for ConA binding (right panel) with [125I]ConA(Koch & Smith, 1982).

but at later stages of spreading these structures dispersed throughout the entirecytoplasm. These studies clearly confirmed the intracellular location of the glyco-protein.

Immunoelectron microscopic localization of the glycoprotein to the endoplasmicreticulum

The identity of the intracellular structures in which the glycoprotein was localizedwas determined by immunoelectron microscopy on thin frozen sections with a colloidgold marker. This is probably the most reliable approach since it provides equalaccess to all intracellular compartments (Tokuyasu, 1973, 1980). The results show(Fig. 6) that most of the gold particles are located within the elongated membranestructures characteristic of endoplasmic reticulum. In the example shown, which isaccompanied by a diagram to facilitate identification of gold particles and mem-branes, 118 particles were clearly within the ER and 12 clearly outside, with about 28being of doubtful location. When the density of gold label associated with variousorganelles was examined (Table 1), the ER was found to have about 100 times that inany other compartment. It was notable that labelling of the Golgi region was notabove the background level, consistent with the results of the endo H sensitivity and


cell fractionation experiments. Because of the localization of the glycoprotein to theER, it is now referred to as endoplasmin.

Hyperexpression of the major glycoprotein in secretory cells and tissues

Secretory cells such as plasma and pancreatic cells contain large amounts ofrough endoplasmic reticulum (Alberts etal. 1983). A survey of the expressionof the endoplasmin showed that it was most abundant in such cells (Fig. 7).Hyperexpression in plasma cells permits the detection of such cells by directimmunofluorescence on spleen cell smears (Fig. 8). Comparison of the cultured celllines derived from B lymphocytes (WEH 1-231) with plasma cells also shows thesignificant increase in the glycoprotein on development of the secretory phenotype(Fig. 7).

The 100 Kglycoprotein is the major calcium-binding protein of the ER

When total cell lysates from the MOPC-315 cell line were probed for calcium-binding proteins after SDS—polyacrylamide gel electrophoresis and electroblotting,

I 2 | o

Fig. 4. Monospecific antibody to endoplasmin. Immunoblotting analysis of whole celllysates (1) and purified glycoprotein (2) with antibodies to the 100 K glycoprotein (seeMaterials and Methods). Left panel: whole anti-serum; right panel: affinity-purifiedantibodies. The molecular weight markers (arrowed) are 95, 65, 45, 30 and 25 K,respectively.


Fig. 5. Immunofluorescence analysis of 3T3 fibroblasts with monospecific antibody toendoplasmin. Cells were prepared as described in Materials and Methods. A,B- Cellsafter 2h spreading. C-F. Cells after 16h spreading. A-E were stained with antibody tothe glycoprotein and F is the same field as E, double-labelled for tubulin with monoclonalanti-tubulin antibody.

Table 1. Distribution of endoplasmin in the different cellular compartments

CompartmentTotal

area examined (cm2)

65140260

6613S44

Particle density cm

0-020-0030-110-02

12-20-06

- 2

Cell surfaceNucleusCytosolMitochondriaEndoplasmic reticulumGolgi apparatus

Immunoelectron micrographs were prepared as described for Fig. 6, and the density of goldparticles in each compartment was measured. The cell surface included the plasma membrane andan area 1 ^m outside it. The cytosol is the space not clearly included within any specificcompartment.

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one of the major calcium-binding proteins detected was a 100 K protein (Fig. 9A).This protein was retained in a crude membrane fraction (Fig. 9B) and was shownto be endoplasmin by purification with ConA (Fig. 9C). Using the purified un-denatured glycoprotein in a calcium-binding assay (Fig. 10) revealed that thebinding kinetics were complex, with evidence of two classes of binding site, one

Tissues

Muscle

Heart

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MOPC

X63

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J558

A37*

P815

WEHI 3B

EL4

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Fig. 7. Analysis of endoplasmin expression in secretory tissues and cells. Samples (1 mg)of protein from tissue or cell extracts were analysed by SDS-polyacrylamide gel electro-phoresis and immunoblotting with antibody to endoplasmin. The immuno-positive bandfrom each sample is shown. •, plasmacytoma cell lines.

I

MM G. Koch and others

involving about four calcium sites with half-maximal binding at 0-4 mM and a secondinvolving about 8-10 calcium sites with half-maximal binding at 6mM. Thusendoplasmin has the properties of a relatively low-affinity high-capacity calcium-binding protein localized in the endoplasmic reticulum.

Endoplasmin is a stress-related protein

Numerous studies have shown that cells subjected to stresses, such as treatmentwith calcium ionophores, tunicamycin or glucose depletion, overexpress a 100 Kprotein (Subjeck & Shyy, 1986; Lee, 1981). To determine whether endoplasmin wasthe same as this protein, it was subjected to amino-terminal sequence analysis.The sequence obtained was: Asp-Asp-Glu-Val-Asp-Val-Asp-Gly-Thr-Val-Glu-Glu-Asp-, which is identical to that obtained for the corresponding stress-related protein(heeetal. 1984).

