2388 | Y. Sun et al. Molecular Biology of the Cell
MBoC | ARTICLE
Orm protein phosphoregulation mediates
transient sphingolipid biosynthesis response
to heat stress via the Pkh-Ypk and Cdc55-PP2A
Yidi Suna,*, Yansong Miaoa,*, Yukari Yamaneb, Chao Zhangc, Kevan M. Shokatc, Hiromu Takematsub,
Yasunori Kozutsumib, and David G. Drubina
aDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720; bLaboratory of
Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida-Shimoadachi,
Sakyo, Kyoto 606-8501, Japan; cDepartment of Cellular and Molecular Pharmacology, University of California,
San Francisco, San Francisco, CA 94143
ABSTRACT Sphingoid intermediates accumulate in response to a variety of stresses, includ-
ing heat, and trigger cellular responses. However, the mechanism by which stress affects
sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphin-
golipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in hu-
mans are potential risk factors for childhood asthma. Here we demonstrate that Orm phos-
phorylation status is highly responsive to sphingoid bases. We also demonstrate, by
monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells
inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to
the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-
induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making
the induction transient. We identified Cdc55–protein phosphatase 2A (PP2A) as a key phos-
phatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation.
In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade
and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress
through Orm protein phosphoregulation. We propose that this mechanism serves as the ba-
sis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress
in a kinetically coupled manner.
Sphingoid intermediates, including sphingoid bases, sphingoid
base phosphates, and ceramides (Figure 1), play important roles in
regulation of cell growth, differentiation, senescence, and apoptosis
(Hannun and Obeid, 2008; Dickson, 2010; Nikolova-Karakashian
and Rozenova, 2010). Serine palmitoyltransferase (SPT) mediates
the rate-limiting first step in sphingolipid biosynthesis (Figure 1). De-
spite the importance of sphingoid intermediates as bioactive mole-
cules, the regulation of sphingolipid biosynthesis through SPT is not
well understood (Cowart and Hannun, 2007). A recent study re-
vealed that yeast Orm proteins, encoded by ORM1 and ORM2,
form a conserved complex with SPT and that their phosphorylation
status affects sphingolipid production (Breslow et al., 2010; Figure 1).
The authors proposed that sphingolipid levels feedback regulate
Orm protein phosphorylation, thus mediating sphingolipid homeo-
stasis (Breslow et al., 2010). However, several important questions
related to this model need answers. For example, whether and
which sphingolipid species affect Orm phosphorylation are not
known. In addition, how temporal regulation of Orm phosphoryla-
tion relates to dynamic changes in sphingolipid biosynthesis is not
University of Geneva
Received: Mar 13, 2012
Revised: Apr 13, 2012
Accepted: Apr 18, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-03-0209) on April 25, 2012.
*These authors contributed equally to this work.
Address correspondence to: David G. Drubin (firstname.lastname@example.org).
Abbreviations used: Cg, Candida glabrata; DHS, dihydrosphingosine; DMSO,
dimethyl sulfoxide; HPLC, high-performance liquid chromatography; PHS,
phytosphingosine; PP2A, phosphatase 2A; Sph, sphingosine; SPT, serine palmi-
toyltransferase; TCA, trichloroacetic acid; Ypk1, yeast protein kinase 1.
© 2012 Sun et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
Volume 23 June 15, 2012 Sphingolipid regulation upon stress | 2389
kinases were previously considered to be downstream of heat-in-
duced sphingolipid base accumulation (Sun et al., 2000; Friant et al.,
2001; Liu et al., 2005; Hannun and Obeid, 2008; Dickson, 2010).
Thus, testing for a possible role for Ypk kinase activity in the sphingo-
lipid biosynthesis response to heat stress is an important goal.
Of importance, the Orm phosphorylation state is set not only by
protein kinases, but also by phosphatases. A reasonable prediction
is that the phosphatases acting on the Orm proteins are likely to be
involved in and/or regulated by sphingolipid levels. Protein phos-
phatase 2A (PP2A) was identified as an attractive candidate for a
ceramide-activated protein phosphatase in yeast (Nickels and
Broach, 1996). Inactivation of the regulatory subunits (such as Cdc55)
or the catalytic subunits of PP2A was reported previously to sup-
press the endocytic defects of a mutant with impaired SPT activity
(Friant et al., 2000), suggesting that PP2A may be involved in sphin-
golipid production. Because PP2A was suggested previously to be
involved in multiple cellular signaling pathways (Jiang, 2006), the
challenge is to identify a specific relationship between Cdc55-PP2A
and the Orm proteins for regulation of sphingolipid biosynthesis.
In this article, by addressing the questions just raised, we demon-
strate how Orm phosphorylation regulation controls sphingolipid bio-
synthesis in response to heat stress in a kinetically coupled manner. We
identify a signaling pathway by which the conserved Pkh-Ypk signaling
cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid pro-
duction upon heat stress through precise regulation of Orm protein
phosphorylation. Our study will therefore provide a foundation for fu-
ture studies of sphingolipid-related responses to other stimuli.
Orm2 is the major Orm species in budding yeast
A 3XFLAG sequence was inserted at the 5′ end of the genes ORM1
and ORM2, which encode the two homologous yeast Orm proteins.
Because Orm2 expression was at least 10 times higher than Orm1
expression (Supplemental Figure S1a) and because 3XFLAG-Orm1
in an orm2∆ strain, but not 3XFLAG-Orm2 in an orm1∆ strain,
caused slow cell growth (Supplemental Figure S1, b and c), we con-
cluded that Orm2 is the major Orm protein. Consistently, orm2∆
cells but not orm1∆ cells showed perturbation in sphingolipid ho-
meostasis (Han et al., 2010). These results suggested that Orm2
provides the majority of the Orm protein function. Thus, we decided
to monitor Orm2 phosphorylation in our studies. As shown in
Figure 2a, four groups of Orm2 bands were observed when resolved
by phosphate-affinity gel, which is widely used for the analysis of
phosphoprotein isotypes (Kinoshita et al., 2006). As previously re-
ported (Breslow et al., 2010), the slower-migrating bands in the top
three groups were confirmed to be phosphorylated forms of Orm2
(Supplemental Figure S2). On the basis of the principle responsible
for protein separation on phosphate-affinity gels (Kinoshita et al.,
2006), the bands in the top three groups are likely to be phosphory-
lated to different extents, with the slowest-migrating species being
the most highly phosphorylated. The bands in the third group ob-
served by Western blotting are often overexposed under the condi-
tions that allow us to observe bands in all four groups (Figure 2a).
Thus, we monitored changes in intensity of groups 1, 2, and 4 as
clear indicators of changes in Orm2 phosphorylation status.
