Endoplasmin is a reticuloplasmin

G. L. E. KOCH, D. R. J. MACER
Medical Research Council Laboraloiy of Molecular Biology, Hills Road, Cambridge CB2 2QH, Englandand F. B. P. WOODING
Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England

Summary
The location of endoplasmin in the endoplasmic
reticulum was investigated by biochemical andimmunoelectron microscopic analyses. The protein could be obtained in a soluble form byprocedures that do not involve the use of any
detergents. The soluble protein has the aminoand carboxy-terminal sequences of the intact
molecule, showing that it has not been proteolysed. Application of the Triton X-114 phaseseparation 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 thelumen, 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.

Introduction

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

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 immunocytochemical studies that this glycoprotein is detectable only in the endoplasmic reticulum (Koch et al.
1986). However, there is disagreement about its precise
location within the endoplasmic reticulum (ER).
Studies on the protease sensitivity of the protein in
isolated microsomes (Lewis et al. 1985) led to theconclusion that the protein is a transmembrane proteinwith a large cytoplasmic domain, and a putative transmembrane 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. a
reticuloplasmin (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. A
Journal of Cell Science 90, 485-491 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988

Keywords: endoplasmin, ERP99, reticuloplasmin.

To this end, we have examined several aspects of thebiochemical properties of endoplasmin and re-examined its location in the ER directly. Integral membraneproteins require detergents for solubilization from cells
as well as during and after purification. Therefore wehave examined whether endoplasmin can be released
from cells and purified without detergent. Second, we
have applied the phase-separation test (Bordier, 1981)
to whole-cell samples to try to determine whether asignificant fraction of endoplasmin is membraneassociated in the cell. Finally, we have used immuno485

M

D

Fig. 2. Analysis of cellular endoplasmin forhydrophobicity. MOPC-315 cells were lysed at low
temperature with Triton X-114 as described by Bordier(1981). The lysate was subjected to phase separation by
incubation 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 electrophoresis
followed by immunoblotting with antibody to endoplasmin(Koch el a!. 1986).

Materials and methods
Purification of endoplasmin

Fig. 1. Comparison of endoplasmin isolated in thepresence (M) and absence (S) of detergent. The M form
was purified as described by Koch el al. (1986), and the S
form as described in Materials and methods. The yields
were > 9 0 % 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 (XlO 3 )M r from the top).

gold labelling on cells with a dilated ER lumen to
examine directly whether the ER is luminal or
membrane-associated.
486

Koch et al.

Murine endoplasmin was purified from MOPC-315 cells
grown as described (Koch et al. 1985). Cells were lysed
hypotonically by suspension in 10mM-Tris-HCl, pH7-5,
with protease inhibitor mix (Koch et al. 1985) at 4°C. The
lysed cells were separated from the soluble contents bycentrifugation at 1000g and the pellet was re-suspended in
PBS with protease inhibitors. The suspension was syringed
through an 18 gauge needle until all the lysed cells (shells)
had been disrupted. The sample was centnfuged at 100 000g
for 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 mannoside as described (Koch et al. 1985, 1986). The eluate wassubjected to gel filtration on Sephadex G150 in 100 mMT r i s H C l , pH7-5, with protease inhibitors, and the peak
containing endoplasmin (monitored by SDS-polyacrylamide
gel electrophoresis) pooled. The material containing endoplasmin was >95 % pure.

N-terminal sequence analysis
Purified endoplasmin was dialysed into water, freeze-dried
and applied to an Applied Biosystems 470A Protein
Sequencer.

