The Rockefeller University Press $30.00
J. Cell Biol. Vol. 187 No. 4 525–536
Correspondence to Sebastian Schuck: firstname.lastname@example.org
Abbreviations used in this paper: CV, coefficient of variation; ERAD, ER-associated
degradation; IE, index of expansion; UPR, unfolded protein response.
Eukaryotic cells contain a rich variety of membrane-bound
organelles. Each organelle has a unique set of functions and
is distinguished by a characteristic morphology. As the need
for certain cellular functions changes during growth, differen-
tiation, or disease, cells adjust the amounts, compositions, and
shapes of their organelles accordingly. To make these adjust-
ments, cells need to be able to sense an imbalance between
demand and capacity for a particular function and restore
homeostasis by rearranging, synthesizing, or degrading organelle
components. Molecular mechanisms underlying such homeo-
static regulation have been discovered, but how they coordinate
the comprehensive remodeling of entire organelles is only par-
tially understood (Haynes et al., 2007; Ron and Walter, 2007;
Sardiello et al., 2009).
The ER is a large, continuous membrane system. It is re-
sponsible for the folding of all proteins that enter the secretory
pathway and is the main site of lipid biosynthesis. The ER con-
sists of the perinuclear ER, which constitutes the nuclear enve-
lope, and the peripheral ER, which extends throughout the
cytoplasm (Voeltz et al., 2002; Borgese et al., 2006; Shibata
et al., 2006). The perinuclear ER is a closed membrane sheet (or
cisterna), whereas the peripheral ER is a network of sheets and
tubules. Sheets are typically decorated with ribosomes, whereas
tubules are mostly ribosome free. ER tubules are formed by the
action of reticulon and reticulon-like proteins (Voeltz et al.,
2006). These morphogenic proteins contain reticulon domains,
which fold into hydrophobic hairpin structures and insert into
the cytoplasmic leaflet of the ER membrane. By means of their
unusual mode of membrane association and their ability to
oligomerize, reticulons tubulate membranes (Hu et al., 2008;
Shibata et al., 2008). Reticulons thus localize to ER tubules as
they generate them. Morphogenic proteins that shape ER sheets
are not known, but ribosome binding to the ER membrane may
stabilize sheets (Shibata et al., 2006; Puhka et al., 2007).
ER size and shape can change dramatically (Federovitch
et al., 2005; Borgese et al., 2006). Perhaps the most impressive
example of the great plasticity of the ER is observed during the
differentiation of B lymphocytes into plasma cells, which syn-
thesize, fold, and secrete their own weight in antibodies every
day. To cope with this enormous folding load, differentiating
lymphocytes drastically increase their levels of ER chaper-
ones (van Anken et al., 2003). Concomitantly, they massively
expand their ER membrane, leading to a more than threefold
pansion during the unfolded protein response (UPR) in the
yeast Saccharomyces cerevisiae. We find that membrane
expansion occurs through the generation of ER sheets, re-
quires UPR signaling, and is driven by lipid biosynthesis.
Uncoupling ER size control and the UPR reveals that mem-
ells constantly adjust the sizes and shapes of their
organelles according to need. In this study, we ex-
amine endoplasmic reticulum (ER) membrane ex-
brane expansion alleviates ER stress independently of an
increase in ER chaperone levels. Converting the sheets of
the expanded ER into tubules by reticulon overexpression
does not affect the ability of cells to cope with ER stress,
showing that ER size rather than shape is the key factor.
Thus, increasing ER size through membrane synthesis is
an integral yet distinct part of the cellular program to
overcome ER stress.
Membrane expansion alleviates endoplasmic
reticulum stress independently of the unfolded
Sebastian Schuck,1,2 William A. Prinz,3 Kurt S. Thorn,2 Christiane Voss,3 and Peter Walter1,2
1Howard Hughes Medical Institute and 2Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158
3Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
© 2009 Schuck et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 187 • NUMBER 4 • 2009 526
to house newly synthesized ER-resident folding machinery.
However, these notions have not been tested experimentally.
In this study, we investigate ER biogenesis in response to
acute ER stress in the budding yeast Saccharomyces cerevisiae
to address the role of UPR signaling in mediating ER mem-
brane expansion, define the pathways involved in synthesizing
new ER membrane, and gain insight into the physiological sig-
nificance of the resulting increase in ER size.
To visualize the yeast ER, we fluorescently labeled Sec63, an
abundant ER transmembrane protein that localizes to both sheets
and tubules and has been used extensively as an ER marker
(Prinz et al., 2000; Voeltz et al., 2006). We generated cells in
which a functional Sec63-GFP fusion protein replaced the en-
dogenous Sec63. Optical sections through the middle of these
cells showed the evenly labeled nuclear envelope and the pe-
ripheral ER, which in yeast lies just underneath the plasma
membrane (Fig. 1 A, left). Peripheral and perinuclear ER are
connected by a small number of tubules, which were only occa-
sionally captured in single optical sections. In midsections, the
peripheral ER appeared as a dotted line because its tubular net-
work is seen in cross sections (Fig. 1 A, top). Its netlike mor-
phology was more evident in cortical sections (Fig. 1 A, bottom).
We estimate that 40% of the cytoplasmic face of the plasma
membrane is covered with ER (see Materials and methods).
ER stress induces ER expansion through
the generation of membrane sheets
Exposure of cells to ER stress by treatment with DTT, which
prevents disulfide bond formation, or tunicamycin, which in-
hibits protein glycosylation, caused massive ER expansion
(Fig. 1 A, right). In midsections, the Sec63-GFP signal along
the cell periphery had a more continuous appearance. Corti-
cal sections showed that this reflected the generation of large
membrane sheets so that the expanded peripheral ER covered
85% of the plasma membrane. In addition, extensions of
the peripheral ER into the cytoplasm were frequently seen,
whereas the nuclear envelope retained its size and shape.
Using electron microscopy, we have shown previously that
the membrane area of the peripheral ER (including cytoplasmic
extensions) increases more than threefold during DTT treatment,
whereas ER volume increases approximately fivefold (Bernales
et al., 2006b). To quantify expansion of the peripheral ER from
light microscopy images, we exploited the fact that Sec63-GFP,
as seen in midsections, became more evenly distributed along the
cell cortex as the expanding ER covered an increasing portion
of the plasma membrane. We determined an index of expansion
(IE) by measuring the variation of the cortical Sec63-GFP signal
and normalizing it to the variation of the perinuclear Sec63-GFP
signal, which represents maximally expanded ER (see Materials
and methods). The resulting index does not provide an absolute
measure for overall ER membrane area or volume but proved
to be a sensitive and reproducible metric for the characteristic
spreading of the cortical Sec63-GFP signal during ER expansion.
