Copyright Ó 2007 by the Genetics Society of America

DOI: 10.1534/genetics.106.064527

SVF1 Regulates Cell Survival by Affecting Sphingolipid Metabolism in

Saccharomyces cerevisiae

Jennifer L. Brace,* Robert L. Lester,† Robert C. Dickson† and Charles M. Rudin*,1

*Johns Hopkins University, Baltimore, Maryland 21231 and †Department of Molecular and Cellular Biochemistry and the
Lucille P. Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536
Manuscript received August 8, 2006
Accepted for publication October 13, 2006

ABSTRACT
Sphingolipid signaling plays an important role in the regulation of central cellular processes, including
cell growth, survival, and differentiation. Many of the essential pathways responsible for sphingolipid
biogenesis, and key cellular responses to changes in sphingolipid balance, are conserved between mam-
malian and yeast cells. Here we demonstrate a novel function for the survival factor Svf1p in the yeast
sphingolipid pathway and provide evidence that Svf1p regulates the generation of a specific subset of
phytosphingosine. Genetic analyses suggest that Svf1p acts in concert with Lcb4p and Lcb3p to generate a
localized pool of phytosphingosine distinct from phytosphingosine generated by Sur2p. This subset is
implicated in cellular responses to stress, as loss of SVF1 is associated with defects in the diauxic shift and the
oxidative stress response. A genetic interaction between SVF1 and SUR2 demonstrates that both factors are
required for optimal growth and survival, and phenotypic similarities between svf1Dsur2D and ypk1D suggest
that pathways controlled by Svf1p and Sur2p converge on a signaling cascade regulated by Ypk1p. Loss of
YPK1 together with disruption of either SVF1 or SUR2 is lethal. Together, these data suggest that com-
partmentalized generation of distinct intracellular subsets of sphingoid bases may be critical for activation of
signaling pathways that control cell growth and survival.

S PHINGOLIPIDS are important structural compo-

nents of cellular membranes. The role of sphingo-
lipid metabolites as key second messengers is becoming
also known as phytosphingosine (PHS), by the hydrox-
ylase Sur2p. DHS and PHS are phosphorylated by the
long-chain base kinases Lcb4p or Lcb5p. The phosphor-
increasingly clear. Sphingoid base signaling has been ylated bases can be dephosphorylated by the phospha-
shown to regulate diverse cellular processes, including tases Lcb3p or Ysr3p or broken down to generate C-16
cell proliferation, apoptosis, angiogenesis, and differ- aldehydes and ethanolamine phosphate by the lyase
entiation (for reviews, see Pyne and Pyne 2000; Hannun Dpl1p (Figure 1). While mammalian cells primarily
et al. 2001; Cuvillier 2002; Spiegel and Milstien 2003; generate sphingosine (SPH), S. cerevisiae lack the en-
Saba and Hla 2004). Since these processes play a central zyme to make SPH and contain relatively high levels of
role in carcinogenesis, these observations have gener- PHS, which appears to function analogously to SPH.
ated increasing interest in sphingolipid signaling path- PHS and SPH can be converted to phytoceramide and
ways as offering a series of attractive targets for novel ceramide, respectively. Ceramide species can be further
cancer therapies (Toman et al. 2001; Spiegel and metabolized to generate complex sphingolipids, includ-
Kolesnick 2002; Ogretmen and Hannun 2004). ing inositolphosphorylceramide (IPC), mannosylated
Evolutionary conservation of sphingolipid metabolic IPC (MIPC), and mannosyl-diinositolphosphorylcera-
pathways emphasizes the importance of these lipids. mide [M(IP)2C]. Several recent reviews provide detailed
Analysis of these pathways in Saccharomyces cerevisiae may summaries of sphingolipid metabolism (Dickson and
provide insight into a central regulatory mechanism for Lester 1999, 2002; Obeid et al. 2002; Alvarez-Vasquez
cell death in mammalian systems. et al. 2005).
Generation of sphingoid bases begins with the con- Several studies have demonstrated that efficient in-
densation of serine and palmitoyl-CoA to yield 3- corporation of the exogenous sphingoid bases DHS or
ketodihydrosphingosine, followed by conversion to PHS into ceramide and the complex sphingolipids
sphinganine, commonly called dihydrosphingosine requires both phosphorylation and subsequent dephos-
(DHS). In yeast, DHS can be used to generate dihydro- phorylation (Mao et al. 1997, 1999; Qie et al. 1997;
ceramide or can be converted to 4-hydroxysphinganine, Mandala et al. 1998; Funato et al. 2003). Yeast lacking
the phosphatase Lcb3p do not convert 3H-DHS into
ceramide or into the complex sphingolipids IPC, MIPC,
1
Corresponding author: Johns Hopkins University, 1550 Orleans St., and M(IP)2C (Mao et al. 1997, 1999; Qie et al. 1997;
CRBII 544, Baltimore, MD 21231. E-mail: [email protected] Mandala et al. 1998). Similarly, cells lacking the kinases

Genetics 175: 65–76 ( January 2007)

