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
. 13: 1009–1020 (1997)
Accumulation of Misfolded Protein Aggregates Leads
to the Formation of Russell Body-like Dilated
Endoplasmic Reticulum in Yeast
KYOHEI UMEBAYASHI1, AIKO HIRATA2, RYOUICHI FUKUDA1, HIROYUKI HORIUCHI1,
AKINORI OHTA1 AND MASAMICHI TAKAGI1*
1
Department of Biotechnology and 2Institute of Molecular and Cellular Biosciences, The University of Tokyo,
Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received 17 January 1997; accepted 18 February 1997
RNAP-I, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in
Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its
amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct
folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, Äpro, was not secreted but was
retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation
of Äpro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that Äpro is recognized as
an unfolded protein species in the ER. Our biochemical data demonstrated that Äpro formed aggregates which
contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed
that the Äpro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such
‘chaperone-associated misfolded protein bodies’ were observed for the first time in yeast. Morphologies of the ER
and nucleus were drastically altered by the accumulation of the Äpro aggregates. The ER lost its flat cisternal shape;
the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded Äpro proteins are
probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly
impaired, and the dilated ER may represent an ER subcompartment where the Äpro aggregates are degraded.
? 1997 John Wiley & Sons, Ltd.
Yeast 13: 1009–1020, 1997.
No. of Figures: 7. No. of Tables: 0.
No. of References: 42.
  — Saccharomyces cerevisiae; endoplasmic reticulum; chaperone; unfolded protein response
INTRODUCTION
In eukaryotic cells, the endoplasmic reticulum
(ER) is the first organelle that secretory proteins
pass through, and is known as a protein-folding
compartment (Helenius et al., 1992). To assist
protein folding and assembly, many kinds of
peculiar enzymes and molecular chaperones reside
in the ER (Gething and Sambrook, 1992). Yet,
under some conditions that perturb protein folding, secretory proteins are unable to fold correctly
and as a result unfolded proteins accumulate in the
ER. In response to such an emergency, various
cellular events are known to take place.
*Correspondence to: Masamichi Takagi.
CCC 0749–503X/97/111009–12 $17.50
? 1997 by John Wiley & Sons, Ltd
First, the synthesis of ER resident chaperones
and enzymes is induced when unfolded proteins
accumulate in the ER. The induced proteins are
considered to facilitate protein folding and/or to
prevent protein aggregation. In mammalian cells,
transcription of the genes encoding BiP (GRP78:
Glucose Regulated Protein), GRP94 and GRP170
is induced by glucose starvation or by the addition
of glycosylation inhibitors, reducing agents, or
calcium ionophores (Lee, 1987; Lin et al., 1993).
These conditions are thought to cause accumulation of unfolded proteins in the ER. Transcription of the gene encoding protein disulfide
isomerase (PDI) is also induced by the addition of
tunicamycin or a calcium ionophore A23187
.   .
1010
(Dorner et al., 1990). Similarly, in Saccharomyces
cerevisiae, transcription of the KAR2 (Normington
et al., 1989; Rose et al., 1989), the PDI1 (LaMantia
et al., 1991) and the EUG1 genes (Tachibana and
Stevens, 1992), which encode yeast BiP, PDI and
Eug1p, whose overproduction can rescue the
growth defect of a Äpdi1 strain, respectively, is
induced by the addition of tunicamycin. Thus,
recent analyses revealed that accumulation of
unfolded proteins in the ER triggers a signal,
which is then transmitted to the nucleus. Ern1p
(Ire1p), a protein kinase located in the ER membrane and required for inositol prototrophy
(Nikawa and Yamashita, 1992), was shown to be
involved in this ER-to-nucleus signal transduction
(Cox et al., 1993; Mori et al., 1993; Shamu and
Walter, 1996).
Second, along with the accumulation of aggregated protein bodies in the ER, enlargement of the
ER occurs (reviewed in Sitia and Meldolesi, 1992).
