Endoplasmin is a reticuloplasmin

G. L. E. KOCH, D. R. J. MACER

Medical Research Council Laboraloiy of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

and F. B. P. WOODING

Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England

Summary

The location of endoplasmin in the endoplasmicreticulum was investigated by biochemical andimmunoelectron microscopic analyses. The pro-tein could be obtained in a soluble form byprocedures that do not involve the use of anydetergents. The soluble protein has the amino-and carboxy-terminal sequences of the intactmolecule, showing that it has not been proteo-lysed. Application of the Triton X-114 phase-separation test does not reveal significanthydrophobicity in the molecule. Immunogold

labelling studies on cells with a dilated endoplas-mic reticulum (ER) lumen show that endoplas-min is uniformly distributed throughout thelumen, with no evidence of a preferential associ-ation with the membrane. These studies clearlydemonstrate that endoplasmin is a luminal pro-tein of the ER, i.e. a reticuloplasmin, and not anintegral membrane protein.

Keywords: endoplasmin, ERP99, reticuloplasmin.

Introduction

Several independent studies have shown that nucleatedcells contain a major glycoprotein variously referred toas GP100 (Koch et al. 1985), GRP94 (Lee et al. 1984),ERP99 (Lewis et al. 1985) or endoplasmin (Koch et al.1986). It has been formally demonstrated by immuno-cytochemical studies that this glycoprotein is detect-able only in the endoplasmic reticulum (Koch et al.1986). However, there is disagreement about its preciselocation within the endoplasmic reticulum (ER).Studies on the protease sensitivity of the protein inisolated microsomes (Lewis et al. 1985) led to theconclusion that the protein is a transmembrane proteinwith a large cytoplasmic domain, and a putative trans-membrane segment was identified in the sequence ofthe protein (Mazzarella & Green, 1987). In contrast,studies on the purified protein indicated that it is asoluble protein located in the lumen of the ER, i.e. areticuloplasmin (Koch, 1987).

The formal resolution of this question is importantfor several reasons. There is considerable interest in thefunction of ER proteins such as endoplasmin, and fromthis standpoint it is important to know whether suchproteins are integral membrane proteins or not. AJournal of Cell Science 90, 485-491 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

particular feature of endoplasmin is the fact that it isretained in the ER by a mechanism that probablyinvolves the C-terminal KDEL sequence (Munro &Pelham, 1986). Elucidating the nature of the mechan-ism involved clearly requires an unambiguous distinc-tion between endoplasmin being a luminal or trans-membrane protein. Finally, it has been proposed(Koch, 1987) that endoplasmin could serve as theprototype marker for reticuloplasm itself. However,such a use also depends on the formal elimination ofany uncertainty about the precise location of theprotein in the ER.

To this end, we have examined several aspects of thebiochemical properties of endoplasmin and re-exam-ined its location in the ER directly. Integral membraneproteins require detergents for solubilization from cellsas well as during and after purification. Therefore wehave examined whether endoplasmin can be releasedfrom cells and purified without detergent. Second, wehave applied the phase-separation test (Bordier, 1981)to whole-cell samples to try to determine whether asignificant fraction of endoplasmin is membrane-associated in the cell. Finally, we have used immuno-

485

M D

Fig. 2. Analysis of cellular endoplasmin forhydrophobicity. MOPC-315 cells were lysed at lowtemperature with Triton X-114 as described by Bordier(1981). The lysate was subjected to phase separation byincubation at 30°C for lOmin. Phases (S, aqueous (>95 %total endoplasmin); D, detergent) were subjected to threecycles each of the phase separation and the phases wereanalysed by SDS-polyacrylamide gel electrophoresisfollowed by immunoblotting with antibody to endoplasmin(Koch el a!. 1986).

Materials and methods

Fig. 1. Comparison of endoplasmin isolated in thepresence (M) and absence (S) of detergent. The M formwas purified as described by Koch el al. (1986), and the Sform as described in Materials and methods. The yieldswere >90% in both cases. Samples were run on a 10%polyacrylamidc-SDS gel (Laemmli, 1970), and stainedwith Coomassie Brilliant Blue. P, protein standards (95,67, 45, 30, 20, 15 (XlO3)Mr from the top).

gold labelling on cells with a dilated ER lumen toexamine directly whether the ER is luminal ormembrane-associated.