66 J. L. Brace et al.

further enhanced through the addition of Pkh1p to the

reaction (Liu et al. 2005b). The analogous pathway in
mammalian cells is controlled by PDK1, and it has been
shown that SPH can similarly activate PDK1 in mamma-
lian cells (King et al. 2000).
We identified SVF1 in a screen for yeast factors reg-
ulating cell survival that could be partially comple-
mented by expression of mammalian Bcl-xL (Vander
Heiden et al. 2002). We have shown that while Svf1p and
Bcl-xL have distinct functional properties, Svf1p func-
tions to regulate survival under a variety of stress con-
ditions. In particular, cells lacking SVF1 are defective in
responding to the metabolic changes that occur during
the diauxic shift (Vander Heiden et al. 2002) and are
hypersensitive to cold stress, H2O2, menadione, and ace-
tic acid, conditions that elevate production of, or ex-
Figure 1.—A schematic overview of yeast sphingolipid syn- posure to, reactive oxidative species (Brace et al. 2005).
thesis and metabolism. Deletion strains of the genes in bold- The mechanism of action of Svf1p has not been defined.
face type were examined.
Here we demonstrate that Svf1p affects cellular survival
in part via modulation of the sphingolipid metabolic
Lcb4p and Lcb5p also demonstrate a deficiency in pathway. Using a genetic approach, we place SVF1 in the
conversion of 3H-DHS to IPC and M(IP)2C (Funato sphingolipid pathway and suggest a model in which
et al. 2003). The apparent requirement for concerted Svf1p regulates the generation of a localized pool of
action of the sphingoid base kinase and phosphatase for sphingoid bases affecting Ypk1p-dependent signaling
production of downstream lipids in the pathway has pathways.
suggested a model in which sphingolipid species are
differentially compartmentalized within the cell. With MATERIALS AND METHODS
prolonged incubation, downstream sphingolipids in
fact can be made at low levels in lcb4Dlcb5D cells but Yeast strains and plasmids: The S. cerevisiae W303 (MATa
ade2-1 can1-100 his3-11.15 leu2-3.112 trp1-1 ura3-1) or BY4741
not in lcb3D cells (Funato et al. 2003). This implies the (MATa his3D1 leu2D0 met15D0 ura3D0) strain backgrounds
existence of an alternative bypass mechanism for inef- were used as indicated in the text (see also Table 1). Strains
ficient conversion of exogenous bases that do not cy- were maintained on synthetic complete (SC) media: 0.17%
cle through phosphorylation/dephosphorylation. The yeast nitrogen base without ammonium sulfate, 1.0% ammo-
phosphorylation/dephosphorylation pathway may fa- nium sulfate (Fisher), amino acid dropout mixture, and 2%
dextrose (Fisher), supplemented with 300 mm adenine hemi-
cilitate proper localization of exogenous lipids for sulfate. Selection media were used as necessary by using the
metabolism by other proteins in the pathway. appropriate amino acid dropout mixture. Counterselection of
Analyses of yeast mutants in the sphingolipid pathway the URA3 containing plasmid pOW4 was achieved by plating
have demonstrated that sphingoid base signaling in yeast to SC media containing 1 mg/ml 5-fluroorotic acid (FOA).
regulates cellular processes, including the heat stress Kanamycin selection was performed with YPD [2% peptone,
1% yeast extract (Fisher), 2% dextrose (Fisher), supplemented
response (Dickson et al. 1997; Jenkins et al. 1997; Mao with 0.15 mg/ml l-tryptophan (Fisher), 2% agar] plates con-
et al. 1999; Skrzypek et al. 1999; Jenkins and Hannun taining 0.2 mg/ml G418. Yeast media reagents were obtained
2001; Ferguson-Yankey et al. 2002), nutrient uptake from QBioGene, unless noted otherwise.
(Skrzypek et al. 1998; Chung et al. 2000, 2001; Hearn Deletion strains of SVF1, LCB4, LCB5, LCB3, YSR3, SUR2,
et al. 2003), endocytosis (Friant et al. 2000, 2001; and DPL1 of the BY4741 background and YPK1 of the BY4743
background were obtained from the Research Genetics
Zanolari et al. 2000), and cytoskeletal organization (Huntsville, AL) knockout collection (kanamycin resistance).
(Friant et al. 2001; Balguerie et al. 2002). Most recently, An SVF1-null strain marked with the HIS3 gene was generated
it has been demonstrated that sphingoid bases activate in the wild-type parental strain by homologous replacement
the yeast kinases Pkh1p and Pkh2p (Friant et al. 2001). using methods previously described (Bahler et al. 1998). In
Pkh1/2p regulate several pathways, including a cell in- the W303 background, replacement of the coding region of
SVF1 with the HIS3 and kanamycin resistance gene was per-
tegrity pathway via activation of the AGC family of ki- formed as described (Bahler et al. 1998; Vander Heiden et al.
nases that includes Pkc1p, Ypk1p, Ypk2p, and Sch9p 2002). All genomic deletions were confirmed by PCR to re-
(Roelants et al. 2002, 2004). These kinases regulate es- gions surrounding the deletion as well as phenotypic confir-
sential pathways, as cells lacking both PKH1 and PKH2 mation after tetrad dissection. Double and triple knockouts
are nonviable (Casamayor et al. 1999). Sphingoid bases were generated through mating of single or double knock-
outs, selection of diploids, and tetrad dissection. All tetrad dis-
may also activate the downstream kinases Ypk1/2p and sections were performed on YPD media with incubation at 30°.
Sch9p directly. In vitro kinase assays show that activity of Marker segregation and PCR analysis were used to confirm the
these kinases increases in the presence of PHS and is generation of double and triple knockouts. To avoid selection
 SVF1 Affects Sphingolipid Metabolism 67

TABLE 1
Yeast strains used in this study

Strain Genotype Reference or source

Wild type W303 MATa ade2-1 can1-100 his3-11.15 leu2-3.112 trp1-1 ura3-1 Vander Heiden et al. (2002)
svf1D W303 a svf1DTHIS3 Vander Heiden et al. (2002)
sur2D W303 a sur2DTkanMX This study
svf1D/1sur2D/1 W303 a/a svf1DTHIS3 sur2DTkanMX a sur2DTkanMX 3 a svf1DTkanMX
Wild type BY4741 MATa his3D1 leu2D0 ura3D0 met15D0 Research Genetics
svf1D BY4741 a svf1DTkanMX Research Genetics
sur2D BY4741 a sur2DTkanMX Research Genetics
lcb3D BY4741 a lcb3DTkanMX Research Genetics
dpl1D BY4741 a dpl1DTkanMX Research Genetics
lcb4D BY4741 a lcb4DTkanMX Research Genetics
lcb5D BY4741 a lcb5DTkanMX Research Genetics
ysr3D BY4741 a ysr3DTkanMX Research Genetics
ypk1D/1 BY4743 a/a ypk1DTkanMX Research Genetics
svf1D BY4741 a svf1DTkanMX This study
svf1D BY4741 a svf1DTHIS3 This study
sur2D BY4741 a sur2DTkanMX This study
svf1D/1sur2D/1 BY4741 a/a svf1DTkanMX sur2DTkanMX a svf1DTkanMX 3 a sur2DTkanMX
lcb3D/1sur2D/1 BY4741 a/a lcb3DTkanMX sur2DTkanMX a sur2DTkanMX 3 a lcb3DTkanMX
lcb4D/1sur2D/1 BY4741 a/a lcb4DTkanMX sur2DTkanMX a sur2DTkanMX 3 a lcb4DTkanMX
lcb5Dsur2D BY4741 a lcb5DTkanMX sur2DTkanMX a sur2DTkanMX 3 a lcb5DTkanMX
dpl1Dsur2D BY4741 a dpl1DTkanMX sur2DTkanMX a sur2DTkanMX 3 a dpl1DTkanMX
lcb3Dsvf1D BY4741 a lcb3DTkanMX svf1DTHIS3 a svf1DTHIS3 3 a lcb3DTkanMX
lcb3Dsvf1D BY4741 a lcb3DTkanMX svf1DTkanMX a svf1DTkanMX 3 a lcb3DTkanMX
lcb4Dsvf1D BY4741 a lcb4DTkanMX svf1DTkanMX a svf1DTkanMX 3 a lcb4DTkanMX
ypk1D/1svf1D/1 BY4741 a/a ypk1DTkanMX svf1DTHIS3 a svf1DTHIS3 3 a ypk1DTkanMX
ypk1D/1sur2D/1 BY4741 a/a ypk1DTkanMX sur2DTkanMX a sur2DTkanMX 3 a ypk1DTkanMX
lcb3D/1dpl1D/1svf1D/1 BY4741 a/a lcb3DTkanMX dpl1DTkanMX svf1DTHIS3 a lcb3DTkanMX svf1DTHIS3 3
a dpl1DTkanMX
lcb3D/1dpl1D/1svf1D/1 BY4741 a/a lcb3DTkanMX dpl1DTkanMX svf1DTkan a lcb3DTkanMX svf1DTkanMX 3
a dpl1DTkanMX
lcb3D/1dpl1D/1sur2D/1 BY4741 a/a lcb3DTkanMX dpl1DTkanMX sur2DTkanMX a dpl1DTkanMX sur2DTkanMX 3
a lcb3DTkanMX

