In eukaryotic cells, all transport between the nucleus and cytoplasm
takes place through nuclear pore complexes (NPCs), very large,
macromolecular channels that span the inner and outer nuclear
membranes (INM and ONM, respectively) (for reviews, see Tran
and Wente, 2006, Lim et al., 2008). NPCs have eightfold symmetry
perpendicular to the nuclear envelope (NE) and contain multiple
copies of approximately 30 proteins called nucleoporins. Relatively
little is known about how NPC assembly occurs, which requires
fusion of the INM and ONM to create a membrane tunnel within
which is embedded an NPC. In metazoan cells, two types of NPC
assembly take place: re-assembly late in mitosis as the NE
reassembles, and de novo assembly during interphase, when the
number of NPCs doubles (for reviews, see Antonin et al., 2008;
D’Angelo and Hetzer, 2008). Saccharomyces cerevisiaehas a closed
mitosis and all NPC biogenesis therefore occurs de novo.
Several factors in addition to nucleoporins have been implicated
in NPC biogenesis. These include components of the Ran GTPase
system (Ryan and Wente, 2002), whose role in coordinating
assembly and disassembly of karyopherin-cargo complexes during
nucleocytoplasmic transport has been well studied (for reviews, see
Conti et al., 2006; Cook et al., 2007; Stewart, 2007; Terry et al.,
2007). Cells carrying specific mutant alleles affecting these proteins
showed mislocalization of nucleoporins as well as accumulation of
nucleoporin-containing cytoplasmic vesicles (Ryan and Wente,
2002; Ryan et al., 2003; Ryan et al., 2007). Proteins involved in
endoplasmic reticulum (ER) to Golgi trafficking, including
components of the COPII coat, have also been implicated in NPC
assembly (Ryan and Wente, 2002).
Previously, we identified Apq12 in a synthetic genetic array
(SGA) screen starting with the rat8-2allele of DBP5, which encodes
a key mRNA export factor (Scarcelli et al., 2007). Apq12 is an
integral membrane protein of the ER and NE, and is required for
efficient assembly of NPCs (Scarcelli et al., 2007). Although Apq12
is not essential for viability, apq12? cells are cold sensitive (16°C)
for growth and grow best at elevated (>30°C) temperatures. In the
absence of Apq12, a subset of nucleoporins mislocalize to
cytoplasmic foci, particularly those comprising the cytoplasmic
filaments of the NPC. Electron microscopy revealed that apq12?
cells contain many defective pores that contact the INM but not the
ONM, where the cytoplasmic filaments are located (Scarcelli et al.,
Addition of the membrane-fluidizing agent benzyl alcohol to
apq12? cells suppressed mislocalization of cytoplasmic filament
nucleoporins and defects in mRNA export (Scarcelli et al., 2007).
Interestingly, increasing the amount of benzyl alcohol beyond the
level used to suppress apq12? phenotypes causes nucleoporin
mislocalization in wild-type cells (Izawa et al., 2004). This suggests
that NPC assembly is sensitive to the fluidity of the NE and that
the observed defects in NPC assembly in apq12? cells could result
from improper regulation of the lipid composition of the nuclear
membrane in response to changes in temperature. Cells respond to
changes in temperature and environmental conditions by adjusting
the composition of their membranes to maintain the fluidity and
Integral membrane proteins Brr6 and Apq12 link
assembly of the nuclear pore complex to lipid
homeostasis in the endoplasmic reticulum
Christine A. Hodge1,*, Vineet Choudhary2,*, Michael J. Wolyniak1,*,‡, John J. Scarcelli1,§, Roger Schneiter2,¶
and Charles N. Cole1,3,¶
1Department of Biochemistry and 3Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA and 2Division of Biochemistry,
Department of Medicine, University of Fribourg, CH-1700 Fribourg, Switzerland
*These authors contributed equally to this work
‡Present address: Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA 23943, USA
§Present address: Tetragenetics, Inc., One Broadway, 14th Floor, Cambridge, MA 02142, USA
¶Authors for correspondence (email@example.com; firstname.lastname@example.org)
Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are
defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6,
which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12?. Cells carrying the temperature-
sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron
microscopy revealed that brr6-1cells have gross NE abnormalities and proliferation of the ER. brr6-1cells were hypersensitive to compounds
that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions
with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of
the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously.
Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production.
These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation
and nucleocytoplasmic transport.
Key words: Membrane dynamics, Nucleocytoplasmic transport, Lipid metabolism, Nucleoporins, Nuclear envelope
Published in "Journal of Cell Science 123(): 141-151, 2010"
which should be cited to refer tothis work.
flexibility required for optimal function (Nishida and Murata, 1996).
For example, following a shift to lower temperatures, cells adjust
the phospholipid composition of their membranes to contain an
elevated proportion of unsaturated and shorter acyl chains. Relative
levels of other membrane lipid components including sterols also
change in response to temperature shifts (Buttke et al., 1980;
To gain further insight into the interplay between biophysical
properties of the NE and NPC assembly, we conducted a screen
for dosage suppressors of the cold-sensitive growth defect of
apq12?. This screen identified BRR6, an essential gene that was
originally identified in a screen for cold-sensitive mutants defective
in mRNA export (de Bruyn Kops and Guthrie, 2001). Brr6 encodes
an integral membrane protein of the ER and NE, with two
predicted transmembrane domains. Cells carrying the cold-sensitive
brr6-1 allele are defective for proper localization of some
nucleoporins (de Bruyn Kops and Guthrie, 2001). Interestingly,
overexpression of APQ12 partially suppressed the growth defect
of brr6-1 cells, indicating that Apq12 and Brr6 share some
Here, we report that cytoplasmic filaments but not nuclear basket
nucleoporins are mislocalized in brr6-1 cells at low temperatures
(16°C-23°C), in a similar fashion to apq12? cells. In contrast to
apq12?, however, brr6-1 cells are hypersensitive to agents that
increase membrane fluidity such as benzyl alcohol and oleic acid,
and to drugs that inhibit sterol or fatty acid synthesis. Consistent
with this drug sensitivity, brr6-1displays strong genetic interactions
with mutants that have defects in the late part of the sterol
biosynthetic pathway or in fatty acid elongation. Biochemical
analyses revealed that brr6-1 cells contain dramatically elevated
levels of total sterols (particularly steryl esters and episterol) and
triacylglycerols (TAGs). Moreover, overproduction of these non-
essential storage lipids is functionally important, because brr6-1
cells are non-viable if neutral lipid synthesis is blocked by deletion
of the respective biosynthetic genes. The data indicate that Apq12
and Brr6 are likely to have an important role in regulating lipid
homeostasis in the ER, thereby impacting NPC biogenesis, nuclear
transport and nuclear mRNA metabolism.
Genetic interactions between BRR6 and APQ12
To understand better the function of Apq12, we conducted a
genetic screen to identify dosage suppressors of the cold-sensitive
growth phenotype of apq12?. We found that high-copy-number
plasmids containing BRR6 restored growth of apq12? at 16°C to
a similar degree as did direct complementation with wild-type
APQ12 (Fig. 1A). Conversely, overexpression of APQ12 partially
suppressed the growth defect of brr6-1 at both low and high
temperatures (Fig. 1A). A double mutant carrying brr6-1 and
apq12? was unable to grow at all temperatures tested (data not
shown). brr6-1 cells show a modest (30% of cells) mRNA export
defect at 30°C, and this was also partially suppressed by
overexpression of APQ12 (Fig. 1B). Interestingly, overexpression
of BRR6 completely suppressed the mRNA export defect of
apq12? cells (Fig. 1B).
To determine whether overexpression of BRR6 could restore
proper localization of nucleoporins in apq12? cells, we examined
the subcellular distribution of GFP-tagged Nup82 and Nup188.