Expansion occurred within 1 h of DTT treatment and reached
increase in ER volume (Wiest et al., 1990). Similarly, induc-
tion of the ER-localized cytochrome P450 detoxification sys-
tem in hepatocytes leads to a pronounced expansion of the
ER membrane, which forms tightly packed whorls (Feldman
et al., 1981). In more artificial settings, ectopic expression of
ER transmembrane proteins in both yeast and mammalian cells
frequently produces ordered arrays of unusually shaped ex-
panded ER (Anderson et al., 1983; Wright et al., 1988; Snapp
et al., 2003; Lingwood et al., 2009).
The main signaling pathway controlling ER homeostasis
is the unfolded protein response (UPR), whose basic features
are conserved from yeast to humans (Bernales et al., 2006a;
Ron and Walter, 2007). All eukaryotes possess IRE1, which acts
as a sensor for protein-folding problems in the ER; metazoans
have two additional sensors, PKR-like ER kinase and ATF6.
When misfolded proteins accumulate in the ER, which signals
that the folding capacity of the ER is exceeded and constitutes a
condition called ER stress, IRE1 is activated. In turn, IRE1 ac-
tivates a transcription factor called Hac1 in yeast and XBP1 in
metazoans that induces a large number of genes encoding parts
of the ER-resident folding machinery. Import of these gene
products into the ER augments the organelle’s folding capacity.
In addition, the UPR activates related functionalities such as
ER-associated degradation (ERAD) and lipid biosynthesis (Cox
et al., 1997; Travers et al., 2000). ERAD is responsible for the
retrotranslocation of terminally misfolded proteins from the ER
into the cytoplasm for proteasome-mediated degradation (Vembar
and Brodsky, 2008). Without the UPR, cells cannot adjust their
levels of ER chaperones according to need and are unable to
maintain ER homeostasis. Knockout of IRE1 or XBP1 is le-
thal in mice, XBP1-deficient B lymphocytes fail to develop into
plasma cells, and yeast lacking Ire1 or Hac1 are hypersensitive
to ER stress (Cox et al., 1993; Mori et al., 1993, 1996; Cox and
Walter, 1996; Reimold et al., 2000, 2001; Urano et al., 2000).
The role of the UPR in regulating the amount of ER mem-
brane is less clear. Expression of active XBP1 stimulates lipid
biosynthesis and enlarges the ER in fibroblasts and B lympho-
cytes (Shaffer et al., 2004; Sriburi et al., 2004). Conversely,
XBP1 deficiency impairs the characteristic ER membrane ex-
pansion during the development of specialized secretory cells
(Reimold et al., 2001; Lee et al., 2005). However, it is unknown
whether removal of XBP1 abolishes the ability of these cells
to expand their ER or prevents them from reaching the stage
of development at which ER expansion would normally take
place. In addition, experiments using UPR-deficient yeast have
yielded conflicting results as to whether the UPR is needed for
ER expansion upon overexpression of ER transmembrane pro-
teins (Cox et al., 1997; Menzel et al., 1997; Takewaka et al.,
1999; Larson et al., 2002). Moreover, the physiological role of
ER membrane expansion has not been explored. The ER of spe-
cialized secretory cells has to process an unusually large amount
of cargo, and ER stress in any cell type increases cargo load as
proteins stay longer in the ER before they are correctly folded
or degraded. It seems reasonable that a larger ER is needed
under these circumstances to accommodate increased amounts of
ER client proteins. In addition, membrane expansion during ER
stress may occur concomitantly with UPR target gene activation
527ER membrane expansion • Schuck et al.
the absence of ER stressors (unpublished data). In contrast,
DTT-induced ER membrane expansion was strongly impaired
(Fig. 2 A). Quantification showed that expansion of the pe-
ripheral ER was normal up to 1 h of treatment but then stalled
(Fig. 2 B). In addition, large ER patches formed in the prox-
imity of the nucleus and at the cell periphery. Colocalization
with Rtn1-Cherry and electron microscopy revealed that these
patches are tangles of irregularly shaped, ribosome-free ER
(Fig. 2, C and D). Given that the membrane elements in these
tangles cannot be resolved by light microscopy, we could not
include them in the quantification of ER membrane expansion.
Therefore, it is not possible to determine quantitatively to what
extent peripheral and total ER membrane amounts are reduced
in DTT-treated hac1 and ire1 mutants. Nevertheless, the ER
patches clearly show that cells cannot generate morphologically
normal expanded ER without UPR signaling.
Proper ER membrane expansion requires
the Ino2/4 complex
Next, we investigated the role of lipid biosynthesis in ER mem-
brane expansion. In yeast, many phospholipid synthesis enzymes
are controlled at the transcriptional level by two transcription
factors, Ino2 and Ino4, which form a heterodimer and require
each other to function (Ambroziak and Henry, 1994; Schwank
et al., 1995). We first analyzed the expression of the Ino2/4 target
gene OPI3, which is involved in phosphatidylcholine synthesis.
its maximum after 2 h (Fig. 1 B and Fig. S1). Tunicamycin-
induced expansion was slower and less pronounced. Because
of its more rapid and stronger effects, we mostly used DTT in
To further characterize the morphology of the expanded ER,
we used the reticulon Rtn1 as an additional marker. As expected,
Rtn1-Cherry localized mainly to the peripheral ER in untreated
cells (Fig. 1 C). After DTT treatment, Rtn1-Cherry remained as-
sociated with the peripheral ER. Notably, it was largely excluded
from cytoplasmic ER extensions, suggesting that these structures
are sheets. Using electron microscopy, we confirmed the expansion
of peripheral ER underlying the plasma membrane (Fig. S2) and
followed ribosome-studded cytoplasmic ER extensions through
sequential serial sections for >300 nm and sometimes up to 800 nm
(Fig. 1 D). This demonstrates that the cytoplasmic strands seen in
single sections are not tubules, which are <100 nm in diameter
(Voeltz et al., 2002) but part of large ER sheets. The magnitude of
ER expansion suggests that it involves the synthesis of new mem-
brane, for which we provide further evidence below (see Proper
ER membrane expansion requires the Ino2/4 complex).
Proper ER membrane expansion
requires UPR signaling
To determine the contribution of UPR signaling to ER mem-
brane expansion, we analyzed hac1 and ire1 mutants express-
ing Sec63-GFP. These cells have a normal ER morphology in
Figure 1. ER stress induces ER membrane
expansion. (A) Sec63-GFP cells (SSY139) un-
treated or treated with DTT or tunicamycin for
2 h. Both ER stressors induce expansion of the
peripheral ER and appearance of cytoplasmic
ER extensions (arrows). (B) Quantification of
ER expansion from images obtained as in A.