Exogenously provided sphingoid bases are sufficient
to induce rapid Orm2 dephosphorylation
We examined how exogenously provided sphingoid bases, which
are early sphingoid intermediates (Figure 1), affect Orm2 phosphory-
lation status. As shown in Figure 2, b and c, dephosphorylated Orm2,
seen as the fastest-migrating bands (the fourth group), increased in
known. Answers to these questions are required to better under-
stand Orm-mediated sphingolipid homeostasis.
Another important question concerns how Orm-mediated sphin-
golipid homeostasis may function in a physiological context. In-
creasing evidence suggests that various stimuli trigger accumulation
of sphingoid intermediates, which in turn function as bioactive mol-
ecules mediating cellular responses (Hannun and Obeid, 2008;
Dickson, 2010). For instance, heat stress–induced sphingoid inter-
mediates act as signaling molecules to induce cellular responses
such as translation initiation of heat shock proteins, gene regulation,
and cell cycle arrest (Dickson et al., 1997; Mao et al., 1999; Jenkins
and Hannun, 2001; Cowart and Hannun, 2005; Cowart et al., 2010;
Meier et al., 2006; Han et al., 2010). It is striking that several groups
demonstrated that heat stress induces sphingolipid biosynthesis in
a rapid and transient manner (Dickson et al., 1997; Jenkins et al.,
1997; Jenkins, 2003; Wells et al., 1998; Mao et al., 1999; Skrzypek
et al., 1999), suggesting that biosynthesis of sphingoid intermedi-
ates in response to stresses requires precise temporal regulation.
We hypothesize that the dynamic changes in sphingoid intermedi-
ate levels upon heat stress may be caused by and/or may lead to
changes of Orm phosphorylation. If this is the case, heat stress could
serve as a model system to address the mechanism of how Orm
phosphoregulation functions in the response of sphingolipid bio-
synthesis to stress in general.
A recent study showed that yeast protein kinases 1 and 2 (Ypk1/2),
the homologues of mammalian serum- and glucocorticoid-inducible
kinase (Casamayor et al., 1999), directly phosphorylate Orm proteins
in vitro (Roelants et al., 2011). This result suggests that Ypk kinase may
regulate sphingolipid homeostasis through its phosphorylation of the
Orm proteins. However, whether Ypk kinase activity indeed affects
sphingolipid production has not been examined. Of interest, the Ypk
FIGURE 1: Schematic diagram of yeast sphingolipid biosynthesis from
endogenous and exogenous precursors. Myriocin is a potent inhibitor
of SPT (Sun et al., 2000), which is the first and rate-limiting enzyme of
the sphingolipid biosynthesis pathway (Buede et al., 1991). Previous
studies proposed that Orm proteins are dynamic negative regulators
of SPT and their activities are inversely proportional to sphingolipid
levels (Breslow et al., 2010). Exogenous sphingoid bases (DHS and
PHS) can be converted into ceramides in the endoplasmic reticulum
after phosphorylation and dephosphorylation by the indicated
enzymes and can then be incorporated into complex sphingolipids
(Qie et al., 1997; Nagiec et al., 1998; Funato et al., 2003).
Sphingoid Bases (DHS/PHS)
(IPC, MIPC, M(IP)2C
2390 | Y. Sun et al. Molecular Biology of the Cell
on Orm2 dephosphorylation (unpublished
data). Dephosphorylated Orm2 greatly in-
creased within 2 min of treatment with 5 μM
PHS at 25°C (Figure 2, d and e). In contrast,
adding 5 μM sphingosine (Sph), which is a
mammalian sphingoid base that cannot res-
cue a yeast sphingolipid deficiency (Wells
and Lester, 1983), did not affect Orm2 phos-
phorylation (Figure 2, d and f), indicating that
the effect of PHS is highly specific. Further-
more, stearylamine (Jenkins and Hannun,
2001), which is a long-chain primary amine,
also did not induce Orm2 dephosphoryla-
tion (unpublished data), providing further
evidence for chemical specificity.
The rapid response to the exogenous
addition of PHS (Figure 2e) suggests that
the sphingoid base itself, rather than its
downstream sphingolipid metabolites (Nag-
iec et al., 1997; Figure 1), triggers Orm2 de-
phosphorylation. To explore this possibility
further, we used a knockout mutant of the
LCB3 gene, which encodes the sphingosine-
1P phosphatase required for ceramide syn-
thesis from exogenous PHS (Mao et al.,
1997, 1999; Qie et al., 1997; Figure 1). In
the absence of Lcb3, exogenous PHS can-
not be incorporated into ceramides (Qie
et al., 1997; Funato et al., 2003). However,
exogenous PHS still rapidly induced Orm2
dephosphorylation in the lcb3∆ strain (Fig-
ure 2g), suggesting that exogenous PHS in-
duces Orm2 dephosphorylation without be-
ing converted to ceramides. A previous
study indicated that lack of Orm2 results in
increased PHS but decreased ceramides in
vivo (Han et al., 2010). Here we found that
Orm1 phosphorylation is greatly reduced in
a strain lacking Orm2 (Supplemental Figure
S1a), in agreement with PHS being sufficient
to induce Orm dephosphorylation. In addi-
tion, exogenous PHS still triggered Orm2
dephosphorylation in mutant cells lacking
both Lcb4 and Lcb5 (Figure 2h), which are
sphingoid base kinases required for phos-
phorylation of exogenously provided PHS
(Nagiec et al., 1998; Funato et al., 2003; Fig-
ure 1). Thus, exogenous PHS itself, without
being converted to phosphorylated sphin-
goid bases (Figure 1), can induce Orm2 de-
phosphorylation. However, compared with
the wild-type cells (Figure 2e), Orm2 de-
phosphorylation in lcb4∆ lcb5∆ cells re-
sponds to PHS to a lesser extent (Figure 2h),
suggesting that phosphorylated sphingoid
bases may also contribute to Orm2
Taken together, our data demonstrate that exogenous addi-
tion of PHS is sufficient to rapidly induce Orm2 dephosphoryla-
tion, indicating that Orm phosphorylation status is highly respon-
sive to levels of sphingoid bases. Although we cannot exclude the
possibility that ceramide and complex sphingolipids also
response to a 10-min treatment at 25°C with a natural yeast sphin-
goid base, phytosphingosine (PHS), in a concentration-dependent
manner. PHS at 5 μM is sufficient to cause maximal effects on Orm2
dephosphorylation (Figure 2c), whereas 20 μM of exogenous dihy-
drosphingosine (DHS), another sphingoid base, had a similar effect
FIGURE 2: Exogenously provided sphingoid bases are sufficient to induce rapid Orm2
dephosphorylation. (a, b, d–h), Western blots showing Orm2 phosphorylation patterns after
separation of the indicated yeast cell extracts on phosphate-affinity gels (top). P-Orm2 indicates
phosphorylated forms of Orm2. We used 3-phosphoglycerate kinase 1 (Pgk1) as a loading
control (bottom). All cells were grown to early log phase in YPD at 25°C before the indicated
treatments. Cell extracts were prepared as described in Materials and Methods. (a) The majority
of the Orm2 is moderately phosphorylated. Western blotting analysis of cell exacts from
wild-type cells expressing 3XFLAG-Orm2 at its endogenous locus with different exposure times:
30 s and 1, 3, and 6 min (left to right). (b, c) Exogenous phytosphingosine (PHS) induces Orm2
dephosphorylation in a concentration-dependent manner. Wild-type cells expressing 3XFLAG-
Orm2 at its endogenous locus were cultured in media containing the indicated concentrations of
PHS (dissolved in methanol) for 10 min. The intensities of the fastest-migrating 3XFLAG Orm2
bands (the fourth group) in each lane (b) were analyzed and plotted in the graph shown in c.
