Molecular Biology of the Cell
Vol. 21, 1556–1568, May 1, 2010
Yip1A Structures the Mammalian Endoplasmic Reticulum
Kaitlyn M. Dykstra, Jacqueline E. Pokusa, Joseph Suhan, and Tina H. Lee
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213
Submitted December 4, 2009; Revised March 2, 2010; Accepted March 8, 2010
Monitoring Editor: Benjamin S. Glick
The structure of the endoplasmic reticulum (ER) undergoes highly regulated changes in specialized cell types. One
frequently observed type of change is its reorganization into stacked and concentrically whorled membranes, but the
underlying mechanisms and functional relevance for cargo export are unknown. Here, we identify Yip1A, a conserved
membrane protein that cycles between the ER and early Golgi, as a key mediator of ER organization. Yip1A depletion led
to restructuring of the network into multiple, micrometer-sized concentric whorls. Membrane stacking and whorl
formation coincided with a marked slowing of coat protein (COP)II-mediated protein export. Furthermore, whorl
formation driven by exogenous expression of an ER protein with no role in COPII function also delayed cargo export.
Thus, the slowing of protein export induced by Yip1A depletion may be attributed to a proximal role for Yip1A in
regulating ER network dispersal. The ER network dispersal function of Yip1A was blocked by alteration of a single
conserved amino acid (E95K) in its N-terminal cytoplasmic domain. These results reveal a conserved Yip1A-mediated
mechanism for ER membrane organization that may serve to regulate cargo exit from the organelle.
The endoplasmic reticulum (ER) in most cell types is an
interconnected membrane network of flattened sheet-like
cisternae and narrow-diameter tubules dispersed through-
out the cell cytoplasm (Baumann and Walz, 2001). The net-
work is continuous with the outer nuclear envelope and
extends outward toward the cell periphery. It is the largest
membrane-bound organelle in animal cells and houses a
wide array of essential cellular processes, including secre-
tory and membrane protein biosynthesis and quality con-
trol, coat protein (COP)II-mediated secretory protein export,
lipid synthesis, detoxification, and the regulation of intracel-
lular Ca?2(Baumann and Walz, 2001).
To accommodate its varied functions, the ER is further
subcompartmentalized into discrete subdomains (Baumann
and Walz, 2001; Voeltz et al., 2002; Borgese et al., 2006;
Shibata et al., 2006). Rough ER, enriched in membrane-
bound ribosomes, is the primary site of protein biosynthesis
and translocation, whereas smooth ER is the major site of
Ca?2exchange, lipid synthesis, and detoxification. Between
rough and smooth regions, the transitional ER comprises the
subdomain where COPII vesicles bud to facilitate protein
and lipid export. Each ER subdomain is generally thought to
adopt the morphology most suited to its primary function.
Thus, the flattened sheets of the rough ER may better ac-
commodate large arrays of actively translating and translo-
cating polysomes, whereas the greater surface-to-volume
ratio associated with the highly curved tubules of the
smooth ER may facilitate rapid transport of ions and lipids
(Shibata et al., 2006).
In addition, the ER can adopt wide-ranging variations in
structure and organization in specialized cell types (Borgese
et al., 2006). Examples include the striking tubular morphol-
ogy of the sarcoplasmic reticulum in muscle (Baumann and
Walz, 2001; Rossi et al., 2008) and the dense parallel arrays of
flattened rough ER sheets in the liver or pancreas (Rajasekaran
et al., 1993; Baumann and Walz, 2001). The ER can also
undergo dramatic expansion. For example, B lymphocytes
undergo a severalfold increase in ER volume during differ-
entiation (Wiest et al., 1990). Another type of frequently
observed ER reorganization is the formation of tightly
stacked membrane arrays of distinct architectures that yet
retain continuity with the rest of the network (Chin et al.,
1982; Yamamoto et al., 1996). These arrays can take on the
appearance of compressed bodies of sinusoidal ER (Anderson
et al., 1983), ordered arrays of tubules and sheets with
hexagonal or cubic symmetry (Chin et al., 1982; Yamamoto et
al., 1996), or concentric membrane whorls (Koning et al.,
1996). Often, membrane packing or stacking occurs as a
consequence of drug treatments (Jones and Fawcett, 1966;
Hwang et al., 1974; Singer et al., 1988) or of up-regulation of
a variety of membrane-anchored ER proteins such as cyto-
chrome P450 (Koning et al., 1996), HMG-CoA reductase
(Chin et al., 1982), microsomal aldehyde dehydrogenase
(Yamamoto et al., 1996), cytochrome b5(Pedrazzini et al.,
2000), or the inositol 1,4,5-triphosphate receptor (Takei et al.,
1994). Significantly, tightly stacked and concentrically whorled
ER membranes also have been observed in a large number of
normal tissues (King et al., 1974), including testicular inter-
stitial cells (Carr and Carr, 1962; Christensen and Fawcett,
1966), adrenocortical cells (Nickerson and Curtis, 1969), mel-
anoma cells (Hu, 1971), cells of the anterior pituitary (Dubois
and Girod, 1971), and hypothalamic arcuate neurons (King
et al., 1974). In some instances, the whorled ER membranes
are primarily smooth, but in others the whorled membranes
also include ribosome-studded rough ER (King et al., 1974).
The prevalence of such large-scale ER reorganization is
suggestive that form follows function. Indeed, ER mem-
brane expansion in differentiating B lymphocytes increases
the protein-folding capacity of the ER that enables massive
up-regulation of antibody secretion (Wiest et al., 1990). The
functional consequences of other types of ER reorganization,
including concentrically whorled ER, is less well under-
This article was published online ahead of print in MBoC in Press
on March 17, 2010.
Address correspondence to: Tina H. Lee (email@example.com).
1556© 2010 by The American Society for Cell Biology
stood. One possibility is that the tight packing of either
smooth or rough membranes provides a means of seques-
tration and storage of either lipid or protein cargo for later
use. Of note, many of the specialized cell types that have
been observed to undergo ER stacking have roles in either
peptide or steroid hormone secretion (King et al., 1974). In
gonadotropin-releasing hormone (GnRH)-secreting hypo-
thalamic arcuate neurons, the formation of ER whorls seems
to also be regulated by the estrous cycle (King et al., 1974).
Thus, large-scale ER reorganization may serve as a mecha-
nism for regulating lipid or protein export in response to
Here, we identify a novel ER-structuring role for Yip1A,
the mammalian homologue of a yeast protein implicated
previously in the biogenesis of COPII transport vesicles
from the ER (Heidtman et al., 2003). In its absence, the ER
network is reorganized into concentrically whorled, stacked
membranes. Stacked whorl formation is in turn sufficient to
delay COPII-mediated ER export, consistent with the idea
that whorled ER formation provides a means of delaying
lipid or protein export. Together, our results suggest a role
for Yip1A in the structural organization of the ER and raise
the possibility that reorganization of ER membranes into
whorls under certain physiological conditions may provide
a means of transiently down-regulating cargo exit without
directly modifying components of the COPII machinery.
MATERIALS AND METHODS
Cell Culture, Constructs, and Transfections
HeLa cells were maintained in minimal essential medium (Sigma-Aldrich, St.