The dilated cisternae of the ER which resembled
Russell Bodies (RBs) was induced by the protein aggregates consisting of aberrantly folded
immunoglobulins (Valetti et al., 1991). Similarly,
formation of intracisternal granules (ICGs), protein aggregates consisting of pancreatic enzymes,
led to the dilation of the ER (Tooze et al., 1989). In
both cases, the protein aggregates are sorted from
other ER proteins, such as ER chaperones. The
dilation of the ER may have a role in forming an
ER subcompartment to which insoluble protein
aggregates are sorted from the ordinary rough ER.
Cells may avoid the blockage of the secretory
pathway in such a way, but the mechanism of ER
dilation remains unclear.
We have shown that the prosequence of
Rhizopus niveus aspartic proteinase-I (RNAP-I;
Horiuchi et al., 1988) is required both for proper
folding in vitro and for the secretion in S. cerevisiae
(Horiuchi et al., 1990; Fukuda et al., 1994), and
that Äpro, a prosequence deleted derivative, is not
secreted (Fukuda et al., 1994) but instead it is
retained and degraded in the yeast ER (Fukuda
et al., 1996). From these results, we reasoned that
Äpro could not fold correctly in the ER due to the
absence of the prosequence. In this paper, we first
examined whether Äpro is recognized as an unfolded protein species in the yeast ER and bound
by ER chaperones. We found that the overproduction of Äpro caused elevated synthesis of BiP
and PDI. Moreover, it was revealed that Äpro
formed large aggregates associated with BiP in
the ER lumen as well as in the nuclear envelope

. 13: 1009–1020 (1997)
lumen, which caused a significant morphological
alteration of the ER and nuclear envelope.
MATERIALS AND METHODS
Strains and media
Escherichia coli strain JA221 (F* lacY leuB6
trpE5 hsdR recA1 ë " ) was used as a host for
plasmid construction. Strain MV1190 (Ä(lacproAB) thi supE Ä(srl-recA) 306::Tn10 tetr (F*
traD36 proAB + lacIq lacZÄM15)) was used as a
host for production of plasmids pYPR3831X and
pYPR3841.
S. cerevisiae strain R27-7C-1C (MATa his3 leu2
ura3 trp1) was transformed with either plasmid
pYPR3831X or pYPR3841 using the lithium
acetate procedure (Ito et al., 1983).
Yeast cells were cultured aerobically at 30)C.
For selection of yeast transformants, YNBD
medium (0·17% yeast nitrogen base without amino
acids and ammonium sulfate (DIFCO), 0·5%
ammonium sulfate, 2% glucose) was used with
appropriate supplements. Transformed yeast cells
were cultured in YNBDCU medium (YNBD
medium containing 2% casamino acids (DIFCO)
and 0·05% uracil), or YNBGCU medium (same as
YNBDCU except that glucose was replaced with
2% galactose).
Plasmid construction
Nucleic acid modification enzymes were purchased from Takara Shuzo Co. (Kyoto, Japan)
and used under conditions suggested by the manufacturer, and recombinant DNA manipulations
were done according to the standard methods
(Maniatis et al., 1982).
Plasmid pYPR3831X, which contains the GAL1
promoter and the glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) terminator but not the
RNAP-I coding sequence, was constructed as
follows. Plasmid pYPR3831 (Fukuda et al., 1994)
was digested with EcoRI and SalI to remove
the wild-type RNAP-I coding sequence, and the
terminal EcoRI and SalI sites of the linearized
vector were filled in with T4 DNA polymerase.
After these blunt ends were ligated with XbaI
linker d (pCTCTAGAG) and digested with XbaI,
the linearized vector was self-ligated to form
plasmid pYPR3831X. Plasmid pYPR3841 contains the Äpro coding sequence, in which the whole
prosequence was deleted from the wild-type
RNAP-I sequence, between the GAL1 promoter
? 1997 John Wiley & Sons, Ltd
    
and the GAPDH terminator. This plasmid was
constructed as follows. Plasmid pYPR2841
(Fukuda et al., 1994) was digested with EcoRI
and partially with SalI, and the resulting 1·2 kb
EcoRI-SalI fragment containing the Äpro coding
sequence was ligated with the 8·7 kb EcoRI-SalI
fragment of plasmid pYPR3831 lacking the
wild-type RNAP-I coding sequence.
