EUKARYOTIC CELL, Apr. 2006, p. 712–722
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 5, No. 4
Endoplasmic Reticulum-Associated Degradation Is Required for Cold
Adaptation and Regulation of Sterol Biosynthesis in the Yeast
Jennifer Loertscher,1Lynnelle L. Larson,2Clinton K. Matson,3Mark L. Parrish,4Alicia Felthauser,3
Aaron Sturm,5Christine Tachibana,6Martin Bard,5and Robin Wright3*
Department of Chemistry, Seattle University, Seattle, Washington 981221; Redmond High School, Redmond, Washington 980522;
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 554553;
Department of Zoology, University of Washington, Seattle, Washington 981954; Department of Biology,
Indiana University Purdue University Indianapolis, Indianapolis, Indiana 462025; and
Department of Biochemistry, University of Washington, Seattle, Washington 981956
Received 15 September 2005/Accepted 16 January 2006
Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded
proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been
associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10
(Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-
3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at
low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or
DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that
assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of
abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity.
Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated
HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-
sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The
sterol metabolite profile of ubc7? cells was altered relative to that of wild-type cells. Since sterol levels are
known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was
examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these
ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.
Cellular membranes are a highly adaptable and their bio-
chemical composition is tightly regulated (47). Unicellular or-
ganisms are able to survive changing environmental conditions
in large part due to their ability to modify the composition of
their membranes in response to environmental stresses such as
low and high temperatures (48). In particular, sterol composi-
tion plays a major role in modulating membrane characteris-
tics, and regulation of the sterol biosynthetic pathway has been
well studied (10, 31, 35).
Wright and others have explored the role of sterol biosyn-
thetic enzymes in membrane formation using a yeast model of
membrane biogenesis. Elevated levels of 3-hydroxy-3-methyl-
glutaryl coenzyme A (HMG-CoA) reductase (HMGR) induce
dramatic endoplasmic reticulum (ER) proliferations (1, 16,
50). In yeast, the membranes induced by the HMGR isozyme,
Hmg1, form a nucleus-associated array of stacked ER mem-
branes called karmellae and represent a well-studied example
of how changes in physiological conditions can result in altered
membrane structures. For example, studies of HMGR-induced
karmellae formation in yeast have provided insights into the
processes of ER proliferation, including the importance of
continued flux though the secretory pathway (17, 26, 36, 38).
However, these studies have not yet provided a comprehensive
understanding of how ER regulatory networks and metabolic
pathways contribute to the modulation of membrane compo-
sition and biogenesis. Nor have they examined how such reg-
ulatory pathways interface with environmental conditions, in-
cluding temperature changes, to affect overall cell physiology
At least two ER quality control mechanisms coordinate cel-
lular physiology with ER-associated protein synthesis and deg-
radation. The unfolded protein response (UPR) induces the
synthesis of ER-localized chaperones such as Kar2 (BiP) in
response to accumulation of abnormal or excess proteins (24,
42). Another ER quality control mechanism, ER-associated
degradation (ERAD), eliminates short-lived or misfolded pro-
teins from the ER (8, 15, 32). ERAD requires the activity of
the ER-associated E2 ubiquitin-conjugating enzymes Ubc6
and Ubc7. Ubc6 is directly anchored to the ER membrane by
a carboxyl-terminal transmembrane domain; Ubc7 is a periph-
eral membrane protein that associates with the ER via its
binding to the ER membrane protein, Cue1 (5, 11, 19). Other
ER proteins that are required for ubiquitination and degrada-
tion of the ERAD substrates include the E3 ubiquitin ligases
Hrd1 (Der3) and Doa10 (4, 6, 45), Hrd3 (14), and Der1 (25).
A synthetic lethality screen of a deletion mutant population
suggests that ERAD has a role in cellular adaptation to chem-
* Corresponding author. Mailing address: Department of Genetics,
Cell Biology, and Development, University of Minnesota, Minneapo-
lis, MN 55455. Phone: (612) 625-1183. Fax: (612) 626-6140. E-mail:
ical and physical changes that affect membranes. These screens
revealed that ubc7? and cue1? mutants have impaired growth
when cells are forced to express high levels of Hmg1 at low
temperatures (51). Here we report results of our investigation
of the relationships among the ubiquitin-proteasome pathway,
ER membrane biogenesis, and low temperature. We analyzed
the potential of more than 50 proteins in ubiquitin-dependent
protein catabolism and related pathways to play a role in cell
viability in the presence of elevated HMGR across a range of
temperatures. This analysis identified Ubc7, Cue1, and Doa10
as being uniquely required for a cell’s normal response to
increased Hmg1at low temperatures. Subsequent experiments
showed that mutants lacking any one of these ERAD genes are
unable to grow at 10°C even when normal levels of HMGR are
expressed. Based on the data reported here, we propose a role
for a specific subset of ERAD enzymes, Ubc7, Cue1, and
Doa10, in allowing cells to adapt membrane characteristics for
survival at low temperatures.
MATERIALS AND METHODS
Yeast strains, media, and plasmids. Most deletion mutant strains examined
were homozygous diploid deletion mutants purchased from Research Genetics (In-
vitrogen Corp., Carlsbad, CA). These deletion strains are in the S288C background,
BY4743 (MATa/? his3?1/his3?1 leu2?0/leu2?0 ura3?0/ura3?0 met15?0/? lys2?0/
?). A full list of mutants screened is given in Table 1. The ubc7?, cue1?, doa10?,
ubc6?, hrd1?, and hrd3? deletions were verified by PCR with primers that hybrid-
ized to sequences flanking the deleted gene and within the KanMX6 gene that was
used to select for the recombination (sequence of oligonucleotides available upon
request). Haploid ubc7?, cue1?, and doa10? strains were generated from another
S288C strain, JRY527 (MATa ade2-101 his3?200 lys2-801 met2- ura3-52) for use in
morphological studies (3). pre1-1 pre2-1 proteasome mutants were generated in the
WCG4 strain (MATa/? his3-11/his3-11 leu2-3/leu2-3 112ura3/112ura3) (18).
