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
Yeast 15, 601–614 (1999)
Systematic Analysis of Yeast Strains with Possible
Defects in Lipid Metabolism
GU
} NTHER DAUM1*, GABRIELE TULLER1, TAMARA NEMEC1, CLAUDIA HRASTNIK1, GIANNI
BALLIANO2, LUIGI CATTEL2, PAOLA MILLA2, FLAVIO ROCCO2, ANDREAS CONZELMANN3,
CHRISTINE VIONNET3, DIANE E. KELLY4, STEVEN KELLY4, ECKHARD SCHWEIZER5,
HANS-JOACHIM SCHU
} LLER6, URSULA HOJAD5, EVA GREINER5 AND KARIN FINGER5
1
Institut für Biochemie und Lebensmittelchemie, Technische Universität, Graz, Austria
Dipartimento di Scienzia e Tecnologica del Farmaco, Torino, Italy
3
Institut de Biochemie, Fribourg, Switzerland
4
Institute of Biological Sciences, University of Wales Aberystwyth, Ceredigion, Wales, U.K.
5
Institut für Mikrobiologie und Biochemie, Lehrstuhl Biochemie, Erlangen, Germany
6
Institut für Genetik und Biochemie, Greifswald, Germany
2
Lipids are essential components of all living cells because they are obligate components of biological membranes,
and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid,
phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene
products have been functionally characterized. Less information is available about genes and gene products
governing the transport of lipids between organelles and within membranes or the turnover and degradation of
complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids
in organellar membranes, a group of five European laboratories established methods suitable to screen for novel
genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within
EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned
open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods
required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and
standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid
metabolism. During these investigations it was demonstrated that different wild-type strains, among them FY1679,
CEN.PK2-1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several
candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their
homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open
reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism.
Copyright 1999 John Wiley & Sons, Ltd.
  — yeast; Saccharomyces cerevisiae; phospholipids; sterols; sphingolipids; fatty acids
INTRODUCTION
The aim of a group of European laboratories
(EUROFAN Node Lipid Metabolism) was to
*Correspondence to: Dr Günther Daum, Institut für Biochemie
und Lebensmittelchemie, Technische Universität, Petersgasse
12/2, A-8010 Graz, Austria. Tel: +43-316-873-6462; fax: +43316-873-6952; e-mail: f548daum@mbox.tu-graz.ac.at.
Contract/grant sponsor: EUROFAN Programme of the EU;
Contract/grant number: BIO4-CT95-0080.
CCC 0749–503X/99/070601–14 $17.50
Copyright 1999 John Wiley & Sons, Ltd.
identify novel genes of the yeast Saccharomyces
cerevisiae that are involved in metabolism,
Contract/grant sponsor: Austrian Ministry of Science and
Transportation; Contract/grant number: 950080.
Contract/grant sponsor: Fonds zur Förderung der wissenschaftlichen Forschung in O
} sterreich; Contract/grant number:
12076.
Contract/grant sponsor: BBW (Switzerland); Contract/grant
number: 95.0191-3.
Received 19 June 1998
Accepted 22 November 1998
.   .
602
turnover, targeting and intracellular transport of
lipids as well as in the regulation of these processes. The specific problem of this project was
that a growth phenotype of mutant strains with
defects in lipid biosynthesis or lipid composition
cannot be generally predicted. Therefore, the
tedious and time-consuming lipid analysis of
total cell extracts or isolated subcellular fractions
had to be performed to obtain fundamental
information.
In the first phase of this project we established
and standardized the methodology for this screening. Tester strains with known defects were used to
demonstrate the reliability and the reproducibility
of the analytical methods. Auxotrophy tests
for lipids or lipid precursors, such as inositol,
fatty acids, phytosphingosine or ergosterol,
were largely omitted from this program because
exhaustive screens of this type had been carried
out in the past. Sensitivity against a number of
drugs and growth under various extreme conditions were tested but most of these tests failed
to correlate with defects in lipid metabolism in
a reliable way. Two exceptions were sensitivity
to fluconazole and Amphotericin B, which did
provide some indications for defects in sterol or
sphingolipid metabolism, respectively. Thus,
complete analysis of the lipid composition of
yeast strains, including qualitative and quantitative detection of fatty acids, phospholipids,
acylglycerols, sterols and sphingolipids by GLC,
HPLC and TLC methods, remained the only
feasible strategy to obtain information about
specific defects.