(d, e) Exogenous PHS induces rapid Orm2 dephosphorylation. Wild-type cells expressing
3XFLAG-Orm2 from its endogenous locus were cultured in media containing methanol alone
(mock) or 5 μM PHS dissolved in methanol for the indicated times. (f) Exogenous sphingosine
(Sph) does not affect Orm2 phosphorylation. Wild-type cells expressing 3XFLAG-Orm2 from its
endogenous locus were cultured in the presence of 5 μM Sph (dissolved in methanol) for the
indicated times. (g, h) Exogenous PHS induces rapid Orm2 dephosphorylation in lcb3∆ cells or
in lcb4∆ lcb5∆ cells. lcb3∆ cells or lcb4∆ lcb5∆ cells expressing 3XFLAG-Orm2 from its
endogenous locus were cultured in the presence of 5 μM PHS for the indicated times.
Increasing Exposure Time
0 0.25 0.51
7.5 10 15
Volume 23 June 15, 2012 Sphingolipid regulation upon stress | 2391
We monitored Orm2 phosphorylation after cells were shifted
from 25 to 39°C. Within 1 min after the temperature shift, dephos-
phorylated Orm2, which is the fastest-migrating band on phos-
phate-affinity gels, started to disappear (Figure 3a). Correspond-
ingly, Orm2 phosphorylation levels increased, as evidenced by the
increased abundance of the slower-migrating bands in the top two
groups (Figure 3a). Orm2 phosphorylation further increased at
2 min (Figure 3a), with the maximal effect observed at ∼5 min, fol-
lowed by a decrease (Figure 3, b and c). Orm2 phosphorylation
was further reduced by 20 min of heat stress and then stabilized
(Figure 3, b and c). Several independent studies demonstrated
contribute to Orm phosphoregulation, the results with lcb3∆ mu-
tants (Figure 2g) support the conclusion that neither of these
sphingolipids is required for Orm phosphoregulation.
Temporal association between Orm phosphorylation
dynamics and de novo sphingoid base production
in response to heat stress
Previous studies suggested that heat stress induces rapid transient
de novo accumulation of sphingoid intermediates, including PHS.
We next asked whether and how Orm protein phosphorylation may
be involved in heat-induced sphingolipid biosynthesis.
FIGURE 3: Orm2 phosphorylation dynamics and sphingoid base production upon heat shock. (a, b, d, e) Western
blots showing Orm2 phosphorylation patterns after separation of the indicated yeast cell extracts on phosphate-
affinity gels (top). P-Orm2 indicates phosphorylated forms of Orm2. Pgk1 was used as a loading control (bottom).
Wild-type cells expressing 3XFLAG-Orm2 from its endogenous locus were used in all experiments and cultured to
early log phase at 25°C before the indicated treatments. Cell extracts were prepared as described in Materials and
Methods. (a–c) Temporal association between Orm phosphorylation dynamics and sphingoid base production upon
heat stress. Cell extracts were prepared after cell cultures were shifted from 25 to 39°C for the indicated times. The
relative intensity of the two slowest-migrating 3XFLAG Orm2 bands (groups 1 and 2) shown in b was quantified.
Time-course experiments shown in b were performed an additional four times. Data collected from the five
independent experiments were plotted in the graph shown in c (black line). Data shown represent the means with
standard deviations. The C18-PHS concentration (c, gray, broken line) was determined by HPLC as described in
Materials and Methods. (d), Orm2 phosphorylation status upon heat stress in the presence of exogenous sphingoid
bases. Cells were shifted from 25 to 39°C for the indicated times. Methanol alone (control) or 5 μM PHS dissolved in
methanol was added exogenously 4 min after cells were shifted from 25 to 39°C. (e) Orm2 phosphorylation upon
heat stress in the presence of myriocin. Left, 0.5 μg/ml myriocin was added to cells upon shift from 25 to 39°C. Right,
cells were treated with 0.5 μg/ml myriocin at 25°C for the indicated times.
Heat stress 39°C
Relative Band intensity
05 10 15 2025
2392 | Y. Sun et al. Molecular Biology of the Cell
phosphorylated form), respectively. The level of sphingoid bases
was greatly reduced in orm2-3A cells and was greatly increased
orm2-3D cells (Supplemental Figure S3), providing evidence that
phosphorylation of these serines plays a role in sphingolipid
production. Furthermore, as shown in Figure 4a, the majority of
orm2-3A protein is in dephosphorylated forms, demonstrating that
phosphorylation of Orm2 residues S46, S47, and S48 is responsible
for the slower migration of phosphorylated Orm2 (Figure 4a; Breslow
et al., 2010). Together, these results strongly suggest that phospho-
rylation of Orm2 by Ypk1 is involved in Orm-mediated sphingolipid
To determine whether the rapid Orm2 phosphorylation induced
by heat stress (Figure 3c, time points between 0 and 4 min) requires
Ypk kinase activity in vivo, we generated an analogue-sensitive
(Bishop et al., 2000) ypk1-as allele in ypk2∆ background. Deletion of
YPK2 has no apparent phenotypic defect, but loss of YPK1 results in
slow growth (Chen et al., 1993). Knocking out both YPK1 and YPK2
causes lethality (Chen et al., 1993). Orm2 phosphorylation in re-
sponse to heat stress in the ypk1-as ypk2∆ mutant was monitored in
the presence of 3-MOB-PP1, which specifically inhibits ypk1-as ki-
nase activity. Heat stress–induced phosphorylation no longer occurs
when Ypk kinase activity is abolished (Figure 4b and Supplemental
Figure S4a), indicating that heat stress facilitates Orm phosphoryla-
tion through Ypk kinase activity. More important, heat induced-
sphingoid base accumulation no longer occurs when Ypk kinase
activity is inhibited (Figure 4c and Supplemental Figure S4b). These
results not only provide strong evidence that Orm phosphorylation
positively regulates sphingolipid production in vivo, but they also
indicate that Ypk kinase transmits the heat stress signals to sphin-
goid intermediate production through phosphorylation of the Orm
Ypk1/2 are phosphorylated and activated by Pkh1/2, the homo-
logues of mammalian PKD1 (Casamayor et al., 1999). Neither Pkh1
nor Pkh2 alone is required for cell growth, but loss of both proteins
causes lethality (Casamayor et al., 1999). Consistently, heat stress–
induced phosphorylation is abolished when Pkh kinase activity is
inhibited (Figure 4d).