Preparation of whole protein extract and
immunoblotting
Yeast cells were collected and washed once
with distilled water. Cells were resuspended in
0·2 -NaOH, 1% â-mercaptoethanol for 10 min
on ice, then trichloroacetic acid was added to a
final concentration of 10%. Following further
incubation for 10 min on ice, precipitates were
collected by centrifugation at 15 000 g for 5 min at
4)C, washed twice with acetone, and resuspended
in Laemmli sample buffer (Laemmli, 1970). Proteins were solubilized by boiling for 5 min, and the
insoluble materials were removed by centrifugation at 15 000 g for 3 min. Aliquots of the samples
were diluted with water, and proteins were
quantitated using Bio-Rad Protein Assay. Samples
were separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE)
and transferred to the nitrocellulose membrane,
Hybond-C (Amersham). The ECL Western
blotting system (Amersham) was used for
detection.
Preparation of polyclonal anti-RNAP-I antibody was described previously (Fukuda et al.,
1994). Anti-BiP (Tokunaga et al., 1992) and antiPDI (LaMantia et al., 1991) antibodies were kindly
provided by M. Tokunaga and T. Mizunaga,
respectively.
RNA preparation and Northern hybridization
Total yeast RNA was prepared as described
(Schmitt et al., 1990). Electrophoresis of denatured
RNA (each 15 ìg) and Northern hybridization
were carried out as described (Maniatis et al.,
1982). The central 1·07 kb EcoRI fragment of
the KAR2 gene, which was kindly provided by
K. Kohno, was used as a radiolabeled probe.
Preparation of microsomes and sedimentation
analysis on sucrose gradients
Spheroplasts, and microsomes were prepared as
described (Franzusoff et al., 1991; Baker et al.,
1990, respectively), except that microsomes
? 1997 John Wiley & Sons, Ltd
1011
were resuspended in phosphate-buffered saline
(PBS) containing 250 m-sorbitol. For solubilization of microsomes, 80 ìl of the microsomal
suspension (corresponding to about 200 ìg of protein) was supplemented with 4 ìl of 800 m-Nethylmaleimide (Sigma), then mixed with an equal
volume of PBS containing 250 m-sorbitol and 4%
Triton X-100, and incubated for 25 min at 4)C.
Detergent solubilized microsomes were loaded
onto the top of 15–90% linear sucrose gradients
(8 ml total volume) containing 0·2% Triton X-100,
150 m-NaCl, 10 m-sodium phosphate (pH 7·5),
and 250 m-sorbitol. Gradients were centrifuged
for 90 min at 4)C in an RPS40T rotor (Hitachi
Koki Co., Ltd) at 28 500 rpm. Gradients were
divided into 15 fractions. Each was diluted with
PBS containing 250 m-sorbitol and 0·2% Triton
X-100, and proteins were precipitated by adding
trichloroacetic acid to a final concentration of
10%. The precipitated proteins were washed once
with acetone, and resuspended in 50 ìl of Laemmli
sample buffer. In each fraction, 5 ìl of the sample
was used for SDS–PAGE and subsequent Western
blotting.
Electron microscopy
Preparation of thin sections of yeast cells by
the freeze-substituted fixation method was carried
out as described (Sun et al., 1992), except that
Reichert KF80 was used to freeze cells and that
thin sections were viewed on a JEOL JEM, 1210
electron microscope (JEOL Tokyo, Japan) at
80 kV. For immunoelectron microscopy, thin
sections were incubated with anti-RNAP-I antibody diluted 1:5000 in PBS containing 1% bovine
serum albumin (BSA) for 30 min at room temperature. After washing six times with PBS, thin
sections were incubated with 15 nm gold particleconjugated secondary antibody for 60 min at room
temperature, then washed twice with PBS and once
with water. Thin sections were stained with uranyl
acetate and lead citrate, and viewed on the electron
microscope described above.
Immunofluorescence microscopy
Cells were grown in YNBDCU medium for
48 h, then washed and transferred to YNBGCU
medium. After 12 h, cells were harvested. Fixation,
permeabilization, and staining of cells were performed as described (Nishikawa and Nakano,
1991) with some modifications. Formaldehyde and
potassium phosphate (pH 6·5) solutions were

. 13: 1009–1020 (1997)
.   .