of suppressor mutations, strains with slow growth phenotypes For confirmation of synthetic lethality, the indicated diploid
were maintained as heterozygous diploids and were sporu- strains were transformed with a plasmid expressing the indi-
lated and dissected just prior to use in experiments. cated gene. Upon tetrad dissection, each resulting genotype
SVF1, DPL1, and YPK1 amplified from yeast genomic DNA containing the plasmid was patched to YPD to allow for plas-
and human Bcl-xL cDNA were cloned into the single-copy high- mid loss. Cells were then struck to plates lacking uracil as a
expression vector pOW4 (uracil selection) and transformed control for viable growth in the presence of the indicated gene.
into yeast using the standard lithium acetate method (Gietz Counterselection was performed on plates containing 1 mg/
et al. 1992). ml FOA and incubation at 30°. Nonviable strains were unable
Growth analysis: In liquid culture, the indicated strains were to grow on plates containing FOA.
grown overnight at 30° in SC media to saturation. Cells were HPLC measurement of sphingoid bases: The indicated
normalized in SC media to an OD600 of 0.08. Normalized cul- strains were grown at 20° in SC media, as described above, until
tures were incubated with rotation at 20°, and samples were midlog phase (22 hr). Where indicated, cells in midlog
removed at the designated times to measure OD600. Three in- phase at 20° were treated with 30 mm C18-SPH (Biomol) for 1
dependent cultures were used for each time point and sample hr prior to harvesting cells, and control cells were treated with
measurement. an equal volume of ethanol. Trichloroacetic acid (TCA) was
For plate analysis, the indicated strains were normalized to added to the cells (10 OD600 units) to a final concentration of
1 3 106 cells/ml (OD650 ¼ 0.1 on a 96-well plate reader) and 5%, and samples were incubated on ice for 5 min. Cells were
5 ml of 10-fold serial dilutions were plated onto SC plates (or harvested by centrifugation and washed once with 10 ml ice-
selection media as necessary) containing the indicated con- cold 5% TCA, followed by two washes in 10 ml ice-cold water.
centration of sphingosine, phytosphingosine, dihydrosphin- Samples were frozen at 80° until further processing. The
gosine, or ethanol control. Stock solutions of sphingosine, sphingoid bases were extracted and converted to fluorescent
phytosphingosine, and dihydrosphingosine (BioMol, Ply- aminoquinoline derivatives, which were separated and ana-
mouth Meeting, PA) were prepared in ethanol and added to lyzed by HPLC as described (Lester and Dickson 2001). C18-
plates at the concentration indicated. NP-40 was added to a S1P (Biomol) was used as a standard to identify the peak
final concentration of 0.0015% to facilitate dispersal of lipids corresponding to phosphorylation of the exogenously added
in the media. Plates were incubated at 20° and were analyzed SPH. Each sample was performed in duplicate. The analysis of
after 6 days. wild-type and svf1D cells was performed in three independent
68 J. L. Brace et al.