A recent study proposed that Orm2 expression levels regulate
sphingolipid synthesis (Liu et al., 2012). However, as shown in
Figure 3a, Orm phosphorylation starts to increase within 1 min af-
ter introduction of heat stress, reaching its maximum at around
5 min, and then decreases. The rapid time scale of these events
strongly suggests that regulation of sphingolipid synthesis in re-
sponse to heat is primarily due to the changes in Orm phosphory-
lation, not to changes in Orm expression.
Together, the results shown in Figures 3 and 4 establish a physi-
ologically relevant context for the association between sphingo-
lipid homeostasis and Orm protein phosphorylation dynamics and
identify a novel feedback pathway for temporal regulation of
sphingolipid biosynthesis during the heat stress response: heat
stress rapidly activates the Pkh-Ypk signaling pathway to induce
Orm phosphorylation, which in turn promotes sphingolipid inter-
mediate production. The accumulated sphingoid intermediates
then trigger Orm dephosphorylation, which in turn down regulates
Cdc55-PP2A is a key phosphatase that counteracts
Ypk1 activity in Orm-mediated sphingolipid biosynthesis
Orm phosphorylation state is set not only by kinases but also
by phosphatases. We next sought to identify the phosphatase in-
volved in Orm dephosphorylation. We hypothesized that the
phosphatase acting on the Orm proteins is likely to be involved in
that heat stress induces rapid de novo accumulation of various
sphingoid intermediates, including sphingoid bases (Dickson et al.,
1997; Jenkins et al., 1997; Jenkins, 2003; Wells et al., 1998; Mao
et al., 1999; Skrzypek et al., 1999). The sphingoid base levels peak
by 10–15 min and decrease to near-basal levels by 30 min of heat
stress (Dickson et al., 1997; Jenkins et al., 1997). Our measurements
of PHS levels confirmed these previous conclusions (Figure 3c, gray,
broken line). Thus, our results demonstrate a striking temporal as-
sociation between Orm phosphorylation dynamics and de novo
sphingoid base production in response to heat stress (Figure 3c).
High levels of Orm phosphorylation induced by heat stress de-
crease when the sphingoid bases (PHS) reach their peak levels
(Figure 3c, time points between 5 and 15 min), suggesting that
heat-induced sphingoid base accumulation and Orm dephosphory-
lation may be mechanistically linked. To investigate this possibility
further, we altered the timing of the sphingoid base accumulation by
adding exogenous PHS 4 min after the temperature shift, when de
novo–synthesized sphingolipids normally just begin to accumulate
(Figure 3c; Dickson et al., 1997; Jenkins et al., 1997; Wells et al.,
1998; Mao et al., 1999; Skrzypek et al., 1999). In control cells, in-
creased Orm2 phosphorylation peaks after 4 min of heat stress and
is only slightly diminished after 6 min of heat stress (Figure 3d, left).
However, addition of exogenous PHS at 4 min reduced Orm2 phos-
phorylation to the basal level at 6 min (Figure 3d, right).
To further test whether de novo synthesis of sphingoid interme-
diates in response to heat stress is required to trigger Orm dephos-
phorylation, we monitored Orm2 phosphorylation in response to
heat stress in the presence of myriocin, a potent SPT inhibitor (Sun
et al., 2000; Figure 1). No obvious changes in Orm2 phosphoryla-
tion were observed up to 5 min after myriocin treatment at 25°C
(Figure 3e), and 2 min of heat stress induced Orm2 phosphoryla-
tion irrespective of myriocin presence (Figure 3, b and e), indicat-
ing that heat stress affects Orm2 phosphorylation before the myri-
ocin-sensitive step. Of interest, in the presence of myriocin, the
increased Orm2 phosphorylation did not decline even after 30 min
of heat stress (compare Figure 3e with Figure 3b), indicating that
de novo synthesis of sphingoid intermediates is required for reduc-
ing the increased Orm2 phosphorylation caused by heat stress.
Together, these results provide strong evidence that increased lev-
els of sphingoid intermediates trigger Orm dephosphorylation
during the heat stress response. Thus, our results suggest that both
exogenously provided sphingoid intermediates (such as PHS) and
the accumulation of physiologically induced sphingoid intermedi-
ates (by heat stress), cause rapid Orm2 dephosphorylation (Figures
2 and 3).
Ypk kinase transmits heat stress signals to the sphingolipid
biosynthesis pathway via Orm phosphorylation
Another example of a striking temporal association between Orm
phosphorylation and sphingolipid intermediate production is the
observation that heat stress triggers Orm phosphorylation in <1 min
(Figure 3a), with phosphorylation reaching its maximum before the
level of sphingoid bases reaches its peak (Figure 3, b and c). This
temporal association raises the important question of determining
how the heat stress signal is transmitted to induce rapid Orm
A recent study suggested that Ypk1, a homologue of mammalian
serum- and glucocorticoid-inducible kinase (Casamayor et al., 1999),
specifically phosphorylates Orm2 residues S46, S47, and S48 in vitro
(Roelants et al., 2011). To examine the role of phosphorylation of
these serine residues on Orm function, we mutated S46, S47,
and S48 of Orm2 to alanine or aspartic acid to generate orm2-3A
(mimicking the dephosphorylated form) or orm2-3D (mimicking the
Volume 23 June 15, 2012 Sphingolipid regulation upon stress | 2393
be much higher than in wild-type cells (Fig-
ure 5b). Moreover, both of these strains have
severe growth defects (Figure 5c). Thus, we
conclude that it is not possible to address
PP2A function, particularly in Orm phospho-
regulation, using these two mutants; the el-
evated Orm2 expression in these mutants
may affect sphingolipid synthesis (Liu et al.,
2012). In contrast, pph21∆, pph22∆, cdc55∆,
and rts1∆ single mutants grow relatively
normally (Figure 5c). Compared to wild-type
cells, Orm2 phosphorylation levels increased
dramatically in cdc55∆ mutants (Figure 5b).
pph21∆ cells also showed higher Orm2
phosphorylation (Figure 5b). In contrast, the
Orm2 phosphorylation patterns in rts1∆
and wild-type cells were indistinguishable
(Figure 5b). These results indicate that
Cdc55-PP2A activity, rather than Rts1-PP2A
activity, mediates Orm dephosphorylation.