Plasmid-expressing yeast strains were grown in rich minimal medium (0.17%
yeast nitrogen base, 0.5% ammonium sulfate, 2% Casamino Acids, and 2% agar,
with 30 ?g of adenine/ml, 20 ?g of histidine/ml, 40 ?g of lysine/ml, 40 ?g of
leucine/ml, 20 ?g of methionine/ml, 30 ?g of tryptophan/ml, and 20 ?g of
tyrosine/ml) lacking uracil to select for retention of plasmids. The carbon source
was 2% glucose or 2% galactose. Yeast strains used in 10°C studies were gown
on YPD (2% yeast extract, 2% proteose peptone, and 2% glucose.) Solid me-
dium contained 2% agar. Agar used in rich minimal medium was washed with
distilled water prior to use to remove contaminating chemicals.
Deletion of UBC7, CUE1, and DOA10 in JRY527 was accomplished by using
appropriate PCR products that carried portions of the 5? and 3? regions of the
gene to be deleted and the Geneticin resistance marker, KanMX6 (49). Disrup-
tion was confirmed via PCR, using primers that hybridized to sequences flanking
the deleted gene and within the KanMX6 sequence.
All plasmids used except p716 have been previously described. The vector
control used in all cases was pBM150 (CEN4, ARS1, URA3, GAL1/10 promoter)
(23). The galactose-inducible HMG1 plasmids, pAK266 (wild-type HMG1 under
GAL1 promoter control in pRS316; CEN6 ARS4 URA3), pDP304 (hmg1,
His1020Gln, catalytically inactive Hmg1, under GAL1 promoter control in
pBM150; CEN4 ARS1 URA3), p558 (hmg1-K442M, L487; the mutant Hmg1
protein is catalytically active but unable to assemble karmellae; see reference 17),
and p260 (encodes galactose-inducible mutant hmg1 protein with a 29-amino-
acid deletion in the catalytic domain; the deletion results in loss of both catalytic
activity and karmellae-inducing ability are described in references 37 and 38).
pRH134-2 was received from Randy Hampton (University of California, San
Diego) and was as described previously (11). To make p716, the GAL1 promoter
was removed from pAK266 with BamHI. The GPDH promoter was PCR am-
plified from plasmid pCR436 [pBluescript KS(?) containing a HindIII-SalI
fragment with the GPDH promoter sequence]. Yeast cells were then trans-
formed with the gapped p716 plasmid and the PCR fragment. The resulting,
stably transformed yeast strains constitutively assembled karmellae and were
lovastatin resistant, confirming that the plasmid encoded functional Hmg1. In
addition, the plasmid was rescued from the yeast and displayed the predicted
restriction enzyme fragment sizes. In all cases, yeast were transformed with the
Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, CA).
Growth assays by serial dilutions. Serial dilutions from an overnight culture
diluted to 0.5 ?600units per ml were made into sterile distilled water at the ratios
of 1:36, 1:5, 1:5, 1:5, and 1:5. Cell suspensions were transferred from the micro-
titer plate to appropriate solid medium using an ethanol-sterilized, 48-pin metal
stamper. Plates were incubated at 10, 16, 26, or 37°C and photographed after an
appropriate period of time (see figure legends). At least three independent
replicate experiments were performed for each set of strains; a representative
experiment is shown in the figures.
Microscopy. For electron microscopy, ubc7? and cue1? cells transformed with
pAK266 were grown in liquid medium containing galactose for 24 h at 16°C.
Cells were then prefixed in glutaraldehyde, postfixed in potassium permanga-
nate, and embedded in Spurr’s resin as previously described (17, 38). Sections
were stained with Reynold’s lead citrate and examined on a Philips CM100
transmission electron microscope operating at 60 kV. Electron microscopic sam-
ples were coded so that observation was made without knowledge of sample
The vital dye, 3,3?-dihexyloxacarbocyanine iodide (DiOC6), was used to visu-
alize karmellae in living cells (27) using a fluorescein filter set (excitation, 480 ?
20 nm; barrier, 535 ? 40 nm) on a Nikon Microphot-FXA fluorescence micro-
scope (Nikon USA, Melville, NY). Cells depicted were grown for 14 h on
galactose at 16°C.
Sterol analysis. Sterolswereisolatedaspreviouslydescribed(33)andanalyzedby
gas chromatography (GC). An HP5890 series II equipped with the Hewlett-Packard
CHEMSTATION software package was used to analyze sterol content. The capil-
lary column (DB-1) was 15 m by 0.25 mm (film thickness; J&W Scientific, Folsom,
CA) and was programmed from 195 to 280°C (1 min at 195°C and then an increase
at 20°C/min to 240°C, followed by 2°C/min until the final temperature of 280°C was
reached). The linear velocity was 30 cm/s using nitrogen as the carrier gas, and all
injections were run in the splitless mode.