Louis, MO) containing 10% fetal bovine serum (Atlanta Biologicals, Norcross,
GA) and 100 IU/ml penicillin and streptomycin (Mediatech, Herndon, VA) at
37°C in a 5% CO2incubator. Knockdown of HeLa cells stably expressing
GalNacT2-green fluorescent protein (GFP) (Storrie et al., 1998) was performed
with Oligofectamine (Invitrogen, Carlsbad, CA) as described previously, us-
ing 40 nM small interfering RNA (siRNA) (Kapetanovich et al., 2005), with the
exception that a single transfection was sufficient for knockdown. The siRNAs
were synthesized using the siRNA construction kit from Ambion (Austin,
TX). The two siRNAs (?1, ?2) used against Yip1A were made against the
following target sequences, respectively: AAGTTACAGCATCGATGATCA
and AATGGTTTTTTGCCTTGCTTT. The siRNA target sequence for Sar1a
was AACCACTCTTCTTCACATGCT and AAGAACTGACCATTGCTGGCA
for Sar1b. The control siRNA used throughout this study targeted bovine
p115 and does not affect p115 in HeLa cells as described previously (Puthen-
veedu and Linstedt, 2004).
Transient cotransfection of HeLa cells with both plasmid DNA and siRNA
was performed with Lipofectamine 2000 (Invitrogen) according to manufac-
turer’s specifications by using 150 ng of DNA and 10 pmol of siRNA per 0.5
ml. Yip1A was cloned out of a HeLa cDNA library using polymerase chain
reaction (PCR) and inserted into the pCS2-MT vector by using the EcoRI and
XbaI sites. The FLAG-tagged Yip1A construct was generated by cutting out
the myc-epitope using the BamHI and EcoRI sites, followed by the addition of
a single FLAG epitope by using a PCR-based loop-in modification of the
QuikChange protocol (Stratagene, La Jolla, CA). The Yip1A rescue construct
was generated by introducing silent mutations into the siRNA-2 target se-
quence using QuikChange (AATGGTCTTCTGTCTTGCTTT). The E95K mu-
tation in the rescue construct was also generated by QuikChange using the
Myc-tagged Sec13 control construct was cloned by PCR amplification from
HeLa cDNA and inserting the PCR product into the pCS2-MT vector by using
BamHI and ClaI. The Myc-ts045 vesicular stomatitis virus glycoprotein
(VSV-G) construct was cloned using PCR to amplify ts045 VSV-G and insert-
ing the product into the pCS2-MT vector at the BamHI site. Myc-tagged DP1
and DP1L1 were generated by PCR amplification from HeLa cDNA and
insertion into EcoRI and XbaI sites in pCS2-MT. Sec61?-GFP (both monomeric
[mGFP] and dimerizing [dGFP] forms) was kindly provided by Dr. E. Snapp
(Albert Einstein University, Bronx, NY).
Antibodies and Other Reagents
Antibodies used include mouse monoclonal antibody (mAb) against protein
disulfide isomerase (PDI) (Abcam, Cambridge, MA); a rabbit polyclonal an-
tibody (pAb) against Calnexin (Abcam); a rabbit pAb against BiP (Abcam); a
rabbit pAb against LCB3 (Cell Signaling Technology, Danvers, MA); a rabbit
pAb against tubulin (Abcam); rabbit pAbs against Giantin, GM130, and
GRASP65 (kindly provided by Dr. A. Linstedt, Carnegie Mellon University,
Pittsburgh, PA); a mouse mAb against ER-to-Golgi intermediate compart-
ment (ERGIC)-53 (kindly provided by Dr. H.-P. Hauri, Biozentrum, Univer-
sity of Basel, Basel, Switzerland), the M2 mouse mAb against the FLAG
epitope (Sigma-Aldrich); and a mouse mAb against the Myc epitope from the
9E10 cell line. Fluorophore-conjugated secondary antibodies were from
Zymed Laboratories (South San Francisco, CA)/Invitrogen. A rabbit pAb was
raised against a 6His-Yip1A (amino acids [aa] 1–89) fusion protein as antigen
(Covance Research Products, Princeton, NJ) and was affinity purified on
Affi-Gel 15 beads (Bio-Rad Laboratories, West Grove, PA) coupled with a
glutathione transferase (GST)-Yip1A N-terminal fusion protein. A rabbit pAb
was raised against GST-Sec13 (Covance Research Products) and affinity pu-
rified on Affi-Gel 10 beads (Bio-Rad Laboratories) coupled with 6His-Sec13.
H89 was from Toronto Research Chemicals (North York, ON, Canada), and
brefeldin A (BFA) was from Sigma-Aldrich.
Immunofluorescence and Immunoblot Assays
siRNA transfections were analyzed 48–72 h after transfection as indicated.
Immunofluorescence procedures were as described previously (Kapetanovich
et al., 2005), except that a 20-min ice-cold methanol fixation was substituted
for paraformaldehyde. Immunoblotting using our affinity-purified Yip1A
antibody, as well as rabbit pAbs against calnexin and tubulin (Abcam), was
performed on siRNA-treated cells harvested from 60-mm dishes as described
previously (Kapetanovich et al., 2005).
Light Microscopy and Photobleaching Experiments
All static images with the exception of those in Figure 4 were obtained using
a Yokagawa spinning disk confocal scranhead (PerkinElmer Life and Analyt-
ical Sciences, Boston, MA) mounted on an Axiovert 200 microscope (Carl
Zeiss, Jena, Germany) with a 100? 1.4 numerical aperture (NA) objective
(Carl Zeiss) and acquired using a 12-bit Orca ER digital camera (Hamamatsu
Photonics, Hamamatsu City, Japan). Maximal value projections of sections at
0.3-?m spacing (4-6/cell) were acquired using Imaging Suite software
(PerkinElmer Life and Analytical Sciences). Quantitation of COPII assembly
was carried out using ImageJ (National Institutes of Health, Bethesda, MD) as
described previously (Kapetanovich et al., 2005). The wide field images in
Figure 4 were obtained with a 63? 1.3 NA objective on an Axioplan micro-
scope (Carl Zeiss) and acquired with QED software and a 12-bit Orca ER
digital camera (Hamamatsu Photonics).
Fluorescence recovery after photobleaching (FRAP) analyses were per-
formed on a 510 Meta/UV Duoscan Spectral Confocal with LSM Zen 2007
software by using a 100? 1.4 NA objective. Regions of interest were subjected
to photobleaching with repeated pulses at 100% laser power with the pinhole
wide open to obtain maximal depth of field. Recovery after photobleaching
was monitored with attenuated laser power. To generate the fluorescence
recovery curves, fluorescence within the photobleached region of interest at
each time point was first normalized to that of a nonbleached reference region
to account for general loss of fluorescence due to image acquisition. Recovery
curves were then generated by setting the fluorescence intensity before
bleaching to 100% and the intensity after the last bleach pulse to 0%.
Thirty-five-millimeter dishes of HeLa cells treated with control and Yip1A
siRNA were fixed 48 or 72 h after transfection with 2% glutaraldehyde/
phosphate-buffered saline (PBS) for 30 min at room temperature. After three
washes in PBS, cells were further fixed in 2% potassium permanganate/H2O
for 45 min, washed three times in distilled H2O, followed by dehydration in
an ascending series of ethanol (10–100%). Samples were then infiltrated in a
1:1 mixture of Epon-Araldite and 100% ethanol. After 30 min, the mixture was
exchanged with 100% Epon-Araldite and held in a desiccator for 60 h. Sam-
ples were then transferred for 24 h each at 30, 40, 50, and 60°C. Epoxy disks
were removed from the dishes, and areas of the disk with cells were cut out
and glued onto a blank embedding capsule with two-part epoxy. Thin (100-
nm) sections were cut using a DDK diamond knife on a Reichert-Jung
Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany) and
stained with lead citrate for 2 min. The grids were viewed on an H-7100
transmission electron microscope (Hitachi High Technologies America, Pleas-
anton, CA) operating at 75 kV. Digital images were obtained using an AMT
Advantage 10 charge-coupled device Camera System (Advanced Microscopy
Techniques, Danvers, MA) and ImageJ (National Institutes of Health).