We next examined how PP2A mutants
respond to the SPT inhibitor myriocin. Pre-
vious studies suggested that Orm proteins
negatively regulate SPT activity and that
Orm phosphorylation relieves their inhibi-
tion of SPT (Breslow et al., 2010; Figure 1).
Thus, impaired PP2A activity, which in-
creases Orm2 phosphorylation, might lead
to enhanced myriocin resistance. Unfortu-
nately, tpd3∆ and pph21∆ pph22∆ mutants
show severe growth defects even without
myriocin (Figure 5c), making assessment of
their myriocin sensitivity difficult. However,
pph21∆, pph22∆, and cdc55∆ single mu-
tants showed resistance to 0.75 μg/ml my-
riocin, a concentration that inhibits growth
of wild-type cells (Figure 5c). The same set
of PP2A mutants could not survive 1 μg/ml
myriocin (Figure 5c), a concentration that
likely causes more complete inhibition of
SPT activity. In addition, cdc55∆ rescued
the growth of the lcb1-100 ts mutant at a
semi restrictive temperature but not at the restrictive temperature
(Figure 5d). Lcb1 is an essential component of yeast SPT (Buede
et al., 1991). Thus, it appears that the myriocin resistance of
the Cdc55-PP2A–deficient mutants is only observable when SPT
activity is not completely blocked and thus is still regulated by
Orm proteins. The rts1∆ mutant shows resistance to even 1 μg/ml
myriocin (Figure 5c), implying that the rts1∆ mutant cells and
cdc55∆ mutant cells are resistant to myriocin through a different
To further explore the relationship between PP2A, the Orm pro-
teins, and SPT regulation, we examined the myriocin sensitivity of
PP2A mutants in an orm-null mutant background. We found that
pph21∆ orm1∆ orm2∆ and cdc55∆ orm1∆ orm2∆ triple mutants
were not resistant to 0.75 μg/ml myriocin (Figure 6, a and b), whereas
rts1∆ orm1∆ orm2∆ mutants were resistant to even 1 μg/ml myriocin
(Figure 6a), establishing that Orm proteins are required for the my-
riocin resistance of Cdc55-PP2A–deficient mutants but not of the
We next tested the role of Ypk phosophorylation of the three
Orm2 serines (Roelants et al., 2011) in the myriocin resistance of
and/or regulated by sphingolipid levels. PP2A (Figure 5a) was
identified as an attractive candidate for a ceramide-activated pro-
tein phosphatase in yeast (Nickels and Broach, 1996). Inactivation
of Cdc55 or the catalytic subunits of PP2A was reported previously
to suppress the endocytic defects of a mutant with impaired SPT
activity (Friant et al., 2000), suggesting that PP2A may be involved
in sphingolipid production. We explored the specific relationship
between Cdc55-PP2A activity and Orm regulation at standard
growth temperatures (25 or 30°C).
The yeast PP2A, similar to its mammalian counterparts, is a het-
erotrimer composed of three distinct subunits, namely A (the struc-
tural subunit, encoded by TPD3), B (the regulatory subunit, encoded
by two distinct genes, CDC55 and RTS1), and C (the catalytic sub-
unit, also encoded by two distinct genes, PPH21 and PPH22)
(Jiang, 2006). If PP2A does dephosphorylate Orm2, lack of PP2A
activity would be expected to result in increased Orm2 phosphory-
lation. tpd3∆ and pph21∆ pph22∆ showed increases in both phos-
phorylated (the two slowest-migrating bands) and dephosphory-
lated forms (the fastest-migrating band) of Orm2 (Figure 5b). The
total Orm2 expression levels in these two mutants also appeared to
FIGURE 4: Ypk kinase transmits heat stress signals to the sphingolipid biosynthesis pathway via
Orm phosphorylation. (a, b, d) Western blots showing Orm2 phosphorylation patterns after
separation of the indicated yeast cell extracts on phosphate affinity gels (top). P-Orm2 indicates
phosphorylated forms of Orm2. Pgk1 was used as a loading control (bottom). All cells were
grown to early log phase in YPD at 25°C before the indicated treatments. Cell extracts were
prepared as described in Materials and Methods. (a) Orm2 residues S46, S47, and S48 are
responsible for slower migration of phosphorylated Orm2. (b) Orm2 phosphorylation in
response to heat stress in the absence of Ypk activity. Dimethyl sulfoxide (DMSO) alone or
50 μM of 3-MOB-PP1 dissolved in DMSO, which specifically inhibits ypk1-as kinase activity, was
added to ypk1as ypk2∆ cells upon shift from 25 to 39°C. (c) Heat-induced sphingoid base
accumulation requires Ypk kinase activity. The C18-PHS concentration was determined in the
ypk1as ypk2∆ cells cultured in the absence (line with circle) or the presence (line with square) of
50 μM 3-MOB-PP1 shifted from 25 to 39°C for the indicated times. (d) Orm2 phosphorylation
status upon heat stress in the absence of Pkh activity. DMSO alone or 100 μM CZ21 dissolved in
DMSO, which specifically inhibits pkh1-as kinase activity, was added to pkh1as pkh2∆ cells upon
shift from 25 to 39°C.
w/o AS inhibitor with AS inhibitor
with AS inhibitor
w/o AS inhibitor
with AS inhibitor
w/o AS inhibitor
2394 | Y. Sun et al. Molecular Biology of the Cell
resistance (Figure 6c). This result estab-
lishes that whereas PP2A may be involved
in multiple cellular signaling pathways (Ji-
ang, 2006), Cdc55-PP2A and Orm protein
activities are linked specifically for sphingo-
lipid biosynthesis. Furthermore, sphingoid
base levels were substantially increased in
cdc55∆ cells (Supplemental Figure S3), in-
dicating that Cdc55-PP2A activity is in-
volved in sphingolipid production.
PP2A activity contributes to the
regulation of Orm phosphorylation
dynamics in response to heat stress
We next examined the role of Cdc55-PP2A
in Orm2 dephosphorylation dynamics in re-
sponse to heat stress. We generated a PP2A
ts mutant, pph21 E102K pph22∆ (Lin and
Arndt, 1995), which allowed us to simultane-
ously introduce heat stress and turn off PP2A
activity. As shown in Figure 7, substantially
less dephosphorylated Orm2, seen as the
fastest-migrating bands on a phosphate-af-
finity gel, appears in the PP2A ts mutant at
both the 10- and 15-min time points com-
pared with pph22∆ cells. The observation
that Orm dephosphorylation was not com-
pletely abolished in the PP2A ts mutant sug-
gests that other phosphatases may also
contribute to Orm protein dephosphoryla-
tion in response to heat stress. It is also pos-
sible that PP2A activity was not completely
lost in the PP2A ts mutant after the short
temperature shift. Nevertheless, our results
indicate that PP2A activity contributes to
regulation of Orm phosphorylation dynam-
ics in response to heat stress.