GC/mass spectrometry analyses of sterols were done by using Thermoquest
Trace 2000 gas chromatograph interfaced to a Thermoquest Voyager mass
TABLE 1. Hmg1-dependent growth inhibition of ubiquitin-proteasome and related mutants
Mutation resulting ina:
Severe growth inhibition
Growth inhibition at one or more
of the temperatures tested
Normal growth at all
cue1?,* doa10?,* ubc7?,*
der1?, hrd1?, hrd3?
ate1?, cdh1?, doa4?, dsk2?, hul5?,
nas6?, rad6?, rad23?, rpn10?,
rub1?, tom1?, ubp2?, ubp3?,
ubp6?, ubp14?, ubp15?, ubr1?,
ubr2?, ufd4?, ump1?, yuh1?
atg10?, cdc26?, hul4?, npl4?,
pex4?, rpn4?, rpn13?,
ubc5?, ubc8?, ubc11?,
ubc13?, ubp1?, ubp5?,
ubp7?, ubp9?, ubp11?,
ubp12?, ubp13?, ubp16?
asi2?, atg7?, uba3?
Other (putative ERAD
substrates or ubiquitin-
aThe temperatures tested were 16, 26, and 37°C. ?, Growth inhibition was observed at 16 and 26°C but not at 37°C; ??, growth inhibition was observed at 37°C but
not at 16 or 26°C.
VOL. 5, 2006ERAD IN COLD ADAPTATION AND STEROL BIOSYNTHESIS 713
spectrometer. The GC separations were done on a fused silica column, DB-5MS
(20 m by 0.18 mm by 0.18 ?m [film thickness; J&W Scientific, Folsom, CA). The
injector temperature was 190°C. The oven temperature was programmed to
remain at 100°C for 1 min, followed by a temperature ramp of 6.0°C/min to a
final temperature of 300°C. The final temperature was held for 25 min. Helium
was the carrier gas with a linear velocity of 50 cm/s in the splitless mode. The
mass spectrometer was in the electron impact ionization mode at an electron
energy of 70 eV, with an ion source temperature of 150°C and scanning from 40
to 850 atomic mass units at 0.6-s intervals.
Elevated expression of Hmg1 in the ubc7?, cue1?, and
doa10? ERAD mutants resulted in cold-sensitive growth. A
competitive growth experiment designed to identify genes in-
volved in karmellae biogenesis revealed that ubc7? and cue1?
mutants divided more slowly under karmellae-inducing condi-
tions than other mutants within the population (51). To con-
firm these results, ubc7? and cue1? strains were individually
transformed with a plasmid carrying a galactose-inducible
HMG1 gene (plasmid AK266) and examined for a galactose-
dependent growth phenotype. Both mutants displayed cold-
sensitive growth on galactose (Fig. 1). Growth was particularly
inhibited at 16°C, with moderate inhibition at 26°C. Thus,
these ERAD genes appeared to be required for low-tempera-
ture viability of yeast cells expressing high levels of HMGR.
This result was consistent with the previously observed slow-
growth phenotype in the population of deletion mutants, since
one of the competitive growth experiments was conducted at
26°C to enable the growth of temperature-sensitive mutants
(51). To determine whether this cold sensitivity was a pheno-
type limited to the BY4743 strain background, we generated
ubc7? and cue1? mutants in another strain background,
JRY527. These new ERAD mutants were similarly cold sen-
sitive in the presence of high levels of Hmg1 (data not shown).
Thus, the cold sensitivity was unlikely to result from cryptic
mutations or other characteristics unique to the BY4743 back-
Only a subset of genes involved in ERAD or the ubiquitin-
proteasome pathway was required for normal growth of cells
with elevated levels of Hmg1. In addition to Ubc7 and Cue1,
other ER-associated proteins have a role in ERAD, including
Doa10, Ubc6, Hrd1, Hrd3, and Der1 (6, 14, 25, 45). Based on
analysis of growth on solid medium of these mutants, only
doa10? mutants exhibited significant cold-sensitive growth in
the presence of increased Hmg1 (Fig. 1 and Table 1). These
results indicated that the ERAD proteins Ubc7, Cue1, and
Doa10 were specifically required for normal growth in re-
sponse to high levels of Hmg1 at low temperatures.
To determine whether the cellular response to increased
Hmg1 requires other components of the ubiquitin-proteasome
pathway, a panel of deletion mutants were transformed with
pAK266 and screened for a growth phenotype at 16, 26, and
37°C (Table 1). Many of the mutants showed slight growth
inhibition in response to increased Hmg1 at one or more of the
temperatures tested. In addition, growth of the doa1? mutant
expressing elevated HMGR was found to be strongly temper-
ature sensitive. However, none of these mutants exhibited the
pronounced cold sensitivity that was observed in ubc7?, cue1?,
or doa10? strains.
ubc7?, cue1?, and doa10? cells displayed defective spatial
and temporal regulation of karmellae assembly. Since the
Hmg1-dependent growth defects of ubc7? and cue1? cells
were originally recognized as part of a screen to identify genes
required for ER membrane biogenesis, we examined whether
these mutants were able to assemble normal karmellae. After
24 h of karmellae induction at the nonpermissive temperature
of 16°C, ubc7? and cue1? mutants contained unusual ER
membrane proliferations that were not observed in wild-type
controls or in previous experiments (50). In contrast to the
highly organized stacks of karmellae membranes that surround
the nucleus, forming a horseshoe pattern in sections (Fig. 2A),
the ER membrane proliferations observed in ubc7? and cue1?
were disorganized and were not always closely associated with
the nucleus (Fig. 2B and C). Thus, although the mutants were
able to proliferate their ER in response to elevated Hmg1, they
were unable to properly modulate the organization and loca-
tion of the resulting proliferations.