For the pull-down of DP1 and DP1L1 with endogenous Yip1A, a 10-cm plate
of HeLa cells was transfected with either Myc-DP1 or Myc-DP1L1 by using
CaPO4. After 72 h, cells were washed with ice-cold PBS and then scraped and
solubilized for 30 min at 4°C for in HKT lysis buffer (100 mM KCl, 1% Triton
X-100, 20 mM KHEPES, pH 7.2, and protease inhibitors). The lysate was then
passed five times through a 25-gauge needle and then centrifuged for 20 min
at 14,000 ? g. The lysates were then incubated with 5 ?l of antibody (immu-
noglobulin [Ig]G or Yip1A) bound to 15 ?l of protein A-Sepharose beads (GE
Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) that had
Yip1A Structures the ER
Vol. 21, May 1, 2010 1557
been washed in lysis buffer. After a 2-h incubation at 4°C, the beads were
washed three times with lysis buffer. The beads were then boiled for 10 min
in 50 ?l of 2? reducing sample buffer and resolved on a 10% SDS gel.
Immunoblotting was performed using anti-Myc antibodies.
For the pull-down of DP1 using the FLAG-Yip1A constructs, 2 ? 10-cm
plates of HeLa cells were transfected with either Myc-DP1 only, FLAG-
Yip1A-wt and Myc-DP1, or FLAG-Yip1A-E95K and Myc-DP1 by using jetPEI
(Polyplus-Transfection, New York, NY) according to manufacturer’s specifi-
cations. The immunoprecipitation was performed as described above; how-
ever, the lysates were incubated with anti-FLAG M2 agarose beads (Sigma-
ts045-VSV-G Transport Assay
To assay ER export in Yip1A knockdown cells, HeLa cells were cotransfected
with either Myc-ts045 VSV-G and a control siRNA, or Myc-ts045 VSV-G and
Yip1A siRNA by using Lipofectamine 2000 (Invitrogen). Forty-eight hours
after transfection, cells were placed at 40°C, the nonpermissive temperature
for ER export of ts045 VSVG, to accumulate VSVG in the ER. After a 24-h
incubation at the nonpermissive temperature, cells were shifted to the per-
missive temperature of 32°C for varying times. To assay ER export in cells
expressing GFP-Sec61?, HeLa cells were cotransfected with Myc-ts045 VSV-G
and either dGFP-Sec61? or mGFP-Sec61?. Twenty-four hours after transfec-
tion, cells were shifted to 40°C and processed for the trafficking assay as
described just above.
In Vitro COPII Assembly Assay in Yip1A Knockdown
Cells in a 10-cm dish were transfected with either control or Yip1A siRNA by
using Oligofectamine (Invitrogen) and passaged onto coverslips after 24 h.
After an additional 24 h, a second siRNA transfection was performed. Knock-
down was monitored 48 h after the second transfection by immunostaining
with Calnexin antibodies. At this time, the whorled ER phenotype was
confirmed in ?95% of cells. The COPII assembly assay was performed pre-
cisely as described previously (Lee and Linstedt, 2000) 72 h after the second
Yip1A Knockdown Alters ER Network Organization
Yip1 in yeast has been established as a cycling membrane
protein required for COPII vesicle biogenesis (Heidtman et
al., 2003). However, the mechanism by which it facilitates ER
export remains largely unknown. There are two Yip1 iso-
forms in mammalian cells, Yip1A and Yip1B. Although each
is 31% identical to yeast Yip1, Yip1B is expressed only in the
heart, whereas Yip1A is ubiquitous (Tang et al., 2001). We
therefore focused on Yip1A. Previous work suggests that
both yeast Yip1 and mammalian Yip1A are ER-to-Golgi
cycling proteins with a steady state pattern of localization
coincident with ER exit sites and/or the ERGIC (Tang et al.,
2001; Heidtman et al., 2003; Yoshida et al., 2008). Staining
with our affinity-purified Yip1A antibodies confirmed a high
degree of colocalization of Yip1A with ERGIC-53, a marker
for the ERGIC (Supplemental Figure 1, A and B). Further-
more, consistent with the idea that Yip1A cycles constitu-
tively through the ER and ERGIC compartments (Tang et al.,
2001; Heidtman et al., 2003; Yoshida et al., 2008), blocking ER
export using the protein kinase inhibitor H89 (Aridor and
Balch, 2000; Lee and Linstedt, 2000), caused redistribution
(t1/2? 10 min) of Yip1A to the ER as observed previously
(Lee and Linstedt, 2000) for the rapidly recycling ERGIC-53
(Supplemental Figure 1, C and D).
To determine the consequences of Yip1A depletion, Yip1A
in HeLa cells was targeted for RNA interference (RNAi).
Two different siRNAs targeting Yip1A yielded substantial
knockdown of the protein as determined by immunoblot
(Figure 1A). By indirect immunofluorescence, siRNA-1 and
-2 both reduced Yip1A to near undetectable levels (compare
Figure 1, D and F, to B), although a higher percentage of cells
treated with siRNA-2 lacked detectable protein. To our ini-
tial surprise, cells lacking Yip1A displayed strikingly altered
ER organization as marked by antibodies against the lumi-
nal ER resident PDI (Mazzarella et al., 1990). In contrast to
control siRNA-treated cells with robust Yip1A staining (Fig-
ure 1B) and a typically dispersed tubular-reticular network
that extended throughout the cell cytoplasm (Figure 1C),
cells with undetectable Yip1A protein exhibited a notable
loss of the peripheral tubular ER network and an apparent
clustering of ER membranes into large micrometer-sized
structures (Figure 1, E and G). The change in ER morphol-
ogy was not marker specific. It was also readily observed
with antibodies against the transmembrane ER resident cal-
nexin (Ahluwalia et al., 1992). Moreover, the observed clus-
tering of ER membranes seemed to reflect a reorganization
of the ER as opposed to membrane proliferation, as shown
by comparable levels of calnexin in control and Yip1A
siRNA-treated cells (Figure 1A).
In addition to alterations in ER morphology, a marked
fragmentation of the Golgi apparatus into mini-stacks was
observed (Supplemental Figures 2, A–F, and 7), as reported
previously (Yoshida et al., 2008). But despite extensive frag-
mentation of the Golgi, light level analysis revealed that
Golgi marker separation was similar to that in control cells
(unpublished data), and Golgi subcompartmentalization
and its overall organization seemed to be maintained in cells
lacking Yip1A (Supplemental Figure 7B). Several observa-
tions suggested that the striking ER rearrangements ob-
served upon Yip1A knockdown were unlikely to have
occurred as an indirect consequence of either Golgi frag-
mentation or redistribution of Golgi tethering and/or stack-
ing proteins to the ER. First, a significant number of cells
with clustered ER were detected as early as 48 h after Yip1A
siRNA addition. At this time, ?50% of cells with clustered
(or whorled; see below) ER showed as yet no noticeable
Golgi fragmentation (Figure 1H). Second, the ER seemed
normal in cells treated with siRNAs targeting either gol-
gin160 (Yadav et al., 2009) or GRASP55 (Feinstein and
Linstedt, 2008), even though the Golgi was fragmented in
both cases (unpublished data). Third, both components of
the Golgi tethering/stacking complex GM130/GRASP65
(Barr et al., 1997; Sengupta et al., 2009) colocalized with other
Golgi markers in Yip1A-depleted cells (Supplemental Figure
2, G–L) in structures largely separate from the ER (Supple-
mental Figures 2, M–O, and 3). Finally, the ERGIC compart-
ment was maintained mostly separate from the ER in cells
lacking Yip1A, as evidenced by costaining with Yip1A and
ERGIC-53 antibodies in control siRNA- and Yip1A siRNA-
treated cells (Supplemental Figures 4 and 5).