Control apq12? cells carrying an empty vector displayed aberrant
localization of both Nup82-GFP and Nup188-GFP, whereas apq12?
cells harboring the APQ12 plasmid displayed normal nucleoporin
localization (Fig. 1C). Overexpression of BRR6completely restored
Nup188-GFP localization, whereas Nup82-GFP localization was
partially restored. In the latter case, most cells showed Nup82-GFP
at both the NE and in cytoplasmic foci, many of which were located
near the nuclear periphery (Fig. 1C, and data not shown).
Overexpression of BRR6 also restored Nup159/Rat7 localization to
about the same extent as that of Nup82-GFP (data not shown).
Overexpression of APQ12 partially suppressed mislocalization of
nucleoporins in brr6-1cells (Fig. 1C). At 30°C and18°C, the fraction
of brr6-1 cells with mislocalized Nup82-GFP was approximately
25% and 60%, respectively. At 18°C, the fraction was reduced to
about 25% in cells overexpressing APQ1. Taken together, these
results indicate that Apq12 and Brr6 probably have related functions
that affect growth at low temperature, mRNA export, and NPC
assembly and distribution.
Fig. 1. Dosage suppression of growth, mRNA export and nucleoporin
mislocalization of brr6-1 and apq12? ? cells by APQ12 and BRR6. (A)?Serial
dilutions of apq12? and brr6-1 cells overexpressing BRR6 and APQ12 were
spotted onto plates and incubated at the temperatures shown. (B)?In situ
hybridization assay to analyze the effect on the mRNA-export defects of
apq12? and brr6-1 cells of over-expressing BRR6 and APQ12 by using
multicopy plasmids. (C)?Direct visualization of Nup82-GFP and Nup188-GFP
in apq12? and brr6-1 cells overexpressing BRR6 and APQ12. Nup82-GFP and
Nup188-GFP were expressed from the NUP82 and NUP188 chromosomal
loci. Scale bars: 5??m.
brr6-1 affects the assembly of NPC filaments
BRR6is essential for viability and encodes a 197 amino acid protein;
this protein, similarly to Apq12, is an integral membrane protein
of the NE-ER, with two predicted transmembrane domains. The
brr6-1allele (R110K) is both cold (16°C) and heat (37°C) sensitive.
However, even at the optimal permissive temperature (30°C), brr6-
1 cells have defects in mRNA export and two core nucleoporins,
Nsp1 and Nup188, are mislocalized (de Bruyn Kops and Guthrie,
2001). Because cytoplasmic filaments but not nuclear basket
nucleoporins are mislocalized in apq12? cells (Scarcelli et al.,
2007), we extended earlier studies by localizing additional
nucleoporins in brr6-1cells. This revealed extensive mislocalization
of Nup82-GFP, a cytoplasmic filament nucleoporin, to cytoplasmic
and perinuclear foci (Fig. 2A), but no mislocalization of Nup60-
GFP, a nuclear basket nucleoporin, at any temperature (data not
shown). At all temperatures, Nup82-GFP was localized properly in
some cells (with all or most Nup82-GFP at the nuclear periphery),
whereas in others, all or most Nup82-GFP was cytoplasmic with
many bright foci. Nup159, another cytoplasmic filament
nucleoporin, was also extensively mislocalized (data not shown),
with phenotypes ranging from normal or nearly normal localization
to mislocalization of most or all Nup159-GFP. We hypothesize that
the heterogeneity seen in the localization of cytoplasmic filament
nucleoporins in the brr6-1background reflects the different numbers
of copies of plasmid-borne brr6-1 in different cells. Because we
were unable to replace BRR6 in the genome with brr6-1, a single
copy of brr6-1 appears to be insufficient to support growth.
We showed earlier that apq12? cells are defective in NPC
biogenesis but not in NPC stability (Scarcelli et al., 2007) and
wanted to determine whether this is also the case for brr6-1 cells.
Two factors complicate this analysis. First, whereas the apq12?
cell population is genetically homogeneous (all cells lack Apq12),
brr6-1 cells are not. The brr6-1 allele is carried on a CEN plasmid
(with the genomic copy of BRR6deleted), and therefore the number
of copies of the mutant gene is not the same in all cells. Second,
in apq12? cells there is no detectable mislocalization of any
nucleoporins at 30°C and extensive mislocalization at 16°C
(Scarcelli et al., 2007). This makes it straightforward to observe
and quantify changes in nucleoporin localization when apq12?cells
are temperature shifted. By contrast, nucleoporins mislocalized in
brr6-1 cells are mislocalized to some extent at all temperatures,
although the defect is more severe at lower temperatures. Fig. 2B
shows a montage of brr6-1 cells grown at 30°C or shifted to 18°C
overnight. The heterogeneity of phenotypes is readily apparent and
indicates that even under optimal growth condition (30°C), Nup82-
GFP is mislocalized in many cells. Because cells are able to grow
and divide at this temperature, the nucleoporin-mislocalization
phenotype indicates that brr6-1cells are defective in NPC assembly.
Fig. 2. Mislocalization of cytoplasmic filament nucleoporins in brr6-1 cells.
(A)?Wild-type and brr6-1 cells expressing Nup60-GFP or Nup82-GFP were
shifted to 20°C overnight and live cells directly visualized. (B)?Wild-type and
brr6-1 cells grown either at 30°C or shifted to 18°C overnight before
visualization. Note the heterogeneity of the distribution of Nup82-GFP in
brr6-1 but not in wild-type cells (right). Scale bars: 5??m.
Fig. 3. Electron microscopy reveals NE abnormalities in brr6-1 cells. Wild-type and brr6-1 cells were grown at 30°C or shifted to 18°C overnight and processed
for thin-section electron microscopy. (A)?Wild-type cells, 18°C; (B) wild-type cells, 30°C; (C-E) brr6-1 cells, 18°C; (F,G) brr6-1 cells, 30°C. Arrows and
arrowheads: electron-dense structures and inclusions [arrowheads (A,B,G), arrow (D)], sometimes clustered [arrows (E,F)] or clustered in additional double
membranes [black arrows (C,G)]; extensive white expansions of the NE and ER [white arrows (C)]. n, nucleus.
Therefore, it is difficult to determine whether NPCs are also unstable
To define the NPC mislocalization and NE phenotype of brr6-
1 at the ultrastructural level, cells were fixed and analyzed by
electron microscopy (Fig. 3). In wild-type cells at both 18°C and
30°C, NPCs appear as electron-dense material that spans the lumen
of the NE (arrowhead in Fig. 3A,B). Essentially all NPCs from
wild-type cells had this normal appearance. By contrast, in brr6-1
cells at both 18°C (Fig. 3C-E) and 30°C (Fig. 3F,G) severe
abnormalities were seen. The overall number of NPCs or related
structures seen in each section was approximately half that seen in
wild-type sections. Of these, about one-third appeared to be normal.
In brr6-1 cells, there were many electron-dense inclusions beneath
the INM or between the INM and ONM (black arrow in Fig. 3D,
white arrowheads in Fig. 3G), often roughly spherical, larger than
NPCs and sometimes clustered (arrows in Fig. 3E,F). At 30°C, there
were about half as many of these inclusions as normal-appearing
NPCs, whereas in cells maintained at 18°C, dense inclusions in
these locations were about twice as common as normal-appearing
NPCs. Interestingly, membrane blebs, most containing electron-
dense inclusions, were seen in brr6-1 cells at 30°C (about as many
blebs near the NE as there were normal-appearing NPCs), but very
rarely in cells maintained at 18°C overnight. We cannot tell whether
these are partially-assembled NPCs, aggregates of nucleoporins or
other material. In about one-third of thin sections, electron-dense
structures were clustered together in additional double-membranes
(black arrows in Fig. 3C,G), often extending linearly a considerable
distance away from the NE (Fig. 3C). Although these structures
resemble NPCs, we do not believe that they are NPCs or NPC-
related structures because GFP-tagged nuclear basket nucleoporins
and some core nucleoporins were detected only at the nuclear
periphery in brr6-1 cells, and never in these projections (data not
shown). The composition of these structures is not known. The
extensive white expansions of the NE and ER (white arrows in Fig.