As previously reported, karmellae can be visualized in living
cells with fluorescence microscopy using the vital dye, 3,3?-
dihexyloxacarbocyanine iodide (DiOC6). Subsequent ex-
amination of homozygous diploid ubc7?, cue1?, and doa10?
mutants using this method yielded unexpected new informa-
tion about variation in karmellae morphology in wild-type
FIG. 1. The ERAD proteins Ubc7, Cue1, and Doa10 are required
for normal growth in the presence of increased Hmg1. Wild-type,
ubc7?, cue1?, and doa10? cells of strain BY4743 were transformed
with a plasmid either encoding a galactose-inducible HMG1 gene
(pAK266) or the vector control. The leftmost spot is inoculated from
a culture at 1.4 ? 106cells per ml, followed by 1:5 serial dilutions.
Images were taken after 2 days of growth on glucose or galactose at 37
and 26°C and after 5 days of growth at 16°C.
714LOERTSCHER ET AL.EUKARYOT. CELL
strains of different backgrounds. All studies of karmellae mor-
phology conducted in our laboratory prior to those presented
here have been in yeast strain JRY527. Because ubc7? and
cue1? mutants were identified from a pool of mutants created
by the Deletion Consortium, initial investigation of ubc7? and
cue1? mutants and other ubiquitin-proteasome mutants was
conducted with mutants derived from the BY4743 parent
strain. The wild-type BY4743 cells were difficult to stain with
DiOC6; the cells were very densely stained and appeared to
contain some disorganized membrane strips and whorls in ad-
dition to normal karmellae. The unusual staining characteris-
tics in this wild-type strain made it difficult to use in vivo
observations to determine the extent of karmellae abnormali-
ties in the mutants. Therefore, we examined karmellae struc-
ture in ubc7?, cue1?, and doa10? mutants that were generated
in the more easily stained JRY527 background.
As expected from past observations, staining of wild-type
JRY527 cells with DiOC6revealed normal karmellae, without
unusual staining observed in the BY4743 background (Fig.
2D). Consistent with our observations of the ubc7?, cue1?,
and doa10? mutants in the BY4743 background, the analogous
mutant strains generated in the JRY527 background assem-
bled both karmellae and abnormal membrane structures (Fig.
2E, F, and G). Thus, the abnormalities in karmellae assembly
in ubc7?, cue1?, and doa10? mutants were unlikely to result
from differences in strain background. In the absence of high
levels of Hmg1, no unusual membrane structures were ob-
served in any cells examined, although the BY4743 strain con-
tinued to be difficult to stain optimally with DiOC6.
Increased HMG-CoA reductase catalytic activity, not kar-
mellae, was responsible for Hmg1 sensitivity in ERAD mu-
tants. Previous studies have shown that Hmg1-dependent in-
duction of karmellae requires a region in the last ER lumenal
region of the membrane domain (“loop G”) and is indepen-
dent of HMGR catalytic activity (36, 37). These observations
allowed us to test the hypothesis that the cold-sensitive phe-
notype and abnormal karmellae assembly in ubc7?, cue1?, and
doa10? mutants were functionally related. To determine
whether the observed Hmg1-dependent cold sensitivity was
due to abnormal karmellae biogenesis, mutants were trans-
formed with plasmids containing mutated or truncated forms
of Hmg1 (Fig. 3A). Wild-type cells transformed with pDP304,
which encodes a catalytically inactive form of Hmg1, make
normal karmellae when grown on galactose-containing me-
dium but do not express elevated HMGR activity. The ubc7?
mutants expressing the catalytically inactive, karmellae-induc-
ing form of Hmg1 (pDP304) grew as well as cells expressing
the vector control plasmid. Thus, the catalytic activity was
essential for the cold-sensitive phenotype. cue1? and doa10?
mutants transformed with pDP304 also grew similarly to wild-
type (data not shown.)
ubc7? cells expressing a galactose-inducible, catalytically in-
active form of Hmg1 that is unable to induce karmellae (p260)
grew as well on galactose-containing media as cells trans-
FIG. 2. ubc7?, cue1?, and doa10? cells make aberrant karmellae. Wild-type, ubc7?, and cue1? cells of strain BY4743 transformed with
pAK266 (carrying a galactose-inducible HMG1 gene) were fixed for electron microscopy after 24 h of growth on galactose at 16°C. (A) A
representative wild-type cell exhibits a normal karmellae structure, which is labeled K. (B and C) Abnormal membrane proliferations observed in
mutants, including disorganized membrane strips that are not closely associated with the nucleus, are indicated with arrows. (D, E, F and G) The
lipophilic dye DiOC6was used to stain wild-type and deletion cells of strain JRY527 after 14 h of growth on galactose at 16°C. A representative
wild-type cell exhibits a normal karmellae structure (D). Mutants show a diversity of abnormal membrane proliferations including loops, whorls,
and membrane strips (E, F, and G).
VOL. 5, 2006 ERAD IN COLD ADAPTATION AND STEROL BIOSYNTHESIS715
formed with pDP304. Conversely, ubc7? cells expressing a
galactose-inducible mutant form of hmg1 that was unable to
induce the formation of karmellae but retained catalytic activ-
ity (p558) was cold sensitive. ubc7? mutants constitutively ex-
pressing HMG1 under the control of the GPDH promoter
(p716) showed a slight cold-sensitive phenotype on both glu-
cose and galactose at 16°C, indicating that the observed phe-
notype is not carbon source dependent (Fig. 3A). Finally,
ubc7? cells expressing galactose-inducible Hmg2 (pRH134-2)
grew more poorly on galactose than vector control transfor-
mants. The Hmg2 protein has identical catalytic activity as
Hmg1 but induces proliferation of short stacks of smooth
membranes that can be associated with the nucleus or plasma
membrane or present in the cytoplasm (28). Collectively, these
observations indicate that increased HMGR activity, not ab-
normal karmellae assembly, was responsible for the cold-sen-
To confirm that the primary cause of Hmg1-dependent cold
sensitivity in ubc7?, cue1?, and doa10? cells was HMGR cat-
alytic activity, mutant cells expressing pAK266 were grown at
16°C in the presence of lovastatin, a competitive inhibitor of
HMGR. As previously observed, the mutants expressing high
levels of Hmg1 displayed cold-sensitive growth when they ex-
pressed elevated HMGR. However, when HMGR activity was
reduced by the presence of lovastatin, nearly normal growth
was restored at 16°C (Fig. 3B.) This result demonstrated that
the increased catalytic activity of Hmg1 is toxic at cold tem-
peratures to ubc7?, cue1?, and doa10? mutant cells.