Yip1A Loss Leads to ER Membrane Stacking
Ultrastructural analysis by thin section electron microscopy
(EM) corroborated the change in ER network organization
seen at the light level. In contrast to control cells with a high
density of ER membrane profiles in the cell periphery (Fig-
ure 2A), the majority of Yip1A knockdown cells were rela-
tively devoid of identifiable peripheral ER membranes (Fig-
ure 2B). Instead, large densely packed membrane clusters
were frequently observed (arrows in Figure 2B). Such
densely packed ER structures were never observed in con-
trol cells. At higher magnification, the membrane clusters,
clearly corresponding to the compacted ER membranes ob-
served at the light level, were visible as stacked membranes
arranged in concentric whorls ranging from 1 to 5 ?m in
outer diameter (Figure 2, C and D). The whorls were ER-
derived because clear connections to the nuclear envelope
were frequently observed (arrows in Figure 2, E and F).
Connectivity of the whorls to the ER network was also
demonstrated by relatively rapid diffusion of mGFP-sec61?
from the general ER into bleached whorls (Supplemental
K. M. Dykstra et al.
Molecular Biology of the Cell1558
Figure 6). Moreover, as expected for ER membranes, mito-
chondria were frequently in close apposition either to the
whorls themselves or to membranes extending from the
whorls. And, as suggested by light level analysis, Golgi
cisternal organization seemed relatively normal in both con-
trol (Supplemental Figure 7A) and Yip1A knockdown cells
(Supplemental Figure 7B), although the Golgi stacks were
shorter in cells lacking Yip1A, as expected from the frag-
mentation seen at the light level.
Two observations suggested that the ER whorls were not
a consequence of stress-induced ER autophagy. First, a lim-
iting membrane (Bernales et al., 2006; Ogata et al., 2006) was
never observed to surround the whorls. Second, immuno-
blotting with an antibody against BiP (Munro and Pelham,
1986), an ER chaperone that is up-regulated by ER stress
(Ron and Walter, 2007), showed no increase in BiP protein
expression (unpublished data). Third, immunostaining with
antibodies against LCB3, a marker of autophagy (Kabeya et
al., 2000), showed no change in knockdown cells (unpub-
lished data). Finally, the tubulin-staining pattern in cells
lacking Yip1A was indistinguishable from that in control
cells (Supplemental Figure 8), rendering it unlikely that the
effect of Yip1A depletion on ER organization was an indirect
consequence of perturbation of the microtubule (MT) cy-
toskeleton (Terasaki et al., 1986).
Serial thin sections prepared from cells relatively early
after knockdown (48 h) revealed that newly forming ER
whorls were probably comprised of flat, sheet-like cisternae,
as well as narrow-diameter tubules. In many whorls, con-
centric tubule-like arrays frequently persisted through mul-
tiple 100-nm serial sections, suggesting a structure made up
predominantly of stacked sheets (Figure 3, A–I). However,
newly forming whorls also contained membranes with more
tubule-like morphology. For example, we observed whorls
(Figure 3J) containing circular profiles that persisted for
more than a single 100-nm section (Figure 3J, i and i’) as well
as tubular profiles that did not persist for more than a single
100-nm section (Figure 3J, ii and ii’ and iii and iii’). Both
were indicative of a tubular morphology. Thus, it seemed
that the ER generally, both flat sheets and curved tubules,
was transformed into a stacked and concentrically whorled
arrangement upon Yip1A loss.
A Conserved Residue in the Cytoplasmic Domain of
Yip1A Is Required for the ER Structuring Function
To ascertain that the large-scale reorganization of the ER
network into whorls was due specifically to Yip1A deple-
tion, a siRNA-immune FLAG-Yip1A rescue construct was
generated and cotransfected into cells along with the siRNA.
Under these conditions, 66 ? 4% (3 independent experi-
HeLa cells stably expressing Golgi-localized GFP-GalNacT2 were transfected with a control siRNA, Yip1A siRNA-1, or Yip1A siRNA-2;
harvested 72 h later; and probed using antibodies against Yip1A, calnexin, and tubulin. (B–G) Yip1A loss correlates with ER morphological
changes. Cells transfected with control siRNA (B and C), Yip1A siRNA-1 (D and E), or Yip1A siRNA-2 (F and G) were fixed 72 h later and
doubly stained with antibodies against Yip1A (B, D, and F) and PDI (C, E, and G). Bar, 10 ?m. (H) ER morphological changes precede Golgi
fragmentation. Cells transfected with a control siRNA or Yip1 siRNA-2 were fixed 48 or 72 h later and stained with antibodies against PDI.
ER and Golgi morphologies were classified as indicated (frag Golgi, fragmented Golgi; whorled ER indicates compacted ER as shown in E
and G; abnormal ER indicates ER morphologies intermediate between normal and whorled). Quantitation of the average percentage of cells
displaying the indicated ER and Golgi morphologies from three independent experiments (?50 cells/condition/experiment) is shown, ?SD.
Single asterisk indicates p ? 0.05 and double asterisk indicates p ? 0.001 (Student’s t test).
RNAi-mediated Yip1A depletion causes ER compaction. (A) Immunoblot demonstrating loss of Yip1A by two distinct siRNAs.
Yip1A Structures the ER
Vol. 21, May 1, 20101559
ments of ?50 cells/experiment) of cells expressing a control
Sec13-Myc construct (Figure 4A) displayed the whorled ER
phenotype (marked by single asterisks in Figure 4B, quan-
tified in G). In contrast, virtually no cells expressing the
FLAG-Yip1A rescue construct displayed whorled ER. That
is, every cell expressing the FLAG-Yip1A rescue construct
(Figure 4C) exhibited a normal dispersed, tubular-reticular
ER network morphology (marked by double asterisk in Fig-
ure 4D, quantified in G). This result confirmed that the
large-scale reorganization of the ER network observed upon
treatment with Yip1A siRNA was indeed a specific conse-
quence of Yip1A loss.
To determine whether the ER structuring function of
Yip1A might depend on conserved and essential residues,
we took advantage of a previously published mutagenesis
study of yeast Yip1 wherein charged Yip1 residues con-
served between yeast and mammals were individually al-
tered and tested for effects on growth (Chen et al., 2004).
Because a charge reversal of a specific Glu residue to Lys
(E76K) in the N-terminal cytoplasmic domain of Yip1 ren-
dered the protein nonfunctional for restoring growth even
though the protein was stably expressed (Chen et al., 2004),
we chose to test this version for its ability to structure the ER.