486 Koch et al.

Purification of endoplasminMurine endoplasmin was purified from MOPC-315 cellsgrown as described (Koch et al. 1985). Cells were lysedhypotonically by suspension in 10mM-Tris-HCl, pH7-5,with protease inhibitor mix (Koch et al. 1985) at 4°C. Thelysed cells were separated from the soluble contents bycentrifugation at 1000g and the pellet was re-suspended inPBS with protease inhibitors. The suspension was syringedthrough an 18 gauge needle until all the lysed cells (shells)had been disrupted. The sample was centnfuged at 100 000gfor 30 min to remove nuclei and membranes, and thesupernatant was used to purify the endoplasmin.

The supernatant was mixed with concanavalin A (ConA)-Sepharose and the resin eluted with 10 % cr-methyl manno-side as described (Koch et al. 1985, 1986). The eluate wassubjected to gel filtration on Sephadex G150 in 100 mM-Tr isHCl , pH7-5, with protease inhibitors, and the peakcontaining endoplasmin (monitored by SDS-polyacrylamidegel electrophoresis) pooled. The material containing endo-plasmin was >95 % pure.

N-terminal sequence analysisPurified endoplasmin was dialysed into water, freeze-driedand applied to an Applied Biosystems 470A ProteinSequencer.

C-terminal sequence analysisPurified endoplasmin was dialysed into water. Both carboxy-

Fig. 3. Immunoelectron microscopic localization of endoplasmin in cells with a dilated ER lumen. Samples were preparedas described in Materials and methods. Sections containing rat gonadotrophin-producing cells (GPC) were labelled withrabbit anti-endoplasmin followed by Protein A-colloidal gold conjugate (15 nm). Grids were stained with uranyl acetate.A—C. Section through a GPC showing the large dilated ER (e). D. Section through a GPC with less-dilated ER (<?). Therectangles on A and B indicate the areas that are shown at higher magnification in B and C, respectively. A, X5000; B,X16000; C, X60 000; D, X60000.

peptidase A-DFP (Sigma C-0514) and carboxypeptidase Y(Sigma C-3888) were used to hydrolyse 300/ig proteinsamples, using incubation times of 0-5, 5, IS and 60min at37°C (Hayashi, 1976). For hydrolysis with carboxypeptidaseA, protein was dissolved in 100 il 0-2M-iV-ethylmorpholine,pH 8-5, then 5 nmol of norleucine and 10;Ug of enzyme wereadded (per sample), as above. Triplicate samples were madeand rabbit muscle aldolase was used as a control.

Carbohydrate analysisThe Triton X-114 phase-separation test was carried out

according to the method of Bordier (1981). The reliability ofthe test was checked with bacteriorhodopsin as the standardintegral membrane protein and haemoglobin as the solublestandard. In all experiments these proteins partitioned intothe detergent and aqueous phases, respectively. For analysisof cellular endoplasmin, cells were lysed directly with TritonX-114 in the cold and then subjected to phase separation asusual. Phases were analysed for endoplasmin by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) andimmunoblotting with anti-endoplasmin (Towbin el al. 1979;Koche? al. 1986).

Endoplasmin localization 487

PS 0 001 0-1-+NP40 -

1-0 0 0-01 0-1- - N P 4 0 -

1-0 mg trypsin ml- 1

-Endoplasmin

\ \

5-8

Fig. 4. Trypsin digestion of microsonial membranes from MOPC-315 cells. Microsomal membranes were prepared asdescribed by Lewis el al. (1985) and treated with trypsin in the presence (lanes 1-4) and absence (lanes 5-8) of O'l %Nonidet P40 in phosphate-buffered saline for 15 min at 20°C. Samples were analysed on 12-5 % SDS—polyacrylamide gels.PS, protein standards (see Fig. 1). Lanes 1-4, 1-0, 0 1 , 0-01, Omgrnl"1 tryps i n in 0-1 % Nonidet P40/PBS; lanes 5-8, 1-0,0 1 , 0 0 1 , Omgmr 1 trypsin in PBS.

