THE BIP PROTEIN AND THE ENDOPLASMIC RETICULUM OF SCHIZOSACCHAROMYCES POMBE: FATE OF THE NUCLEAR ENVELOPE DURING CELL DIVISION
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Journal of Cell Science 105, 1115-1120 (1993) 1115
Printed in Great Britain © The Company of Biologists Limited 1993
The BiP protein and the endoplasmic reticulum of Schizosaccharomyces
pombe: fate of the nuclear envelope during cell division
Alison L. Pidoux* and John Armstrong†
Membrane Molecular Biology Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln’s Inn Fields, London
WC2A 3PX, UK
*Present address: Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720, USA
†Authorfor correspondence at present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
SUMMARY
A polyclonal antibody was raised to the C-terminal temperatures and by treatment with tunicamycin. The
region of fission yeast BiP. The use of this antibody for antibody cross-reacts with proteins of similar molecu-
immunoprecipitation, western blotting and immunoflu- lar weight in the yeasts Kluyveromyces lactis and
orescence has confirmed and extended the observations Schizosaccharomyces japonicus. Immunofluorescence of
made previously with an epitope-tagged BiP molecule. BiP has been used to follow the behaviour of the ER
A fraction of BiP protein is glycosylated in Schizosac - and in particular the nuclear envelope through the cell
charomyces pombe cells. Pulse-chase experiments cycle.
showed that this modification occurs rapidly upon syn-
thesis and that the extent of glycosylation does not then
change with time. BiP protein is induced by elevated Key words: hsp70, glycosylation, cytokinesis
INTRODUCTION genetic analysis, but differs from that organism in many
aspects of its molecular and cell biology. We have previ-
The luminal compartment of the endoplasmic reticulum ously described the cloning and analysis of the BiP gene
(ER) contains a subset of proteins that are soluble but nev- from S. pombe (Pidoux and Armstrong, 1992). Like its
ertheless are not secreted by vesicle transport out of the homologue in S. cerevisiae, it is essential for viability and
cell. One member of this group is BiP, a protein originally its mRNA is induced by various forms of stress. Unlike
identified by its presence in the ER of pre-B cells, bound BiP proteins from most other species, however, the
to heavy-chain immunoglobulin in the absence of light encoded protein contained a predicted site for N-linked
chains (Haas and Wabl, 1983). The protein is now known glycosylation. By overexpressing an altered form of the
to be ubiquitous, and is a member of the hsp70 family of gene encoding an immunological ‘tag’, we presented evi-
stress-regulated proteins (Munro and Pelham, 1986). Unlike dence that this glycosylation site was used, but only in a
other members of this family, BiP is synthesised with a small proportion of the molecules. The same tagged BiP
cleavable N-terminal signal sequence for translocation into protein was used to visualise the ER of S. pombe by
the ER, and a C-terminal sequence specifying its localisa- immunofluorescence, revealing the nuclear envelope as
tion within the ER (Munro and Pelham, 1986, 1987). BiP well as a peripheral reticulum reminiscent of that found
appears to function in regulating the folding and assembly in higher cells. These observations, however, had the
of a variety of membrane and secretory proteins. This potential limitation of requiring the expression of an
process requires binding to the nascent protein followed by altered protein at abnormal and variable levels in the pop-
release coupled to ATP hydrolysis; in the cases of some ulation of cells.