The response of proteasome mutants to increased Hmg1
was similar to that of ubc7?, cue1?, and doa10? cells. Because
Ubc7, Cue1, and Doa10 are part of the molecular machinery
that covalently attaches ubiquitin to target proteins, we hy-
pothesized that the Hmg1-dependent cold sensitivity in these
mutants was due to the failure of these mutants to ubiquitinate
FIG. 3. HMG-CoA reductase catalytic activity, not the presence of karmellae, is the cause of decreased growth in response to increased levels
of Hmg1. (A) Wild-type and ubc7? cells were transformed with vector control or plasmids expressing galactose-inducible HMG1 (pAK266),
galactose-inducible mutant hmg1 with no catalytic activity (pDP304 and p260), galactose-inducible hmg1 mutant that has catalytic activity but is
unable to make karmellae (p558), a constitutively expressed HMG1 (p716), or galactose-inducible HMG2 (pRH134-2). The left-most spot is
inoculated from a culture at 1.4 ? 106cells per ml, followed by 1:5 serial dilutions. Images were taken after 5 days of growth at 16°C. The asterisk
indicates that the expression of high levels of Hmg2 results in the formation of membrane structures that are distinct from karmellae. (B) Wild-
type, ubc7?, cue1?, and doa10? cells were transformed with either galactose-inducible HMG1 (pAK266) or vector control plasmids. Transformants
were grown on glucose, galactose, or galactose plus 400 ?g of lovastatin/ml (lova). The left-most spot is inoculated from a culture at 1.4 ? 106cells
per ml, followed by 1:5 serial dilutions. Images were taken after 5 days of growth at 16°C.
716 LOERTSCHER ET AL.EUKARYOT. CELL
a specific target protein or proteins. Ubiquitination of this
target might result in either activation of a proteasome-inde-
pendent event (41) or degradation of the protein by the pro-
teasome (29). To distinguish between these two possibilities,
we examined the growth characteristics of proteasome mutants
that expressed increased levels of Hmg1. If an inability to
degrade the target protein were the basis for the Hmg1 sensi-
tivity, then cells with defects in proteasome function should
show similar cold-sensitive phenotypes as those observed in
ubc7?, cue1?, and doa10? mutants.
Although genes encoding proteins that compromise the pro-
teasome are essential, partial-loss-of-function mutants are vi-
able. Strain WCG4/11-12 (pre1-1 pre1-2) exhibits partial loss of
function of two essential 20s core particle components, Pre1
and Pre2, and has been shown to have 5% of normal protea-
some activity (18). The growth of this mutant strain and a
congenic wild-type expressing elevated Hmg1 was examined.
Under all conditions tested, pre1-1 pre2-1 mutants grew less
well than the wild type. In addition, mutant transformants
displayed variability in growth that was not observed in the
congenic wild-type transformants. Consequently, to ensure
that conclusions about the effects of increased levels of Hmg1
were not confounded by variability in growth of independent
transformants, 29 randomly selected pre1-1 pre2-1 vector con-
trol transformants and 29 randomly selected pAK266 transfor-
mants were examined. Two representative transformants for
each plasmid are shown in Fig. 4. As expected, all 29 of the
pre1-1 pre2-1 vector control transformants that were examined
grew as well with normal levels of Hmg1 (i.e., on glucose) as
with high levels of Hmg1 (i.e., on galactose). Of the 29 pre1-1
pre2-1 mutants transformed with AK266, 25 exhibited Hmg1-
induced cold sensitivity, a finding consistent with the hypoth-
esis that normal growth of cells with elevated levels of HMGR
requires ubiquitin-mediated protein degradation. Given the
poor growth of pre1-1 pre2-1 mutants, the four transformants
with normal growth may have gained reversion or suppressor
mutations that elevate proteasomal function. Interestingly, the
Hmg1-induced growth inhibition observed in proteasome mu-
tants was not as extreme as that observed in ERAD mutants.
Therefore, the cellular response to increased Hmg1 may re-
quire additional ERAD-dependent events that are proteasome
Deletion of HMG2 did not suppress cold sensitivity in ubc7?