data that Svf1p and Bcl-xL have distinct roles in

regulating cell survival (Brace et al. 2005).
To confirm the role of SVF1 in the response to
elevated levels of SPH, we obtained an SVF1-null strain
in an alternative yeast background. Similar to the result
seen in the W303 background, BY4741 svf1D cells are
resistant to growth on SPH (Figure 2C). Together, these
data confirm a role for SVF1 in regulating growth and
survival in the presence of elevated levels of SPH.
SVF1 demonstrates a genetic interaction with the
dihydrosphingosine hydroxylase gene SUR2: To better
understand the function of SVF1 in the sphingolipid
pathway, we generated double knockouts of SVF1 and
Figure 2.—SVF1-null cells are resistant to growth inhibitory genes regulating key steps in the metabolism of sphin-
concentrations of SPH. (A) W303 wild-type (WT) or svf1D cells goid bases, including LCB4, LCB3, SUR2, and DPL1. We
were normalized and 10-fold serial dilutions were spotted onto identified a genetic interaction between SVF1 and the
plates containing ethanol (control) and 30 or 50 mm SPH.
dihydrosphingosine hydroxylase SUR2. Yeast deficient
Plates were incubated at 20° for 6 days. (B) W303 WT or svf1D
cells were transformed with a control vector, Bcl-xL, or SVF1. in both SVF1 and SUR2 exhibit a slow growth phenotype
Ten-fold serial dilutions of normalized cells were plated onto (Figure 3A). A similar growth defect was observed in the
selection plates containing 50 mm SPH or ethanol (control). W303 background (data not shown). These data dem-
Plates were incubated at 20° for 6 days. (C) BY4741 WTor svf1D onstrate that SVF1 and SUR2 together are required for
cells were normalized and 10-fold serial dilutions were plated
optimal cell proliferation, and their synthetic interac-
onto selection plates containing ethanol (control) and 30 or
50 mm SPH. Plates were incubated at 20° for 6 days. tion suggests that they may act in parallel pathways to
regulate growth and survival.
experiments, each run in duplicate. The analysis of wild-type To determine if SUR2 is necessary for the SPH
and svf1D cells expressing SVF1 and cells treated with SPH was resistance we observed in svf1D cells, we examined
performed once in duplicate. The analysis of wild type, svf1D, growth of svf1Dsur2D cells in the presence of 30 mm
sur2D, lcb3D, and lcb4D was performed twice in duplicate, and SPH. Interestingly, not only were these cells resistant to
the analysis of svf1Dlcb4D and svf1Dlcb3D was performed once
in duplicate. SPH, but also the addition of SPH improved colony
growth and density (Figure 3B). One hypothesis to
explain these data is that the slow growth phenotype of
RESULTS
svf1Dsur2D cells in the absence of SPH may be due to a
Cells lacking SVF1 are resistant to a growth inhibitory relative deficiency in generation of essential sphingoid
concentration of sphingosine: SVF1 was initially identi- bases. Yeast cells generate DHS and PHS and do not
fied in a screen for functional homologs of the produce SPH. To determine if the endogenous sphin-
mammalian survival protein Bcl-xL (Vander Heiden goid bases DHS or PHS could restore growth, we plated
et al. 2002), and cells lacking SVF1 are hypersensitive to cells in the presence or absence of PHS and DHS. In the
conditions associated with oxidative stress (Brace et al. presence of 15 mm PHS, but not of 15 mm DHS, growth
2005). Because of this association, we were interested in was restored to svf1Dsur2D yeast (Figure 3C). Similar
examining how SVF1 might influence other cellular phenotypes were observed in the W303 background
pathways implicated in the regulation of cell survival in (Figure 3, D and E). This suggests that SVF1 and SUR2
yeast. Sphingolipid signaling plays a key role in the act upstream of PHS in the sphingolipid pathway.
regulation of mammalian cell death. We examined the Cells lacking LCB3 and SUR2 exhibit a phenotype
response of yeast lacking SVF1 to a growth inhibitory similar to that of svf1Dsur2D cells: Examination of SPH
concentration of SPH. To our surprise, we observed that resistance in strains deleted for genes encoding known
svf1D cells demonstrate markedly increased resistance regulators of the sphingolipid pathway may provide
to SPH at concentrations as high as 50 mm (Figure 2A). insight into Svf1p function and would allow epistatic
This seemingly paradoxical observation suggested that placement of SVF1 in the sphingoid base pathway. We
Svf1p may have a specific function in regulating sphin- found that cells lacking the sphingosine-1-phosphate
golipid metabolic pathways. phosphatase LCB3, like svf1D cells, were resistant to SPH
We examined the ability of Bcl-xL to complement the (Figure 4A). The lcb3Dsvf1D strain exhibits a similar
SPH response phenotype of svf1D cells. Overexpression phenotype to either single deletion strain (data not
of Bcl-xL is unable to complement the SPH resistance of shown). We also generated a series of double knockouts
svf1D cells. Reintroduction of SVF1 to these cells, with SUR2 and genes encoding known regulators of the
however, restores their sensitivity to SPH, demonstrat- sphingolipid pathway to determine if additional genetic
ing that this phenotype is specific for loss of SVF1 interactions with SUR2 would provide insight into Svf1p
(Figure 2B). This observation supports our previous function. We observed a growth defect in cells lacking
 SVF1 Affects Sphingolipid Metabolism 69

Figure 3.—A genetic interac-

tion is observed between SVF1
and SUR2. (A) Overnight cultures
of BY4741 WT, svf1D, sur2D, and
svf1Dsur2D cells were normalized
to an OD600 of 0.08 and grown at
20°. At the indicated time points,
samples were removed to measure
OD600. Mean OD600 6 standard
deviation of three independent
cultures for each strain is shown.
(B) BY4741WT, svf1D, sur2D, and
svf1Dsur2D cells were normalized
and 10-fold serial dilutions were
spotted onto plates containing
ethanol (control) or 30 mm SPH.
Plates were incubated at 20° for 6
days. (C) BY4741WT, svf1D, sur2D,
and svf1Dsur2D cells were normalized and 10-fold serial dilutions were spotted onto plates containing ethanol (control), 15 mm DHS,
or 15 mm PHS. Plates were incubated at 20° for 6 days. (D) W303WT, svf1D, sur2D, and svf1Dsur2D cells were normalized and 10-fold
serial dilutions were spotted onto plates containing ethanol (control) or 30 mm SPH. Plates were incubated at 20° for 6 days. (E)
W303 WT, svf1D, sur2D, and svf1Dsur2D cells were normalized and 10-fold serial dilutions were spotted onto plates containing
ethanol (control), 15 mm DHS, or 15 mm PHS. Plates were incubated at 20° for 6 days.