An extensive homology search carried out in
collaboration with MIPS (Martinsried Institute
for Protein Sequences, Martinsried, Germany)
revealed that the yeast genome contains approximately 170–200 open reading frames that are
proved, or can be assumed, to be involved in
lipid metabolism. Some of these candidate
open reading frames were selected randomly
and the corresponding deletion strains were
analysed according to the above mentioned
criteria. We are aware, however, that changes in
lipid composition and/or lipid metabolism may be
secondary effects caused by defects in other cellular processes. Thus, results obtained in this
primary screening should be interpreted with
caution and only be used as the basis for more
detailed investigations which may prove or
disprove a direct effect of the candidate genes on
lipid metabolism.
Copyright 1999 John Wiley & Sons, Ltd.
METHODS
Strains and culture conditions
All yeast strains (Saccharomyces cerevisiae) and
media used for these studies are listed in Tables 1
and 7. YJ3C 344 and YJ3C 366 were provided by
T. Dunn, Bethesda, MD, and HLY3 by S. D.
Kohlwein, Graz, Austria. Unless otherwise indicated, strains were grown on YPD medium (2%
glucose) to the late logarithmic phase. Minimal
medium (MM) with 0·1  inositol and without
choline was prepared as described by Klig et al.
(1985). Growth was followed by measuring the
OD at 600 nm. Plate tests were carried out on solid
YPD medium supplemented with additives as
shown in Figure 1 (see below). Deletion strains
used in this study were constructed by the method
described by Wach et al. (1994, 1996).
Lipid analysis
Lipids were extracted by the procedure of Folch
et al. (1957) from whole yeast cells after disintegration with glass beads on a Merckenschlager
homogenizer for 3 min with cooling. Individual
phospholipids were separated by two-dimensional
thin-layer chromatography (TLC) on Silica gel 60
plates (Merck, Darmstadt, Germany) using
chloroform/methanol/25% NH3 (65:35:5 per vol.)
as the first developing solvent, and chloroform/
acetone/methanol/acetic acid/water (50:20:10:10:5
per vol.) as the second. Phospholipids were visualized on TLC plates by staining with iodine vapour,
scraped off the plate, and quantified by the method
of Broekhuyse (1968).
For the analysis of neutral lipids, extracts were
applied to Silica gel 60 plates with the aid of
a sample applicator (Linomat IV; CAMAG,
Muttenz, Switzerland) and chromatograms were
developed by ascending TLC using as a solvent
system either light petroleum/diethyl ether/acetic
acid (20:20:0.8 per vol.) or light petroleum/diethyl
ether (39.2:0.8 v/v). Triacylglycerols were visualized by post-chromatographic staining using a
chromatogram immersion device (CAMAG,
Muttenz, Switzerland). Plates were dipped for 2 s
into a developing reagent consisting of 0·63 g
MnCl2 . 4 H2O, 60 ml water, 60 ml methanol and
4 ml conc. sulphuric acid, briefly dried and heated
to 100C for 30 min. Quantification of triacylglycerols was carried out by densitometric scanning at
400 nm with triolein (NuCheck, Inc., Elysian,
MN) as a standard.
Yeast 15, 601–614 (1999)
Tester strains.
Strain
Medium
ORF/gene deleted
CPR1-Ä1
YPD
JL20
YJ3C 344
YPD
YPD
YJ3C 366
IKY2
YPD
YPDa
YKL182w/FAS1
JS91.15-23
HLY3
YPDa
MMb
YGR157w/CHO2
W303-1A
MMb
FY1679
YPD
CEN.PK2-1C
YPD
a
Genotype
YHR042w/NCPR1 MATa leu2-3, 2-112 his4-519 ade1-100 ura3-52
ncpr1::LEU2
MATa leu2-3, 2-112 his4-519 ade1-100 ura3-52
YBR036c/CSG2
MATa ade2 ade3 his ura3 leu2 lys2 csg2::LEU2
MATa ade2 ade3 his ura3 leu2 lys2
MATá ura3-52 leu2-3,112 trp1-289 his3-Ä1
Äfas1::LEU2
MATá ura3-52 leu2-3,112 trp1-289 his3-Ä1
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1
trp1-1 cho2::LEU2
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1
trp1-1
MATa ura3-52 leu2Ä1 trp1Ä63 his3Ä200
MATa leu2-3,112 ura3-52 trp1-289 his3Ä1
MAL2-8c SUC2
Characterization
Electron donor for squalene epoxidase, sterol
C14-demethylase and sterol C22-desaturase
Wild-type to CPR1-Ä1
Protein required for synthesis of the mannosylated sphingolipids
Wild-type to YJ3C 344
Fatty acyl-CoA synthase, â-chain
Wild-type to IKY2
Phosphatidylethanolamine N-methyltransferase
      
Copyright 1999 John Wiley & Sons, Ltd.
Table 1.
Wild-type to HLY3
Wild-type strain used for yeast genome sequence
analysis
Wild-type strain used in the German Function
Analysis Network
Medium: YEPD plus 1% Tween 40 plus 0·05% palmitic acid.