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

Fig. 3. Immunoelectron microscopic localization of endoplasmin in cells with a dilated ER lumen. Samples were prepared
as 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 (<?). The
rectangles 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 protein
samples, using incubation times of 0-5, 5, IS and 60min at
37°C (Hayashi, 1976). For hydrolysis with carboxypeptidase
A, 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 analysis
The 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 standard
integral membrane protein and haemoglobin as the soluble
standard. In all experiments these proteins partitioned into
the detergent and aqueous phases, respectively. For analysis
of cellular endoplasmin, cells were lysed directly with Triton
X-114 in the cold and then subjected to phase separation as
usual. Phases were analysed for endoplasmin by S D S 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

00-01 0-1--NP40-1-0 mg trypsin ml - 1-Endoplasmin

\ \
5-8

Fig. 4. Trypsin digestion of microsonial membranes from MOPC-315 cells. Microsomal membranes were prepared as
described 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 tryp s i n in 0-1 % Nonidet P40/PBS; lanes 5-8, 1-0,
0 1 , 0 0 1 , O m g m r 1 trypsin in PBS.
Electron microscopy
Animals were killed with an overdose of phenobarbitone
sodium and tissues fixed by procedures described by Wooding (1980) and Wooding et al. (1980). Blocks for electron
microscopy were embedded in Araldite after dehydration inethanol and propylene amide. Samples were not treated with
osmium tetroxide. For immunocytochemistry (Wooding,1981) 70-100nm thick sections on 300mesh uncoated nickel
grids 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
488

Koch et al.

particles, Janssen) for 30 min at room temperature, andwashed with PBS. Sections were stained with uranyl acetate
followed by lead citrate. Controls were carried out with PBS.
Results
Endoplasmin can be solubilized and purified ivithout
detergent
A procedure was developed for isolating endoplasminfrom plasmacytoma cells without using any detergent.
The procedure is based on the earlier observation

(Koch et al. 1986) that when such cells are subjected tohypotonic swelling and lysis, the cytoplasmic contents
are lost but the ER contents are retained within the
resultant shells. However, when the shells are disrupted mechanically, endoplasmin and the other putative reticuloplasmins are released in a completely
soluble form. This contrasts with shells and microsomes prepared under isotonic conditions where theendoplasmin is retained by the microsomal membranes, even after disruption of the shells. The reason
for this difference is not known, but, since the studies
described below show that it does not involve proteolytic modification, it probably reflects a difference inthe orientation of the microsomal membranes obtained
after mechanical disruption of the shells produced bylysis under isotonic and hypotonic conditions, respectively.
The solubilized endoplasmin was purified according
to the identical procedure described previously for
isolating the protein from detergent lysates of cells(Koch et al. 1985). The purified protein appears
identical to that purified from detergent lysates by
SDS-polyacrylamide gel electrophoresis (Fig. 1). Gel
filtration in the absence of detergent gives an apparent
molecular weight of 200X103, showing that the purified protein is dimeric when isolated by either procedure. Amino acid analyses of the detergent-free anddetergent-plus forms of the protein are also identical.
All other analyses used, such as two-dimensional gel
electrophoresis, ConA binding or peptide mapping
reveal 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 conditions had undergone a proteolytic modification thatremoved a hydrophobic segment, and thus rendered itsoluble in detergent-free buffers. Therefore the aminoand carboxy-terminal amino acids of the detergent-freeprotein were determined and compared with that of theintact polypeptide. The amino terminus of the detergent-free form was identified as Asp-Asp-Val-Asp-,
which is the same as that obtained for the detergentplus form (Koch et al. 1986) as well as the intact
polypeptide deduced from cDNA sequencing (Mazzarella & Green, 1987), showing that the amino terminus
had not been proteolysed. Carboxypeptidase digestion
unequivocally showed that the carboxy terminus was
leucine in both forms of the protein. Further digestion
was not significant, so only one residue was obtained.
This is identical with the expected carboxy-terminal
residue 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 easily
detected by SDS-polyacrylamide gel electrophoresis;
since it was not, we conclude that the protein isolated
under detergent-free conditions has not been proteolysed.
Cellular endoplasmin does not exhibit hydmphobic
characterTo examine whether endoplasmin in the intact cellformed an association with hydrophobic moieties, andthereby established membrane associations, the Triton
X-114 phase-separation test (Bordier, 1981) was applied to whole cells. It was expected that such hydrophobic associations could lead to the partitioning of theotherwise hydrophilic enodplasmin into the detergent
phase in significant amounts. However, Fig. 2 showsthat more than 95 % of the endoplasmin remains in the
aqueous phase. Thus this test confirms that endoplasmin does not possess the properties of an intrinsic
membrane protein, and also indicates that there is little
stable association with other intrinsic membrane components such as lipids or membrane proteins.
Immunogold labelling shows that endoplasmin is aluminal protein
The most direct test for the localization of endoplasmin
is to examine its location in cellular ER. To do thisconvincingly, we have used direct immunogold labelling on thin sections with monospecific antibodies toendoplasmin. In order to facilitate the distinction
between membrane-associated and luminal proteins,
cells that contained a dilated ER lumen were examined.
Fig. 3 shows a section of a rat gonadotrophin-producing cell immunogold labelled with anti-endoplasmin.
Fig. 3A shows a cell with an exceptionally dilated
segment of ER. At higher magnification (Fig. 3B andC) it is apparent that the gold particles are uniformly
distributed throughout the luminal region. Fig. 3Dshows a stained section through a cell with less dilated
ER and this also shows no preferential association with
the ER membrane.
Microsomal endoplasmin is not selectively degradedby trypsin
The basis of the previous conclusion that endoplasmin(ERP99) is a trans-membrane protein was its apparent
sensitivity to proteolytic digestion in microsomal preparations. Fig. 4 shows the effect of trypsin on microsomal membranes from the MOPC-315 cell, the same
one as that used previously (Lewis et al. 1985). In
contrast to the previous studies, no digestion of endoplasmin was obtained in the absence of a detergent thatpermeabilizes the membranes and renders the contents
accessible to the trypsin. This observation is also
Endoplasmin localization489