and cue1? cells. The cold sensitivity of the pre1-1 pre2-1 strain
described above suggests that one component of the normal
cellular response to increased levels of Hmg1 is the UBC7-,
CUE1-, and DOA10-dependent degradation of a protein tar-
get. Hmg2 is a known ER-resident target of Ubc7 and the E3
ubiquitin ligase, Hrd1 (11, 16). Although Doa10 and Hrd1
have been shown to have distinct substrate specificity, experi-
mental evidence suggests that Hrd1 and Doa10 have some
overlapping function (45). Therefore, we hypothesized that
under our unique experimental conditions, the inability of the
ubc7?, cue1?, and doa10? mutants to degrade Hmg2 might be
the underlying molecular cause of their sensitivity to elevated
Hmg1 expression. If so, deletion of HMG2 in any of the mutant
strains should suppress the cold-sensitive phenotype. Double-
deletion mutants lacking both UBC7 and HMG2 were gener-
ated in the JRY527 background and transformed with either
pAK266 or a vector control plasmid. As seen in Fig. 5, deletion
of HMG2 did not restore the growth observed in vector con-
trols to ubc7? cells and, therefore, did not suppress the Hmg1-
induced cold sensitivity. Therefore, Hmg2 is unlikely to be the
essential target of UBC7-, CUE1-, and DOA10-dependent
Although Hmg1 is not a substrate for ERAD under standard
growth conditions (11), we hypothesized that it might become
subject to degradation under certain conditions, such as growth
at low temperatures. To test this hypothesis, total protein was
isolated from wild-type, ubc7?, cue1?, and doa10? cells trans-
formed with pAK266 after 24 h of karmellae induction at
permissive and nonpermissive temperatures. Immunoblot
analysis with an antibody specific to the Hmg1 isozyme re-
vealed that total levels of Hmg1 in mutant cells were the same
as or lower than that of wild-type cells (data not shown). Thus,
the loss of these ERAD proteins did not lead to the elevation
FIG. 4. Proteasome function mutants are sensitive to increased levels of Hmg1. A pre1-1, pre2-1 mutant strain and a congenic wild-type strain
were transformed with either pAK266 or vector control plasmids. The left-most spot is inoculated from a culture at 1.4 ? 106cells per ml, followed
by 1:5 serial dilutions. Images were taken after 12 days of growth at 16°C. Two independent pre1-1 pre2-1 transformants with the vector and pAK266
are shown for comparison.
FIG. 5. Deletion of HMG2 does not suppress cold sensitivity in
ubc7? cells. A ubc7? hmg2? double deletion mutant strain in the
JRY527 background was transformed with either pAK266 or vector
control plasmids. The left-most spot was inoculated from a culture at
1.4 ? 106cells per ml, followed by 1:5 serial dilutions. Images were
taken after 5 days of growth at 16°C.
VOL. 5, 2006 ERAD IN COLD ADAPTATION AND STEROL BIOSYNTHESIS 717
FIG. 6. The sterol metabolite profile of ubc7? cells in the presence of normal and increased levels of Hmg1 differs from the isogenic wild-type
control. GC was used to measure the relative amounts of sterol metabolites as a percentage of total cellular sterol. Wild-type or ubc7? cells of strain
JRY527 were transformed with galactose-inducible HMG1 (pAK266) or a vector control plasmid. Expression of Hmg1 was induced in strains
containing pAK266 by growth on galactose for either 24 h at 16°C (A) or 14 h at 30°C (B). The data shown are representative of results observed
in three similar experiments at each temperature range.
718 LOERTSCHER ET AL.EUKARYOT. CELL
of Hmg1 levels, making it unlikely that Hmg1 itself is the
essential target of Ubc7, Cue1, and Doa10.
Sterol metabolite profiles were altered in ubc7? cells.
HMGR catalyzes the formation of mevalonate, the rate-limit-
ing step in the biosynthesis of sterols and other isoprenoids in
animals and fungi (12). A reasonable molecular mechanism for
the Hmg1 sensitivity observed in mutant cells is that increased
flux through the sterol biosynthetic pathway results in accumu-
lation of a toxic metabolite whose levels are normally held in
check via the action of Ubc7, Cue1, and Doa10. For example,
Donald et al. showed that cells overexpressing the Hmg1 cat-
alytic domain accumulate increased squalene levels and show
decreased growth rates (11).
GC was used to analyze sterol metabolite composition of
ubc7? and wild-type cells in the BY4743 background in the
presence of normal and increased levels of Hmg1 (Fig. 6).
Although this method did not measure absolute sterol levels, it
provided quantitative data concerning the relative amounts of
particular sterols within a sample. Interestingly, increased lev-
els of Hmg1 resulted in a decrease in the percentage of ergos-
terol in both wild-type and ubc7? cells at both permissive and
restrictive temperatures. This result suggested that one or
more sterol biosynthetic enzymes that catalyze reactions down-
stream of squalene synthase were downregulated in response
to elevated flux through the sterol biosynthetic pathway. In
addition, this regulation appeared to be intact in ubc7? mu-
The proportion of squalene in wild-type cells expressing
elevated levels of Hmg1 was higher than in the vector control.
However, the proportion of squalene in ubc7? mutants was
actually lower than that of wild-type cells. The observation that
ubc7? cells exhibited lower squalene levels ruled out the pos-
sibility that accumulation of excess squalene in ubc7? cells was
responsible for their Hmg1 sensitivity.
Although the proportion of squalene was lower in ubc7?
cells than in the wild type, the proportion of several other
sterol metabolites was elevated in ubc7? cells grown at 16°C
with high levels of Hmg1. If the Ubc7/Cue1/Doa10 complex
regulates flux through the sterol biosynthetic pathway by tar-
geting ergosterol biosynthetic enzymes for degradation, then
the loss of UBC7 function should lead to elevated levels of
these enzymes, in turn producing inappropriately elevated
amounts of their products. Thus, the elevated proportions of
lanosterol (synthesized by Erg7), 4,4-dimethylzymosterol (syn-
thesized by Erg24), fecosterol (synthesized by Erg6), and
4-methylfecosterol (resulting from incomplete C-4 demethyla-
tion by the Erg25, Erg26, and Erg27 complex) in ubc7? mu-
tants suggest that the essential substrate(s) of the Ubc7/Cue1/
Doa10 complex may be Erg6, Erg7, Erg24, Erg25, Erg26,
and/or Erg27. Of these potential target proteins, only Erg6 and
Erg24 are nonessential. Erg6 is a soluble protein that is asso-
ciated with lipid particles and the ER (2). Erg24 is an ER
transmembrane protein (30). Thus, the localization of both
proteins is consistent with the possibility that they are ERAD
targets. We constructed ubc7? erg6? and ubc7? erg24? double
mutants to test directly whether loss of these erg genes sup-
pressed the cold-sensitive phenotype of ubc7? mutants. In
both cases, the double mutants were as cold sensitive as the
ubc7? mutants alone (data not shown.) Thus, neither the loss
of Erg6 nor the loss of Erg24 individually suppressed the cold
sensitivity of ubc7? mutants.