Strikingly, 64 ? 1% of cells expressing FLAG-Yip1A (E95K)
displayed the whorled ER phenotype despite the properly
localized expression of protein (marked by single asterisks
in Figure 4, E, F, quantified in G). Thus, the Yip1A (E95K)
construct was no better than the negative control Sec13-Myc
construct in its ability to rescue the whorled ER phenotype.
This result indicates that the ER structuring function of
Yip1A in mammals depends critically on a conserved, cyto-
Yip1A Interacts with the ER Structuring Protein DP1
We reasoned that the inability of Yip1A (E95K) to fulfill its
ER structuring function might stem from its inability to
interact with a critical binding partner. Among the several
Yip1 binding proteins identified in yeast (Matern et al., 2000;
Calero et al., 2001; Chen et al., 2004; Heidtman et al., 2005),
the ER integral membrane protein Yop1 stands out as a
potential candidate for mediating the ER structuring func-
tion of Yip1. Deletion of Yop1 along with the structurally
related Rtn1/Rtn2 in yeast resulted in a loss of cortical ER
tubules (Voeltz et al., 2006); and purified Yop1 reconstituted
into synthetic liposomes was sufficient to induce liposome
tubulation (Hu et al., 2008). Furthermore, Yip1 overexpres-
sion in yeast suppressed lethality due to Yop1 overexpres-
sion, suggesting an antagonistic relationship between the
two proteins (Calero et al., 2001). These observations
prompted us to test whether DP1 (Voeltz et al., 2006), the
mammalian homologue of Yop1, binds Yip1A. For this,
Yip1A immunoprecipitation was performed on detergent-
solubilized lysates from cells transfected with Myc-DP1.
Indeed, both Myc-DP1 and the related Myc-DP1L1 (50%
identical) were recovered on Yip1A antibody beads but not
on control antibody beads (Figure 5A).
To next assess whether the inability of Yip1A (E95K) to
structure the ER might be due to an inability to bind DP1, an
antibody against the FLAG epitope tag was used to immu-
noprecipitate FLAG-Yip1A from cells cotransfected with
Myc-DP1 and either wild-type FLAG-Yip1A or Flag-Yip1A
(E95K). As shown (Figure 5B), Myc-DP1 was recovered in
association with both the wild type and the E95K variant of
FLAG-Yip1A. Therefore, the inability of Yip1A (E95K) to
maintain a dispersed ER network cannot be attributed sim-
ply to its failure to bind the ER structuring protein DP1.
Further work is required to elucidate the functional rele-
vance of the Yip1A–DP1 interaction for ER structuring.
Yip1A Depletion Slows VSVG Export from the ER without
Affecting COPII Recruitment
Our results thus far indicated that Yip1A was required
specifically to maintain a dispersed tubular-reticular ER net-
work. Because previous studies in yeast had implicated Yip1
in COPII-mediated ER export, we next compared the kinetics
of ts045 VSV-G ER export (Kreis and Lodish, 1986; Presley et
al., 1997) in the presence and absence of Yip1A. For this, cells
were cotransfected with either Myc-tagged ts045 VSV-G and
a control siRNA, or Myc-ts045 VSV-G and Yip1A siRNA.
Forty-eight hours after transfection, cells were placed at
40°C, the nonpermissive temperature for ER export of ts045
VSV-G, to accumulate VSV-G in the ER. After a 24-h incu-
bation at the nonpermissive temperature, cells were shifted
A low-magnification thin section transmission EM view of cells
treated with a control (A) or Yip1A (B) siRNA. Arrows (B) indicate
dense ER membrane aggregates seen only in Yip1A siRNA-treated
cells. Bar, 10 ?m. (C) A higher magnification view of an entire ER
whorl. Bar, 500 nm. (D) A high magnification view of stacked
membranes of a portion of an ER whorl. Bar, 100 nm. (E and F) ER
whorls are continuous with the nuclear envelope. A low-magnifi-
cation view of two interconnected whorls each connected to the
nuclear envelope (E) and a higher magnification view of a whorl
exhibiting connections to the outer nuclear envelope (F). Arrow-
heads (E and F) indicate the nuclear envelope and arrows (E and F)
indicate membrane continuities between whorl and nuclear envelope.
N, nucleus; C, cytoplasm; W, whorl. Bar, 2 ?m (E) and 100 nm (F).
Ultrastructural analysis of cells lacking Yip1A. (A and B)
K. M. Dykstra et al.
Molecular Biology of the Cell 1560
to the permissive temperature of 32°C for 0, 20, 60, or 120
min. As expected, VSV-G moved synchronously from the ER
(Figure 6A) to the Golgi (Figure 6B) by 20 min and to the
surface (Figure 6C) by 60 min in control siRNA-treated cells
(quantified in Figure 6G). In Yip1A knockdown cells, VSV-G
also accumulated in the ER at the nonpermissive tempera-
ture (Figure 6D). Indeed, staining for Myc-VSV-G in these
cells allowed ready visualization of the whorls induced by
Yip1A loss. In contrast to the control cells, however, a sig-
nificant fraction of Yip1A knockdown cells retained VSV-G
in the ER at 20 min (Figure 6E). Cells lacking Yip1A function
were identified by the presence of whorled ER structures
after doubly staining with calnexin antibodies. By 20 min,
VSV-G had moved out of the ER in only ?20% of cells
displaying whorled ER membranes (Figure 6G). In the re-
maining 80% of Yip1A knockdown cells, VSV-G was clearly
retained in whorled ER membranes (Figure 6E, quantified in
G). By 60 min, the fraction of cells with VSV-G in whorled
ER declined to ?30% and VSVG was detected in the Golgi
and/or surface in ?70% of cells (quantified in Figure 6G). By
120 min, ER export of VSV-G had occurred in ?80% of cells
displaying whorled ER (Figure 6F, quantified in G). Thus,
ER export was significantly delayed, though not altogether
blocked, in the absence of Yip1A. The marked slowing of ER
export upon loss of Yip1A function was in agreement with
studies in yeast (Heidtman et al., 2003).
To determine whether the slowing of ER export might be
due to defects in the recruitment and assembly of COPII
subunits to ER exit sites, the steady state distribution of the
COPII subunit Sec13 was examined in cells with and with-
out Yip1A. For this, cells were cotransfected with Sec13-Myc
and either a control siRNA or Yip1A siRNA. As expected,
control cells exhibited a robust Sec13-Myc distribution con-
sistent with an ER exit site pattern (Figure 7A). Assembled
COPII structures were distributed throughout the dispersed
ER (Figure 7B, merge in C). Surprisingly, Yip1A knockdown
cells also exhibited a robust Sec13-Myc distribution (Figure
7D), even in cells with extensive ER whorling (Figure 7E).
Notably, Sec13 positive structures seemed to be largely ex-
cluded from the whorled ER (arrows in merge in Figure 7F).
This result suggested that Yip1A was not required for the
recruitment of COPII subunits to presumptive ER exit sites,
although the exit sites formed in its absence seemed altered
in their distribution.
To further assess the ability of cells lacking Yip1A to
recruit COPII proteins to the ER, an in vitro COPII assembly
and tubules. (A–I) Serial (100-nm) transmission EM
thin sections through an ER whorl 48 h after Yip1A
siRNA treatment. Bar, 100 nm. (J) A thin section
micrograph through a different ER whorl 48 h after
Yip1A siRNA treatment. Arrows (i, ii, and iii) indi-
cate three different examples of tubular morphology.