Electron microscopyAnimals were killed with an overdose of phenobarbitonesodium and tissues fixed by procedures described by Wood-ing (1980) and Wooding et al. (1980). Blocks for electronmicroscopy were embedded in Araldite after dehydration inethanol and propylene amide. Samples were not treated withosmium tetroxide. For immunocytochemistry (Wooding,1981) 70-100nm thick sections on 300mesh uncoated nickelgrids were initially jet washed with phosphate-buffered saline(PBS) and floated on a drop of antibody solution for 30 min atroom temperature. The grid was washed and floated on adrop of colloidal gold label (lOnm protein A-coated gold

particles, Janssen) for 30 min at room temperature, andwashed with PBS. Sections were stained with uranyl acetatefollowed by lead citrate. Controls were carried out with PBS.

Results

Endoplasmin can be solubilized and purified ivithoutdetergentA procedure was developed for isolating endoplasminfrom plasmacytoma cells without using any detergent.The procedure is based on the earlier observation

488 Koch et al.

(Koch et al. 1986) that when such cells are subjected tohypotonic swelling and lysis, the cytoplasmic contentsare lost but the ER contents are retained within theresultant shells. However, when the shells are dis-rupted mechanically, endoplasmin and the other puta-tive reticuloplasmins are released in a completelysoluble form. This contrasts with shells and micro-somes prepared under isotonic conditions where theendoplasmin is retained by the microsomal mem-branes, even after disruption of the shells. The reasonfor this difference is not known, but, since the studiesdescribed below show that it does not involve proteo-lytic modification, it probably reflects a difference inthe orientation of the microsomal membranes obtainedafter mechanical disruption of the shells produced bylysis under isotonic and hypotonic conditions, respect-ively.

The solubilized endoplasmin was purified accordingto the identical procedure described previously forisolating the protein from detergent lysates of cells(Koch et al. 1985). The purified protein appearsidentical to that purified from detergent lysates bySDS-polyacrylamide gel electrophoresis (Fig. 1). Gelfiltration in the absence of detergent gives an apparentmolecular weight of 200X103, showing that the puri-fied protein is dimeric when isolated by either pro-cedure. Amino acid analyses of the detergent-free anddetergent-plus forms of the protein are also identical.All other analyses used, such as two-dimensional gelelectrophoresis, ConA binding or peptide mappingreveal no differences between the protein isolated in thepresence or absence of detergent.

Thus, these studies show that detergents are notrequired for the solubilization and purification of theapparently native protein. However, it was possiblethat the protein obtained under detergent-free con-ditions had undergone a proteolytic modification thatremoved a hydrophobic segment, and thus rendered itsoluble in detergent-free buffers. Therefore the amino-and carboxy-terminal amino acids of the detergent-freeprotein were determined and compared with that of theintact polypeptide. The amino terminus of the deter-gent-free form was identified as Asp-Asp-Val-Asp-,which is the same as that obtained for the detergent-plus form (Koch et al. 1986) as well as the intactpolypeptide deduced from cDNA sequencing (Mazzar-ella & Green, 1987), showing that the amino terminushad not been proteolysed. Carboxypeptidase digestionunequivocally showed that the carboxy terminus wasleucine in both forms of the protein. Further digestionwas not significant, so only one residue was obtained.This is identical with the expected carboxy-terminalresidue for the intact polypeptide (Sorger & Pelham,1987; Mazzarella & Green, 1987). Examination of theprotein sequence (Mazzarella & Green, 1987) showsthat the next leucine residue is 55 residues in from the

C terminus. If the protein had been cleaved at this site,it would have yielded a polypeptide chain shortened by=6XlO3.fl/r. Such a change would have been easilydetected by SDS-polyacrylamide gel electrophoresis;since it was not, we conclude that the protein isolatedunder detergent-free conditions has not been proteo-lysed.

Cellular endoplasmin does not exhibit hydmphobiccharacterTo examine whether endoplasmin in the intact cellformed an association with hydrophobic moieties, andthereby established membrane associations, the TritonX-114 phase-separation test (Bordier, 1981) was ap-plied to whole cells. It was expected that such hydro-phobic associations could lead to the partitioning of theotherwise hydrophilic enodplasmin into the detergentphase in significant amounts. However, Fig. 2 showsthat more than 95 % of the endoplasmin remains in theaqueous phase. Thus this test confirms that endoplas-min does not possess the properties of an intrinsicmembrane protein, and also indicates that there is littlestable association with other intrinsic membrane com-ponents such as lipids or membrane proteins.