1012
Figure 1. Accumulation of Äpro induced by the GAL1 promoter. Wild-type cells harboring plasmid pYPR3841 were
grown in YNBDCU medium for 48 h, then washed and transferred to YNBGCU medium to overproduce Äpro. At the
indicated times after the start of overproduction, aliquots of the
culture were withdrawn and whole protein extract was prepared
as described in Materials and Methods. In each lane, 5 ìg of
protein was loaded, separated by SDS–PAGE and transferred
to the nitrocellulose membrane. Äpro was detected with antiRNAP-I antibody.
added directly to YNBGCU medium to a final
concentration of 5% (v/v) and 100 m, respectively, and cells were fixed at 30)C for 2 h. Cells
were suspended in spheroplasting buffer containing 100 ìg/ml Zymolyase 100T at a concentration
of 1#107 cells/ml. Anti-RNAP-I antibody and
anti-BiP antibody were diluted 1:500 and 1:100,
respectively, and the secondary antibody, fluorescein isothiocyanate-conjugated anti-rabbit antibody, was diluted 1:200 in PBS containing
1% BSA. After the final wash with PBS containing 0·1% BSA, mounting medium (1 mg/ml
p-phenylenediamine, 10% (v/v) PBS, 90% (v/v)
glycerol) was dropped onto the slide. For staining
of BiP, cells were observed at 1000-fold magnification. For Äpro, cells were observed at 400-fold
magnification because the intensity of staining
was weaker than in the case of BiP, and the
photographs were enlarged.
RESULTS
Synthesis of BiP and PDI is induced by the
overproduction of Äpro
We examined whether synthesis of BiP and PDI
is induced when Äpro accumulates in the ER. For
this purpose, we constructed a YEp-type plasmid
pYPR3841, in which Äpro production was controlled by the GAL1 promoter. Plasmid
pYPR3831X, which is the same as pYPR3841
except for the absence of the Äpro coding sequences, was used for the control experiments.
Wild-type cells harboring pYPR3841 were grown
in glucose-containing medium, then switched to
galactose-containing medium to overproduce

. 13: 1009–1020 (1997)
Figure 2. Induction of KAR2 mRNA by the overproduction
of Äpro. Wild-type cells harboring plasmid pYPR3831X (lanes
1 and 3) or pYPR3841 (lanes 2 and 4) were cultured as
described in Figure 1. At 9 and 20 h after the start of overproduction, total RNA was extracted for Northern hybridization
using a probe specific for the KAR2 gene.
Äpro. Figure 1 shows the time course of Äpro
accumulation; Äpro began to accumulate at 9 h
after induction. Marked accumulation was not
observed during the first 6 h, probably because the
quantity of intracellular Äpro was below the
detectable level under our experimental conditions.
When wild-type cells harboring pYPR3831X were
examined in the same manner, no bands were
detected (data not shown).
Then, we examined whether transcription of the
KAR2 gene is induced by the accumulation of
Äpro. The Northern analysis shows that, in comparison with the control experiment, KAR2
mRNA level increased about 2·2-fold (Figure 2;
compare lanes 1 and 2) and 2·0-fold (Figure 2;
compare lanes 3 and 4) at 9 h and 20 h after the
start of Äpro overproduction, respectively. To test
whether the amount of BiP actually increased, we
detected BiP in the same protein extracts as in
Figure 1 using anti-BiP antibody. As shown in
Figure 3A, when Äpro was overproduced, the
amount of BiP continuously increased (upper
panel), whereas such an increase was not observed
in the control experiment (lower panel).
Next, whether the amount of BiP is higher in
Äpro-overproducing cells than in control cells was
examined by quantitating the intensity of bands in
Figure 3A. In Figure 3B, it was shown that the
amount of BiP in Äpro-overproducing cells was
from 1·3- to 1·8-fold higher than that in the control
cells throughout the time examined except for 0 h.