3C) might be lipids, reflecting abnormalities in lipid metabolism
(see below). These were observed in approximately 30% of thin
sections of brr6-1 cells, but in only about 5% of sections of wild-
type cells, and those seen in wild-type cells were much smaller than
those in brr6-1 cells. In apq12? cells, there were very few normal
NPCs and a large number of what appeared to be partial NPCs
extending from the INM part of the way across the lumen of the
NE (Scarcelli et al., 2007). Partial NPCs were rare in brr6-1 cells
and very few normal-appearing NPCs were present. This might
indicate a more severe NPC assembly defect in these cells compared
withapq12??cells. These aberrant phenotypes were seen very rarely
in wild-type cells.
brr6-1 is hypersensitive to treatments that affect
membrane fluidity and to drugs that inhibit lipid
A key finding made earlier was that the growth of apq12? cells in
medium containing benzyl alcohol partially suppressed defects in
nucleoporin localization and mRNA export (Scarcelli et al., 2007).
The growth of brr6-1 cells was inhibited by benzyl alcohol at all
temperatures (Fig. 4A). Since the cold-sensitive phenotype conferred
by the brr6-1 and apq12? mutations might reflect a loss of ability
to make proper adjustments in membrane fluidity, we wondered
whether supplementing the medium with the unsaturated fatty acid
oleic acid could rescue the defect. Surprisingly, 5 mM oleic acid
was detrimental to the growth of both brr6-1andapq12?cells (Fig.
We examined the effects on brr6-1 and apq12? cells of
terbinafine (5-50 ?g/ml) and ketoconazole (1-2 ?g /ml), which are
compounds that inhibit different enzymes in the ergosterol
biosynthesis pathway (Petranyi et al., 1984; Sheets and Mason,
1984; Meredith et al., 1985). The two drugs had little or no effect
on growth of wild-type or apq12? cells (Fig. 4B). Strikingly, the
brr6-1 strain was hypersensitive to both. Cerulenin is an inhibitor
of fatty acid synthase (Omura, 1981). Growth of both brr6-1 and
apq12? was inhibited by cerulenin under conditions where growth
of wild-type cells was not (Fig. 4B). We conclude that the brr6-1
mutation confers sensitivity to inhibitors of multiple lipid
Synthetic genetic interactions between lipid biosynthesis
mutants and brr6-1 and apq12? ?
We described earlier the genetic interaction between apq12? and
acc1-7-1 (Scarcelli et al., 2007). ACC1 encodes acetyl-CoA
Fig. 4. Effect of compounds that alter membrane properties or inhibit
lipid biosynthetic pathways on growth of wild-type, brr6-1 and apq12? ?
cells. Serial dilutions of wild-type, brr6-1 and apq12? cells were grown on
YPD plates or on plates containing: (A) the membrane fluidizers benzyl
alcohol or oleic acid, or (B) drugs that inhibit sterol synthesis (terbinafine and
ketoconazole), or fatty acid synthesis (cerulenin) and incubated at the
carboxylase, the rate-limiting enzyme in fatty acid synthesis. The
apq12? acc1-7-1 double-mutant strain grew considerably less well
than either single mutant at lower temperatures, and considerably
better at 34°C than the temperature-sensitive acc1-7-1mutant alone.
We extended this analysis by testing for synthetic interactions of
bothapq12?andbrr6-1with deletions of non-essential genes whose
products act in diverse lipid biosynthetic pathways. The results are
summarized in Table 1. brr6-1 displayed extensive synthetic
lethality, with mutations affecting sterol and fatty acid metabolism
(e.g. erg2,erg3,erg5,erg6; elo1,elo2,elo3; mga2, spt23). These
genetic interactions are consistent with the hypersensitivity of brr6-
1 cells to inhibitors of sterol and fatty acid biosynthesis. Although
there were fewer cases where apq12? showed synthetic lethality
or growth defects when combined with lipid biosynthetic mutants,
we did observe synthetic growth defects between apq12?anderg2?.
Nem1 and Spo7 are subunits of a heterodimeric phosphatase
(Siniossoglou et al., 1998; Santos-Rosa et al., 2005) that regulates
indirectly several phospholipid biosynthetic genes by modulating
the phosphorylation state of Pah1, a transcriptional repressor whose
targets are phospholipid biosynthetic genes (Siniossoglou et al.,
1998; Santos-Rosa et al., 2005). The morphology of the NE is
abnormal in cells lacking Spo7, Nem1 or Pah1/Smp2, which is also
the case in brr6-1 and apq12? cells. brr6-1 displayed synthetic
lethality with spo7? but not with nem1?. This is discussed further
apq12? ? and brr6-1 cells have aberrant lipid profiles
The observations that brr6-1 and, to a lesser extent, apq12? cells,
are sensitive to drugs that inhibit lipid biosynthetic pathways,
together with the fact that these mutants exhibit synthetic
interactions with mutations affecting multiple lipid biosynthetic
pathways, suggest that Apq12 and Brr6 might directly or indirectly
regulate membrane lipid composition and/or lipid synthesis. We
examined the phospholipid and neutral lipid composition of apq12?
and brr6-1 cells by labeling with [3H]palmitic acid for 6 hours at
20, 24 or 37°C. Lipids were extracted and analyzed by thin layer
chromatography (TLC). This revealed that wild-type and mutant
strains had the same phospholipid composition (data not shown).
By contrast, there was a twofold increase in the level of neutral
triacylglycerols (TAGs) in brr6-1 cells at 37°C (Fig. 5A). This
hyper-accumulation was rescued completely by the presence of
wild-type BRR6, indicating that Brr6 function is important for
neutral lipid homeostasis at 37°C.
Drug sensitivity and genetic interactions also suggested a possible
role for Brr6 in sterol function and/or metabolism. To examine
whether apq12? and brr6-1 affect sterol metabolism, lipids were
Table 1. Genetic interactions of apq12? ? and brr6-1 with mutations affecting lipid biosynthesis
Lipid mutant tested
Function of encoded protein
Sterol homeostasis, GPI-anchor synthesis
Acyl-CoA sterol acyltransferases
Acyltransferases required for triacylglycerol synthesis
Phosphatase as dimer with Nem1p
Phosphatase as dimer with Spo7p
Fatty acid elongation
Fatty acid elongation
Fatty acid elongation
Transcription factor for OLE1
Transcription factor for OLE1
Abbreviations: SL, synthetically lethal; SS, synthetically sick; NT, not tested.
Fig. 5. Analysis of TAGs and sterols in apq12? ? and brr6-1 cells. (A)?brr6-1
cells accumulate triacylglycerols. Cells were labeled with [3H]palmitic acid for
6 hours at 37°C. Lipids were extracted, separated by thin layer
chromatography and levels of radiolabeled fatty acids in the different lipid
classes were quantified by radio-scanning. (B)?brr6-1 cells have elevated
levels of steryl esters. (C)?brr6-1 and apq12? cells accumulate the sterol
precursor episterol. Cells were cultivated overnight at 24°C. Lipids were
extracted and sterols were analyzed and quantified by GC-MS, using
cholesterol as an internal standard. These analyses were performed
independently two (B,C) or three (A) times and results shown are means ±
extracted from cells grown overnight at 24°C, and the sterol profile
was determined by gas chromatography and mass spectrometry
(GC-MS). This revealed a sevenfold increase in the level of total
sterols in brr6-1 compared with wild-type cells (Fig. 5B), which
was due primarily to increased levels of steryl esters, because the
levels of free sterols in the brr6-1 strain were not significantly
different from the wild type (data not shown). In addition, this
analysis revealed a three- to fourfold increase in the level of the
sterol episterol in both apq12? and brr6-1 cells (Fig. 5C). Episterol
is an intermediate in ergosterol biosynthesis, but is normally present
at a very low level.This phenotype was also rescued by the presence
of plasmid-borne APQ12(inapq12?cells) or BRR6(inbrr6-1cells).