A model for how Orm phosphorylation
regulation controls sphingolipid
biosynthesis in response to stress
in a kinetically coupled manner
As bioactive molecules, sphingoid interme-
diates transmit signals when their cellular lev-
els change in response to various stresses
(Hannun and Obeid, 2008; Dickson, 2010;
Nikolova-Karakashian and Rozenova, 2010).
However, both lack of sphingolipids and con-
stitutive high levels of sphingolipids compromise cell viability (Buede
et al., 1991; Chung et al., 2001). Thus, biosynthesis of sphingoid in-
termediates in response to stresses requires precise temporal control.
Previously, very little was known about the regulatory mechanism.
Here we provided evidence supporting a model in which the
conserved Pkh-Ypk signaling cascade and Cdc55-PP2A facilitate/
ensure rapid, transient sphingolipid production upon heat stress
through regulation of Orm protein phosphorylation (Figure 8): 1) A
Pkh-Ypk cascade is rapidly activated upon heat stress. 2) Orm phos-
phorylation rapidly increases. 3) Orm phosphorylation releases inhi-
bition of SPT activity. 4) Activated SPT promotes de novo synthesis
of sphingoid intermediates. 5) Accumulated sphingoid intermedi-
ates act as signaling molecules to initiate the cellular heat shock
Cdc55-PP2A deficient mutants. As shown in Figure 6, b and d, the
pph21∆ orm2-3D orm1∆ and cdc55∆ orm2-3D orm1∆ strains show
myriocin resistance, whereas the pph21∆ orm2-3A orm1∆ and
cdc55∆ orm2-3A orm1∆ strains do not. These results support the
conclusion that Cdc55-PP2A counteracts Ypk1 phosphorylation of
the three indicated Orm2 serines in regulation of sphingolipid pro-
duction. In contrast, the rts1∆ orm2-3A orm1∆ strain is resistant to
myriocin (Figure 6d), which further confirms our conclusion that
myriocin resistance of rts1∆ is independent of the Orm proteins.
A previous study demonstrated that cdc55∆ confers resistance
to rapamycin (Jiang and Broach, 1999), which is an inhibitor of
TOR signaling. Strikingly, we found that Orm proteins are required
for myriocin resistance of cdc55∆ cells but not for their rapamycin
FIGURE 5: Orm2 phosphorylation status and myriocin resistance in various PP2A mutants.
(a) Subunit composition of yeast PP2A. (b) Orm2 phosphorylation status in cells lacking the
indicated PP2A subunits. Western blots showing the phosphorylation patterns for Orm2 after
separation on phosphate-affinity gels. All cells were grown to early log phase in YPD at 25°C.
Cell extracts were prepared as described in Materials and Methods. Pgk1 was used as a loading
control. (c) Growth of PP2A mutants in the presence of myriocin. Various mutants were grown
on plates containing the indicated concentrations of myriocin at 30°C for 3 d. (d) Cdc55 absence
rescued the growth of an lcb1-100 ts mutant at semirestrictive temperature but not restrictive
temperature. Mutants were grown at the indicated temperature for 3 d. Note that cdc55∆
lcb1-100 cells grow better than lcb1-100 at 30 and 33°C.
Control0.5µg/ml Myriocin 0.75µg/ml Myriocin 1µg/ml Myriocin
Volume 23 June 15, 2012 Sphingolipid regulation upon stress | 2395
are inhibited by micromolar concentrations of sphingoid bases (Friant
et al., 2001). Thus, the most harmonious interpretation of our results
and previous findings is that the Pkh-Ypk cascade is also regulated by
heat-induced sphingolipid intermediate accumulation via a negative
feedback mechanism by which increased sphingolipids inhibit Pkh-
Ypk kinase–mediated phosphorylation of Orm proteins, thereby re-
storing SPT inhibition.
How could heat stress activate Ypk kinases to trigger Orm2 phos-
phorylation in such a rapid manner (Figure 3a)? Breslow et al. (2010)
showed that disruption of sphingolipid biosynthesis by myriocin
results in an increase in Orm phosphoryla-
tion. However, the increase in Orm phos-
phorylation in response to heat stress ap-
parently is not due to low levels of
sphingolipids, because both the levels of
Orm phosphorylation and the levels of
sphingolipid intermediates actually con-
tinue to increase at these early time points
after heat stress (Figure 2C, time points be-
tween 0 and 4 min; Dickson et al., 1997;
Jenkins et al., 1997; Jenkins, 2003; Wells
et al., 1998; Mao et al., 1999; Skrzypek
et al., 1999). Moreover, our data suggest
that heat stress affects Orm phosphoryla-
tion before the myriocin-sensitive step (Fig-
ure 3e). Ypk1 activation is known to require
its phosphorylation by both Pkh kinases
and the TORC2 complex (Casamayor et al.,
1999; Kamada et al., 2005). Recently
Slm1/2 was demonstrated to play impor-
tant roles in recruiting Ypk1 to the plasma
membrane for activation by the TORC2
responses, as described in the Introduction. Sphingoid intermediate
accumulation also feeds back to dephosphorylate Orm proteins,
possibly by inhibiting Pkh-Ypk kinase activity or/and activating
Cdc55-PP2A phosphatase activity. 6) Dephosphorylated Orm pro-
teins inhibit SPT activity.
The Pkh-Ypk cascade was previously proposed to be downstream
of heat-induced sphingolipid accumulation (Dickson, 2010). However,
our data indicate that the Pkh-Ypk cascade is activated in response to
heat stress and induces de novo sphingolipid biosynthesis (Figure 4).
Of interest, a previous in vitro study indicated that Pkh1/Pkh2 kinases
FIGURE 7: Orm phosphorylation status in PP2A-deficient mutants upon heat shock. Western
blots showing the phosphorylation patterns for Orm2 after separation on phosphate-affinity
gels. P-Orm2 indicates phosphorylated forms of Orm2. Pgk1 was used as a loading control. Cell
extracts were prepared from cells shifted from 25 to 39°C for the indicated times as described
in Materials and Methods. The pph21E102K pph22∆ mutant is temperature sensitive.
FIGURE 6: PP2A-deficient mutants confer myriocin resistance specifically through effects on the Orm proteins. (a, b, d)
Impaired Cdc55-PP2A activity results in myriocin resistance dependent on Orm phosphorylation status. Various mutants
were grown at 30°C for 3 d on plates containing the indicated myriocin concentrations. (c) Orm proteins are required for
myriocin resistance but not rapamycin resistance in cdc55∆. Various mutants were grown on plates containing 0.5 μg/ml
myriocin or 100 nM rapamycin at 30°C for 3 d.