The IE was determined as described in Ma-
terials and methods. Statistical significance
compared with t = 0 of P ≤ 102 (*) and
P ≤ 106 (***) is shown. Error bars indi-
cate SEM. (C) Cytoplasmic ER extensions in-
duced by ER stress exclude the reticulon Rtn1.
Sec63-GFP Rtn1-Cherry cells (SSY421) un-
treated or treated with DTT for 2 h. The cyto-
plasmic extensions in DTT-treated cells (arrows)
are almost devoid of Rtn1. (D) Cytoplasmic
ER extensions induced by ER stress are large
membrane sheets. Electron micrographs of
wild-type cells (SSY139) treated with DTT for
2 h. (top) Low magnification images of cells
with cytoplasmic ER extensions (arrows) are
shown. (bottom) Sequential 50-nm sections are
shown at a higher magnification correspond-
ing to the boxed area (top). The 0-nm image
is the same as that shown in the top panel.
The ER membrane is traced in blue. The ER
sheet shown extends for at least 350 nm in
the z direction. M, mitochondrion; N, nucleus;
V, vacuole. Bars: (A and C) 2 µm; (D) 250 nm.
JCB • VOLUME 187 • NUMBER 4 • 2009 528
requires the Ino2/4 complex, likely because it is needed for the
induction of lipid synthesis genes. In addition, these results
help explain the membrane expansion defects in hac1 and
ire1 mutants: without UPR signaling, the induction of lipid
synthesis genes through activation of Ino2/4 is reduced, likely
resulting in an insufficient supply of lipids to support full ER
ER membrane expansion is driven by
The identification of the Ino2/4 complex as an important
player in regulating ER size enabled us to test whether ac-
tivation of Ino2/4 is not only necessary but also sufficient
for ER membrane expansion. When lipids are plentiful, the
Ino2/4 complex is inhibited by Opi1, which binds to Ino2
(Fig. 4 A). When more lipids are needed, Opi1 dissociates
from Ino2, allowing Ino2/4 to activate its target genes (Carman
and Henry, 2007). To activate Ino2/4 constitutively, we
either deleted OPI1 or deleted INO2 and provided mutant
ino2(L119A) on a plasmid. The ino2(L119A) mutant protein
cannot be bound by Opi1 and is always active (Heyken et al.,
2005). Both approaches resulted in expansion of the periph-
eral ER (Fig. 4, B and C). The size of the nuclear envelope
remained unchanged, as observed previously (O’Hara et al.,
2006). ER expansion by deletion of OPI1 required both INO2
and INO4, confirming that opi1 mutants have an expanded
ER because the inhibition of Ino2/4 is relieved (Fig. S3). In
addition, constitutive ER expansion in opi1 mutants was ob-
served in both lipid-free medium and lipid-containing YPD
medium (unpublished data). Like DTT-treated wild-type cells,
DTT treatment caused a substantial induction of Opi3 pro-
tein levels (Fig. 3, A and B) in line with the previously
reported induction of OPI3 mRNA levels during the UPR
(Travers et al., 2000). In contrast, the increase in Opi3 protein
levels was strongly reduced in hac1 or ino2 mutants. The same
pattern was observed for INO1, an Ino2/4 target gene involved
in phosphatidylinositol synthesis (unpublished data). Thus, ER
stress activates Ino2/4-dependent expression of lipid synthesis
genes through UPR signaling. We then tested whether ER mem-
brane expansion required the Ino2/4 complex. DTT-induced
expansion was diminished in both ino2 and ino4 mutants, the
peripheral ER retained its dotted appearance in midsections,
and cytoplasmic ER extensions did not form (Fig. 3 C). In addi-
tion, aberrant ER patches were observed occasionally. Whether
these are related to the patches seen in DTT-treated hac1 and
ire1 mutants remains to be determined, but their appearance
emphasized that proper ER membrane expansion fails in the
absence of Ino2/4. Quantification confirmed this conclusion and
additionally showed that untreated ino2 and ino4 mutants had
a smaller ER than wild-type cells (Fig. 3 D), underscoring the
role of Ino2/4 in ER size control.
If the Ino2/4 complex is important for ER membrane
expansion because it regulates lipid biosynthesis, it may be
possible to bypass the need for Ino2/4 by providing lipids in
the growth medium. Therefore, we tested DTT-induced ER
expansion of ino2 and ino4 mutants kept in YPD, a rich me-
dium that includes lipids. Under this experimental regimen,
ER expansion no longer required Ino2 or Ino4, and the defect
in basal ER size was rescued (Fig. 3, E and F). Thus, unless
lipids are supplied exogenously, full ER membrane expansion
Figure 2. Proper ER membrane expan-
sion requires UPR signaling. (A) Wild-type,
hac1∆, and ire1∆ cells expressing Sec63-
GFP (SSY139, SSY161, and SSY467) treated
with DTT for 2 h. Expansion of the peripheral
ER is impaired in UPR mutants, and aberrant
cytoplasmic ER patches form at the cell pe-
riphery and adjacent to the nucleus (arrows).
(B) Quantification of ER expansion from
images was obtained as in A. Statistical sig-
nificance compared with the wild type at the
same time point of P ≤ 106 (***) is shown.
Error bars indicate SEM. (C) Sec63-GFP
Rtn1-Cherry hac1∆ cells (SSY429) untreated
or treated with DTT for 2 h. The cytoplasmic
ER patches in DTT-treated cells contain both
Sec63 and Rtn1. (D) Cytoplasmic ER patches
induced by ER stress in hac1∆ cells are tan-
gles of smooth ER. Electron micrographs of
hac1∆ cells (SSY161) treated with DTT for
2 h. (top) Low magnification images of hac1∆
cells with cytoplasmic ER patches (arrows),
which are found either at the cell periphery or
close to the nucleus, are shown. (bottom) Se-
quential 50-nm sections are shown at a higher
magnification corresponding to the boxed
area (top). The 0-nm image is the same as
shown in the top panel. The ER tangle shown
consists of numerous randomly arranged
ribosome-free elements. N, nucleus; V, vacuole.
Bars: (A and C) 2 µm; (D) 250 nm.
529ER membrane expansion • Schuck et al.
ER membrane expansion alleviates
During a normal UPR in wild-type cells, ER membrane expan-
sion goes hand in hand with an increase in the levels of ER
chaperones. Accordingly, the protein levels of the most abun-
dant ER chaperone Kar2, the Kar2-related chaperone Lhs1, and
the essential protein disulfide isomerase Pdi1 increased upon
opi1 mutants and ino2(L119A)-expressing cells displayed
ribosome-studded cytoplasmic ER extensions, which electron
microscopy showed to be large ER sheets (Fig. 4 D and not
depicted). These results indicate that the activation of lipid
biosynthesis by Ino2/4 drives ER membrane expansion and
produces an ER morphology closely resembling that gener-
ated after ER stress.