Immunogold labelling shows that endoplasmin is aluminal proteinThe most direct test for the localization of endoplasminis to examine its location in cellular ER. To do thisconvincingly, we have used direct immunogold label-ling on thin sections with monospecific antibodies toendoplasmin. In order to facilitate the distinctionbetween membrane-associated and luminal proteins,cells that contained a dilated ER lumen were examined.Fig. 3 shows a section of a rat gonadotrophin-produc-ing cell immunogold labelled with anti-endoplasmin.

Fig. 3A shows a cell with an exceptionally dilatedsegment of ER. At higher magnification (Fig. 3B andC) it is apparent that the gold particles are uniformlydistributed throughout the luminal region. Fig. 3Dshows a stained section through a cell with less dilatedER and this also shows no preferential association withthe ER membrane.

Microsomal endoplasmin is not selectively degradedby trypsinThe basis of the previous conclusion that endoplasmin(ERP99) is a trans-membrane protein was its apparentsensitivity to proteolytic digestion in microsomal prep-arations. Fig. 4 shows the effect of trypsin on micro-somal membranes from the MOPC-315 cell, the sameone as that used previously (Lewis et al. 1985). Incontrast to the previous studies, no digestion of endo-plasmin was obtained in the absence of a detergent thatpermeabilizes the membranes and renders the contentsaccessible to the trypsin. This observation is also

Endoplasmin localization 489

consistent with the conclusion that endoplasmin is nota trans-membrane protein.

Discussion

These studies clearly demonstrate that endoplasmin isa constituent of the luminal space of the ER, andtherefore belongs to the class of proteins termedreticuloplasmins (Koch, 1987). Since previous studieshave formally shown that endoplasmin does not exist inany other membrane compartment, such as the Golgiapparatus (Koch, 1986), endoplasmin can be reliablyemployed as a marker for reticuloplasm from mam-malian cells.

Although endoplasmin is a reticuloplasmin, there isthe possibility that a small fraction is associated withthe membrane via an integral membrane componentsuch as the putative KDEL receptor that has beenproposed as part of a mechanism for ensuring thatproteins such as endoplasmin are confined to theendoplasmic reticulum (Munro & Pelham, 1986).Interestingly, we have observed a small fraction ofendoplasmin that does partition into the detergentphase in the Triton X-114 test. However, it appearsthat the steady-state level of any complex betweenendoplasmin and integral membrane proteins is ex-tremely small, and most of the protein is strictlyluminal.

One of the main purposes of this study was toexamine the claim that endoplasmin is a trans-membrane protein (Lewis et al. 1985; Mazzarella &Green, 1987), and it is pertinent to consider theexperimental basis for this claim. It was originallybased on the observation that microsomal membranesexhibit differential sensitivity of endoplasmin to pro-teolytic digestion. Thus endoplasmin was found to bemuch more sensitive than some of the other proteins,and this was interpreted to mean that endoplasmincontained luminal and cytoplasmic domain, and musttherefore be a trans-membrane protein. However, thisis not the only possible explanation, since it is knownthat endoplasmin is itself hypersensitive to proteolysis,and the differential sensitivity of endoplasmin couldsimply reflect this property. The other line of evidenceis the interpretation of the sequence of endoplasmindeduced from the cDNA sequencing. However,neither of these criteria for trans-membrane proteins isdirect, and we conclude that the interpretation wasincorrect.

The need to clarify this question arose from itsrelevance to the whole concept of reticuloplasm itself.Until recently this was a poorly defined material, and itwas generally assumed that the major protein constitu-ents of the ER lumen were the soluble secretoryproteins. We have suggested that this is not a true

reflection of the nature of reticuloplasm, and proposedthat it consists of a protein-rich medium (Koch, 1987).The main basis for this proposal was that endoplasminwas itself an abundant (>10mgml~') luminal ER-specific protein. The confirmation that endoplasmin isindeed a luminal protein completes the formal estab-lishment of its classification as a reticuloplasmin. Itseems reasonably certain then that the other putativereticuloplasmins, e.g. BiP (Munro & Pelham, 1986)and PDI (Edmanef al. 1985), will also satisfy the samecriteria. Thus there seems to be no remaining obstacleto the hypothesis that the ER luminal material (reticu-loplasm) consists of a family of abundant proteins(reticuloplasmins) not significantly associated with themembrane (Koch, 1987).

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{Received 17 February 19SS - Accepted 12 April 19SS)

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