Taken together, these results indicate that apq12? and brr6-1 cells
have defects in sterol synthesis and that brr6-1cells possess a strong
defect in neutral lipid homeostasis, resulting in the accumulation
of both triacylglycerols and steryl esters.
Overexpression of Brr6 restores normal episterol levels to
apq12? ? cells
We next tested how the altered sterol levels of apq12? and brr6-
1 cells were affected by overexpression of BRR6 or APQ12,
respectively. The data in Fig. 6A show that overexpression of
BRR6 restored the normal level of episterol in apq12? cells. By
contrast, only a modest reduction in the level of episterol was
seen when APQ12 was overexpressed in brr6-1 cells (Fig. 6B)
and this was not statistically significant. Most likely, the difference
in the ability of Apq12 and Brr6 to suppress the defects in episterol
levels reflects the fact that Brr6 is essential and Apq12 is not.
This could also underlie the partial suppression of the brr6-1
growth defect by overexpression of APQ12, in contrast to the
complete suppression of the apq12? growth defect by BRR6.
Importantly, the suppression of both the growth defects and
elevated episterol level of apq12? by BRR6 supports the
hypothesis that Apq12 and Brr6 have direct roles in modulating
or sensing lipid levels or membrane properties.
Production of neutral lipids is essential for viability of brr6-1
To examine whether the increased levels of TAGs and steryl esters
in brr6-1 are of functional importance, we tested the viability of
brr6-1 and apq12? cells in combination with deletions of ARE1
and ARE2, which encode the two yeast sterol acyltransferases, or
LRO1andDGA1,which encode two acyltransferases that synthesize
triacylglycerol. Because an are1? are2? double mutant is viable
(Yang et al., 1996; Yu et al., 1996), synthesis of steryl esters is
dispensable in an otherwise wild-type background. Similarly,
because a lro1? dga1? double mutant is viable, synthesis of
triacylglycerides is also dispensable (Oelkers et al., 2002; Sorger
and Daum, 2002). However, combining are1? are2? with brr6-1
caused a strong synthetic growth defect (Fig. 7A), indicating that
conversion of free sterols to steryl esters is important for growth
of brr6-1 cells. Synthetic lethality was seen when brr6-1 was
combined with lro1?dga1?(Fig. 7B), indicating that TAG synthesis
is essential for growth of brr6-1 cells.
The fact that brr6-1 cells accumulate elevated levels of steryl
esters and require steryl ester formation for survival could indicate
that these cells have elevated levels of free sterols in the ER that
might result from a defect in export of free ergosterol from the
ER. We monitored the subcellular distribution of sterols using the
fluorescent sterol analogue NBD-cholesterol, which we have
previously shown to reflect the known natural subcellular
distribution of ergosterol (Reiner et al., 2006). Mutant cells
deficient in heme production (to block endogenous sterol synthesis
and allow uptake of exogenous sterols) were incubated with NBD-
cholesterol for 1 hour at 24°C and its distribution was analyzed
by fluorescence microscopy. This revealed prominent ring-like
staining of the plasma membrane and punctuate intracellular
staining of lipid droplets in wild-type cells (Fig. 8A). By contrast,
the brr6-1 mutant displayed substantially elevated staining of
aberrantly large lipid droplets that were frequently in close
proximity to or possibly associated with the nuclear ER (Fig. 8A,
arrowheads), as revealed by visualization of the ER with the ER
Fig. 6. Overexpression of BRR6 suppresses the elevated level of episterol
in apq12? ? cells. apq12? and brr6-1 cells containing an empty vector or
overexpressing BRR6 (A) or APQ12 (B), respectively, from multicopy
plasmids, were cultivated overnight at 24°C. Lipids were extracted and sterols
were analyzed and quantified by GC-MS. These analyses were performed
independently two times and results shown are means ± s.e.m.
Fig. 7. Production of steryl esters and triacylglycerides is essential in brr6-
1 cells. (A)?Analysis of brr6-1 synthetic interactions with are1? are2?. ARE1
and ARE2 were deleted in a brr6-1 background to produce the are1? are2?
brr6-1 triple mutant. Growth of the indicated strains on YPD plates is shown.
(B)?Analysis of brr6-1 synthetic interactions with lro1? dga1?. LRO1 and
DGA1 were deleted in a brr6-1 strain containing a wild-type copy of BRR6 on
a URA3 plasmid. Cells were plated on YPD plates and then replicated to YPD
plates and to plates containing 5-fluoro-orotic acid (5-FOA) to select for
colonies that have lost the URA3 BRR6 plasmid. Plates were incubated for 4
days at 24°C.
luminal marker Kar2-mRFP-HDEL (Gao et al., 2005).
Endogenously produced sterols showed similar localization, as
revealed by staining with filipin (data not shown). Lipid droplets
are dedicated storage organelles for neutral lipids and contain
primarily steryl esters and triacylglycerides. These observations,
together with the observed elevated levels of steryl esters, indicate
that brr6-1 hyperesterifies sterols that are synthesized
endogenously, as well as sterols taken up from the outside.
To monitor the sterol hyperesterification phenotype more directly,
heme-deficient cells were incubated with [14C]cholesterol and the
conversion of free cholesterol to steryl esters was analyzed by TLC
analysis of radiolabeled sterols. This revealed that brr6-1 cells
accumulated approximately one-third more free sterol (Fig. 8B) than
wild-type cells. Furthermore, brr6-1 cells had a strongly elevated
rate of steryl ester formation as they produced three times as many
steryl esters than wild-type cells after 8 hours of incubation in
medium containing [14C]cholesterol (Fig. 8B).
The observation that brr6-1 has strongly elevated levels of the
two neutral lipids, steryl esters and TAGs, and the fact that steryl
ester and TAG formation is essential in brr6-1 point to a possible
function of Brr6 in neutral lipid homeostasis. The enzymes that
synthesize neutral lipids (Are1, Are2, Dga1 and Lro1) are localized
in ER membranes (for a review, see Czabany et al., 2007). The
resulting neutral lipids are then deposited into lipid droplets. These
lipid droplets are believed to be formed from the ER membrane,
from which they might bud off to become independent organelles
(Murphy and Vance, 1999). It is interesting to note that apq12?
was identified as an mld (many lipid droplets) mutant in a screen
of the yeast knockout strains to identify mutants with altered lipid-
droplet morphology (Fei et al., 2008).
To examine whether brr6-1 displayed an aberrant lipid-droplet
phenotype, lipid droplets were visualized using Erg6-RFP as a
marker protein. Erg6-RFP was mislocalized to the NE in brr6-1
cells, and lipid droplets were frequently seen encircling the NE of
these cells (Fig. 9, arrowheads). Quantification of this lipid droplet
phenotype revealed that in 72.5% of brr6-1 cells, lipid droplets
were arranged in a circular pattern around the DAPI-stained
nucleus. In wild-type or apq12? cells, by contrast, lipid droplets
were observed in a circular pattern around the nucleus in fewer
than 30% of cells. These observations suggest that the
overproduction of neutral lipids in brr6-1 results in over-
accumulation of neutral lipids in the NE and perinuclear ER, where
the enzymes for TAG and steryl ester synthesis are located, and
that these neutral lipids are not efficiently partitioned into lipid
droplets. Accumulation of neutral lipids in the ER, as suggested
by ultrastructural analysis (Fig. 3C), could then result in altered
membrane properties and defects in NPC assembly.
Fig. 8. brr6-1 cells accumulate free sterols and steryl esters. (A)?Subcellular
distribution of NBD-cholesterol. Heme-deficient cells of the indicated
genotype and expressing the ER marker Kar2-mRFP-HDEL were incubated
with the fluorescent sterol analog NBD-cholesterol for 1 hour at 24°C and the
distribution of NBD-cholesterol was analyzed by fluorescence microscopy.