Figure 3. Proper ER membrane expansion
requires the Ino2/4 complex. (A) ER stress in-
creases the levels of lipid synthesis enzymes.
Western blot of HA tag from Opi3-HA–
expressing wild-type, hac1∆, and ino2∆ cells
(SSY477, SSY485, and SSY484) treated with
DTT for up to 3 h. Pgk1 served as a loading
control. (B) Quantification of Opi3 levels nor-
malized to Pgk1 from Western blots obtained
as in A. Error bars indicate SEM from three
independent experiments. (C) ER expansion
requires both Ino2 and Ino4. Sec63-GFP–
expressing wild-type, ino2∆, and ino4∆ cells
(SSY139, SSY369, and SSY460) treated with
DTT for 2 h. (D) Quantification of ER expan-
sion from images obtained as in C. Statistical
significance compared with wild-type cells of
P ≤ 104 (**) and P ≤ 106 (***) is shown.
Error bars indicate SEM. (E) The requirement for
Ino2 and Ino4 is bypassed in lipid-containing
medium. Wild-type, ino2∆, and ino4∆ cells
(SSY139, SSY369, and SSY460) treated with
DTT for 2 h in YPD medium. Normal ER expan-
sion is observed in all three strains. (F) Quanti-
fication of ER expansion from images obtained
as in E. Neither ino2∆ nor ino4∆ cells show
statistically significant differences compared
with wild-type cells. Expansion in wild-type cells
is less pronounced here because of the lower
effectiveness of DTT in YPD than in SC medium.
Error bars indicate SEM. Bars, 2 µm.
Figure 4. ER membrane expansion is driven
by Ino2/4 activity. (A) Schematic depiction of
the negative regulation of Ino2/4 by Opi1.
(B) Activation of Ino2/4 results in constitutive
ER expansion. Untreated wild-type, opi1∆,
and ino2(L119A) cells expressing Sec63-GFP
(SSY433, SSY290, and SSY400). Bar, 2 µm.
(C) Quantification of ER expansion from images
obtained as in A. Statistical significance com-
pared with wild-type cells of P ≤ 106 (***) is
shown. Error bars indicate SEM. (D) Activation
of Ino2/4 produces expanded ER morphologi-
cally similar to that generated after ER stress.
Electron micrographs of untreated opi1∆ cells
(SSY290) are shown. (left) A low magnifica-
tion image is shown. (right) Sequential 50-nm
sections are shown at a higher magnification
corresponding to the boxed area. The 0-nm
image is the same as that shown in the low
magnification image. The ER sheet shown ex-
tends for at least 350 nm in the z direction.
LD, lipid droplet; V, vacuole. Bars, 500 nm.
JCB • VOLUME 187 • NUMBER 4 • 2009 530
DTT treatment (Fig. 5, A and B). Strikingly, this increase was
completely lacking in OPI1-deficient or ino2(L119A)-expressing
cells (Fig. 5, C and D). Thus, these cells have a dilute ER with
basal chaperone levels but an expanded ER membrane and volume.
This finding shows that activation of Ino2/4 uncouples mem-
brane expansion from an increase in ER chaperone levels.
In addition, ER membrane expansion by deletion of OPI1
or expression of ino2(L119A) still occurred in hac1 mutants
(Fig. 5, E and F), showing that membrane expansion can occur
independently of UPR signaling.
The ability to experimentally uncouple ER membrane ex-
pansion and chaperone induction allowed us to address the phys-
iological role of ER size regulation. We assessed the sensitivity
of cells to ER stress by growing them on plates containing sub-
lethal concentrations of tunicamycin for 2–3 d. For growth on
plates, tunicamycin is preferable over DTT, which is quickly ren-
dered inactive as a result of oxidation by air. To directly compare
various strains with very different tunicamycin sensitivity, we
chose a relatively low tunicamycin concentration that even al-
lowed some growth of hac1 mutants. As expected, hac1 mutants,
which cannot properly expand their ER membrane or raise their
chaperone levels in response to ER stress, showed hypersensitiv-
ity to tunicamycin. opi1 mutants were indistinguishable from
wild-type cells. Revealingly, hac1∆ opi1∆ cells tolerated ER
stress much better than hac1∆ cells (Fig. 6 A). Similarly, over-
expression of Ino2 and especially Opi1-insensitive ino2(L119A)
enhanced ER stress tolerance of hac1 mutants (Fig. 6 B). These
results indicate that enlarging the ER alleviates ER stress inde-
pendently of an increase in chaperone levels.
If ER membrane expansion were indeed important for
cells to withstand ER stress, deletion of INO2 should render
them hypersensitive to ER stressors. Consistent with this predic-
tion, ino2 mutants grew more slowly in the presence of tunica-
mycin than wild-type cells (Fig. 6 C). However, ino2 mutants
showed an obvious growth defect only at relatively high tunica-
mycin concentrations. An explanation for this phenotype is pro-
vided by the observation that hac1∆ ino2∆ cells already grew
slowly in the absence of tunicamycin (not depicted) and were
completely unable to grow at low tunicamycin concentrations
(Fig. 6 D). Thus, ino2 mutants can resist considerable ER stress
with the help of the UPR but cannot overcome even mild stress
when additionally deprived of HAC1. Furthermore, ino2 mu-
tants grown for an extended period of time in lipid-free medium
had elevated levels of ER chaperones even in the absence of ex-
ternal ER stressors (Fig. 6 E). Thus, cells compensate defective
ER size control after deletion of INO2 by raising ER chaperone
levels. If they are unable to do so, as in the case of hac1∆ ino2∆
cells, they become exquisitely sensitive to ER stress.
ER membrane expansion alleviates stress
as a result of increased ER size rather
than altered ER shape
Finally, to extend the morphological analysis of ER expansion,
we sought to understand the transition from a tubular to a cister-
nal peripheral ER. We focused on the reticulons, which are the
morphogenic proteins responsible for tubule formation. During
expansion, Rtn1 retained its localization to the peripheral ER
Figure 5. ER membrane expansion can be uncoupled from the UPR.
(A) ER stress induces the levels of ER protein folding enzymes. Western
blot of Kar2, Lhs1, and Pdi1 from wild-type cells (SSY139) treated with
DTT for up to 4 h. Pgk1 served as a loading control. (B) Quantifica-
tion of Western blotting shown in A with values normalized to Pgk1.