mutant proteins, the binding is irreversible (reviewed by We describe here the direct observation of BiP in wild-
Gething and Sambrook, 1992). In the budding yeast Sac - type S. pombe, using an antibody raised to part of the pro-
charomyces cerevisiae the BiP protein, which is encoded tein expressed in bacteria. We have compared the alter-
by the gene KAR2, is additionally required for transloca- ations in protein levels under different forms of stress to
tion of nascent proteins across the ER membrane, as well those of the corresponding mRNA, and monitored the effi-
as in an undefined role in fusion of nuclei after mating ciency and kinetics of glycosylation. The antibody is shown
(Rose et al., 1989; Normington et al., 1989; Vogel et al., to recognise an appropriately sized protein in two other
1990). yeast species. The immunofluorescence analysis of the ER
The fission yeast Schizosaccharomyces pombe is a of S. pombe has been extended to show the behaviour of
simple eukaryote that, like S. cerevisiae, is amenable to the nuclear envelope during the cell cycle.1116 A. L. Pidoux and J. Armstrong
MATERIALS AND METHODS staining with Ponceau S before immuno-labelling. Glutathione-S-
transferase-ypt5 fusion protein (Armstrong et al., 1993) was pro-
Yeast strains vided by Dr S. Ponnambalam. To demonstrate specificity of the
S. pombe strain 972 and Schizosaccharomyces japonicus were antibody, each fusion protein was added during western blotting
from Prof. Jeremy Hyams, University College, London; at a concentration of 1 µg/ml. Conventional and confocal immuno-
Kluyveromyces lactis and Pichia pastoris from Dr Kevin Hard- fluorescence were as described (Pidoux and Armstrong, 1992)
wick, Laboratory for Molecular Biology, Cambridge. except that cells were fixed by addition of 1/10 volume 37%
formaldehyde to growing cultures and incubation continued at
30°C for 30 min. The anti-BiP antibody was used at a dilution of
Preparation of anti-BiP antibody 1:100 for immunoprecipitation, 1:20000 for western blotting, and
A 400 bp HindIII fragment from the 3′ region of the BiP gene 1:100 for immunofluorescence. Protein A-Sepharose was used
(Pidoux and Armstrong, 1992) was inserted into the vector pATH3 directly for immunoprecipitation (Pidoux and Armstrong, 1992),
for production of trpE-BiP fusion protein (Spindler et al., 1984). while peroxidase-conjugated and fluorescein-conjugated goat anti-
Escherichia coli containing the pATH3-BiP plasmid were grown rabbit antibodies (both Tago) were used for western blotting and
overnight at 37°C in 5 ml M9CA medium containing 20 mg/ml immunofluorescence, at dilutions of 1:1000 and 1:100, respec-
tryptophan, 30 µg/ml ampicillin, diluted into 50 ml of the same tively.
medium without tryptophan and grown at 30°C with vigorous aer-
ation for 1 h. Then, 250 µl of 1 mg/ml indole acrylic acid was
added and the incubation continued for 3 h. Cells were pelleted RESULTS
and resuspended in 1 ml 10 mM NaPO4, pH 7.2, 1% β-mercap-
toethanol, 1% SDS, 6 M urea and incubated at 37°C for 2-3 h. Production and characterisation of anti-BiP
An equal volume of 2× sample buffer was added and the sample
heated to 95°C for 5 min before loading onto a 10% SDS-PAGE antibodies
gel. Proteins were transferred from the gel to nitrocellulose mem- The C-terminal 110 to 130 amino acids of S. pombe BiP
brane by semi-dry blotting and visualised by Ponceau S staining. were used to make fusion proteins for immunisation of a
The trpE-BiP fusion protein band was excised from the filter, rabbit. This region of the protein was chosen because it
washed, dried and dissolved in dimethyl sulphoxide. shows minimal homology to other members of the hsp70
For production of glutathione-S-transferase-BiP fusion protein, family. Serum was tested for cross-reactivity against S.
the vector pGEX-2T (Smith and Johnson, 1988) was first digested pombe proteins by western blotting (Fig. 1). Two species
with BamHI and EcoRI and a synthetic polylinker was inserted. of 75-80 kDa were detected by the antibody in wild-type
The resulting plasmid, pGEX20T, contained adjacent restriction
sites for BamHI, EcoRI, XhoI, ClaI, SpeI and XbaI, while the orig- cells (lane 2), but not by pre-immune serum (lane 1). Bind-
inal EcoRI site was destroyed. An EcoRI-XhoI fragment from the ing of the antibody could be inhibited by incubation with
3′ end of the BiP gene (Pidoux and Armstrong, 1992) was then glutathione-S-transferase-BiP fusion protein (lane 3) and
inserted. A saturated 100 ml culture of E. coli containing the was specific to the BiP portion of the fusion protein since
pGEX-20T-BiP plasmid was inoculated into 1 l of L-broth con- incubation with glutathione-S-transferase-ypt5 fusion pro-
taining ampicillin and incubated for 1 h. Fusion protein was
induced by addition of IPTG to 100 mM and the culture grown
for a further 5 hours. Cells were pelleted and resuspended in 15
ml ice-cold 1% (v/v) Triton X-100 in phosphate-buffered saline
(PBS-TX) and placed on ice. Cells were lysed by sonication (sev-
eral 3 s bursts at full power). Then 2 ml of pre-swollen glutathione-
agarose beads (Sigma) in PBS-TX was added to the lysate and
incubated for 5 min at room temperature with inversion. Beads
were pelleted by centrifugation at 1700 r.p.m. for 2 s and washed
5 times with ice-cold PBS-TX. The beads were gently transferred
to a 2 ml tube and washed 3 times. Fusion protein was eluted by
washing three times with 1 ml 0.5 M glutathione (Sigma), 50 mM
Tris-HCl, pH 7.0, for 2 min at room temperature, and dialysed
against PBS.