Serial 100-nm thin sections through the correspond-
ing regions (i, ii, and iii) of the whorl in J are shown
in i’, ii’, and iii’.
Early ER whorls consist of both sheets
Yip1A Structures the ER
Vol. 21, May 1, 20101561
assay was also performed. To ensure that most of the Yip1A
siRNA-treated cells used in the assembly assay were de-
pleted of Yip1A, a double sequential knockdown was per-
formed. Then, to confirm efficient depletion, cells were
stained with antibodies against calnexin 24 h before the
assembly assay. At this time, ?95% of the doubly trans-
fected Yip1A knockdown cells displayed the whorled ER
morphology, confirming efficient Yip1A depletion. To mon-
itor the ability of the ER to support COPII assembly in the
absence or near complete absence of Yip1A, cells were sub-
sequently permeabilized with digitonin to extract endoge-
nous COPII proteins (Kapetanovich et al., 2005). The perme-
abilized cells were then incubated in the presence or absence
of rat liver cytosol. As expected, COPII assembly did not
occur in the absence of added cytosol (Figure 7G) but was
robust in the presence of added cytosol (Figure 7H) in con-
trol siRNA-treated cells. Also as expected, cells depleted of
Yip1A were incapable of supporting COPII assembly in the
absence of added cytosol (Figure 7I). However, assembly
was again surprisingly robust in the presence of added
cytosol (Figure 7J, quantified in K). Thus, although COPII-
mediated ER export was significantly delayed in the absence
of Yip1A, the delay seemed not to be a simple consequence
of inhibition of coat protein recruitment to ER membranes.
Blocking ER Export Does Not Induce ER Stacking
Two straightforward models could account for both the loss
of normal ER network morphology and the slowing of ER
export in response to Yip1A loss: 1) Yip1A is required pri-
marily for COPII vesicle biogenesis but the inhibition of
COPII function resulting from Yip1A loss secondarily cause
changes in ER structure. 2) Yip1A is required primarily for
ER morphogenesis, but the structural rearrangements result-
ing from Yip1A depletion secondarily inhibit COPII func-
tion. To distinguish between these models, we first asked
whether an ER export delay similar to that caused by Yip1A
loss, but induced by independent means, would lead to ER
organizational changes such as those seen in Yip1A knock-
down cells. ER export blockade was imposed by two inde-
pendent means. In the first, cells were treated with siRNAs
targeting both Sar1a and Sar1b (Kuge et al., 1994), a GTPase
required for COPII assembly and vesicle formation (Nakano
and Muramatsu, 1989). After confirming a block in ER ex-
port in Sar1a/b double knockdown cells (Figure 8A), ER
morphology was assessed. As expected, the ER network in
control cells with normal levels of assembled Sec13 (Figure
not mutant siRNA-immune Yip1A construct. Cells cotransfected
with Yip1A siRNA-2 and either a control Sec13-Myc construct (A
and B), a siRNA-immune wild-type FLAG-Yip1A construct (C and
D), or a siRNA-immune mutant (E95K) FLAG-Yip1A construct (E
and F) were fixed 72 h later and doubly stained with antibodies
against the Myc epitope (A) and calnexin (B) or the FLAG epitope (C
and E) and calnexin (D and F). Single asterisks (A–F) indicate
expressing cells that exhibit ER whorls. Double asterisks (A–F)
indicate expressing cells that do not exhibit ER whorls. Bar, 10 ?m.
(G) Quantitation of the percentage of Sec13-Myc or wild type or
whorled ER phenotype from three independent experiments (?50
cells per experiment), ?SD. Single asterisks indicate p ? 0.0001
(Student’s t test). Double asterisk indicates no statistically signifi-
The whorled ER phenotype is rescued by a wild type but
cells transfected with either Myc-DP1 or Myc-DP1L1 were solubi-
lized in 1% Triton X-100 and subjected to immunoprecipitation with
either a control antibody (IgG) or Yip1A antibody. Bound protein
was subjected to immunoblotting with an antibody against the Myc
epitope. (B) HeLa cells cotransfected with Myc-DP1 and either
wild-type FLAG-Yip1A or FLAG-Yip1A (E95K) were solubilized in
1% Triton X-100 and subjected to immunoprecipitation with M2
FLAG antibody beads. Bound protein was subjected to immuno-
blotting with the Myc epitope antibody. As a control, the Myc-DP1
recovered on M2 beads in the absence of FLAG-Yip1A is also
Both wild-type and E95K Yip1A bind to DP1. (A) HeLa
K. M. Dykstra et al.
Molecular Biology of the Cell1562
8B) was typically dispersed (Figure 8C). Significantly, cells
with only background levels of assembled Sec13 (Figure 8D)
also had a relatively normal dispersed ER network morphol-
ogy (Figure 8E). Similar results were obtained when COPII
assembly was blocked by another means, this time by treat-
ment with the protein kinase inhibitor H89 (Aridor and
Balch, 2000; Lee and Linstedt, 2000). Treatment of cells with
H89 caused a rapid redistribution of ERGIC-53 from its
typical ERGIC localization (Figure 8F) to an ER-like pattern
(Figure 8H), indicating efficient ER export blockade by H89.
Nonetheless, the morphology of the ER, as marked by PDI
staining, was similar in both untreated (Figure 8G) and
H89-treated (Figure 8I) cells. Thus, neither of two indepen-
dent means of blocking ER export yielded an ER morpho-
logical resembling that obtained by Yip1A depletion. There-
fore, the whorled ER phenotype observed upon Yip1A
depletion was unlikely to have occurred as a secondary
consequence of inhibiting ER export.
ER Stacking Is Sufficient to Slow ER Export
We next considered the alternate model, that ER membrane
stacking caused by Yip1A loss might be sufficient to delay
ER export and account for the diminishment of COPII func-
tion in cells lacking Yip1 function. In support of this hypoth-
esis, membrane stacking induced by an unrelated method
has been shown previously to delay VSV-G export from the
ER (Amarilio et al., 2005). In that case, overexpression of the
integral ER membrane-anchored VAP-B/Nir2 complex was
suggested to lead to membrane stacking through trans-
dimerization of VAP-B/Nir2 complexes on opposing mem-
branes, leading to a membrane zippering effect (Amarilio et
al., 2005). One caveat, however, is that VAP-B, like Yip1A,
also has been suggested to function in ER-to-Golgi traffick-
ing (Soussan et al., 1999). Thus, it remained possible that the
delay in ER export induced by VAP-B/Nir2 overexpression
could be attributed to a direct role for VAP-B in ER-to-Golgi
trafficking. To address this caveat, we sought a means of
inducing ER stacking that was unlikely to impact ER export
directly. For this, we took advantage of a previous study
demonstrating that overexpression of a dimerizing GFP-
Sec61? fusion protein (dGFP-Sec61?) could drive ER stacking
through head-to-head interactions between ER membrane-an-
chored GFP monomers on opposing ER membranes (Snapp et
al., 2003). Because neither Sec61? nor GFP have any role in
COPII function, we reasoned that this method of inducing ER
membrane stacking provided a way of testing whether ER
membrane stacking per se might be sufficient to delay
To assess ER export efficiency, the time course of VSV-G
ER export in cells expressing dGFP-Sec61? was compared
with that in cells expressing mGFP-Sec61?, a variant inca-
pable of inducing ER stacking due to a mutation in a residue
required for GFP dimerization (Snapp et al., 2003). As antic-
ipated, cells expressing dGFP-sec61? exhibited compacted
ER structures (Figure 9, B, E, H, and K) shown previously to
correspond to stacked and whorled ER membranes by EM
(Snapp et al., 2003). Also, as anticipated, Myc-ts045 VSV-G
was predominantly in the compacted ER structures before
shift to the permissive temperature (Figure 9, A–C). Even 20
min after the shift, at a time when VSV-G had exited the ER
in nearly all cells expressing the control mGFP-Sec61? con-
struct (Figure 9M), nearly 80% of cells with compacted ER
retained VSV-G in the compacted ER structures (Figure 9,
D–F, quantified in M). Therefore, the export of VSV-G from
the ER was slowed markedly by ER membrane stacking.