SUR2 and LCB3 (Kim et al. 2000; Figure 4), as well as decrease in the amount of C18-PHS and C18-phytosphin-
in cells lacking SUR2 and LCB4. Interestingly, like gosine phosphate (PHSP) in svf1D cells (Figure 5A). To
svf1Dsur2D cells, sur2Dlcb3D cells exhibit increased confirm that this decrease in C18-PHS and C18-PHSP was
resistance to PHS (Figure 4B). The phenotypic similar- due to loss of SVF1, we overexpressed SVF1 in these cells
ities between Svf1p- and Lcb3p-deficient cells suggest and measured sphingoid base levels. SVF1-null cells
that these factors may act in concert in the same pathway expressing SVF1 exhibit C18-PHS and C18-PHSP levels
and/or may perform a similar function. similar to wild-type cells. Wild-type cells overexpressing
To determine if Svf1p has a biological function similar SVF1 generate higher levels of these lipids than wild-type
to that of the lipid phosphatase Lcb3p, we used HPLC to cells containing a control vector (Figure 5B). Together,
directly measure levels of various sphingoid bases in these data demonstrate that Svf1p regulates cellular levels
svf1D and wild-type cells. We observed a significant of C18-PHS and C18-PHSP. While we observe a slight
reduction of other lipids—specifically, C20-PHS and C-20
PHSP—in the svf1D strain, these lipids either were not
consistently reduced or were not restored to wild-type
levels by reintroduction of SVF1 (data not shown).
Cells lacking SVF1 demonstrate improved growth in
the presence of SPH. To determine how addition of SPH
alters sphingoid base generation, we added 30 mm SPH
to growing wild-type or svf1D cells and measured sphin-
goid base levels by HPLC. SPH suppresses the generation
of C18-PHS in wild-type cells to a level comparable to that
seen in svf1D cells and increases the production of C18-
PHSP in svf1D cells to that of wild-type cells (Figure 6A).
Therefore, addition of SPH appears to normalize the
levels of these sphingoid bases in wild-type and svf1D
cells. Wild-type and svf1D cells treated with SPH also
generate similar levels of sphingosine-1-phosphate, S1P
Figure 4.—Cells lacking LCB3 exhibit a phenotype similar to (Figure 6A). These data demonstrate that cells lacking
svf1D. (A) BY4741 WT, lcb4D, lcb5D, lcb3D, ysr3D, sur2D, dpl1D, SVF1 are not resistant to SPH through deficiencies in
and svf1D cells were normalized and 10-fold serial dilutions were uptake of the sphingoid base from the media nor
spotted onto plates containing ethanol (control) or 30 mm SPH. through an inability to phosphorylate SPH and confirm
Plates were incubated at 20° for 6 days. (B) BY4741 WT, sur2D, that treatment of cells with SPH can alter endogenous
svf1D, sur2D svf1D, sur2Dlcb4D, sur2Dlcb5D, sur2Dlcb3D, and
sur2Ddpl1D cells were normalized and 10-fold dilutions were lipid levels. These data further suggest that the relative
spotted onto plates containing ethanol (control), 20 mm DHS, deficiencies of C18-PHS and C18-PHSP in svf1D cells may
or 20 mm PHS. Plates were incubated at 20° for 6 days. contribute to the growth defect of this strain: addition of
70 J. L. Brace et al.

Figure 5.—Sphingoid base measurement in svf1D cells demonstrates a reduction of C18-PHS and C18-PHSP in the mutant. (A)
Overnight cultures of WT and svf1D cells were normalized to an OD600 of 0.08 and grown at 20°. Samples were collected in midlog
phase and sphingoid base levels were determined by HPLC. The average picomoles/A600 of the indicated sphingoid base 6 stan-
dard deviation of six independent samples is indicated for each strain. The fold change in svf1D cells compared to wild type for
each sphingoid base is shown below the graph. (B) WT and svf1D cells were transformed with a control vector or SVF1. Overnight
cultures were grown in selection media, normalized to an OD600 of 0.08, and incubated at 20°. Samples were collected in midlog
phase and sphingoid base levels were determined by HPLC. The average picomoles/A600 of the indicated sphingoid base 6 stan-
dard deviation of two independent samples is indicated for each sample.

SPH leads to both improved survival and relative nor- growth. The genetic interaction observed between SVF1
malization of levels of these lipids. and SUR2 suggests that Svf1p may play a role in the
The sphingolipid profile of svf1D cells does not re- phosphorylation/dephosphorylation pathway, and we
semble that of sur2D, lcb4D, or lcb3D cells (Figure 6B). hypothesize that Svf1p might regulate this pathway to
Sur2p is the only known dihydrosphingosine hydroxylase generate a subset of PHS. To explore this hypothesis, we
in the yeast genome, and SUR2-null cells manifest a examined sphingoid base profiles of cells lacking LCB3
striking reduction of PHS and elevation of DHS. Consis- or LCB4 in combination with loss of SVF1. We observe, as
tent with the known functions of Lcb4p and Lcb3p as a expected, that cells lacking LCB4 accumulate unphos-
lipid kinase and phosphatase, respectively, LCB4- and phorylated sphingoid bases (Figure 6B). While svf1D
LCB3-null cells accumulate markedly elevated levels of cells exhibit reduced generation of C18-PHS, lcb4D and
unphosphorylated and phosphorylated sphingoid bases, lcb4Dsvf1D cells accumulate similar levels of C18-PHS
respectively. While loss of SUR2, LCB4, or LCB3 alters (Figure 6C). This suggests that Svf1p does not prevent
ratios of phosphorylated-to-unphosphorylated sphingoid generation of C18-PHS upstream of Lcb4p. In contrast,
bases dramatically, in svf1D cells these ratios are main- while cells lacking LCB3 exhibit increased levels of both
tained in a range similar to that of wild-type cells. Taken C18-PHSP and C18-PHS, cells additionally lacking SVF1
together, these data suggest that unlike Sur2p, Lcb4p, do not accumulate these lipids to the same extent (Fig-
and Lcb3p, Svf1p may not have a primary enzymatic ure 6C). This suggests that Svf1p acts on, or upstream of,
function in the sphingolipid pathway, but may influence Lcb3p to generate PHSP and PHS. Together, these data
the production of a discrete subset of PHS and PHSP. support a model in which Svf1p regulates the concerted
Generation of ceramide from exogenous sphingoid action of Lcb4p and Lcb3p to generate a subset of PHS
bases requires phosphorylation by Lcb4p followed by and PHSP.
dephosphorylation by Lcb3p (Mao et al. 1997, 1999; Qie Loss of SVF1 but not SUR2 suppresses the lethality of
et al. 1997; Mandala et al. 1998; Funato et al. 2003). the lcb3Ddpl1D strain: If Svf1p regulates the generation
While these experiments test the exogenous addition of of a subset of sphingolipids, loss of SVF1 might be ex-
sphingoid bases, we hypothesize that this coupled reac- pected to suppress the growth defect of a strain that
tion occurs on lipids generated intracellularly. As pre- accumulates excessive amounts of this subset. One such
viously suggested, this coupling may serve to localize mutant might be the lcb3Ddpl1D strain. Lcb3p and
sphingolipids to specific intracellular compartments Dpl1p regulate different metabolic pathways reducing
(Funato et al. 2003). Similar genetic interactions be- PHSP. Yeast lacking both LCB3 and DPL1 are nonviable,
tween LCB3 or LCB4 and SUR2 suggest that disruption suggesting that although the analogous sphingolipid
of this coupling and loss of PHS are detrimental to cell S1P is generally considered to be a prosurvival factor in
 SVF1 Affects Sphingolipid Metabolism 71