A rabbit was injected with 0.5 mg of trpE fusion protein in
complete Freund’s adjuvant. After several boosts of 0.5 mg of 92 kDa
fusion protein in incomplete Freund’s adjuvant at 4-week inter-
vals, no antibodies reactive against BiP had appeared. Therefore
the same rabbit was immunised by a similar protocol with the glu- 69 kDa
tathione-S-transferase fusion protein.
Methods for induction of BiP, endoglycosidase H digestion,
35S-labelling of cells and immunoprecipitation, electrophoresis,
fluorography and western blotting, were as before (Pidoux and
Armstrong, 1992), with the exception that protein extracts were
prepared by disruption with glass beads. Yeast cells in log phase
were pelleted, resuspended in 2× sample buffer, an equal volume Fig. 1. Detection of S. pombe BiP protein by western blotting
of acid-washed glass beads (425-600 µm; Sigma) was added and (lanes 1-4) and immunoprecipitation (lane 5). A doublet of the
the cells were disrupted by vigorous vortexing. Extracts were predicted size is recognised by immune (lane 2) but not pre-
immediately heated to 95°C for 5 min, centrifuged briefly and the immune (lane 1) serum. Binding is prevented by BiP fusion
supernatants taken for gel electrophoresis. Efficient transfer of protein (lane 3) but not by ypt5 fusion protein (lane 4). GST,
proteins to membranes for western blotting was confirmed by glutathione-S-transferase.BiP from fission yeast 1117
Fig. 3. Induction of BiP protein by heat
shock and tunicamycin treatment. Equal
numbers of cells were incubated at 30°C
(lane 1) or 39°C for 30 min (lane 2) or
with 1 µg/ml tunicamycin at 30°C for 2
h (lane 3). In each case cells were
labelled for the last 30 min of
incubation, and the BiP protein
immunoprecipitated. Both treatments
cause some induction of protein; in the
presence of tunicamycin only the
unglycosylated form is observed.
Tm, tunicamycin
Fig. 2. Partial glycosylation of S. pombe BiP. The upper band of
the BiP doublet detected by western blotting (lane 1) is removed
by digestion with endoglycosidase H (lane 2). Pulse-chase
tein. The proportion of glycosylated protein did not change
analysis of 35S-labelled protein (lanes 3-7) shows no change in the throughout the chase period up to 2 h (lanes 4-7). These
proportion of the glycosylated form in protein immunoprecipitated observations indicate that BiP is glycosylated concomi-
after 5, 10, 30, 60 and 120 min after the start of the chase. tantly with or very shortly after its synthesis.
In mammalian cells BiP is induced by agents such as
tunicamycin and calcium ionophores but not by heat shock.
tein showed no such effect (lane 4). The anti-serum could S. pombe BiP mRNA is induced by heat shock and by treat-
also immunoprecipitate both BiP species from metaboli- ment with tunicamycin (Pidoux and Armstrong, 1992). To
cally labelled cells (lane 5). Therefore the antiserum appears determine whether BiP protein levels were also affected by
to be specific for S. pombe BiP protein. these treatments, BiP was immunoprecipitated from
The BiP protein labelled cells. Cells subjected to 30 min heat shock at 39oC
contain a higher level of the glycosylated and unglycosy-
S. pombe BiP contains a single potential N-linked glyco- lated forms of BiP (Fig. 3, lane 2) than untreated cells (lane
sylation site near the N terminus of the protein. We have 1). In cells treated with 1 µg/ml tunicamycin for 2 h (lane
previously shown that a fraction of epitope-tagged BiP 3) only the unglycosylated form is present, as expected for
expressed from a plasmid receives core carbohydrate mod- an inhibitor of N-linked glycosylation. The level of the ung-
ifications (Pidoux and Armstrong, 1992). To confirm that lycosylated form is higher in these cells than in the
this phenomenon was not restricted to the altered version untreated control.