White arrowheads indicate staining of the NE-ER. The numbers indicate the
proportion of NBD-cholesterol-stained lipid droplets that colocalize with the
Kar2-mRFP-HDEL-stained NE-ER (n?100 cells). Scale bar: 5??m. (B)?Heme-
deficient cells of the indicated genotype were incubated with [14C]cholesterol.
Samples were removed at the time points indicated, lipids were extracted and
the levels of free and esterified cholesterol were quantified. Results shown are
means ± s.e.m.
Fig. 9. brr6-1 cells have aberrant lipid droplets. Cells were transformed with
a plasmid expressing the lipid droplet marker protein Erg6-RFP and analyzed
by fluorescence microscopy. White arrowheads indicate lipid droplets that
encircle the NE-ER. The numbers indicate the proportion of cells that display
circular arrangement of Erg6-RFP marked lipid droplets around the nucleus
(n?100 cells). DNA was revealed by staining with DAPI. Scale bar: 5??m.
Genetic interactions and similar mutant phenotypes for
brr6-1 and apq12? ?
We identified BRR6 as a dosage suppressor of apq12?. brr6-1 and
apq12? are synthetically lethal, and APQ12 and BRR6 were able
to suppress the cold-sensitive growth defects of each other (Fig. 1).
Although the phenotypes seen in brr6-1 cells are similar to those
of apq12?, brr6-1 defects were generally much stronger. brr6-1
was more sensitive to chemical and pharmacological agents that
impact lipid biosynthesis and membrane fluidity, showed more
extensive alterations in the levels of neutral lipids, and was
synthetically lethal with more lipid biosynthetic mutants than was
apq12?. These differences are consistent with a more important
role for Brr6 in the maintenance of membrane fluidity, than for
Apq12, which is not essential.
A third gene, encoding a Brr6-related protein, BRL1(BRR6-like),
was identified as a suppressor of a temperature-sensitive xpo1
(exportin 1) mutant (Saitoh et al., 2005). Brl1 is essential, has
substantial homology to Brr6 (24% identical and 46% similar) and
is also found in the NE. Proteins containing the domain shared
between Brr6 and Brl1 are present in fungi that undergo closed
mitoses, including Candida glabarata
neoformans. brl1 mutants share phenotypes of nucleoporin
mislocalization and mRNA-export defects with brr6-1 and apq12?
(Saitoh et al., 2005). BRR6 was able to suppress the growth defect
of brl1 mutants, which were synthetically lethal with brr6-1. Brl1
and Brr6 interacted with each other in a two-hybrid analysis (Saitoh
et al., 2005). However, we found that BRL1 could not suppress
apq12? (unpublished results). Future studies will investigate the
possibility that these three proteins function together.
Normal nuclear morphology is dependent upon membrane
Many earlier studies indicated that the shape of the nucleus is related
to lipid composition of cellular membranes. Nem1 and Spo7 are
the catalytic and regulatory subunits, respectively, of an ER-NE-
associated protein phosphatase (Siniossoglou et al., 1998; Santos-
Rosa et al., 2005). The target of this enzyme is Pah1/Smp2 (Santos-
Rosa et al., 2005), which acts as a transcriptional repressor of several
phospholipid biosynthesis genes and is a homologue of mammalian
Lipin, expressed at a high level in adipose tissue (Peterfy et al.,
2001). Pah1 has phosphatidic acid phosphatase activity and thereby
controls the synthesis of triacylglycerols (Han et al., 2006). NEM1,
SPO7 and PAH1 are non essential, but strains lacking any one have
abnormal nuclear morphology including extensions of the NE that
contain NPCs (Siniossoglou et al., 1998; Tange et al., 2002).
Overproduction of Pah1 restores normal nuclear membrane structure
to nem1? and spo7? cells. In cells lacking Pah1, defects in lipid
metabolism affect multiple classes of lipids including
triacylglycerols (Han et al., 2006). These studies demonstrate that
nuclear shape and the distribution of NPCs depends upon proper
regulation of lipid biosynthesis. The finding that brr6-1 is
synthetically lethal with spo7? but not nem1? suggests that
deletions of NEM1 and SPO7 affect certain aspects of TAG
synthesis differently from each other. Spo7 is the regulatory subunit
for the Spo7/Nem1 heterodimer (Siniossoglou et al., 1998; Santos-
Rosa et al., 2005) and could be important for localized activation
of Pah1. This might explain why brr6-1 is lethal when combined
with spo7? but viable when combined with nem1?. We also found
that PAH1 is essential in our wild-type strain background. PAH1,
SPO7 and NEM1 have also been linked genetically to NUP84
(Siniossoglou et al., 1998; Santos-Rosa et al., 2005). Together, these
results are consistent with our finding that apq12? and brr6-1cells
have alterations in lipid metabolism, NE structure and shape, and
Other studies have suggested that the lipid composition of NE
itself is important for NPC biogenesis. A mutation in ACC1/MTR7,
which encodes acetyl CoA carboxylase, was isolated in a screen
for mRNA-export mutants (Schneiter et al., 1996). Acc1 is essential,
and is responsible for synthesis of malonyl CoA, the key building
block for synthesis of fatty acids. mtr7-1 acc1-7-1 cells have been
shown to have abnormal NPCs and mislocalized nucleoporins, and
were defective in the formation of very-long-chain fatty acids,
something not observed with other acc1 alleles (Schneiter et al.,
1996). This suggested that very-long-chain fatty acids might have
a role in NPC assembly, perhaps by helping to bring together the
INM and ONM at points where fusion is to occur, as NPCs are
assembled (Schneiter et al., 1996; Schneiter et al., 2004).
brr6-1 and apq12? ? might affect the functioning of a
An interesting possibility to account for our results is that the
apq12? and brr6-1 mutations impact the known ability of cells
to sense environmental changes that normally trigger
modifications in membrane composition needed to maintain
membrane homeostasis (for reviews, see Murata and Los, 1997;
Los and Murata, 2004; Zhang and Rock, 2008). Little is known
about how fluidity and other biophysical properties of membranes
are sensed and how this information is transduced to adjust lipid
synthesis and membrane composition. Fluidity sensors are thought
to respond to decreases in temperature, in part through activating
fatty acid desaturases, which introduce double-bonds into acyl
chains of fatty acids and phospholipids, and thereby have a large
impact on the melting temperature of membranes. To function
properly, cells must be able to sense the need to induce changes
in membrane composition and to prevent changes of too great a
Yeast has a single fatty acid desaturase, Ole1 (Stukey et al., 1989),
whose production is tightly regulated. Mga2 and Spt23, two
homologous membrane-associated transcription factors related to
mammalian NF-kB, are released from membrane association by
proteolysis when cells sense the need to activate OLE1 (Hoppe et
al., 2000). Neither Mga1 nor Spt23 is essential, but cells lacking
both are not viable (Zhang et al., 1999). brr6-1 showed synthetic
lethality with both mga2? and spt23? (Table 1). Morphological
defects in the NE were seen following a shift of an mga2? spt23-
ts mutant to non-permissive temperature (Zhang et al., 1999). Cells
lacking Ole1 are able to grow when supplemented with unsaturated
fatty acids (UFAs) but the membrane abnormalities seen following
depletion of UFAs are substantially more severe than those seen in
mga2?spt23-tscells (Zhang et al., 1999). One possibility to account
for this is that membrane anchoring of Mga2 and Spt23 might be
sensitive to changes in membrane fluidity, such that changes in
fluidity could promote proteolytic activation of Mga2 and Spt23,
thereby leading to inappropriate activation of OLE1.