The increase in protein level was similar to that of
mRNA level shown above. These results indicate
that the accumulation of Äpro in the ER caused
the elevated amount of BiP through the transcriptional induction of the KAR2 gene. Similarly to
BiP, the amount of PDI increased when Äpro was
? 1997 John Wiley & Sons, Ltd
    
Figure 3. Elevated amount of BiP in response to the accumulation of Äpro in the ER. (A) In each lane, 20 ìg of the same
protein extract as in Figure 1 was separated by SDS–PAGE and
transferred to the nitrocellulose membrane. BiP was detected
with anti-BiP antibody. For the control experiment, wild-type
cells harboring plasmid pYPR3831X were treated in the
same way. (B) The intensity of bands corresponding to BiP
was quantitated using NIH Image Analysis software, and the
ratios of the band intensity between the Äpro-overproducing
cells and the control cells were calculated. Values from four
independent experiments were averaged. Bars indicate standard
deviations.
overproduced (data not shown). It is quite possible
that the elevation of PDI is due to the induced
transcription of the PDI1 gene, because the PDI1
gene is transcriptionally induced via Ern1p in
response to unfolded proteins in the ER (Cox
et al., 1993). Therefore, we conclude that the
accumulation of Äpro in the ER elevates the
synthesis of at least two ER resident chaperones,
BiP and PDI.
? 1997 John Wiley & Sons, Ltd
1013
Äpro forms large aggregates associated with BiP
in the ER
There are many reports that BiP binds to unfolded or unassembled proteins. Because the above
results indicate that Äpro is recognized as an
unfolded protein species in the ER, it is quite
possible that BiP binds to Äpro. To test this
possibility, we performed immunoprecipitation
experiments using anti-RNAP-I antibody. As a
result, BiP indeed co-precipitated with Äpro, however, this was also observed when preimmune
serum, the irrelevant antibody or even no antibody
was used (data not shown). From this result, we
suspected that Äpro in the ER forms aggregates
large enough to precipitate in the absence of
antibody and protein A-Sepharose, and that BiP
binds to these aggregates. In contrast to BiP, PDI
was not co-precipitated (data not shown), suggesting that PDI does not associate, or only weakly
associates with Äpro. To test these possibilities,
microsomes were prepared from the cells overproducing Äpro, solubilized by detergent and analysed
for Äpro, BiP and PDI by density gradient centrifugation on 15–90% linear sucrose gradients.
Äpro was shown to be present almost exclusively in
the heavy fractions (Figure 4A; fractions 4–11),
suggesting that Äpro forms large aggregates.
Although part of BiP (approximately 40%) was
detected in the top fractions (Figure 4A; fractions
13, 14 and 15), the remaining showed similar
distribution to that of Äpro in the heavy fractions
(Figure 4A; fractions 4–11). In both proteins, the
peak coincides at fraction 6 and both gradually
decrease in fractions 7–11. Without the overproduction of Äpro, BiP was present almost
exclusively in the top fractions (Figure 4B; fractions 13–15). These results indicate, in accordance
with the above idea, that Äpro forms large
aggregates which are associated with BiP in the
ER. In contrast to BiP, PDI was detected only in
the top fractions (Figure 4C; fractions 14 and 15),
again suggesting that PDI does not bind to the
aggregates containing Äpro.
Electron microscopical analysis of the Äpro
aggregates
To examine the structures of the Äpro aggregates in vivo, we observed the cells overproducing
Äpro by electron microscopy using the freezesubstituted fixation method (Sun et al., 1992).
As shown in Figure 5A–C, there were many
characteristic cisternal structures that contained

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.   .
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Figure 4. Sedimentation analysis of Äpro, BiP and PDI. Wild-type cells harboring plasmid
pYPR3831X or pYPR3841 were grown in YNBDCU medium for 48 h, then washed and transferred to
YNBGCU medium. After 12 h, cells were harvested and microsomes were prepared as described in
Materials and Methods. Detergent-solubilized microsomes were resolved by centrifugation on 15–90%
linear sucrose gradients as described in Materials and Methods and gradients were divided into 15
fractions. After precipitation by adding trichloroacetic acid, proteins were separated by SDS–PAGE
and transferred to the nitrocellulose membrane. Äpro, BiP and PDI were detected with anti-RNAP-I,
anti-BiP and anti-PDI antibodies, respectively.