of the BiP protein, S. pombe protein extracts were incu-
bated overnight in the presence or absence of endoglycosi-
dase H and analysed by western blotting (Fig. 2, lanes 1 Cross-reactivity of anti-BiP antibody
and 2). The upper band disappears upon endoglycosidase Although BiP proteins from different species show low
H treatment, indicating that it contains N-linked carbohy- homology in their C-terminal regions, it was possible that
drate. A pulse-chase experiment was performed to investi- the antibody raised against S. pombe BiP would cross-react
gate whether glycosylation occurs immediately upon syn- with other yeast BiP proteins and might therefore be useful
thesis or gradually, with all BiP molecules eventually for their study. Protein extracts were made from
becoming glycosylated. After 5 min of labelling, immuno- Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia
precipitated BiP was present as both the glycosylated and pastoris, Schizosaccharomyces japonicus and S. pombe
unglycosylated forms (Fig. 2, lane 3), with the glycosylated cells, and analysed by western blotting using the anti-BiP
form accounting for approximately 10% of the total pro- antibody (Fig. 4A). Cross-reactive species of the expected
A B
92 kDa
69 kDa
Fig. 4. (A) Reaction of the antiserum with other yeast species. Equivalent amounts of extracts from S. pombe (lane 1), K. lactis (lane 2), S.
japonicus (lane 3), S. cerevisiae (lane 4) and P. pastoris (lane 5) were analysed by western blotting with the BiP antibody. K. lactis and S.
japonicus have a faint cross-reacting band of the appropriate mobility. (B) Alignment of the C-terminal regions of BiP protein sequences
from K. lactis (K. l.) and S. pombe (S. p.), showing regions of consensus (con).1118 A. L. Pidoux and J. Armstrong
Fig. 5. Immunofluorescence of BiP.
(A) Conventional epifluorescence
microscopy shows labelling of the
nuclear envelope and elements close
to the plasma membrane.
(B,C) Confocal microscopy. A
section through the centre of cells
(B) shows similar structures to (A).
An image from approximately
2 µm above the centre of the cells (C)
reveals in addition a network of
peripheral tubules. Bars: 10 µm (A);
2 µm (B, C).
mobility for BiP were detected in K. lactis and S. japonicus wild-type BiP is the same as that previously reported for
extracts, though the bands were fainter than for S. pombe epitope-tagged protein (Pidoux and Armstrong, 1992).
BiP. S. japonicus and S. pombe are related fission yeasts. In The behaviour of the ER during the cell cycle, and par-
contrast, the budding yeast K. lactis , whose BiP gene has ticularly the nuclear envelope, could be followed using the
been cloned (Lewis and Pelham, 1990), is much more dis- anti-BiP antibody (Fig. 6). The cell in Fig. 6A has just
tantly related to S. pombe. However, a comparison of the divided from its sister cell; staining of the nuclear envelope
C-terminal amino acid sequences of BiP from the two and peripheral ER is apparent. Cell growth occurs at the
species revealed regions of sequence conservation (Fig. 4B). ends of the cell (Fig. 6B, C) with cell length increasing
from 7 to 14 µm through the cell cycle. Early in anaphase,
Immunofluorescence with the anti-BiP antibody when the chromosomes begin to separate, the nucleus elon-
Wild-type cells were fixed with formaldehyde and gates as is shown by the shape of the nuclear envelope stain-
processed for immunofluorescence using the anti-BiP anti- ing in Fig. 6D. As the spindle elongates in anaphase B the
body. The nuclear envelope, elements near the plasma nuclei move apart (Fig. 6E, F). In Fig. 6E labelling that
membrane and strands through the cytoplasm were labelled appears to correspond to two thicknesses of nuclear enve-
(Fig. 5A). This distribution was also observed by confocal lope membrane is seen stretching between the nuclei. The
microscopy of the mid-section of cells (Fig. 5B); in addition nuclei in fact move almost to the ends of the cell before
images from above or below the mid-section revealed a they return to the centre of the two incipient daughter cells
peripheral reticulum (Fig. 5C). Thus the distribution of (Fig. 6G). By this time the nuclear envelope, which wasBiP from fission yeast 1119
Fig. 6. Confocal immunofluorescence of BiP in cells at different stages of the division cycle. The nuclear envelope elongates (B,C) until
the daughter nuclei are connected by a thick strand of BiP-labelled membrane (D-F). Just before and during cell division BiP protein
appears concentrated at the equator (G). Bar, 2 µm.
stretched between the two nuclei, has virtually disappeared.