Export was not blocked though, as VSV-G reached post-ER
structures in ?50% of cells with stacked ER by 60 min
(Figure 9, G–I and M), and the number increased further to
?70% at the 120-min time point (Figure 9, J–L and M).
Interestingly, the kinetics with which VSV-G exited the ER
in these cells mirrored closely the kinetics with which it
exited the ER in Yip1A knockdown cells. The results support
the notion that membrane stacking per se is sufficient to
reduce ER export kinetics. Altogether, the results described
herein suggest that Yip1A plays a novel ER network dis-
persal function. ER network dispersal by Yip1A in turn is
required for rapid ER export.
whorling. (A–F) Cells cotransfected with Myc-ts045 VSV-G and
either a control siRNA (A–C) or Yip1A siRNA-2 (D–F) were shifted
48 h after transfection to 40°C to accumulate VSV-G in the ER. After
an additional 24 h, cells were shifted to 32°C for 0 (A and D), 20 (B
and E), 60 (C), or 120 (F) min to allow ER export. Cells were fixed
and doubly stained with antibodies against the Myc epitope and
calnexin (only the Myc staining is shown). Arrows (D–F) indicate
the positions of ER whorls as marked by Calnexin staining. Bar, 10
?m. (G) Quantitation of the percent of cells expressing Myc-ts045
VSV-G with the protein in post-ER structures. For cells transfected
with Yip1A siRNA, only cells with whorled ER, as marked by
Calnexin staining, were quantified. Shown are the averages from
three independent experiments, ?SD (p values obtained using the
Student’s t test).
Export of ts045 VSV-G from the ER is slowed by ER
Yip1A Structures the ER
Vol. 21, May 1, 20101563
Yip1A depletion in mammalian cells led to dramatic alter-
ations in ER network organization unlike any previously
documented for a specific loss of function perturbation.
Based on the combined data, we hypothesize that Yip1A
plays a role in maintaining a dispersed ER network. In its
absence, ER membranes undergo a stacking and whorl for-
mation reaction that in turn slows protein export. Our re-
sults also provide experimental support for the notion that a
general change in the organization of the ER network can
significantly impact rates of protein export from the or-
ganelle. Furthermore, they suggest the possibility that cer-
tain specialized cells might use large-scale ER reorganiza-
tion to elicit transient changes in the secretory capacity of
Previous studies have reported variable degrees of Golgi
fragmentation but no ER morphological change or delay in
ER-to-Golgi trafficking after Yip1A RNAi (Yoshida et al.,
2008; Kano et al., 2009). A potential underlying explanation
for the observed differences in the knockdown phenotype is
the extent of Yip1A depletion obtained with distinct siRNAs.
Indeed, we have observed a strong correlation between ER
morphology and the level of residual Yip1A protein re-
maining after knockdown. Cells without detectable Yip1A
almost always displayed the whorled ER phenotype,
whereas cells with residual Yip1A did not, suggesting that
low levels of Yip1A are able to maintain a relatively dispersed
The Basis for Inhibition of ER Export by ER Membrane
Stacking and Whorling
Why does ER membrane stacking and whorling delay
COPII-mediated protein export? COPII proteins were re-
cruited efficiently to presumptive ER exit sites even in the
absence of Yip1A, and yet ER export of VSVG was markedly
slowed. One potential explanation is that reorganization of a
large fraction of the ER into stacked whorls sequesters cargo
molecules away from cytoplasmic coat proteins, thereby
reducing the efficiency of cargo capture by the COPII coat.
Thus, COPII vesicle formation per se might proceed in the
absence of Yip1A, but the resulting vesicles might contain
reduced amounts of cargo. Although our FRAP data suggest
that the diffusion of mGFP-Sec61? into and presumably out
of whorls is relatively unrestricted (Supplemental Figure 6),
the diffusional mobility of VSV-G has not yet been examined
and may be preferentially hindered. An alternative possibil-
ity is that a late step in COPII vesicle biogenesis, subsequent
to the initial steps of Sar1 activation and coat protein recruit-
ment, is sensitive to the membrane morphological changes
associated with ER stacking and whorling. Generation of
either positive or negative membrane curvature or the mix-
ing of phospholipids ultimately required for vesicle budding
may comprise this late step.
Yip1A May Regulate the Ability of One or More ER
Proteins to Interact In Trans
Although the MT cytoskeleton and associated motor pro-
teins are acknowledged to play a key role in extending ER
cells lacking Yip1A. (A–F) Cells cotransfected
with Sec13-Myc and either a control siRNA
(A–C) or Yip1A siRNA-2 (D–F) were fixed 72 h
later and doubly stained with antibodies
against the Myc epitope (A and D) and caln-
exin (B and E). The merge is also shown (C and
F). Arrows (D–F) indicate the lack of Sec13-
Myc in ER whorls. Bar, 10 ?m. (G–K) Cytosol
dependent COPII assembly does not require
Yip1A. Cells doubly transfected with a control
(G and H) or Yip1A siRNA-2 (I and J) were
permeabilized with 30 ?g/ml digitonin 72 h
after the second transfection and incubated with
an ATP-regenerating system and guanosine 5?-
presence (H and J) of 4 mg/ml rat liver cytosol.
After 20 min at 37°C, cells were fixed and stained
with antibodies against Sec13. Bar, 10 ?m. (K)
Quantitation of the total fluorescence in Sec13-
positive structures under the indicated condi-
tions is shown, three independent experiments,
COPII recruitment is not blocked in
K. M. Dykstra et al.
Molecular Biology of the Cell1564
tubules toward the cell periphery (Waterman-Storer and
Salmon, 1998), they are unlikely to account for all of the
observed variations in ER morphology. Indeed, ER mem-
brane stacking is readily experimentally inducible with-
out perturbation of the MT cytoskeleton. High level
expression of several distinct ER membrane anchored
proteins including HMG-CoA reductase (Chin et al.,
1982), microsomal aldehyde dehydrogenase (Yamamoto
et al., 1996), cytochrome b5(Pedrazzini et al., 2000), the
inositol 1,4,5-triphosphate receptor (Takei et al., 1994), and
the ER-anchored VAP-B along with its binding partner
Nir2 (Amarilio et al., 2005) have each been shown to drive
ER stacking. In many if not all cases, membrane stacking
seems to be driven by the ability of the cytoplasmic do-
main of the overexpressed protein to interact with itself
in-trans, in effect “zippering” apposing membranes to-
gether. Even low affinity (100 ?M) interactions between
membrane-anchored GFP monomers were sufficient to
drive formation of stacked structures as long as the mono-
mers were expressed at high enough concentrations to
drive the head-to-head binding reaction (Snapp et al.,
2003). Thus, it seems that the ER network has an inherent
propensity to undergo stacking. As such, a general mech-
anism for preventing undesired stacking reactions might
contribute significantly to the morphogenesis of a dis-
persed ER network. Conversely, down-regulation of a
factor such as Yip1A that promotes network dispersal
might provide a mechanism for generating stacked and
whorled membranes in accordance with a physiological
need, for example, to transiently slow the export of lipid
and protein from the ER.