ubc7?, cue1?, and doa10? mutants are cold sensitive in the
absence of elevated levels of HMGR. The results presented
thus far suggested that the Ubc7-Doa10 ERAD pathway is
important for regulating sterol biosynthesis. In addition, it
appears that this regulation is particularly important at low
temperatures. Interestingly, Fig. 6 shows that expression of
increased levels of HMGR in wild-type cells growing at 30°C
results in sterol profile changes that are similar to those seen in
wild-type cells growing with normal levels of HMGR at 16°C.
In addition, the sterol profiles of ubc7? mutants were abnor-
mal under all conditions tested. Given these results, we hy-
pothesized that increased levels of HMGR produce cellular
responses similar to low temperature growth. According to this
scenario, cells growing at 16°C with high HMGR levels would
respond physiologically as if they were growing at a lower
temperature. To begin exploring this hypothesis, we examined
the ability of ubc7?, cue1?, and doa10? mutants expressing
normal levels of HMGR to grow at 10°C. As predicted, these
ERAD mutants, but not others, are cold sensitive, although
ubc7? and cue1? mutants are more cold sensitive than the
doa10? mutant (Fig. 7.)
ERAD as a regulator of the sterol biosynthetic pathway at
low temperature. Our results demonstrate that the ability of
yeast cells to thrive at low temperature requires three specific
ERAD enzymes: Ubc7, Cue1, and Doa10. The growth rate of
cells lacking any one of these enzymes is decreased at 10°C.
Impaired growth is also observed at 16°C when HMGR activity
is expressed at high levels. In addition, ubc7? mutants have
altered sterol composition profiles compared to the wild type
in the presence of both normal and elevated levels of Hmg1.
These results suggest that proper regulation of ergosterol bio-
synthesis in response to cold is an essential physiological ad-
aptation that enables yeast to survive at low temperatures.
FIG. 7. The ERAD proteins Ubc7, Cue1, and Doa10 are required
for normal growth at low temperature in the absence of high levels of
Hmg1. Wild-type yeast strains and cue1?, doa10?, and ubc7? mutants
were serially diluted (1:5) and plated onto YPD medium. After growth
at 10°C for 12 days, significant slow growth was observed in the
cue1? and ubc7? mutants. The doa10? mutant is slightly cold
sensitive at 10°C.
VOL. 5, 2006ERAD IN COLD ADAPTATION AND STEROL BIOSYNTHESIS 719
Therefore, we propose that ERAD is needed for cold adapta-
tion because it regulates ergosterol biosynthesis.
Unicellular organisms possess a remarkable ability to adjust
to environmental changes including temperature extremes.
Studies in yeast and bacteria have shown that these organisms
adapt to low temperature in part through altering the lipid and
sterol content of their membranes (see references 13 and 46).
Interestingly, S. cerevisiae has a single desaturase that can in-
troduce one double bond into the fatty acid chain (7, 43, 44).
Thus, the fatty acids present in the phospholipids of budding
yeast are either unsaturated or monounsaturated (10). Conse-
quently, modifications of sterols may play a much more impor-
tant role in the cold adaptation responses of yeast than in other
organisms that produce a wider variety of phosopholipids.
Published reports by Rodriguez-Vargas et al. and Schade et
al. provide evidence that alterations in membrane composition
at 10°C may be due to changes in global transcriptional pat-
terns (39, 40). Although changes in gene expression certainly
play a role in altering membrane composition in response to
cold, evidence presented in this report suggests that posttrans-
lational regulation is also involved in cold adaptation.
The first indication that ubc7?, cue1?, and doa10? mutants
might have defects in sterol metabolism was our observation
that these mutants are hypersensitive to miconazole (unpub-
lished results). Miconazole belongs to the azole class of anti-
fungals that inhibit ergosterol synthesis by interfering with the
function of Erg11, lanosterol demethylase (for a review, see
reference 31). Mutations that result in altered sterol profiles
can cause either hypersensitivity or resistance to azoles. Inter-
estingly, the hypersensitivity of ubc7?, cue1?, and doa10? mu-
tants to miconazole was exacerbated by increased HMGR lev-
els; in contrast, increased expression of HMGR in the wild type
resulted in decreased sensitivity to miconazole (unpublished
results). These observations led us to examine the whether
defects in ERAD affected sterol metabolism.
As suggested initially by their miconazole sensitivity, ubc7?
mutants have altered sterol metabolite profiles, leading to the
hypothesis that altered flux through the sterol biosynthetic
pathway is the basis of the cold-sensitive phenotype. Based on
the inability of erg6? or erg24? to suppress the cold sensitivity
of ubc7? mutants, Erg6 and Erg24 are unlikely to be the
essential targets of Ubc7-dependent ubiquitination. However,
other ergosterol biosynthetic enzymes remain candidates.