(C) The levels of ER protein–folding enzymes are unchanged by activa-
tion of Ino2/4. Western blot of Kar2, Lhs1, and Pdi1 from untreated
wild-type, opi1∆, and ino2(L119A)-expressing cells (SSY433, SSY290,
and SSY400). (D) Quantification of Western blots obtained as in C
with values normalized to Pgk1. Error bars indicate SEM from three
independent experiments. (E) ER expansion by activation of Ino2/4
can occur independently of UPR signaling. Untreated hac1∆, hac1∆
opi1∆, and hac1∆ ino2(L119A) cells expressing Sec63-GFP (SSY434,
SSY364, and SSY441). Bar, 2 µm. (F) Quantification of ER expansion
from images obtained as in E. Statistical significance compared with
hac1∆ cells of P ≤ 104 (**) and P ≤ 106 (***) is shown. Error bars
531ER membrane expansion • Schuck et al.
physiology of ER membrane expansion by dissociating it not
only from an increase in ER chaperone levels but also from
changes in ER shape. hac1∆ opi1∆ cells, which have an ex-
panded ER but no UPR, were more resistant to tunicamycin than
hac1∆ cells, and this increased tunicamycin resistance was un-
affected by Rtn1 overexpression (Fig. 7 E). Therefore, under
these conditions, ER size rather than shape was important for
cells to overcome ER stress.
Our results show that membrane expansion in response to ER
stress involves the generation of large ER sheets, is restricted to
the peripheral ER, and is impaired by disruption of the UPR. ER
stress also induces lipid synthesis enzymes through the UPR
and the Ino2/4 transcription factor complex. In the absence of
Ino2/4, stress-induced membrane expansion is diminished, likely
because of reduced lipid biosynthesis. Conversely, activation
of Ino2/4 causes constitutive ER membrane expansion. Impor-
tantly, membrane expansion by activation of Ino2/4 occurs
without a concomitant increase in ER chaperone levels and is
independent of Hac1, showing that ER expansion and the UPR
can be uncoupled. ER membrane expansion on its own allevi-
ates ER stress, indicating that enlarging the ER is an integral
part of an effective UPR. Furthermore, the predominantly
(Fig. 1 C). In addition, Rtn1 protein levels did not change dur-
ing ER stress or upon activation of Ino2/4 (Fig. 7, A and B).
This observation is consistent with earlier data showing con-
stant mRNA levels for all three yeast reticulon and reticulon-
like proteins under these conditions (Travers et al., 2000). Thus,
the tubulation capacity of the reticulons may remain unchanged
during ER expansion. If so, the growth of the ER membrane
may overwhelm the reticulons’ capacity to induce tubules, re-
sulting in the generation of sheets. This scenario predicts that
augmenting reticulon capacity should confer a tubular morphol-
ogy onto the expanded ER. To test this prediction, we tagged
Rtn1 with GFP to label ER tubules and additionally expressed
dsRed-HDEL, which is targeted to the ER lumen and labels
both tubules and sheets (Fig. 7 C, left). In opi1 mutants, sheets
were greatly expanded (Fig. 7 C, middle). Rtn1-GFP lined the sheet
edges, which have a high membrane curvature. Overexpression
of untagged Rtn1 in opi1 mutants led to extensive conversion of
sheets into tubules (Fig. 7 C, right). We conclude that reticulon
capacity limits the amount of tubules and determines the shape
of the expanded ER.
Quantification showed that the peripheral ER in opi1 mu-
tants remained expanded after overexpression of Rtn1 (Fig. 7 D),
despite its altered shape. Thus, combining activation of Ino2/4
and reticulon overproduction uncoupled membrane expansion
and sheet formation. This allowed us to further dissect the
Figure 6. ER membrane expansion allevi-
ates ER stress. (A) Deletion of OPI1 increases
resistance of hac1∆ cells to ER stress. Tunica-
mycin sensitivity of wild-type, hac1∆, opi1∆,
and hac1∆ opi1∆ cells (SSY139, SSY161,
SSY290, and SSY364) as assessed by plat-
ing dilution series of cells onto solid SC
medium containing 0.05 µg/ml tunicamycin.
Series represent fivefold dilutions from one
step to the next. (B) Overexpression of Ino2
and especially ino2(L119A) increases resis-
tance of hac1∆ cells to ER stress. Tunicamycin
sensitivity of wild-type, hac1∆, hac1∆ ino2∆
pINO2, and hac1∆ ino2∆ pino2(L119A) cells
(SSY433, SSY434, SSY439, and SSY441)
assessed as in A except that leucine was omit-
ted from the plates. (C) ino2 mutants show
increased sensitivity to ER stress. Tunicamy-
cin sensitivity of wild-type and ino2∆ cells
(SSY139 and SSY430) assessed as in A.
(D) ino2 mutants depend on HAC1 to over-
come even mild ER stress. Tunicamycin sensi-
tivity of wild-type, hac1∆, ino2∆, and hac1∆
ino2∆ cells (SSY139, SSY161, SSY369, and
SSY430) assessed as in A. (E) Untreated ino2
mutants show chaperone induction indicative
of constitutive UPR signaling. Western blot of
Kar2, Lhs1, and Pdi1 from untreated wild-type
and ino2∆ cells (SSY139 and SSY369) grown
for 24 h in lipid-free SC medium. Pgk1 served
as a loading control. Numbers indicate fold
induction in ino2 mutants compared with wild
type and normalized to Pgk1.
JCB • VOLUME 187 • NUMBER 4 • 2009 532
The observation that the shape of the expanded ER can be
shifted from cisternal to tubular by simple overexpression of a re-
ticulon suggests that ER shape depends on the balance between
the amount of ER membrane and the reticulons’ capacity to gen-
erate tubules. According to this view, the sheet morphology of
the expanded ER during the UPR results from membrane growth
without a corresponding increase in reticulon activity so that their
tubulation capacity is exceeded and sheets form either by default
or through the action of sheet-stabilizing proteins. A similar model
invoking limiting reticulon capacity has been proposed recently to
explain the formation of an appropriately sized nuclear envelope at
the end of mitosis (Webster et al., 2009). We note that raising re-
ticulon levels may only be a crude experimental substitute for how
their capacity is normally regulated. Reticulon capacity could be
controlled posttranslationally, perhaps by changes in oligomeriza-
tion behavior (Shibata et al., 2008). The physiological significance
of the transition from a tubular to a cisternal ER during the UPR
remains an open question. It is not obvious whether the tubule to
sheet conversion contributes to the up to fivefold increase in ER
volume, and accurate measurements of the dimensions of sheets
and tubules before and after ER stress by electron tomography are
likely needed to answer this question. Also, it is unknown whether
sheets and tubules have different functions relevant for mitigating
ER stress. In any event, forcing a tubular morphology onto the ex-
panded ER by reticulon overexpression did not affect membrane
expansion or sensitivity to ER stress. Therefore, the main benefit of
ER remodeling during the UPR appears to lie in the increase in ER
size rather than the conversion of tubules into sheets.
cisternal expanded ER is converted to mainly tubular by over-
expression of Rtn1, suggesting that ER shape is determined by
reticulon capacity. However, changing the ratio of tubules to
sheets does not affect the alleviation of ER stress by membrane
expansion. These findings reveal an important role of ER size
control in the maintenance of ER homeostasis.