Ifbrr6-1 or apq12?cells were defective in their ability to induce
modifications in membrane composition in response to a shift to
a lower temperature, it might be expected that supplementing the
medium with oleic acid would at least partially suppress the
defects. The double bond in the oleate acyl chain introduces a
kink that disrupts acyl chain packing in the lipid bilayer. An
increase in oleate incorporation into phospholipids therefore can
increase membrane fluidity (Hazel, 1995). Suppression of brr6-
1 and apq12? by oleic acid was not observed. Rather, both strains
showed sensitivity to oleic acid at 23°C and brr6-1 cells were
also sensitive to oleic acid at 30°C (Fig. 4A). This suggests that
the observed defects might reflect an excess of membrane fluidity
at lower temperatures, as opposed to a deficit, which is consistent
with the hypersensitivity of brr6-1 cells to benzyl alcohol. If this
were the case, then the primary defect might be the inability of
cells to sense when proper membrane composition and dynamics
had been restored following a temperature shift.
Lipid esterification is essential for viability of brr6-1
Our finding that the viability ofbrr6-1cells depends on esterification
of sterols (Fig. 7) is particularly intriguing and suggests that these
cells accumulate elevated levels of free sterols in the ER that need
to be neutralized by esterification so that they do not adversely affect
ER and NE function. Elevated levels of free cholesterol in the ER
of animal cells result in depletion of ER calcium stores, induction
of the unfolded protein response (UPR) and ultimately apoptosis
(Feng et al., 2003). Mechanistically, this has been explained by an
inhibition of the normal conformational freedom of ER by the
cholesterol-induced increase in membrane lipid order (Li et al.,
2004). In yeast, Arv1, which is a conserved ER integral membrane
protein, is required for viability of cells that cannot esterify free
sterols. Previous work has shown that arv1? mutant cells
accumulate sterols in the ER and display elevated levels of steryl
esters (Tinkelenberg et al., 2000). Arv1 is also required for the
maturation of glycosylphophatidylinositol (GPI) anchors, which are
attached to the C-terminus of proteins in the ER lumen. The
alterations in sphingolipid and sterol levels observed in arv1? have
been proposed to be a secondary consequence of a reduced flux of
lipids out of the ER (Swain et al., 2002; Kajiwara et al., 2008). Our
observation that arv1? is synthetically lethal with brr6-1 indicates
that Brr6 provides an overlapping function with Arv1 required for
lipid homeostasis in the ER. Although we did not observe defects
in ER function in apq12? cells (Scarcelli et al., 2007), growth of
brr6-1 is sensitive to tunicamycin (our unpublished results), a
characteristic of mutants with defects in ER function.
Because brr6-1 cells contain elevated levels of TAGs and steryl
esters, they would be expected to be more resistant to cerulenin
than wild-type cells are. Fatty acids can be released from TAGs
and steryl esters to bypass and compensate for the block in fatty
acid synthesis caused by cerulenin. Because brr6-1 cells are more
sensitive than the wild type to cerulenin, they appear to depend
more strongly on ongoing fatty acid synthesis. This might be needed
to move another lipid (free sterol, diacylglycerol, or phosphatidic
acid) into the neutral lipid pool, where any excess would be less
harmful than in a non-acylated form. The finding that brr6-1 is
synthetically lethal with elo1?, elo2? and elo3?, supports the idea
that the ability to make a range of fatty acids has increased
importance when cells are mutant for brr6.
The data presented here provide strong support for the hypothesis
that Apq12 and Brr6 have a direct role in lipid and membrane
homeostasis and that the observed defects in NPC biogenesis and
mRNA export are consequences of the resulting altered membrane
properties. Because many strains with defects in mRNA export (e.g.
rat8-2/dbp5, mex67-5, prp20-1) have normal NPCs and NE (Aebi
et al., 1990; Segref et al., 1997; Tseng et al., 1998), an mRNA-
export defect cannot be the underlying cause of the NE and lipid
metabolism abnormalities seen in apq12? and brr6-1 cells. Defects
in NPCs and the NE are seen in some strains that lack a non-essential
nucleoporin or produce a mutant form of an essential nucleoporin.
This might indicate that unsuccessful attempts to assemble NPCs
can lead to defects in the NE, perhaps resulting from abnormal
interactions between the NE and integral membrane nucleoporins
or nucleoporins that have a role in curvature of the NE at sites of
NPC insertion. However, the fact that neither Apq12 nor Brr6 are
components of NPCs makes it unlikely that the primary direct defect
from mutations affecting these proteins is in NPC assembly. It will
be interesting to see whether there are defects in lipid metabolism
in these nucleoporin mutant strains.
It is striking that the NE and NPC defects seen in brr6-1 and
apq12? cells are considerably more severe than in cells lacking
individual non-essential lipid metabolism genes or those treated with
a range of compounds that affect membrane properties and lipid
biosynthesis. Further studies will be required to understand the
mechanism by which Brr6 and Apq12 affect lipid metabolism and
the fluidity of the nuclear membrane, and how this impacts NPC
Materials and Methods
Yeast strains and plasmids
The strains and plasmids used in these studies are listed in supplementary material
Table S1. All strains were grown and media prepared using standard methods
(Sambrook et al., 1989). For growth assays, strains were grown overnight, then diluted
back to OD600?0.3. Strains were then serially diluted 1:10, and 3 ?l of each dilution
Live-cell fluorescence microscopy was performed using cells grown and mounted in
SCD medium. Images were acquired using a Nikon TE2000-E microscope fitted with
a Nikon ?100 Plan Apochromat oil objective (NA 1.4), Orca-ER CCD camera
(Hamamatsu, Bridgewater, NJ) and Phylum Live Software, version 3.5.1 (Improvision,
Lexington, MA) or a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen,
Germany) equipped with an AxioCam CCD camera and AxioVision 3.1 software.
GFP, Cy3 and Cy5 were visualized by using an X-cite 120 UV lamp and Chroma
filter sets. Images were processed using Adobe Photoshop (Adobe Systems, San Jose
CA). The fluorescence in situ hybridization assay (FISH) was performed as described
previously (Cole et al., 2002).
Electron microscopy was performed as previously described (Scarcelli et al., 2007).
In brief, the cells were grown to an OD600of 0.5-1.0 in YPD medium, pelleted and
resuspended in 0.1 M cacodylate buffer, pH 6.8. Primary fixation was performed
with 3% glutaraldehyde, 1% paraformaldehyde and 0.1% tannic acid in 0.1 M
cacodylate buffer, pH 6.8, at room temperature for 1 hour and then overnight at 4°C.
Cells were washed twice with 0.1 M cacodylate buffer, pH 6.8, and twice with 0.1
M phosphate buffer, pH 7.5, and treated with zymolyase 100T (10 mg/ml) to produce
spheroplasts. After washing with phosphate buffer, pH 7.5, and cacodylate buffer,
pH 6.8, the cells were retreated with 3% glutaraldehyde, 1% paraformaldehyde and
0.1% tannic acid in 0.1 M cacodylate buffer, pH 6.8, for 1 hour at room temperature,
washed three times with 0.1 M cacodylate buffer, pH 6.8, and embedded in low melting
temperature agarose (SeaPrep; FMC). Post fixation was performed with 2% osmium
tetroxide in 0.1 M cacodylate buffer, pH 6.8, for 1 hour on ice. Subsequently, the
cells were washed in cacodylate buffer, pH 6.8, and deionized water, en bloc stained
with 0.5% uranyl acetate overnight, dehydrated with ethanol, and embedded in Spurr’s
resin (medium grade). Thin sections were cut on an ultramicrotome (MT5000; Sorvall)
with a section thickness of 100 nm. Sections were poststained with uranyl acetate
and Venable and Coggleshell’s lead citrate and examined on a transmission electron
microscope (JEM 1010; JEOL) at 100 kV.
A Yep13 2 ? LEU2 library (Rose and Broach, 1991) was utilized to identify genes
that could suppress apq12? cold-sensitivity at 16°C. The library was transformed
into apq12? cells, and cells were plated on leucine dropout medium and incubated
at 16°C for 14 days. Candidate suppressor colonies were retested for growth at 16°C.
apq12? cells carrying an empty LEU2 vector were included as a negative control.