Figure 6.—Sphingoid base measurement in deletion strains supports a role for Svf1p in the Lcb3p and Lcb4p pathway. (A)
Overnight cultures of WT and svf1D cells were grown to midlog phase at 20°. SPH, or an equal volume of ethanol as a control, was
added to a final concentration of 30 mm. Samples were collected after 1 hr and sphingoid base levels were determined by HPLC.
The average picomoles/A600 of the indicated sphingoid base 6 standard deviation of duplicate samples is indicated for each
strain. (B) Overnight cultures of WT, svf1D, sur2D, lcb3D, and lcb4D were normalized to an OD600 of 0.08 and incubated at
20°. Samples were collected in midlog phase and sphingoid base levels were determined by HPLC. The average picomoles/
A600 of the indicated sphingoid base 6 standard deviation of four independent samples is indicated. (C) Overnight cultures
of WT, svf1D, lcb3D, lcb3Dsvf1D, lcb4D, and lcb4Dsvf1D were normalized to an OD600 of 0.08 and incubated at 20°. Samples were
collected in midlog phase and sphingoid base levels were determined by HPLC. The average picomoles/A600 of the indicated
sphingoid base 6 standard deviation of duplicate samples is indicated.

mammalian cells, excessive PHSP in yeast can be toxic control. In the absence of DPL1 expression (FOA-
(Kim et al. 2000; Zhang et al. 2001). That this lethality is containing plates), lcb3Ddpl1D cells were unable to grow,
due to accumulation of phosphorylated bases is further while, although growth was slow, lcb3Ddpl1Dsvf1D cells
supported by the observation that viability can be could form colonies (Figure 7B). This suggests that re-
restored by deletion of the kinase LCB4 (Kim et al. duced generation of a subset of sphingoid bases de-
2000; Zhang et al. 2001). We hypothesized that if loss of pendent on Svf1p can partially suppress lethality of the
LCB4 restores viability by preventing excessive genera- dpl1Dlcb3D strain.
tion of a discrete subset of phosphorylated sphingoid It has been observed that the ability to obtain viable
bases dependent on Svf1p, then loss of SVF1 might lcb3Ddpl1D mutants depends on the auxotrophic mark-
exhibit a similar phenotype. Triple knockouts were gen- ers of the background strain. While prototrophic dou-
erated through mating of lcb3Dsvf1D with dpl1D. While ble mutants are viable (Skrzypek et al. 1999; Birchwood
in the control crosses of lcb3D and dpl1D, lcb3Ddpl1D et al. 2001), double knockouts generated in strains auxo-
cells were not recovered (data not shown), we were able trophic for some amino acids are lethal (Kim et al. 2000;
to consistently obtain triple lcb3Ddpl1Dsvf1D cells (Fig- Zhang et al. 2001). This may be a consequence of the fact
ure 7A). The triple knockout cells demonstrate a slow that sphingolipids play a role in nutrient uptake from
growth phenotype, but were recovered at the expected the media (Chung et al. 2000, 2001; Hearn et al. 2003).
ratios. To confirm this lethal interaction, diploid strains Since we are using the HIS3 gene to replace the SVF1
were transformed with a vector containing DPL1. Upon gene, we wanted to confirm that the isolation of viable
tetrad dissection, viable colonies expressing DPL1 were lcb3Ddpl1Dsvf1D cells was due to loss of SVF1, not to ad-
obtained. Cells were grown in the absence of selection dition of the HIS3 gene. Therefore, we generated a strain
to allow for plasmid loss and then plated onto counter- in which the SVF1 gene was replaced with the kanamycin
selection plates containing FOA or selection media as a resistance gene. Using this strain, we were still able to
72 J. L. Brace et al.