Network Dispersal by Yip1A May Involve DP1
Although transinteractions between ER membrane proteins
may underlie ER stacking and whorled ER formation in
Yip1A knockdown cells, alternate mechanisms can be envi-
sioned. In this regard, several putative Yip1 binding partners
have been reported in yeast (Calero et al., 2001; Heidtman et al.,
2003; Chen et al., 2004; Heidtman et al., 2005). Among the
group, the conserved integral ER membrane protein Yop1
(DP1 in mammalian cells) is unique in its steady-state local-
ization to the ER as well as its previously proposed role in
tubulating ER membranes (Voeltz et al., 2006). Moreover, an
antagonistic relationship between Yip1 and Yop1 has been
established in yeast (Calero et al., 2001). As such, DP1 stands
out as a possible mediator of Yip1A function in ER network
dispersal. Indeed, we have observed that both DP1 and the
related DP1L1 protein can be coimmunoprecipitated with
mammalian Yip1A. Furthermore, DP1 is present in ER mem-
brane whorls (Supplemental Figure 9). However, assessing
the functional significance of the interaction will require
mapping the DP1 binding determinant in Yip1A and testing
whether the binding interaction is required for the network
dispersal function of Yip1A. If indeed the ER dispersal func-
tion of Yip1A were to involve DP1, it would be of interest to
determine whether the underlying mechanism of network
dispersal involves the proposed membrane curvature-in-
ducing activity of DP1 (Voeltz et al., 2006; Hu et al., 2008). For
example, Yip1A might be envisioned to negatively regulate
DP1, perhaps through competitive inhibition of the homo-
oligomerization of DP1 that has been suggested to tubulate
ER membranes. In the absence of Yip1A, excessive oligomer-
to cause ER whorling (A–E). (A) Sar1 knock-
down blocks ER export. HeLa cells expressing
with a control siRNA or siRNAs targeting both
Sar1a and Sar1b isoforms were treated with 2.5
?g/ml BFA for 30 min to redistribute GFP-Gal-
NacT2 to the ER and subsequently incubated
without drug to allow ER export. At the indi-
cated times, cells were fixed, and the percentage
of cells with GFP-GalNacT2 in post-ER struc-
tures was counted. (B–E) ER export blockade by
Sar1 knockdown does not affect ER morphology.
Control (B and C) or Sar1 knockdown cells (D
and E) were fixed 72 h after transfection and
doubly stained with antibodies against Sec13 (B
and D) and PDI (C and E). Bar, 10 ?m. (F–I) ER
ER morphology. Untreated cells (F and G) or
cells treated for 20 min with 100 ?M H89 (H and
I) were fixed and stained singly for ERGIC-53 (F
and H) or PDI (G and I). Bar, 10 ?m.
ER export blockade is not sufficient
Yip1A Structures the ER
Vol. 21, May 1, 20101565
ization of DP1 would lead to excessive tubulation of the ER,
resulting in ER whorls.
Additional Roles for Yip1A
Although our results indicate a novel role for Yip1A in ER
membrane morphogenesis, Yip1A may play additional roles
in the early secretory pathway. Indeed, although a signifi-
cant pool of Yip1 is in the ER at steady state (Lorente-
Rodriguez et al., 2009), it also seems to cycle rapidly between
the ER and Golgi (Heidtman et al., 2003; Yoshida et al., 2008).
Therefore, Yip1A seems to exit the ER at a significant rate
and may travel as far as the early Golgi before being re-
trieved back to the ER. Interestingly, Yip1A knockdown has
been reported to lead to a reduction in membrane-associated
Rab6, suggesting that Yip1A somehow stabilizes Rab6 on
membranes (Kano et al., 2009). The loss of membrane-bound
Rab6 in turn was associated with a slowing of Shiga toxin
trafficking from the Golgi to the ER, suggesting a possible
role for Yip1A in Rab6-mediated retrograde trafficking (Sun
et al., 2007; Kano et al., 2009). Consistent perhaps with those
observations, we have detected a delay in the retrograde
redistribution of ERGIC-53 to the ER upon ER export block-
ade in Yip1A knockdown cells (unpublished data). Thus
there may be an additional role for Yip1A in ERGIC-to-ER
retrograde transport that might help to explain the Golgi
fragmentation observed in Yip1A knockdown cells. Indeed,
additional roles for the protein as it cycles through post-ER
compartments may account for the ability of Yip1A to inter-
act with specific proteins within the early Golgi. Further
work is required to elucidate these other potential roles of
Yip1A. Nonetheless, it is important to note that the ER
structuring defects observed herein are unlikely to occur as
a consequence of Rab6 membrane dissociation or a block in
retrograde transport, because previous studies have shown
that targeted Rab6 (Sun et al., 2007) and COPI (Guo et al.,
2008) knockdown has no obvious effect on ER network
ER Whorling Is Reversible and Regulated
Concentrically whorled and stacked ER membranes have
been documented in a surprising number and variety of
normal tissues, although the functional significance of the
membrane whorls remains largely unknown (Carr and Carr,
1962; Nickerson and Curtis, 1969; King et al., 1974). Intrigu-
ingly, the majority of the tissues in which ER whorls have
been documented serve to secrete either peptide or steroid
hormones (King et al., 1974). In GnRH-secreting hypotha-
lamic arcuate neurons, ER whorls peak during the diestrus
phase of the estrous cycle, diminishing during the proestrus,
when an increase in GnRH secretion signals the pituitary to
release leutinizing hormone (King et al., 1974). Thus, ER
whorling is a reversible process that is regulated by specific
signaling pathways. It is tempting to speculate that rear-
rangement of the ER into whorls may serve to reversibly
regulate hormone secretion. Finally, whether the Yip1A-
dependent ER dispersal/ER whorling pathway might pro-
vide a regulatory mechanism contributing to the formation
of such structures is an intriguing possibility that remains to
We thank Dr. E. Snapp for the kind contribution of GFP-Sec61? constructs,
members of the Lee and Linstedt laboratories for fruitful discussions, and Drs.
to slow ER export. Cells cotransfected with
Myc-ts045 VSV-G and either a control mGFP-
Sec61? construct (not shown but quantified in
M) or the ER stack-inducing dGFP-Sec61? con-
struct were shifted to 40°C to accumulate
VSV-G in the ER. Thereafter, cells were shifted
to 32°C for 0 (A–C), 20 (D–F), 60 (G–I), or 120
(J–L) min. At the indicated times, cells were
fixed and stained with Myc epitope antibodies.
The corresponding VSV-G (A, D, G, and J) and
GFP-Sec61? (B, E, H, and K) as well as merged
images (C, F, I, and L) are shown. Bar, 10 ?m.
(M) Quantitation of the kinetics of VSV-G ex-
port under each condition, the average of two
independent experiments, ?SD is shown.
ER membrane stacking is sufficient
K. M. Dykstra et al.
Molecular Biology of the Cell1566
M. Puthenveedu and A. Linstedt for helpful comments on the manuscript.
This work was supported by a American Cancer Society Research Scholar
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