electron-dense materials. They seemed to be continuous with the nuclear envelope and remarkably
accumulated, forming irregularly proliferated network structures. Most cells observed exhibited
such aberrant membranous structures, though the
degree of their accumulation varied among the
cells. In the control cells that did not produce
Äpro, such marked accumulation of membranous
structures with the electron-dense materials was
not observed (Figure 5E). These observations
strongly suggest that the accumulated membranous structures were derived from the ER and
that the electron-dense materials represented
the Äpro aggregates. Electron-dense materials
were also found in the peripheral ER close to the
plasma membrane (Figure 5D, arrowheads) and in
the nuclear envelope lumen (Figure 5B–D, thick
arrows). Besides the abnormal cytoplasmic ER
morphology, the cells overproducing Äpro also
exhibited aberrantly-shaped nuclei. In contrast to

. 13: 1009–1020 (1997)
the typically-shaped round nucleus in the control
cells (Figure 5E), the nuclei in the cells overproducing Äpro were bumpy and not uniform among
the cells (Figure 5A–D).
Next, cells were observed by immunoelectron
microscopy to detect Äpro. Thin sections prepared
by the freeze-substituted fixation method were
incubated with anti-RNAP-I antibody and then
with a colloidal gold-conjugated secondary antibody. As shown in Figure 6A, gold particles
clustered in the aberrant membranous structures
described above. In these structures, most gold
particles could be seen on the electron-dense
materials (Figure 6B, C), indicating that these
materials indeed represent the Äpro aggregates.
We found gold particles also on the electron-dense
materials in the nuclear envelope lumen (Figure
6C, arrow), again indicating that the Äpro aggregates are present in the nuclear envelope as well as
in the enlarged ER.
? 1997 John Wiley & Sons, Ltd
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1015
Figure 5. Electron micrographs of the cells overproducing Äpro (A–D) and of the control cell (E).
Wild-type cells harboring plasmid pYPR3841 (A–D) or pYPR3831X (E) were grown as in Figure 4
and fixed by the freeze substitution technique. (B) Magnification of the region boxed in (A) where
proliferated ER containing electron-dense materials was observed. Thick arrows in (B)–(D) indicate
the nuclear envelope lumen containing electron-dense materials. Arrowheads in (D) indicate the
peripheral ER that contains electron-dense materials beneath the plasma membrane. Small arrows
in (D) indicate nuclear microtubules, demonstrating that the cell is mitotic. N, nucleus; V, vacuole;
M, mitochondrion; MT, microtubule.
Immunofluorescence staining of Äpro and BiP
The enlarged ER containing the Äpro aggregates was also observed by indirect immunofluor? 1997 John Wiley & Sons, Ltd
escence microscopy. When stained with anti-BiP
antibody, the control cells exhibited the typical
ER-staining pattern; the nuclear envelope and the

. 13: 1009–1020 (1997)
.   .
1016
Figure 6. Immunoelectron micrographs of the cells overproducing Äpro. Thin sections prepared in
Figure 5 were incubated with anti-RNAP-I antibody and then with a colloidal gold-conjugated
secondary antibody. (B) Magnification of the region boxed in (A) where clusters of gold particles were
observed. The arrow in (C) indicates the gold particles found on the electron-dense materials in the
nuclear envelope lumen. N, nucleus; V, vacuole.
peripheral ER were stained (Figure 7A). In
addition to such staining, cytoplasmic regions were
stained in the cells overproducing Äpro (Figure
7B). Because these regions seemed to be continuous with the nuclear envelope, they most likely
represent the aberrantly enlarged ER observed

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above. In addition, this result indicates that BiP is
distributed in the enlarged ER. The cytoplasmic
staining which represents the enlarged ER was also
observed with anti-RNAP-I antibody (Figure 7C).
Taken together, we concluded that Äpro builds
up large aggregates in vivo, in agreement with our
? 1997 John Wiley & Sons, Ltd
    
Figure 7. Localization of BiP and Äpro by indirect immunofluorescence. Wild-type cells harboring plasmid pYPR3831X
(A) or pYPR3841 (B and C) were grown as in Figure 4, then
fixed and stained with anti-BiP antibody (A and B) or antiRNAP-I antibody (C), and then the fluorescein isothiocyanateconjugated secondary antibody.
biochemical data, and that the aggregates are
present in the aberrantly proliferated membranous
structures which derived from the ER as well as in
the nuclear envelope lumen.