loss and then plated onto counterselection plates

containing FOA or selection media as a control. While
wild-type cells could form colonies in the absence of
DPL1 (FOA-containing plates), lcb3Ddpl1Dsur2D cells
were unable to grow in the absence of DPL1 (Figure
7D). These data further indicate that Svf1p and Sur2p
have distinct functions and support the hypothesis that
Svf1p regulates the generation of a subset of sphingoid
bases that controls survival.
Loss of YPK1 mimics the phenotypes observed in the
svf1Dsur2D strain and demonstrates a synthetic in-
teraction with SVF1 and SUR2: We have suggested that
SVF1 and SUR2 regulate the generation of separate sub-
sets of sphingoid bases; however, this does not explain
the growth defect seen in cells lacking these genes. It has
been shown that PHS can activate the Pkh1/2p pathway
(Friant et al. 2001). Since svf1Dsur2D cells are deficient
in PHS, these cells may not properly activate this path-
Figure 7.—Loss of SVF1, but not SUR2, suppresses the le-
thality of lcb3Ddpl1D. (A) Diploid lcb3D/1dpl1D/1svf1D/1
way. We therefore considered whether inadequate
cells were dissected onto YPD plates and incubated at 30°. activation of downstream kinases in this pathway might
A representative dissection plate with phenotypes (K, kanamy- contribute to the phenotypes observed in the svf1D
cin resistance; H, histidine prototrophic; W, wild type) is sur2D strain. A key downstream component of this sig-
shown. The triple knockout is shaded. (B) Diploid lcb3D/1 nal transduction pathway is the kinase Ypk1p.
dpl1D/1svf1D/1 cells were transformed with a vector ex-
pressing DPL1 and dissected onto YPD plates at 30°.
Similar to svf1Dsur2D cells, ypk1D cells exhibit a slow
lcb3Ddpl1Dsvf1D (1), lcb3Ddpl1D (2), and WT (3) cells express- growth phenotype (Chen et al. 1993; Roelants et al.
ing DPL1 were patched to YPD twice to allow for plasmid loss. 2002; Figure 8A). Since svf1D and svf1Dsur2D cells are
Viable cells in the absence of DPL1 grew on counterselection resistant to growth on SPH, we examined the growth of
media containing FOA. Cells were plated to uracil as a con- ypk1D cells on 30 mm SPH. Interestingly, similarly to
trol. Knockouts were confirmed by PCR. (C) Diploid lcb3D/1
dpl1D/1sur2D/1 cells were dissected onto YPD plates and in-
svf1Dsur2D, not only are ypk1D cells highly resistant to
cubated at 30°. A representative dissection plate with pheno- what is typically a toxic dose of SPH, but also SPH
types (K, kanamycin resistance; H, histidine prototrophic;W, markedly improves the growth of these cells (Figure
wild type) is shown. The expected triple knockout is shaded. 8B). These data suggest that YPK1 functions in the same
(D) Diploid lcb3D/1dpl1D/1sur2D/1 cells were transformed pathway as SVF1 and SUR2.
with a vector expressing DPL1 and dissected onto YPD plates
at 30°. WT (5, 7) and lcb3Ddpl1Dsur2D (4, 6) cells expressing
To further understand the epigenetic relationship
DPL1 were patched to YPD twice to allow for plasmid loss. Vi- among YPK1, SVF1, and SUR2, we attempted to generate
able cells in the absence of DPL1 grew on counterselection double knockouts among these mutants. YPK1-null cells
media containing FOA. Cells were plated to uracil as a con- were mated to svf1D or sur2D cells. Upon tetrad dis-
trol. Knockouts were confirmed by PCR. section, we were able to obtain small colonies corre-
sponding to ypk1D; however, we were unable to recover
any svf1Dypk1D or sur2Dypk1D cells. To confirm this
isolate lcb3Ddpl1Dsvf1D triple knockouts (data not lethal interaction, diploid svf1D/1ypk1D/1 or sur2D/
shown), demonstrating that the loss of SVF1 and not 1ypk1D/1 strains were transformed with a vector con-
the incorporation of HIS3, is responsible for suppressing taining YPK1. Upon tetrad dissection, viable colonies
the lethality of lcb3Ddpl1D cells. expressing YPK1 were obtained for each genotype. Cells
It has been suggested that PHS, not DHS, is the were grown in the absence of selection to allow for plas-
‘‘active’’ sphingoid base in the cell and is responsible for mid loss and then plated onto counterselection plates
growth inhibition (Chung et al. 2001; Friant et al. containing FOA or selection media as a control. While
2001). To determine if the reduced levels of PHS in the wild-type, svf1D, sur2D, and ypk1D cells could form
SVF1-null strain is responsible for suppressing lethality colonies on FOA-containing plates, svf1Dypk1D and
of the lcb3Ddpl1D double knockout, we attempted to sur2Dypk1D cells were unable to grow in the absence of
generate a triple knockout with the enzyme responsible YPK1 (Figure 8C). This confirms genetically the inter-
for the generation of PHS, SUR2. In this case, we were action between the generation of sphingoid bases and
unable to recover any triple knockouts (Figure 7C). the activation of the YPK1 pathway and supports the
Again, diploid strains were transformed with a vector hypothesis that SVF1 and SUR2 regulate the generation
containing DPL1. Upon tetrad dissection, we obtained of different subsets of sphingoid bases that affect the
viable lcb3Ddpl1Dsur2D cells expressing DPL1. Cells were activity of YPK1-dependent signaling pathways. This
grown in the absence of selection to allow for plasmid synthetic interaction also illustrates the importance of
 SVF1 Affects Sphingolipid Metabolism 73

growth and survival with relevance to mammalian sys-

tems. We previously demonstrated that the yeast survival
factor Svf1p plays a role in survival responses and is
required for survival in the presence of reactive oxygen
species (Vander Heiden et al. 2002; Brace et al. 2005).
Here, we demonstrate a role for Svf1p in the sphingoid
base pathway.
Exogenously added sphingoid bases require sequen-
tial phosphorylation and dephosphorylation for the cell
to utilize these bases in the generation of downstream
ceramide metabolites, and it is believed that these
reactions serve to properly localize the sphingoid base
to allow efficient action by downstream enzymes (Qie
et al. 1997; Mao et al. 1997, 1999; Mandala et al. 1998;
Funato et al. 2003). We hypothesize that these coupled
phosphorylation/dephosphorylation reactions are re-
quired for the localized generation of a subset of PHS
and PHSP regulated by Svf1p. We demonstrate through
genetic manipulation and sphingoid base measurement
that Svf1p functions to regulate the generation of C18-
PHS and C18-PHSP and may do so through control of
the concerted action of Lcb4p and Lcb3p. The suppres-
sion of the lethal dpl1Dlcb3D strain through additional
loss of SVF1 suggests that preventing flow through this
pathway can partially restore viability to a strain that
accumulates this subset. The observation that loss of
SUR2 does not suppress this lethality demonstrates that
Svf1p and Sur2p have distinct functions and further
supports the model that Svf1p regulates the generation
Figure 8.—Cells lacking YPK1 exhibit phenotypes similar of a subset of sphingoid bases distinct from Sur2p.
to those of svf1Dsur2D. (A) Overnight cultures of WT, svf1D, One possible function of Svf1p, and of the concerted
and ypk1D cells were normalized to an OD600 of 0.08 and
grown at 20°. At the indicated time points, samples were re-
action of Lcb3p and Lcb4p, may be to generate a
moved to measure OD600. Mean OD600 6 standard deviation localized pool of sphingoid bases to activate targets,
of three independent cultures for each strain is shown. (B) including the Ypk1p pathway. Cells lacking SUR2 are
WT, svf1D, ypk1D, and svf1Dsur2D cells were normalized and unable to generate PHS (Figure 6B; Haak et al. 1997),
10-fold serial dilutions were spotted onto plates containing yet they do not exhibit a growth defect and are able
ethanol (control) or 30 mm SPH. Plates were incubated at
20° for 6 days. (C) WT, ypk1D, svf1D, svf1Dypk1D, and
to generate complex sphingolipids with DHS as the
sur2Dypk1D expressing YPK1 were patched to YPD twice to al- sphingoid base backbone (Haak et al. 1997). In sur2D
low for plasmid loss. Viable cells in the absence of YPK1 grew cells, Svf1p, Lcb3p, and Lcb4p may generate a localized
on counterselection media containing FOA. Cells were struck pool of DHS that activates the Ypk1/2p pathway. DHS
to selection media as a control. can stimulate Ypk1 activity, albeit with less efficiency
than PHS (Liu et al. 2005a). However, loss of Sur2p
in combination with the inability to localize sphingoid
YPK1 activation and sphingoid base generation for bases through the additional loss of Svf1p, Lcb3p, or
growth and viability. Lcb4p may significantly reduce Ypk1p activation leading
to growth arrest similar to that observed when YPK1 is
deleted. Supporting this model, we do not observe
DISCUSSION
growth defects in cells lacking Sur2p in combination
Cellular growth and survival are highly regulated pro- with Lcb5p or Dpl1p, proteins not thought to be part of
cesses that become disrupted in human diseases, in- this coupling reaction (Figure 4).
cluding cancer. Understanding the function of proteins These synthetic interactions with SUR2 support a
and pathways regulating growth and survival may pro- model in which PHS generated by Sur2p and the
vide insight into the process of oncogenesis. The localized generation of PHS by Svf1p, Lcb3p, and
genetic manipulability of the yeast S. cerevisiae and the Lcb4p converge on activation of a pathway required
conservation of survival pathways and enzymes between for growth. Genetic evidence suggests that Svf1p and
yeast and mammalian cells supports the use of S. cere- Sur2p influence activity of Ypk1p or a downstream
visiae as a model in which to study the processes of pathway regulated by Ypk1p. We have demonstrated
74 J. L. Brace et al.