? suppressor screen
All compounds were purchased from Sigma and dissolved in dimethyl sulfoxide
(DMSO) prior to addition to media. Terbinafine from Neil Ryder (Novartis Research
Institute, Vienna, Austria) was used in some experiments. With the exception of oleic
acid and palmitic acid, the appropriate compound was added to YPD medium
following autoclaving. Oleic acid and palmitic acid were added before autoclaving
along with 0.5% Tween-40 to facilitate dissolving. Compounds were used at the
following concentrations: 0.2% benzyl alcohol, 5 mM oleic acid, 5 ?g/ml terbinafine,
1 ?g/ml ketoconazole, 2.5 ?g/ml cerulenin. For growth analysis in the presence of
these agents, strains were diluted tenfold four times across rows of a 96-well plate,
and 4 ?l samples from the original and diluted samples were spotted onto YPD plates
supplemented with the appropriate agent. Plates were grown for 5 days at 23°C, 30°C
and 37°C and photographed after 3 and 5 days or at 16°C for 14 days and photographed
after 7 and 14 days.
Total lipids were analyzed following [3H]palmitic acid labeling of cells. Cells were
cultivated in YPD at 24°C, diluted in fresh medium and incubated at 20°C, 24°C or
37°C for 1 hour before addition of 10 ?Ci/ml of [9,10-3H]palmitic acid (10 mCi/ml;
American Radiolabeled Chemicals, St Louis, MO). After 6 hours of labeling, cells
were collected, lipids were extracted with chloroform and methanol (1:1, v/v), lipids
were dried under a stream of nitrogen and aliquots of the lipid extract containing
equal counts were analyzed by thin layer chromatography (TLC) using silica gel 60
plates (Merck, Darmstadt, Germany). Phospholipids were separated using the solvent
system chloroform, methanol and potassium-chloride (0.25%) (55:45:5, v/v/v).
Neutral lipids were separated using petroleum ether, diethyl ether and glacial acetic
acid (70:30:2, v/v/v). Plates were then analyzed by radio-TLC scanning (Tracemaster
20, Berthold Technologies, Bad Wildbad, Germany) and exposure to a
phosphorimaging screen (Bio-Rad).
For sterol analysis, cells were cultivated in SC medium overnight at 24°C and
processed essentially as described (Quail and Kelly, 1996) using cholesterol as internal
standard. Free sterols and base-hydrolyzed total sterols were derivatized by TMS
[N?O?-bis-(trimethylsilyl)-trifluoroacetamide] and analyzed by GC-MS on a Voyager
Trace 2000 Series GC-MS (Thermo Fisher Scientific, Waltham, MA) equipped with
a Zebron ZB-35 capillary column (35% phenyl-methyl polysiloxane; dimensions:
30 m ? 0.25 mm ? 0.25 ?m film thickness). Sterols were identified based on their
mass fragmentation pattern and by comparison to commercially available standards.
Staining cells with NBD-cholesterol, sterol uptake and esterification were performed
as previously described (Reiner et al., 2006).
We thank Catherine Heath for excellent technical assistance, Christine
Guthrie and Anne de Bruyn Kops for a brr6-1 mutant strain, and Susan
Wente, Charlie Barlowe and T. Y. Chang for valuable discussions and
advice on the manuscript. This work was supported by grants from the
National Institute of General Medical Sciences, National Institutes or
Health (GM33998) to C.N.C. and from the Swiss National Science
Foundation (3100-120650) to R.S. M.J.W. and J.J.S. were supported
by a training grant from the National Institute of Arthritis and
Musculoskeletal and Skin Disorders, National Institutes of Health.
M.J.W. also received support from an undergraduate science education
grant to Dartmouth College from the Howard Hughes Medical Institute.
Deposited in PMC for release after 12 months.
Supplementary material available online at
Aebi, M., Clark, M. W., Vijayraghavan, U. and Abelson, J. (1990). A yeast mutant,
PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is
defective in a gene homologous to the human gene RCC1 which is involved in the
control of chromosome condensation. Mol. Gen. Genet. 224, 72-80.
Antonin, W., Ellenberg, J. and Dultz, E. (2008). Nuclear pore complex assembly through
the cell cycle: regulation and membrane organization. FEBS Lett. 582, 2004-2016.
Buttke, T. M., Jones, S. D. and Bloch, K. (1980). Effect of sterol side chains on growth
and membrane fatty acid composition of Saccharomyces cerevisiae. J. Bacteriol. 144,
Cole, C. N., Heath, C. V., Hodge, C. A., Hammell, C. M. and Amberg, D. C. (2002).
Analysis of RNA export. Methods Enzymol. 351, 568-587.
Conti, E., Muller, C. W. and Stewart, M. (2006). Karyopherin flexibility in
nucleocytoplasmic transport. Curr. Opin. Struct. Biol. 16, 237-244.
Cook, A., Bono, F., Jinek, M. and Conti, E. (2007). Structural biology of
nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647-671.
Czabany, T., Athenstaedt, K. and Daum, G. (2007). Synthesis, storage and degradation
of neutral lipids in yeast. Biochim. Biophys. Acta. 1771, 299-309.
D’Angelo, M. A. and Hetzer, M. W. (2008). Structure, dynamics and function of nuclear
pore complexes. Trends Cell Biol. 18, 456-466.
de Bruyn Kops, A. and Guthrie, C. (2001). An essential nuclear envelope integral
membrane protein, Brr6p, required for nuclear transport. EMBO J. 20, 4183-4193.
Fei, W., Shui, G., Gaeta, B., Du, X., Kuerschner, L., Li, P., Brown, A. J., Wenk, M.
R., Parton, R. G. and Yang, H.(2008). Fld1p, a functional homologue of human seipin,
regulates the size of lipid droplets in yeast. J. Cell Biol. 180, 473-482.
Feng, B., Yao, P. M., Li, Y., Devlin, C. M., Zhang, D., Harding, H. P., Sweeney, M.,
Rong, J. X., Kuriakose, G., Fisher, E. A. et al. (2003). The endoplasmic reticulum is
the site of cholesterol-induced cytotoxicity in macrophages. Nat. Cell Biol. 5, 781-792.
Gao, X. D., Tachikawa, H., Sato, T., Jigami, Y. and Dean, N. (2005). Alg14 recruits
Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite
UDP-N-acetylglucosamine transferase required for the second step of N-linked
glycosylation. J. Biol. Chem. 280, 36254-36262.
Gorsch, L. C., Dockendorff, T. C. and Cole, C. N. (1995). A conditional allele of the
novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation
of mRNA export and reversible clustering of nuclear pore complexes. J. Cell Biol. 129,
Han, G. S., Wu, W. I. and Carman, G. M. (2006). The Saccharomyces cerevisiae Lipin
homolog is a Mg2+-dependent phosphatidate phosphatase enzyme. J. Biol. Chem. 281,
Hazel, J. R. (1995). Thermal adaptation in biological membranes: is homeoviscous
adaptation the explanation? Annu. Rev. Physiol. 57, 19-42.
Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H. D. and Jentsch, S.
(2000). Activation of a membrane-bound transcription factor by regulated
ubiquitin/proteasome-dependent processing. Cell 102, 577-586.
Izawa, S., Takemura, R. and Inoue, Y. (2004). Gle2p is essential to induce adaptation
of the export of bulk poly(A)+ mRNA to heat shock in Saccharomyces cerevisiae. J.
Biol. Chem. 279, 35469-35478.
Kajiwara, K., Watanabe, R., Pichler, H., Ihara, K., Murakami, S., Riezman, H. and
Funato, K. (2008). Yeast ARV1 is required for efficient delivery of an early GPI
intermediate to the first mannosyltransferase during GPI assembly and controls lipid
flow from the endoplasmic reticulum. Mol. Biol. Cell 19, 2069-2082.