Control0.5µg/ml Myriocin 100nM Rapamycin
2396 | Y. Sun et al. Molecular Biology of the Cell
Orm phosphorylation is primarily regulated by sphingoid bases and
sphingoid base phosphates. In agreement with these conclusions,
our experiments indicate that exogenously provided sphingoid
bases are sufficient to induce Orm dephosphorylation without be-
ing converted to ceramide or to complex sphingolipids (Figure 2).
A recent study proposed that Orm1 and Orm2 may regulate
sphingolipid synthesis via two different mechanisms (Liu et al., 2012).
We observed the phosphorylation dynamics of Orm1 and Orm2
upon heat stress to be very similar (unpublished data), suggesting
that both of the Orm proteins function similarly in regulation of the
sphingolipid biosynthesis response to heat stress. However, we
found that Orm2 expression was at least 10 times higher than Orm1
expression (Supplemental Figure S1a). It is also worth mentioning
that neither C-terminally tagged Orm1 nor Orm2 is functional
(unpublished data) and that Orm1 tagged at its N-terminus with
3XFLAG cannot fulfill Orm1 function in the orm2∆ background
(Supplemental Figure S1A). Our studies indicate that Orm function
is preserved in N-terminally tagged Orm2.
Phosphate-affinity gels separated Orm2 protein into four mobil-
ity groups. Western blotting analysis revealed that the majority of
Orm proteins are moderately phosphorylated (bands in the third
group, Figure 2a) under standard growth conditions. This large pool
of moderately phosphorylated Orm protein may explain how cells
rapidly respond to various environmental stresses in a graded man-
ner. In addition, the majority of orm2-3A proteins were detected as
dephosphorylated forms, and levels of sphingoid bases were greatly
reduced in orm2-3A cells, further supporting the conclusion that de-
phosphorylated Orm proteins negatively regulate sphingolipid bio-
synthesis. Given that the reduction of dephosphorylated Orm2 and
the increase in hyperphosphorylated Orm2 occur at the same time
in response to heat stress (Figure 3b, time points 2 and 5 min), SPT
activation in response to heat stress could be caused by release of
SPT inhibition by dephosphorylated Orm2, by positive regulation of
SPT by hyperphosphorylated Orm2, or both.
Cdc55-PP2A counteracts Ypk1 activity in Orm-mediated
sphingolipid biosynthesis regulation
In comparison to kinases, phosphatases often function in a less spe-
cific manner. In this study, we performed a series of experiments to
demonstrate a specific relationship between Cdc55-PP2A and the
Orm proteins in regulating sphingolipid biosynthesis. First, sphingo-
lipid biosynthesis increased in cdc55∆ mutants. Second, Orm proteins
were required for myriocin resistance but not for rapamycin resistance
of cdc55∆ cells. Finally, genetic analysis suggested that Cdc55-PP2A
functions to counteract Ypk1 kinase–mediated Orm phosphorylation.
In contrast, we found that Rts1, another PP2A regulatory subunit,
is involved in sphingolipid regulation in an Orm protein–indepen-
dent manner. Previous studies suggested that Sac1 binds and mod-
ulates SPT activity in a pathway independent of Orm proteins
(Breslow et al., 2010). Future investigations are needed to test
whether Rts1-PP2A is involved in Sac1 modulation of SPT activity.
PP2A was previously identified as an attractive candidate for a
ceramide-activated protein phosphatase in yeast (Nickels and
Broach, 1996), although direct evidence that ceramides (or other
sphingoid intermediates) activate PP2A has not been reported. It is
not clear whether other phosphatases are also involved in Orm phos-
phoregulation. Loss of Ypk kinase activity abolished sphingolipid
biosynthesis in response to heat stress (Figure 4c), but Orm phos-
phorylation still decreased (Figure 4b, right), suggesting that Cdc55-
PP2A may affect Orm phosphorylation at least partially in a constitu-
tive manner. Because both Pkh kinases and the TORC2 complex are
required for Ypk kinase activity, an alternative explanation for our
complex (Niles et al., 2012). Of interest, both Pkh kinases and
Slm1/2 localize in punctate eisosome plasma membrane domains
(Walther et al., 2007; Grossmann et al., 2008), and the TORC2
complex localizes in small foci on the plasma membrane but not in
eisosomes (Berchtold and Walther, 2009). Another recent study
suggested that plasma membrane stress might induce relocaliza-
tion of Slm1/2 and activation of TORC2 (Berchtold et al., 2012).
Thus, it is possible that heat stress may rapidly decrease the rigid-
ity of plasma membrane, resulting in release of Pkh kinase and
Slm1/2 from eisosomes, thereby triggering Ypk kinase activation.
Both the lipids and the proteins involved in this regulatory circuit
(Figure 8) are highly conserved between yeast and mammalian cells.
Thus, the mechanism we proposed here may serve a general basis
for how Orm phosphoregulation controls sphingolipid biosynthesis
in response to stress in a kinetically coupled manner.
Orm phosphorylation status is highly responsive
to sphingoid bases
In this study, we demonstrated that the high levels of Orm phospho-
rylation induced by heat stress decline while the sphingoid bases
(PHS) reach their peak levels (Figure 3c, time points between 5 and
15 min). Several previous studies established that sphingoid bases
and sphingoid base phosphates accumulate with similar timing
(Dickson et al., 1997; Jenkins et al., 1997). However, the heat-in-
duced ceramides peak ∼10 min after the sphingoid bases reach
their peak (Dickson et al., 1997; Jenkins et al., 1997), and no obvious
changes in the levels of complex sphingolipids were observed dur-
ing 2 h of heat stress (Jenkins et al., 1997). Thus, the decrease of
Orm phosphorylation induced by heat stress is kinetically coupled
to changes in sphingoid base and sphingoid base phosphate levels
but not ceramide or complex sphingolipid levels, suggesting that
FIGURE 8: A feedback regulation pathway in which Orm protein
phosphorylation dynamics rapidly and precisely regulate sphingolipid
biosynthesis in response to heat stress. See the text for a description.
Sphingoid base 1 phosphate
Heat Stress Response
Volume 23 June 15, 2012 Sphingolipid regulation upon stress | 2397
pRS306-PPH21 E102K plasmid was integrated into the URA3 locus
of a pph21∆::CgHIS3 strain.
Phytosphingosine purified from Saccharomyces cerevisiae was ob-
tained from Avanti Polar Lipids (Alabaster, AL). Sphingosine was ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA), and
stearylamine was obtained from Sigma-Aldrich (St. Louis, MO). All
lipids were dissolved in methanol.