There is a striking concentration of ER staining at the equa-
tor of cells that are about to divide or are dividing (Fig. 6E,
G). Further images of this phenomenon are shown in Fig.
7. The reticular structures are present around the cell periph-
ery throughout the cell cycle and do not appear to break
down at any stage (data not shown).
DISCUSSION
The BiP protein is of interest for several reasons: its role
in folding and assembly of membrane and secretory pro-
teins, its inducibility following a variety of cellular stresses,
and as a model protein retained in the ER lumen. Previ-
ously we reported the cloning of the BiP gene from the fis-
sion yeast S. pombe, and its unusual retention signal, ADEL
(Pidoux and Armstrong, 1992). Here we have described an
immunological analysis of the BiP protein, using an anti-
body raised to bacterially expressed C-terminal fragments
of the protein.
An unexpected feature of the predicted amino acid
sequence was a site for N-linked glycosylation. Such sites
are generally absent from BiP sequences; in contrast, they
are present but necessarily unused in cytoplasmic members
of the hsp70 family. In an epitope-tagged BiP the site was
Fig. 7. Confocal immunofluorescence of BiP in cells undergoing
used, but in only a small proportion of the molecules cytokinesis. Before cell division (A-E) there is a concentration of
(Pidoux and Armstrong, 1992). We have shown here that labelling at the cell equator, appearing as a patch or spot. This
the same applies to the natural protein, and in addition that material appears to be shared between the two daughter cells upon
the extent of glycosylation does not increase with time, in division (F). Bar, 2 µm.
spite of the co-localisation of BiP with the glycosylation
machinery in the ER (Fig. 2). Thus the glycosylation site
presumably is inaccessible after the protein has been cosylation is a late event related to degradation of the pro-
translocated, folded and released into the ER lumen. One tein. Hence the functional significance of the partial glyco-
possibility apparently eliminated by these results is that gly- sylation remains unknown.1120 A. L. Pidoux and J. Armstrong
We investigated the ability of the antibody to react with ture comprises two concentric tubules separately connected
BiP from other species of yeast. Antibodies to conserved to the inner and outer nuclear envelopes, do the separate
luminal proteins of the secretory pathway in general show layers have different compositions reflecting their distinct
quite a restricted specificity, thereby avoiding reaction with origins? Is the process of membrane breakage purely
the homologous protein of the host species within the ER mechanical, or does it require a distinct scission activity?
or Golgi of the antibody-producing cell. The antibody did Is the ER material at the equator specifically involved in
not react with mammalian cells (not shown), or with the generating the new plasma membrane that subsequently
budding yeasts S. cerevisiae or Pichia pastoris (Fig. 4A). forms at the same site? Future work may help to integrate
Conversely, an antibody to BiP of S. cerevisiae (Rose et the answers to these questions with the wealth of informa-
al., 1989) did not cross-react with S. pombe (not shown). tion already available concerning other aspects of the cell
The S. pombe antiserum did detect a protein in the related cycle in S. pombe.
fission yeast S. japonicus and also, surprisingly, in the bud-
ding yeast K. lactis (Fig. 4A). The sequence of BiP from We thank Jeremy Hyams and Kevin Hardwick for yeast strains,
the latter species is known (Lewis and Pelham, 1990). Com- Mark Rose for antibody, Vas Ponnambalam for fusion protein,
parison of the sequences revealed a short region of sequence and Kathryn Ayscough, Sally Bowden, Mark Craighead, Kevin
Hardwick and Vas Ponnambalam for helpful discussions during
conservation near the C terminus (Fig. 4B). Previously we the course of this work.
showed that the C-terminal sequence of S. pombe BiP,
ADEL, acted as an ER retention signal in this species, and
that variants from other species, including the K. lactis
sequence DDEL, could also function but less efficiently REFERENCES
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The appearance of these structures raises numerous ques-
tions concerning their topology, function and coordination (Received 22 October 1992 - Accepted, in revised form,
with other events in the cell cycle. If the filamentous struc- 27 April 1993)You can also read