The discovery that a larger ER alone alleviates ER stress ar-
gues that the role of membrane expansion during a normal UPR,
when ER size and chaperone levels increase simultaneously, goes
beyond merely providing space to accommodate newly synthe-
sized folding machinery. One possibility is that a larger ER can
tolerate more misfolded proteins before essential functions break
down. Moreover, a larger ER could promote protein folding itself.
During folding, proteins expose hydrophobic residues that are
buried in their final conformations. This makes folding intermedi-
ates vulnerable to aggregation should they encounter one another.
Increasing ER volume lowers the concentration of folding inter-
mediates, which may give proteins more time to fold by avoiding
aggregate formation. This idea is consistent with in vitro experi-
ments, showing that the unassisted folding of proteins is more effi-
cient at low concentrations because aggregation is reduced (Apetri
and Horwich, 2008). A similar mechanism might apply for
membrane-associated proteins, whose dilution caused by an in-
crease in membrane area could help avoid detrimental interactions.
Finally, membrane-associated processes that support protein fold-
ing or remove misfolded proteins, such as protein glycosylation or
ERAD, could operate more efficiently when a larger membrane area
is available. None of these possibilities are mutually exclusive.
Figure 7. ER membrane expansion alleviates
stress as a result of increased ER size rather
than altered ER shape. (A) Rtn1 levels are un-
changed by ER stress or activation of Ino2/4.
Western blot of Cherry tag from Rtn1-Cherry–
expressing wild-type cells treated with DTT for
up to 4 h and from untreated Rtn1-Cherry–
expressing wild-type, opi1∆, and ino2(L119A)
cells (SSY421, SSY478, and SSY473). Pgk1
served as a loading control. (B) Quantifica-
tion of Western blots shown in A with values
normalized to Pgk1. (C) Overexpression of
Rtn1 converts sheets (arrows) into tubules
(arrowheads). Untreated wild-type and opi1∆
cells expressing dsRed-HDEL and Rtn1-GFP
were used to mark the entire ER lumen and ER
tubules, respectively, and carrying an empty
vector or an expression plasmid encoding un-
tagged Rtn1 (SSY523, SSY531, and SSY532).
(D) ER expansion is unaffected by Rtn1 over
expression. Quantification of ER expansion from
images of untreated wild-type, opi1∆, and
Rtn1-overexpressing opi1∆ cells (SSY523,
SSY531, and SSY532). Statistical signifi-
cance compared with wild-type cells of P ≤
106 (***) is shown. Error bars indicate SEM.
(E) Rtn1 overexpression does not affect sen-
sitivity to ER stress. Tunicamycin sensitivity of
wild-type, hac1∆, and hac1∆ opi1∆ cells car-
rying empty vectors and hac1∆ opi1∆ cells
carrying an expression plasmid encoding un-
tagged Rtn1 (SSY510, SSY533, SSY535, and
SSY536) as assessed by plating dilution series
of cells onto solid SC medium (without uracil)
containing 0.05 µg/ml tunicamycin. Series
represent fivefold dilutions from one step to
the next. Bar, 2 µm.
ER membrane expansion • Schuck et al.
Fourth, the residual expansion seen in hac1 and ire1 mutants and
the residual increase in Opi3 and Ino1 protein levels in HAC1-
deficient cells suggest that another signaling pathway exists in
yeast that can sense ER stress and induce membrane expansion.
This putative second pathway, which may correspond to the re-
cently described super–UPR pathway (Leber et al., 2004), could
also help explain why overexpression of ER transmembrane
proteins can still trigger ER expansion in IRE1-deficient yeast
(Menzel et al., 1997; Larson et al., 2002). Perhaps this alternative
pathway is sufficient to allow long-term adaptation of ER size but
is overwhelmed by the acute ER stress caused by DTT or tunica-
mycin. Finally, it is intriguing that the size of the nuclear envelope
does not change during UPR. Unlike mammalian cells, yeast do
not have nuclear lamins that could act as a scaffold to restrict nu-
clear size during ER membrane expansion. However, it has been
found that at least part of the yeast nuclear envelope can resist
expansion by an unknown mechanism (Campbell et al., 2006).
Similar to yeast, full ER expansion in mammals requires
the Hac1 homologue XBP1, but some residual expansion still
seems possible in its absence (Lee et al. 2005). This points to
additional signaling pathways that can regulate ER size, and
the ATF6 pathway has recently been suggested to play such a
role (Bommiasamy et al., 2009). Also, similar to yeast, UPR
signaling activates lipid biosynthesis in fibroblasts, and ex-
perimentally activating phosphatidylcholine synthesis leads to
ER membrane expansion without an accompanying increase
in ER chaperone levels (Sriburi et al., 2004, 2007). Although
the expansion elicited by increased phosphatidylcholine pro-
duction was modest compared with that achieved by expres-
sion of active XBP1, these results indicate that ER membrane
expansion may be driven by lipid biosynthesis also in mam-
malian cells. Because there is no known mammalian master
regulator of lipid biosynthesis analogous to the yeast Ino2/4
complex, it is difficult to test whether a more comprehensive
activation of lipid biosynthesis would recapitulate UPR-mediated
ER membrane expansion, as is the case in yeast. Nevertheless,
it would be interesting to further explore the poorly under-
stood regulation of mammalian lipid biosynthesis by the UPR
(Acosta-Alvear et al., 2007).
In summary, ER stress induces membrane expansion
through UPR-mediated activation of lipid biosynthesis, and the
subsequent increase in ER size on its own is sufficient to allevi-
ate stress. Thus, the UPR maintains ER homeostasis by two in-
timately connected but distinct mechanisms: by providing new
ER-folding machinery and by providing more ER surface area
and lumenal space.