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

Discussion
These studies clearly demonstrate that endoplasmin is
a constituent of the luminal space of the ER, andtherefore belongs to the class of proteins termed
reticuloplasmins (Koch, 1987). Since previous studies
have formally shown that endoplasmin does not exist in
any other membrane compartment, such as the Golgi
apparatus (Koch, 1986), endoplasmin can be reliably
employed as a marker for reticuloplasm from mammalian cells.
Although endoplasmin is a reticuloplasmin, there is
the possibility that a small fraction is associated withthe membrane via an integral membrane component
such as the putative KDEL receptor that has been
proposed as part of a mechanism for ensuring that
proteins such as endoplasmin are confined to theendoplasmic reticulum (Munro & Pelham, 1986).
Interestingly, we have observed a small fraction of
endoplasmin that does partition into the detergent
phase in the Triton X-114 test. However, it appears
that the steady-state level of any complex between
endoplasmin and integral membrane proteins is extremely small, and most of the protein is strictly
luminal.
One of the main purposes of this study was to
examine the claim that endoplasmin is a transmembrane 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 membranes
exhibit differential sensitivity of endoplasmin to proteolytic digestion. Thus endoplasmin was found to bemuch more sensitive than some of the other proteins,
and this was interpreted to mean that endoplasmin
contained luminal and cytoplasmic domain, and must
therefore be a trans-membrane protein. However, thisis not the only possible explanation, since it is known
that endoplasmin is itself hypersensitive to proteolysis,
and the differential sensitivity of endoplasmin could
simply reflect this property. The other line of evidence
is the interpretation of the sequence of endoplasmin
deduced from the cDNA sequencing. However,neither of these criteria for trans-membrane proteins is
direct, and we conclude that the interpretation wasincorrect.
The need to clarify this question arose from its
relevance to the whole concept of reticuloplasm itself.
Until recently this was a poorly defined material, and it
was generally assumed that the major protein constituents of the ER lumen were the soluble secretory
proteins. We have suggested that this is not a true
490

Koch et al.

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 endoplasmin
was itself an abundant (>10mgml~') luminal ERspecific protein. The confirmation that endoplasmin isindeed a luminal protein completes the formal establishment of its classification as a reticuloplasmin. It
seems reasonably certain then that the other putative
reticuloplasmins, e.g. BiP (Munro & Pelham, 1986)
and PDI (Edmanef al. 1985), will also satisfy the same
criteria. Thus there seems to be no remaining obstacle
to the hypothesis that the ER luminal material (reticuloplasm) 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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