DISCUSSION
In this paper, we have demonstrated that the
accumulation of Äpro in the ER caused elevated
synthesis of BiP and PDI, indicating that Äpro is
recognized as an unfolded protein species in the
ER. In addition, our biochemical and morphological analyses have demonstrated that Äpro built up
large aggregates associated with BiP in the ER and
nuclear envelope, and that the presence of the
Äpro aggregates led to the remarkable alteration in
the morphologies of the ER and nucleus.
When Äpro was moderately produced by the
GAPDH promoter, marked enlargement of
the ER/nuclear envelope was not observed by
immunofluorescence staining of Äpro nor PDI
(Fukuda et al., 1996). Assuming that Äpro tends to
aggregate, overproduction of Äpro by the GAL1
promoter might result in grosser aggregates of
? 1997 John Wiley & Sons, Ltd
1017
Äpro that enlarged the ER and nuclear envelope.
Such morphological changes are of interest in view
of organelle biogenesis and integrity.
First, overproduction of Äpro induced aberrant
proliferation of the membrane probably derived
from the ER. Proliferation of the yeast ER membrane is known to be caused by the overproduction
of ER membrane proteins, such as HMG-CoA
reductase (Wright et al., 1988), cytochrome b5
(Vergères et al., 1993), cytochrome P450 (Schunck
et al., 1991; Ohkuma et al., 1995), and Sec12p
(Nishikawa et al., 1994). Our results presented here
differ from these previous observations in that
Äpro is completely translocated into the ER lumen
(Figures 5 and 6 in this article and Fukuda et al.,
1994), demonstrating that the accumulation of
insoluble aggregates in the ER lumen can also
induce membrane proliferation.
Second, Äpro built up large aggregates that
were visible as electron-dense regions in the ER
and nuclear envelope. Localization of the Äpro
aggregates to both the ER and nuclear envelope
is in agreement with other ER resident proteins
(Preuss et al., 1991; Strambio-de-Castillia et al.,
1995). As in the cases of RBs and ICGs, the ER
enlarged in response to the presence of the Äpro
aggregates. It should be noted that like cells containing RBs, yeast cells were mitotic even when
Äpro was overproduced (K. Umebayashi, unpublished observation). This suggests that the secretory function of the ER was not grossly impaired
in spite of the presence of the large, insoluble Äpro
aggregates. As reported for RBs (Valetti et al.,
1991), formation of insoluble protein aggregates
may have the good fortune not to inhibit the ER
secretory function, because the aggregates are
sorted away from the functional ER. Thus, yeast
and mammals may share the conserved response to
the accumulation of insoluble protein aggregates
in the ER, that is, dilation of the ER and sorting of
the aggregates. Therefore, we regard the Äpro
aggregate as a yeast counterpart for RB. However,
there are some differences between the Äpro aggregates and its mammalian counterparts. While RBs
are reported to be devoid of ER resident proteins
(Valetti et al., 1991), we have shown that BiP is
included in the Äpro aggregates. This may reflect
the binding specificity of BiP with the misfolded
proteins. Alternatively, the sorting mechanism of
misfolded protein aggregates may not be completely the same between yeast and mammals.
Another difference is that while ICGs are known
to be degraded by autophagy (Tooze et al., 1990),

. 13: 1009–1020 (1997)
.   .
1018
Äpro seems not to be degraded in the vacuoles.
Äpro was not localized in the vacuoles in vacuolar
proteinase-deficient pep4prb1 cells (Fukuda et al.,
1996). Moreover, in our present study, sequestration of the enlarged ER containing the Äpro
aggregates into the vacuoles was not observed by
electron microscopy. It is expected that further
studies will reveal the components that participate
in the degradation of Äpro in the ER.