Materials and methods
Antibodies and plasmids
The following primary antibodies were used: mouse anti-HA (Covance),
mouse anti-Pgk1 (Invitrogen), rabbit anti-Kar2 (Walter laboratory, Uni-
versity of California, San Francisco, San Francisco, CA), sheep anti-
Lhs1 (provided by C.J. Stirling, University of Manchester, Manchester,
England, UK; Tyson and Stirling, 2000), rabbit anti-Pdi1 (provided by
J. Winther, Carlsberg Laboratory, Copenhagen, Denmark), rabbit anti-
dsRed (MBL International), and rabbit anti-Sec61 (Walter laboratory).
Secondary antibodies conjugated to alkaline phosphatase were obtained
from Millipore. Centromeric expression plasmids pRS415-MET25-INO2
The appearance of tangles of smooth tubular ER in UPR-
deficient cells exposed to ER stress is intriguing. These tangles
could reflect disruption of ER structure by misfolded proteins.
Alternatively, they could arise from the lack of a sheet-stabilizing
protein. A candidate for such a protein is Sec61, which forms
the translocation channel for protein import into the ER. Sec61
is also needed for the binding of ribosomes to the ER mem-
brane, and ribosome binding has been suggested to stabilize ER
sheets (Shibata et al., 2006; Puhka et al., 2007). In addition,
Sec61 is induced by ER stress in a UPR-dependent manner
(Travers et al., 2000). However, opi1 mutants and cells express-
ing ino2(L119A) have expanded rough ER sheets despite nor-
mal Sec61 protein levels (Fig. S4), indicating that Sec61 is not
limiting for the generation of new ER sheets.
We have proposed previously that the Hac1 transcription
factor coordinates the induction of chaperone genes and membrane
biogenesis (Cox et al., 1997). The finding that Hac1-dependent
Ino2/4 activity is needed for proper ER membrane expansion
strengthens this model. In fact, the relationship between Hac1 and
Ino2/4 is remarkably similar to that between the UPR and ERAD.
The ERAD machinery operates at a basal level at all times but is
activated during ER stress by Hac1-dependent induction of ERAD
components. ERAD-deficient yeast are hypersensitive to ER stress
and show constitutive activation of the UPR. Deletion of either
IRE1 or an ERAD component is tolerated well, but combined de-
letion causes severe synthetic phenotypes (Travers et al., 2000).
Likewise, Ino2/4 activity is stimulated during ER stress through
Hac1, and ino2 mutants show increased sensitivity to tunica-
mycin, have elevated ER chaperone levels indicative of constitu-
tive UPR signaling, and display synthetic sickness upon additional
deletion of HAC1. Thus, Ino2/4 is another functional module that
is recruited by Hac1 to help cells mount an effective UPR.
Nevertheless, many questions remain concerning the cas-
cade of events that culminates in ER membrane expansion. First,
it is unclear how UPR signaling activates Ino2/4-dependent tran-
scription. A plausible mechanism is that Hac1 inhibits Opi1, thereby
derepressing Ino2/4 (Cox et al., 1997; Brickner and Walter, 2004).
There are several ways in which Hac1 could inhibit Opi1, e.g.,
by directly binding to Opi1 to promote dissociation from Ino2
or by inducing the transcription of an Opi1 inhibitor. Interest-
ingly, Opi1 translocates from the nucleus to the peripheral ER
after inositol depletion (Loewen et al., 2004) but not after DTT
treatment (unpublished data), indicating different mechanisms of
Ino2/4 derepression. Second, we do not know which Ino2/4 target
genes are critical for ER membrane expansion. Given that the re-
quirement for Ino2/4 is bypassed when lipids are provided exoge-
nously, lipid synthesis genes are probably a key. We tested several
Ino2/4-regulated lipid synthesis genes, including INO1, PSD1,
CHO2, and OPI3, but no single deletion phenocopied the ER ex-
pansion defect seen in ino2 and ino4 mutants (unpublished data).
Third, it remains to be determined whether deletions of INO2 and
INO4 are truly equivalent. It is generally accepted that neither
transcription factor can function without the other, but some gene
promoters appear to be bound by only one of the two proteins
(Lee et al., 2002), and a previous study concluded that membrane
proliferation after expression of the canine ribosome receptor in
yeast required INO2 but not INO4 (Block-Alper et al., 2002).
JCB • VOLUME 187 • NUMBER 4 • 2009 534
divided by the CV for cortical signal of cells of interest so that ER expan-
sion resulted in an increase of IE. The IE is not as intuitive as estimates of
the coverage of the plasma membrane with ER but has several advan-
tages. It is very reproducible, can be obtained without image manipula-
tion of any kind, and can be calculated from midsections, which are
easier to acquire than well-focused cortical sections. 30–40 cells were
quantified per condition, the mean of IE ± SEM was determined, and
Student’s t test was used to assess statistical significance of differences
between conditions. The MatLab script we used is provided in the Online
Cells were grown to early log phase at 30°C in 100 ml YPD, washed
with SC, and resuspended in 100 ml fresh SC. Cells were left untreated
or treated with 8 mM DTT for 2 h and processed essentially as described
previously (McDonald and Müller-Reichert, 2002; Bernales et al., 2006b).
In brief, cells were filtered and rapidly frozen using a high pressure freezer
(EM PACT; Leica). Samples were transferred onto frozen fixative (1% os-
mium tetroxide, 0.1% uranyl acetate, and 3% water in acetone), freeze
substituted with a freeze substitution system (EM AFS2; Leica), and em-
bedded in epon resin. 50-nm-thin sections were cut, stained with 2% aque-
ous uranyl acetate for 3 min and Reynold’s lead citrate for 1 min, and
viewed under an electron microscope (Tecnai 12; FEI).
Cells were grown at 30°C in YPD or SC lacking leucine to maintain plas-
mid selection where appropriate. Where indicated, cells were treated with
DTT as described in the previous paragraph. For the experiment shown in
Fig. 6 E, cells were grown in YPD, washed, switched to SC, and grown for
another 24 h. Cells were harvested by centrifugation, washed twice with
water to remove DTT that would interfere with the subsequent protein deter-
mination, and were disrupted by bead beating. Proteins were extracted
with urea and SDS, and protein concentrations were measured using the
bicinchoninic acid protein assay kit (Thermo Fisher Scientific). 10–50 µg
total protein was resolved on Bis-Tris gels (NuPAGE; Invitrogen) and trans-
ferred onto polyvinylidene difluoride membranes. Membranes were
blocked, probed with primary and secondary antibodies, and incubated
with enhanced chemifluorescence substrate (GE Healthcare). Fluorescence
was detected with a variable mode imager (Typhoon 9400; GE Health-
care) and quantified using ImageQuant (GE Healthcare).