that YPK1-null cells exhibit a similar phenotype to that nents regulated by Ypk1p has not been defined, and
of sur2Dsvf1D, suggesting that lack of Ypk1p activity con- other cellular processes may be regulated that influence
tributes to the phenotype of these cells. Alternatively, survival. Genetic suppression of phenotypes observed in
Svf1p and Sur2p may regulate the homologous kinase ypk1D or svf1Dsur2D strains may provide insight into
Ypk2p. YPK2 demonstrates a synthetic lethality with these processes.
YPK1 (Chen et al. 1993), and SVF1 and SUR2 also exhibit Although Svf1p affects PHS levels, Svf1p lacks se-
lethality in combination with YPK1. These genetic inter- quence homology to domains conserved in hydroxy-
actions affecting viability are specific to YPK1: double lases, and it is unlikely that Svf1p generates PHS
knockouts between SVF1 and YPK2 or the upstream enzymatically. One possibility is that Svf1p may regulate
kinases PKH1/2 are viable and exhibit no growth defect the hydrolysis of ceramide or complex sphingolipids in
(data not shown). Studies are ongoing to further under- the membrane, resulting in a localized production of
stand the relationship between the Svf1p/Sur2p and the PHS. Svf1p contains a predicted transmembrane do-
Ypk1/2 pathways. main by EMBOSS (Rice et al. 2000) and may reside in
High levels of SPH are toxic to wild-type cells, but the cellular membranes. Sphingoid bases, generated in the
mechanism of cell death is not clear. The resistance to cytosol, might be recognized by the cell in the same way
SPH seen in svf1D and the reversal of SPH toxicity seen as exogenous addition. Evidence from mammalian cells
in both sur2Dsvf1D and ypk1D strains suggest that cell suggests that SPH generated de novo and via hydrolysis of
viability is dependent on a common pathway regulated complex sphingolipids plays distinct roles in respond-
by Sur2p/Svf1p and Ypk1p. In vitro, it has been dem- ing to cellular stress (Levade and Jaffrezou 1999;
onstrated that Ypk1p can be activated by SPH and that Sawai and Hannun 1999), and recent data suggest that
SPH is more potent than PHS at inducing Ypk1p activity a similar response exists in yeast (Cowart et al. 2006). In
(Liu et al. 2005a). In addition, it has been shown that fact, the hydrolysis pathway in yeast appears to play a
constitutive overexpression of the catalytically active ki- more important role in regulation of carbon source
nase domain of Ypk1p is growth inhibitory (Roelants utilization and yeast reproduction. This is interesting,
et al. 2002). These data support a model in which given that svf1D cells exhibit a defect in the diauxic shift.
overactivation of Ypk1p by SPH leads to growth arrest. Cellular localization of second messengers is impor-
One hypothesis is that SPH requires phosphorylation/ tant for proper activation of signaling pathways. Gener-
dephosphorylation prior to activation of targets in the ation of PIP3 on mammalian cell membranes recruits
cell, and lack of SVF1 prevents this activation. This phos- kinases such as Akt to the membrane, where it can be
phorylation/dephosphorylation step may be required acted on by its upstream kinase, PDK1. Localization is an
to properly localize SPH promoting Ypk1p activation. important component regulating specificity in activa-
Specific localization of SPH and other sphingolipids by tion of signaling cascades. A similar situation may occur
Svf1p may be required to recruit kinases, including in yeast, where generation of PHS in defined intracel-
Ypk1p, to specific cellular locations. Therefore, in the lular locations could activate kinases in a specific man-
absence of Svf1p, SPH may be unable to activate targets ner and result in different outcomes on the basis of the
that induce growth arrest. We demonstrate here that location of the signal and the surrounding proteins. It
addition of SPH to wild-type cells reduces the intracel- has been demonstrated that phosphorylation and local-
lular level of C18-PHS, but has the opposite effect in ization of Ypk1p is regulated by sphingoid bases, where
svf1D cells. Adaptation of svf1D cells to survival with addition of PHS recruits Ypk1p to the plasma membrane
lower C18-PHS may contribute to the SPH resistance ob- (Sun et al. 2000). A regulated subset of PHS dependent
served in this strain. on Svf1p may influence Ypk1p localization, resulting in
We present a model in which SVF1 regulates the lo- differential responses to stress. Further characterization
calized generation of a pool of PHS. Previously observed of these pathways may shed light on the signaling pro-
phenotypes of svf1D suggest that a function of Svf1p may perties of sphingoid bases in both mammalian and yeast
be to allow cells to adapt to rapid changes in environ- cells.
mental conditions, including the diauxic shift, temper- We thank S. J. Kron and J. D. Boeke for yeast strains. C.M.R. is sup-
ature downshift, and oxidative stress (Vander Heiden ported by the Flight Attendant Medical Research Institute and by the
et al. 2002; Brace et al. 2005). The hypothesized subset Burroughs Wellcome Fund. Work in R.C.D.’s laboratory was supported
of PHS dependent on Svf1p may activate specific sig- by a grant (GM41302) from the National Institutes of Health and by
a grant (P20-RR020171) from the National Center for Research
naling pathways, including the Ypk1p pathway, that
Resources.
protect cells from these stresses. It has been suggested
that Ypk1p plays a role in the cell wall integrity (CWI)
pathway that is implicated in maintenance of viability
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