Hitchcock et al. identified 211 membrane-associated proteins
that are ubiquitinated, 83 of which are potential targets of
Ubc7-dependent ubiquitination (20). This analysis showed that
several ergosterol biosynthetic proteins were ubiquitinated
(Erg1, Erg2, Erg5, Erg9, Erg11, Erg25, and Erg27), and three
were potential ERAD substrates (Erg1, squalene monooxy-
genase; Erg9, squalene synthetase; and Erg27, 3-keto sterol
reductase). Because all of these genes are essential for viabil-
ity, we were unable to test whether their loss suppressed the
cold-sensitive phenotype of ubc7? mutants. Nevertheless, the
data of Hitchcock et al. confirm that ergosterol biosynthetic
enzymes are targets of ERAD, a finding consistent with our
Previous work by other labs has also suggested a relationship
among cold adaptation, membrane composition, and ERAD.
For example, transcription of OLE1, an essential gene that
encodes the only ?9 fatty acid desaturase found in yeast, is
regulated by the transcription factors Mga2 and Spt23 (52).
The activity of these transcription factors is, in turn, regulated
by proteasomal processing initiated by an ERAD complex con-
taining Npl4, Ufd1, and Cdc48 (21, 22). Importantly, Naka-
gawa et al. found that activation of Mga2 requires proteasomal
processing that it is activated by cold temperatures, leading
them to conclude that Mga2 is a low temperature sensor in
yeast (34). In addition to regulating transcriptional activators
of the OLE1 gene, ERAD components, including Ubc7, Ubc6,
and Cue1, but not Doa10, directly regulate the stability of the
Ole1 protein (7).
Taking all of these data into account, the essential nature of
Ubc7, Cue1, and Doa10 at low temperatures and in the pres-
ence of elevated levels of HMGR could be explained in several
ways. First, it is possible that improper regulation of Ole1
levels in the ERAD mutants could be the direct cause of cold
sensitivity. However, given that mga2? mutants are not cold
sensitive and are only slightly sensitive to elevated HMGR
(Table 1) and that Braun et al. (7) have shown that Doa10 is
not involved in modulating Ole1 protein stability, this simple
explanation seems unlikely. A more likely explanation is that
the ultimate cause of cold sensitivity in ubc7?, cue1?, and
doa10? cells results from a combination of altered sterol levels
and failure to regulate Ole1 levels.
ERAD as a process that integrates levels of proteins, lipids,
and membranes. The question of how cells sense the need to
alter existing or synthesize additional membranes and how they
subsequently coordinate production of the proteins and lipids
required for these changes has long puzzled biologists. Cox et
al. proposed that the unfolded protein response (UPR) is the
nexus for coordinating the functional demand for membrane
biogenesis with increased protein and lipid production (9).
Part of this hypothesis is based on their observation that kar-
mellae biogenesis requires the UPR. However, subsequent
studies conducted in our laboratory could not confirm this
observation and, instead, showed that the UPR is neither re-
quired for nor induced upon karmellae biogenesis (26). These
observations, together with the results described here, suggest
that the crucial coordination of membrane biogenesis, enzyme
activity, and lipid production in yeast may be a specialized
function of ERAD instead of the UPR. Specifically, deletion of
UBC7, CUE1, or DOA10 resulted in a variety of defects, in-
cluding altered sterol composition, inability to regulate the
amount and morphology of the ER, and cold sensitivity. Thus,
the ERAD functions carried out by Ubc7, Cue1, and Doa10
appear to be required for integration of protein stability, mem-
brane lipid composition, and three-dimensional membrane
The possibility of a broader role of ubiquitination in sterol
metabolism and cold adaptation. In addition to the strong
Hmg1-induced cold sensitivity observed in ubc7?, cue1?, and
doa10? mutants, we also observed less severe growth defects in
22 other mutants with defects in genes involved in ubiquitina-
tion or proteasome function. Few patterns arose among mu-
tants found to be slightly sensitive versus insensitive to Hmg1.
Both groups contained several ubiquitin-like proteins and sev-
eral ubiquitin-specific proteases. Although both groups con-
tained E2 and E3 proteins, the slightly sensitive group had five
E3 and one E2 protein, while the nonsensitive group had one
E3 and seven E2 proteins. Since many E3 proteins were only
720LOERTSCHER ET AL.EUKARYOT. CELL
recently identified or remain unknown, the observed distribu-
tion could simply be due to selection criteria for mutants to
include in this screen. Alternatively, this observation may sug-
gest that the function of E3 enzymes may be less redundant
than that of E2 enzymes. Regardless of the particular charac-
teristics of each group, the fact that so many ubiquitination
mutants were at least slightly sensitive to increased Hmg1
confirms the importance of the ubiquitin/proteasome pathway
in allowing cells to properly respond to changes in sterol bio-
synthetic capacity. The impaired growth of ubc7?, cue1?, and
doa10? mutants at 16°C expressing abnormally high levels of
Hmg1 resembles the observed cold sensitivity of these mutants
at 10°C expressing normal levels of Hmg1. Based on results
presented here, we hypothesize that engineered overexpres-
sion of Hmg1 mimics the physiological consequences of low
temperature. Thus, we predict that ubiquitination genes re-
quired for normal growth in the presence of elevated HMGR
will also be required for survival at 10°C.
We acknowledge the assistance of Emily Cadera, Dangelei Fox,
Brian Rezvani, and Jeff Merkel, talented undergraduates and good
friends who not only provided experimental assistance but also were
fearless in asking questions and who helped us laugh at both appro-
priate and inappropriate times. We also thank Jeff Simonson for pro-
viding the data for the ubc7? hmg2? double mutant experiment and
Peter Jauert for assistance with strain construction.
This study was supported by National Science Foundation grant
MBC-0078287 (R.W.) and National Institutes of Health grants
GM62104 (M.B.) and GM67368-01 (J.L.).
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