Cells were grown at 30°C in YPD or SC lacking leucine or uracil until they
had reached log phase (OD600 = 0.2–0.4). Cultures were diluted to 2.5 ×
106 cells/ml, dilution series with fivefold dilution steps were prepared, and
2 µl of each dilution, i.e., 5 × 103 cells at the highest concentration, were
spotted onto SC plates containing the indicated concentrations of tunica-
mycin. Plates were incubated at 30°C for 2–3 d.
Online supplemental material
Fig. S1 shows Sec63-GFP expressing wild-type cells treated with DTT
for up to 150 min. Fig. S2 shows electron micrographs of untreated
and DTT-treated wild-type cells. Fig. S3 shows Sec63-GFP expressing
opi1∆, opi1∆ ino2∆, and opi1∆ ino4∆ cells. Fig. S4 shows Sec61 pro-
tein levels of untreated and DTT-treated wild-type and hac1∆ cells and
of untreated opi1∆ and ino2(L119A) cells. Table S1 lists all of the yeast
strains used in this study. The MatLab script used for image analysis is also
provided. Online supplemental material is available at http://www.jcb
We thank Colin Stirling and Jacob Winther for antibodies, Hans-Joachim
Schüller and Karsten Weiss for plasmids, and Eugenio Marco for the MatLab
script. We are grateful to Kent McDonald and Mei Lie Wong for help and
advice on electron microscopy, to Diego Acosta Alvear, Georg Borner, Brooke
Gardner, Benoit Kornmann, Han Li, Saskia Neher, Anne-Lore Schlaitz, and
Eelco van Anken for comments on the manuscript, and to all members of the
Walter laboratory, especially Tomas Aragon and Benoit Kornmann, for discus-
sion and ideas.
S. Schuck was supported by the Ernst Schering Foundation and the
International Human Frontier Science Program Organization, W.A. Prinz and
C. Voss were supported by the Intramural Research Program of the National
Institute of Diabetes and Digestive and Kidney Diseases, and P. Walter is an
Investigator of the Howard Hughes Medical Institute.
Submitted: 14 July 2009
Accepted: 19 October 2009
and pRS415-MET25-ino2(L119A) (Heyken et al., 2005), which are
derived from pRS415-MET25 (Mumberg et al., 1994), were provided
by H.J. Schüller (Institut für Genetik und Funktionelle Genomforschung,
Greifswald, Germany). The integrative expression plasmid YIplac-dsRED-
HDEL-NatMX (Madrid et al., 2006) was provided by K. Weiss (University
of California, Berkeley, Berkeley, CA) and encodes dsRed-HDEL that is
targeted to the ER lumen by a signal sequence and retained there by an
HDEL sequence. To make the multicopy expression plasmid YEplac195-
RTN1, which encodes Rtn1 controlled by its own promoter, the coding
sequence of RTN1 plus 400 bps upstream of the start was amplified by
PCR from chromosomal DNA and cloned into YEplac195 (Gietz and
Sugino, 1988) between the SphI and KpnI sites.
All strains used were generated in this study, derived from a W303
wild-type strain (MATa; leu2-3,112; trp1-1; can1-100; ura3-1; ade2-1;
his3-11,15), are listed in Table S1. Gene deletions and modifications
were introduced by a PCR-based method (Longtine et al., 1998). Strains
expressing dsRed-HDEL were created by transformation with EcoRV-
linearized YIPlac-dsRED-HDEL-NatMX. Tagging the essential Sec63 pro-
tein with GFP did not affect cell growth, indicating that the Sec63-GFP
fusion protein was functional.
Strains without plasmids were grown at 30°C in YPD medium unless in-
dicated otherwise. When cultures had reached early log phase (OD600 =
0.15 0.2), cells were washed with 1 vol of SC medium (containing
yeast nitrogen base, amino acids, and 2% dextrose) and resuspended
in the same volume of fresh SC. The only exception to this switch from
lipid-containing YPD to lipid-free SC was the experiment shown in
Fig. 3 E, in which cells were washed and resuspended in fresh YPD.
Strains carrying plasmids were grown, washed, and resuspended in
SC without leucine or uracil as appropriate. Cells were left untreated
or treated with 8 mM DTT (Roche) or 1 µg/ml tunicamycin (EMD) for
the times indicated. Cells from 1 ml culture were pelleted at 10,000 g
for 1.5 min and resuspended in 30 µl SC. 7 µl was transferred onto
a glass slide, covered with a 22 × 50–mm cover glass, and immedi-
ately imaged live at room temperature. Images with an optical thickness
of 700 nm were acquired on a spinning-disk confocal microscope
(provided by the Nikon Imaging Center, University of California, San
Francisco) consisting of an inverted microscope (TE2000U; Nikon), a
spinning-disk confocal microscope (CSU22; Yokogawa), a camera (Cas-
cade II:512; Photometrics), and a Plan Apo VC 100×/1.4 NA oil objec-
tive lens (Nikon), and was controlled by the MicroManager program
(Stuurman et al., 2007). GFP was excited at 488 nm with an argon
laser and imaged using a 525/50 emission filter. Cherry was excited
at 568 nm with an argon krypton laser and imaged using a 615/55
emission filter. For the experiment shown in Fig. 7 C, a widefield micro-
scope (BX61; Olympus), a UPlan Apo 100×/1.35 NA oil objective lens,
a camera (Retiga EX; QImaging), and the iVision program (BioVision,
Inc.) were used. The brightness and contrast of the resulting images was
adjusted using Photoshop (Adobe).
To estimate the coverage of the plasma membrane with ER, unprocessed
16-bit image files of cortical sections were analyzed using MatLab (Math-
Works). A region of interest was defined for at least 15 cells per condi-
tion, and using an identical threshold for all images, the fraction of pixels
with Sec63-GFP signal was determined. To quantify ER expansion without
selecting regions of interest or thresholding, optical sections through the
middle of yeast cells were used. Using a modified version of an existing
script for the quantification of plasma membrane signal (provided by
E. Marco, Harvard Medical School, Boston, MA; Marco et al., 2007),
we first determined the intensity of the Sec63-GFP signal along the cell
cortex. We calculated the intensity’s coefficient of variation (CV), i.e., the
standard deviation of the intensity divided by its mean. Untreated wild-
type cells gave high CV values as a result of the large fluctuations of the
cortical Sec63-GFP signal, which resulted from the dotted appearance of
the cross sectioned tubular ER network. Cells with expanded ER yielded
lower CV values as Sec63-GFP became more evenly distributed along the
cortex within large ER sheets. To calculate the IE of the peripheral ER, we
made use of the fact that the perinuclear Sec63-GFP signal, which repre-
sents the closed membrane sheet that makes up the nuclear envelope, sets
the value for maximally expanded ER and gives the lowest possible CV.
We defined IE as the CV for nuclear envelope of untreated wild-type cells
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