DISCUSSION

The main outcome of these studies has been the first clear demonstration thatone of the major cellular proteins is a glycoprotein localized in the endoplasmicreticulum. Because this glycoprotein is the most abundant protein subunit in the ERof a wide variety of cells in all mammalian (and vertebrate) species examined to date,we refer to it as endoplasmin.

Fig. 8. Identification of endoplasmin-rich plasma cells by immunofluorescence on spleencell smears. Spleen cell smears were prepared as described in Materials and Methods anddeveloped for immunofluorescence with anti-endoplasmin antibody. Note the sub-population of large plasma cells that stain strongly compared with the weakly staininglymphocytes.


b e d a b e d

Fig. 9. Endoplasmin is a major calcium-binding protein. Calcium-binding was examinedafter SDS-polyacrylamide gel electrophoresis and electro-transfer to nitrocellulose paperas described in Materials and Methods. Lanes a, MOPC-315 cell lysate; b, MOPC-315microsomal membrane fraction; c, purified endoplasmin; d, protein standards (95, 65,45, 30, 20 and 15 K). Left panel: protein stain (Ponceau Red); right panel: 4SCaautoradiograph. The calcium-binding protein in the standards (lane d) is ff-lactalbumin.

Numerous studies on microsomal membranes that are thought to be pre-dominantly ER-derived have failed to demonstrate the presence of endoplasmin.However, we have found that endoplasmin is quite easily lost from such membranepreparations upon mechanical disruption (unpublished observations) unless specialcare is taken to prevent excessive vesiculation. Furthermore, endoplasmin is verysensitive to proteolysis and is rapidly degraded in the absence of a mixture of proteaseinhibitors. Finally, the use of a cultured cell line, a plasmacytoma, rich in ER,permits rapid and efficient isolation of ER membranes, overcoming some of theabove-mentioned difficulties. The practical consequence of this ready loss of endo-plasmin from ER membrane preparations is that many of those prepared by con-ventional procedures may not be of sufficient integrity for the analysis of ERfunction. We suggest that retention of endoplasmin should be used as a test for thestructural integrity of ER membranes. It is interesting from this standpoint that dogpancreas microsomal membranes, which have proved particularly amenable to the


0-01 005 0-1 0-5 1Calcium concn (HIM)

10

Fig. 10. Calcium-binding kinetics of endoplasmin. ConA affinity-purified endoplasminwas analysed for calcium-binding as described in Materials and Methods.

analysis of protein translocation across ER membranes, do contain substantialamounts of endoplasmin (unpublished observation).

As mentioned in the Introduction, several cellular functions have been found toinvolve the endoplasmic reticulum and endoplasmin could participate in one or moreof these. In this study we have obtained evidence that one such function might be aspart of the calcium store of the ER. Current estimates indicate that the overallcalcium pool of cells such as those used in this study is around 1 mM (Alberts et al.1983). Endoplasmin exists at total concentrations of around 1X 10~5 M but there areat least 10 calcium-binding sites in the millimolar range on the protein. Therefore,the protein could account for a significant proportion of the stored calcium within theER. The corollary of this is that endoplasmin actually exists to increase the calciumstorage capacity of the ER and thereby provide the calcium required for intracellularsignalling. In fact, if the results shown in Fig. 9 reflect the situation in vivo,endoplasmin would indeed comprise the major calcium storage protein in ER andthereby perform the function ascribed to calsequestrin in sarcoplasmic reticulum(MacLennan & Holland, 1975).

The demonstration that endoplasmin is the same as one of the major stress-relatedproteins (GRP) in cells is interesting for at least two reasons. First, it has beenclaimed that the GRP is localized in the Golgi complex and also at the plasmamembrane and even in the nucleus of heat-treated cells (Lin & Queally, 1982; Linet al. 1982; Welch et al. 1983). It is clear that in the plasmacytoma cells, the onlycompartment showing significant amounts of the glycoprotein is the ER. Further-more, in other cell lines such as fibroblasts, no evidence for endoplasmin in thenucleus or at the plasma membrane was obtained. Thus the variable results obtainedin the previous study could reflect the anomalous behaviour of the monoclonalantibody used.


The apparent increase in the levels of endoplasmin (GRP) as a result of treatmentof cells with calcium ionophores (Wu et al. 1981) is also interesting in view of theevidence that the protein might be part of the calcium store of the ER. Thusincreases in endoplasmin could assist in the modulation of cellular calcium levelsgenerally. One can speculate that the increases in endoplasmin upon glucose star-vation and other stresses are also consistent with such a role since increased calciumuptake and ER hypertrophy appear to be amongst the earliest responses of cells tostress (Trump et al. 1981).

Finally, it should be emphasized that endoplasmin is one of the most abundantproteins in cells generally. Rough calculations, based on the assumption that the ERis 10 % of the total cell volume, indicate that the concentration in the ER could be asmuch as lOmgml"1. Such high concentrations usually occur with proteins such asactin, which perform a structural role in cells. Thus the possibility that endoplasminalso performs some structural role in the ER should not be ignored.

In summary, these studies have shown that the endoplasmic reticulum contains atleast one abundant glycoprotein, which could play a central role in the functions ofthis organelle, and which should provide a novel approach to the study of ERstructure and function.

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(Received 24 July 1986 -Accepted 20 August 1986)

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