Minimal medium with 0·1  inositol and without choline in the tests; YPD for both strains to grow and maintain.
b
603
Yeast 15, 601–614 (1999)
.   .
604
For quantification of ergosterol, samples and
standards were applied to TLC plates, chromatographed using light petroleum/diethyl ether/acetic
acid (70:30:2 per vol.) as a solvent, and scanned
at 275 nm using a Shimadzu dual-wavelength
chromato scanner CS-930. Individual sterols
were analysed after alkaline hydrolysis (Lewis
et al., 1987) of the lipid extract and silylation
with (trimethylsilyl)Trifluoride (BSTFA) by gas
chromatography–mass spectrometry (VG 12-250;
VG Biotech) using split injections with a split ratio
of 20:1. Sterols were identified by comparison of
retention times to authentic standards.
Sterol biosynthesis in whole yeast cells was
monitored by incorporation of [2-14C]acetate into
non-saponifiable lipids. Washed cells (10–20106
cells) suspended in 5 ml of 25 m Na + /K + phosphate buffer (pH 6·5) containing 1% glucose were
incubated with 2 ìCi of [2-14C]acetate (specific
activity, 50 mCi/m) for 2 h at 30C. Then, cells
were harvested by centrifugation and saponified in
50% ethanol containing 15% KOH for 2 h at 80C.
Non-saponifiable lipids were extracted twice with
light petroleum and separated on silica gel plates
(Merck) developed in n-hexane/ethyl acetate
(85:15; v/v) with authentic references of ergosterol, lanosterol, 2,3-22,23-dioxidosqualene, 2,3oxidosqualene and squalene. Ergosterol and more
polar compounds can be more easily separated by
developing plates in n-hexane/ethyl acetate (75:25,
v/v). Radioactivity in the respective bands was
quantified using a System 2000 Imaging Scanner
(Packard).
Fatty acids were analysed by gas chromatography. Lipids extracted as described above were
subjected to methanolysis using BF3/methanol and
converted to methyl esters (Morrison and Smith,
1964). Fatty acyl methyl esters were separated by
gas chromatography on a Hewlett-Packard 5890 A
gas chromatograph using a 25 m HP5 capillary
column with a temperature gradient (2 min at
150C, 10C/min to 300C, 5 min at 300C). Fatty
acids were identified by comparison to commercial
fatty acyl methyl ester standards (NuCheck, Inc.,
Elysian, MN).
To identify inositol-containing yeast sphingolipids cells were grown in minimal medium supplemented with 1% casein hydrolysate and labelled
with [3H]inositol. Cells were harvested at the logarithmic phase and incubated at 10 OD/ml in
0·25 ml of the same medium for 20 min at 30C.
Then 12 ìCi [3H]inositol were added and cells were
incubated for 40 min at 30C. After adding 0·75 ml
Copyright 1999 John Wiley & Sons, Ltd.
fresh medium the incubation was continued for
another 80 min at 30C. Then, 3 m NaN3 and
10 m NaF were added, cells were harvested by
centrifugation and washed twice with 1  sorbitol.
Chloroform/methanol (0·4 ml; 1:1 v/v) was added
and cells were disintegrated by vortexing with glass
beads. The supernatant was saved and the pellet
was re-extracted several times with chloroform/
methanol/water (10:10:3 per vol.). The combined
supernatants were dried and lipid extracts were
desalted routinely by partitioning between
n-butanol and an aqueous solution of 0·1 m
EDTA, 5 m Tris–HCl, pH 7·5, and backextraction of the butanol phase with water.
Desalted lipid extracts were used for TLC analysis
using chloroform/methanol/water (10:10:3 per
vol.) or chloroform/methanol/0·25% KCl (55:45:10
per vol.) as a solvent system. TLC plates were
sprayed with EN3HANCE (NEN, Boston, MA),
bands were detected by autoradiography and
quantified by densitometric scanning. Nomenclature of sphingolipids is as described previously
(Puoti et al., 1991).
All experimental data shown are mean values
from at least three independent experiments with a
deviation of 15%.
RESULTS AND DISCUSSION
Standardizing methods of lipid analysis by using
tester strains
In order to standardize and evaluate methods of
lipid analysis for the EUROFAN programme
yeast strains with known defects in lipid metabolism and the corresponding wild-types were used
as references (see Table 1). Here we summarize the
results of the analysis of phospholipids (Table 2),
sterols and triacylglycerols (Table 3), sphingolipids
(Table 4) and fatty acids (Table 5) of these strains.