Preparation of whole-cell extracts
Yeast cells were grown to early logarithmic phase in YPD. After the
indicated treatments, cold trichloroacetic acid (TCA) was added to
the yeast culture to a final concentration of 20% (vol/vol). The growth
medium was removed after centrifugation, and cell pellets were
flash frozen in liquid nitrogen. The cells were thawed, resuspended
in 5% (vol/vol) TCA, and lysed by bead beating at 4°C for 10 min.
Whole-cell extracts were collected by 5 min of centrifugation at
14,000 × g at 4°C. The pellets were resuspended in SDS–PAGE
sample buffer containing 50 mM dithiothreitol.
Detection of protein phosphorylation
To detect phosphorylation-dependent mobility shifts of FLAG-Orm1
and FLAG-Orm2, whole-cell extracts were loaded onto 8% SDS
polyacrylamide gels containing 25 μM Phos-tag acrylamide (Wako
Chemicals USA, Richmond, VA) and 25 μM MnCl2. Before transfer to
nitrocellulose membranes, gels were washed twice for 10 min
in 2 mM EDTA containing transfer buffer (25 mM Tris-HCl pH 8.3,
192 mM glycine, 20% [vol/vol] methanol) and then once for 10 min
in transfer buffer without EDTA. Membranes were then probed with
1:4000 mouse anti-FLAG (Sigma-Aldrich).
Lipid analysis by high-performance liquid chromatography
Extraction and processing of sphingoid bases from yeast cells for
fluorescence high-performance liquid chromatography (HPLC) anal-
ysis using the AQC reagent (Waters, Milford, MA) was performed as
described previously (Lester and Dickson, 2001). Sampling of heat-
stressed cells was as described. HPLC analysis was performed using
a C18 column (4.6 × 250 mm, XDB-C18; Hewlett-Packard, Palo,
Alto, CA) on a Shimadzu LC10A series liquid chromatography
system. Isocratic elution was carried out for 60 min at a flow rate of
1.0 ml/min. Lipid-reacted AQC reagent was excited with 244-nm UV
radiation, and the resultant emission signal at 398 nm was detected.
C18-PHS reacted with the AQC reagent was used as a standard for
results could be that Cdc55-PP2A controls Orm phosphorylation by
negatively regulating these two upstream effectors of Ypk kinases,
instead of directly dephosphorylating the Orm proteins. However,
this possibility seems unlikely because Orm2 dephosphorylation
should have no longer occurred upon loss of Ypk kinase activity
(Figure 4b, right), which is contrary to our observation.
MATERIALS AND METHODS
Media and strain construction
The yeast strains used in this study are listed in Supplemental Table
S1. They were grown in standard rich media (yeast extract/peptone/
dextrose [YPD]). The primers used for cloning are listed in Supple-
mental Table S2.
To generate a strain expressing the 3XFLAG-fused Orm2 protein
from its genetic locus, we first constructed a vector using following
strategies. Two DNA fragments were obtained by PCR amplifica-
tion, using primer pairs of HindIII-ORM2-F/FLAG-ORM2-interR and
FLAG-ORM2-interF/XhoI-ORM2-R, respectively. The two DNA frag-
ments were then converted into one by PCR amplification, using
primer pairs of HindIII-ORM2-F/XhoI-ORM2-R. The resulting PCR
product was digested and ligated into the pBluescript II KS(+) vec-
tor using the HindIII and XhoI restriction enzymes. In addition, a
NAT1 (nourseothricin acetyltransferase) DNA fragment, amplified
by primer pairs NatR-ORM2-F/NatR-ORM2-R, was inserted into the
same vector using the HindIII and SpeI restriction enzymes. The re-
sulting plasmid, named pBS-NAT1-FLAG-ORM2, was digested with
HindIII and XhoI, and the fragment containing NAT1-FLAG-ORM2
was transformed into a yeast strain in which ORM2 had been re-
placed by Candida glabrata (Cg) URA3. We isolated transformants
that no longer grow on a plate lacking uracil but do grow on a
nourseothricin-dihydrogen sulfate (clonNAT)–containing plate. The
pBS-NAT1-FLAG-ORM2 plasmid was also used as a template to
generate NAT1-FLAG-ORM2-3A and NAT1-FLAG ORM2-3D plas-
mids using the QuikChange Lightning Site-Directed Mutagenesis
Kit (Agilent Technologies, Santa Clara, CA) and primers ORM2-3A-
F/ORM2-3A-R and ORM2-3D-F/ORM2-3D-R, respectively. A strain
expressing 3XFLAG-Orm1 was generated by a similar strategy but
using different primers, listed in Supplemental Table S2.
To obtain strains expressing Ypk1-as (Ypk1 L424A), two fragments
were PCR amplified from genomic DNA using primers OYS227/
OYS268 and OYS228/OYS267. The two fragments were then con-
verted into one by PCR amplification, using primer pairs of OYS227/
OYS228. The resulting fragment was digested and ligated into
pRS306 vector. pRS306-YPK1AS (YPK1 L424A) was linearized using
StuI and transferred into the URA3 locus of a ypk1∆::CgLEU2 strain.
To obtain strains expressing Pkh1-as, a fragment containing 500
base pairs upstream and downstream of PKH1 was PCR amplified
from genomic DNA using primers PKH1-XhoI-F and PKH1-SacII-R.
The PCR product was digested by XhoI/SacII and was ligated into
pRS306 vector, creating pRS306-PKH1. pRS306-PKH1 was used as
template to generate the PKH1-AS (PKH1 F187V, L203A) plasmid
using the QuikChange Lightning Site-Directed Mutagenesis Kit and
primers PKH1-F187V/L203A-F and PKH1-F187V/L203A-R. PKH1-AS
(PKH1 F187V, L203A) plasmid was linearized using StuI and inserted
into the URA3 locus of a pkh1∆::CgLEU2 strain.
PPH21 E102K plasmid was obtained using a similar strategy as
for the PKH1-AS plasmid. Primers PPH21-BamHI-F and PPH21-NotI-
R were used to amplify the PPH21 fragment. The PPH21 fragment
was ligated into the pRS306 vector, creating pRS306-PPH21.
pRS306-PPH21 was used as a template to generate PPH21 E102K
plasmid using the QuikChange Lightning Site-Directed Mutagene-
sis Kit and primers PPH21-E102K-F and PPH21-E102K-R. Linearized
We are grateful to Françoise Roelants and Jeremy Thorner for shar-
ing information before publication on the identity of the kinase that
phosphorylates the Orm proteins. We thank Howard Riezman for
providing the lcb1-100 strain. We gratefully acknowledge Betsy
Wong and Casey Drubin for the preparation of constructs and
strains. The manuscript was improved by the critical comments of
Georjana Barnes, Christa Cortesio, Jonathan Wong, and Nathaniel
Krefman. This work was supported by National Institutes of Health
Grant R01 GM 50399 to D.G.D. Y.M. acknowledges a Human
Frontiers Science Program Long-term Fellowship.
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