Li, Y., Ge, M., Ciani, L., Kuriakose, G., Westover, E. J., Dura, M., Covey, D. F., Freed,
J. H., Maxfield, F. R., Lytton, J. et al. (2004). Enrichment of endoplasmic reticulum
with cholesterol inhibits sarcoplasmic-endoplasmic reticulum calcium ATPase-2b activity
in parallel with increased order of membrane lipids: implications for depletion of
endoplasmic reticulum calcium stores and apoptosis in cholesterol-loaded macrophages.
J. Biol. Chem. 279, 37030-37039.
Lim, R. Y., Ullman, K. S. and Fahrenkrog, B. (2008). Biology and biophysics of the
nuclear pore complex and its components. Int. Rev. Cell Mol. Biol. 267, 299-342.
Los, D. A. and Murata, N. (2004). Membrane fluidity and its roles in the perception of
environmental signals. Biochim. Biophys. Acta 1666, 142-157.
Meredith, C. G., Maldonado, A. L. and Speeg, K. V., Jr(1985). The effect of ketoconazole
on hepatic oxidative drug metabolism in the rat in vivo and in vitro. Drug Metab. Dispos.
Murata, N. and Los, D. A. (1997). Membrane fluidity and temperature perception. Plant
Physiol. 115, 875-879.
Murphy, D. J. and Vance, J.(1999). Mechanisms of lipid-body formation. Trends Biochem.
Sci. 24, 109-115.
Nishida, I. and Murata, N. (1996). Chilling sensitivity in plants and cyanobacteria: The
crucial contribution of membrane lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47,
Oelkers, P., Cromley, D., Padamsee, M., Billheimer, J. T. and Sturley, S. L. (2002).
The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J. Biol.
Chem. 277, 8877-8881.
Omura, S. (1981). Cerulenin. Methods Enzymol. 72, 520-532.
Peterfy, M., Phan, J., Xu, P. and Reue, K. (2001). Lipodystrophy in the fld mouse results
from mutation of a new gene encoding a nuclear protein, lipin. Nat. Genet. 27, 121-124.
Petranyi, G., Ryder, N. S. and Stutz, A. (1984). Allylamine derivatives: new class of
synthetic antifungal agents inhibiting fungal squalene epoxidase. Science 224, 1239-
Quail, M. A. and Kelly, S. L. (1996). The extraction and analysis of sterols from yeast.
Methods Mol. Biol. 53, 123-131.
Reiner, S., Micolod, D., Zellnig, G. and Schneiter, R. (2006). A genomewide screen
reveals a role of mitochondria in anaerobic uptake of sterols in yeast. Mol. Biol. Cell
Rose, M. D. and Broach, J. R.(1991). Cloning genes by complementation in yeast. Methods
Enzymol. 194, 195-230.
Ryan, K. J. and Wente, S. R.(2002). Isolation and characterization of new Saccharomyces
cerevisiae mutants perturbed in nuclear pore complex assembly. BMC Genet. 3, 17.
Ryan, K. J., McCaffery, J. M. and Wente, S. R.(2003). The Ran GTPase cycle is required
for yeast nuclear pore complex assembly. J. Cell Biol. 160, 1041-1053.
Ryan, K. J., Zhou, Y. and Wente, S. R. (2007). The karyopherin Kap95 regulates nuclear
pore complex assembly into intact nuclear envelopes in vivo. Mol. Biol. Cell 18, 886-898.
Saitoh, Y. H., Ogawa, K. and Nishimoto, T. (2005). Brl1p - a novel nuclear envelope
protein required for nuclear transport. Traffic 6, 502-517.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: a Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S. and Siniossoglou, S. (2005).
The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.
EMBO J. 24, 1931-1941.
Scarcelli, J. J., Hodge, C. A. and Cole, C. N. (2007). The yeast integral membrane protein
Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes.
J. Cell Biol. 178, 799-812.
Schneiter, R., Hitomi, M., Ivessa, A. S., Fasch, E. V., Kohlwein, S. D. and Tartakoff,
A. M. (1996). A yeast acetyl coenzyme A carboxylase mutant links very-long-chain
fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.
Mol. Cell. Biol. 16, 7161-7172.
Schneiter, R., Brugger, B., Amann, C. M., Prestwich, G. D., Epand, R. F., Zellnig, G.,
Wieland, F. T. and Epand, R. M.(2004). Identification and biophysical characterization
of a very long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular Download full-text
membranes. Biochem. J. 381, 941-949.
Segref, A., Sharma, K., Doye, V., Hellwig, A., Huber, J., Luhrmann, R. and Hurt, E.
(1997). Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA
and nuclear pores. EMBO J. 16, 3256-3271.
Sharma, S. C. (2006). Implications of sterol structure for membrane lipid composition,
fluidity and phospholipid asymmetry in Saccharomyces cerevisiae. FEMS Yeast Res. 6,
Sheets, J. J. and Mason, J. I. (1984). Ketoconazole: a potent inhibitor of cytochrome P-
450 dependent drug metabolism in rat liver. Drug Metab. Dispos. 12, 603-606.
Siniossoglou, S., Santos-Rosa, H., Rappsilber, J., Mann, M. and Hurt, E. (1998). A
novel complex of membrane proteins required for formation of a spherical nucleus. EMBO
J. 17, 6449-6464.
Sorger, D. and Daum, G. (2002). Synthesis of triacylglycerols by the acyl-coenzyme
A:diacyl glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces
cerevisiae. J. Bacteriol. 184, 519-524.
Stewart, M. (2007). Molecular mechanism of the nuclear protein import cycle. Nat. Rev.
Mol. Cell. Biol. 8, 195-208.
Stukey, J. E., McDonough, V. M. and Martin, C. E.(1989). Isolation and characterization
of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae. J.
Biol. Chem. 264, 16537-16544.
Swain, E., Stukey, J., McDonough, V., Germann, M., Liu, Y., Sturley, S. L. and Nickels,
J. T., Jr (2002). Yeast cells lacking the ARV1 gene harbor defects in sphingolipid
metabolism. Complementation by human ARV1. J. Biol. Chem. 277, 36152-36160.
Tange, Y., Hirata, A. and Niwa, O. (2002). An evolutionarily conserved fission yeast
protein, Ned1, implicated in normal nuclear morphology and chromosome stability,
interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. J. Cell Sci. 115, 4375-
Terry, L. J., Shows, E. B. and Wente, S. R. (2007). Crossing the nuclear envelope:
hierarchical regulation of nucleocytoplasmic transport. Science 318, 1412-1416.
Tinkelenberg, A. H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M. and Sturley,
S. L. (2000). Mutations in yeast ARV1 alter intracellular sterol distribution and are
complemented by human ARV1. J. Biol. Chem. 275, 40667-40670.
Tran, E. J. and Wente, S. R. (2006). Dynamic nuclear pore complexes: life on the edge.
Cell 125, 1041-1053.
Tseng, S. S., Weaver, P. L., Liu, Y., Hitomi, M., Tartakoff, A. M. and Chang, T. H.
(1998). Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export. EMBO
J. 17, 2651-2662.
Winston, F., Dollard, C. and Ricupero-Hovasse, S. L. (1995). Construction of a set of
convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53-
Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl,
T. M., Rothstein, R. and Sturley, S. L. (1996). Sterol esterification in yeast: a two-
gene process. Science 272, 1353-1356.
Yu, C., Kennedy, N. J., Chang, C. C. and Rothblatt, J. A. (1996). Molecular cloning
and characterization of two isoforms of Saccharomyces cerevisiae acyl-CoA:sterol
acyltransferase. J. Biol. Chem. 271, 24157-24163.
Zhang, S., Skalsky, Y. and Garfinkel, D. J. (1999). MGA2 or SPT23 is required for
transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane
integrity in Saccharomyces cerevisiae. Genetics 151, 473-483.
Zhang, Y. M. and Rock, C. O. (2008). Membrane lipid homeostasis in bacteria. Nat. Rev.
Microbiol. 6, 222-233.