Another protein body, called the ‘BiP body’,
has also been found in the yeast ER (Nishikawa
et al., 1994). We suspect, however, that the Äpro
aggregates are different from the BiP bodies for
the following reasons. As shown in Figure 7,
immunofluorescence BiP staining of the cells containing the Äpro aggregates does not exhibit the
bright dots pattern which is typical for the cells
containing the BiP bodies (Nishikawa et al.,
1994). Moreover, formation of the BiP bodies
occurred when ER-to-Golgi transport was
blocked, not when unfolded proteins accumulated
in the ER and BiP synthesis was consequently
induced by the sec53 mutation (Nishikawa et al.,
1994).
It remains to be determined what components
are involved in sensing the accumulation of unfolded proteins in the ER and in transducing the
signal to Ern1p. Currently, it is thought that free
BiP level in the ER is somehow monitored (Kohno
et al., 1993). That is, when BiP is sequestered by
unfolded proteins, the resulting decrease of free
BiP level in the ER finally causes the stress
response. In combination with this model and our
result shown in Figure 4, we speculate the reason
why the accumulation of Äpro in the ER caused
the stress response; association of BiP with the
insoluble Äpro aggregates led to the reduction of
free, soluble BiP level.
Recent studies have revealed that a 22 bp unfolded protein response (UPR) element in the
promoter region of the KAR2 gene is necessary
and sufficient for the induction of KAR2 mRNA in
response to unfolded proteins (Kohno et al., 1993;
Mori et al., 1992). In addition to the KAR2 gene,
the EUG1 gene (Tachibana and Stevens, 1992) and
the SCJ1 gene, whose gene product is a yeast DnaJ
homologue located in the ER and interacts with
BiP (Schlenstedt et al., 1995), are shown to carry
the UPR elements. Therefore, it is quite possible
that the synthesis of Eug1p, Scj1p and other yet
unidentified yeast ER chaperones is also induced
by the overproduction of Äpro. However, all
species of the induced chaperones may not tightly

. 13: 1009–1020 (1997)
associate with the Äpro aggregates. As shown in
the cases of BiP and PDI, some may associate
stably while others may not. What determines the
selectivity of the chaperone species that bind to the
Äpro aggregates? As for BiP, it was shown that
BiP preferentially binds to hydrophobic peptides,
suggesting that unfolded proteins in an extended
conformation are good substrates for BiP (BlondElguindi et al., 1993). From this point of view, we
assume that Äpro intermolecularly aggregated
with its hydrophobic regions to be recognized by
BiP. Such hydrophobic regions would be buried
inside the properly folded protein, but exposed in a
misfolded conformation due to the absence of the
prosequence. We do not know at present the
reason why PDI did not bind to the Äpro aggregates. We found that Äpro proteins in the aggregates were not intermolecularly linked by disulfide
bonds because Äpro was detected on electrophoresis at its monomer position even under nonreducing conditions (K. Umebayashi, unpublished
observation). This may be the reason considering
that the function of PDI is to catalyse the isomerization of disulfide bonds, however, PDI was also
shown to act like a chaperone in the refolding of
GAPDH, a protein with no disulfide bonds (Cai
et al., 1994).
In conclusion, the altered ER and nuclear
envelope structures that were induced along with
misfolded secretory protein aggregates have been
first depicted in yeast. In addition, the findings
presented here further strengthen our previous
notion on the importance of the prosequence in
proper folding and secretion of RNAP-I. Since the
molecular process of ER dilation in response to
the accumulation of misfolded protein aggregates
is not well understood in mammalian cells, it is
expected that this issue will be revealed by using
excellent yeast genetic and biochemical systems.
Furthermore, studies using yeast systems will provide novel insight into how these misfolded protein
aggregates are degraded in the ER.
ACKNOWLEDGEMENTS
We wish to thank Masao Tokunaga for anti-BiP
antibody, Takemitsu Mizunaga and Hiroyuki
Tachikawa for anti-PDI antibody and helpful
comments on the manuscript, Kenji Kohno for the
KAR2 gene, and Akihiko Nakano for helpful
discussion about the BiP bodies. We also thank the
members of the Takagi Lab for their helpful
discussions and support. This work was partly
? 1997 John Wiley & Sons, Ltd
    
performed using the facilities of the Biotechnology
Research Center, The University of Tokyo.
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