As shown in Tables 2–5, tester strains exhibited the
expected lipid patterns as a result of the respective
metabolic defects. Apart from effects due to a
major defect in lipid metabolism, we often
observed additional alterations in cellular lipid
composition compensating for the primary
changes. Such data may help to identify crossconnections between lipid biosynthetic routes and
regulatory processes involved.
Thus, the cho2 strain, with a defect in the second
and third step of methylation of the de novo
biosynthetic pathway of phosphatidylcholine
(Summers et al., 1988), showed a marked decrease
Yeast 15, 601–614 (1999)
      
Table 2.
605
Phospholipid analysis of tester strains.
JL 20 (w.t)
CPR1-Ä1 (ncpr1)
YJ3C 366 (w.t)
YJ3C 344 (csg2)
JS91·15-23 (w.t)
IKY2 (fas1)
W303-1A (w.t)
HLY3 (cho2)
FY1679
CEN.PK2-1C
Percentage of total phospholipids
mg PL/g
CDW
PA
PtdSer
PtdEtn
PtdCho
PtdIns
CL
DMPtdEtn
LYSO-PL
LPtdEtn
31·5
29·8
28·0
29·2
30·2
33·1
26·4
26·6
31·5
31·4
4·0
6·7
2·7
2·5
7·4
7·5
1·9
7·7
2·0
2·2
5·0
3·3
6·9
6·4
3·3
3·7
5·5
3·1
9·2
5·4
24·5
20·4
21·0
21·7
13·6
15·0
21·0
42·3
20·4
19·0
40·6
47·8
41·4
43·9
45·9
46·3
46·2
14·2
40·0
49·4
18·9
16·2
21·6
17·2
23·1
20·8
18·9
28·4
19·1
16·7
2·1
2·2
1·9
3·1
3·1
1·7
3·1
3·1
2·9
2·9
2·3
2·8
3·3
4·3
2·5
3·5
2·7
n.d
4·0
2·3
2·3
0·6
1·1
0·7
1·1
1·3
0·7
0·4
1·0
0·5
n.d
n.d
0·1
0·2
n.d
0·2
0·2
0·7
1·0
0·1
CDW, cell dry weight; PL, phospholipid; PA, phosphatidic acid; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanolamine;
PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; CL, cardiolipin; DMPtdEtn, dimethyl phosphatidylethanolamine;
LYSO-PL, lysophospholipids; LPtdEtn, lysophosphatidylethanolamine. n.d=Not detected. w.t=Wild-type.
Table 3.
Sterol and triacylglycerol analysis of tester strains.
JL 20 (w.t)
CPR1-Ä1 (ncpr1)
YJ3C 366 (w.t)
YJ3C 344 (csg2)
JS91·15-23 (w.t)
IKY2 (fas1)
W303-1A (w.t)
HLY3 (cho2)
FY1679
CEN.PK2-1C
Ergosterol
(mg/g
CDW)
Erg-ester
(mg/g
CDW)
Ergosterol+
Erg-ester
(mg/g CDW)
Ergosterol
(% of total
sterol)
Lanosterol
(% of total
sterol)
Squalene
(% of total
sterol)
Triacylglycerols
(mg/g CDW)
5·7
3·6
4·7
3·7
6·0
5·1
4·1
3·5
6·0
4·0
9·1
1·2
7·8
6·7
8·7
4·5
14·1
8·9
2·6
16·0
14·9
4·8
12·5
10·4
14·8
9·6
18·2
12·4
8·5
20·0
76·5
69·1
94·1
88·7
73·8
82·4
85·4
89·0
75·3
78·9
6·2
6·5
3·9
7·5
17·5
11·7
9·7
7·3
17·0
14·3
16·8
24·4
2·0
3·8
8·7
5·9
4·9
3·7
7·7
6·8
4·7
8·1
3·9
3·7
10·0
1·8
7·8
9·3
2·4
15·2
of the cellular level of phosphatidylcholine (Table
2). This defect was accompanied by two-fold
increase of the level of phosphatidylethanolamine,
which is the precursor of phosphatidylcholine in
this pathway. Besides the increase of phosphatidylethanolamine, cho2 showed a four-fold increase
in phosphatidic acid. In contrast, the glycerophospholipid composition of the other tester strains
was only minimally altered.
Ncpr1p was originally identified as electron
donor for squalene epoxidase, sterol C14demethylase and sterol C22-desaturase (Sutter and
Loper, 1989). As a consequence, the sterol level of
an ncpr1 deletion strain is low (Table 3). Cellular
concentrations of free ergosterol and ergosteryl
esters are affected by the mutation. Similar to
Copyright 1999 John Wiley & Sons, Ltd.
other strains bearing defects in sterol metabolism,
ncpr1 accumulated the sterol precursor squalene at
the expense of ergosterol. It has to be noted,
however, that the corresponding wild-type
strain, JL20, has already an exceptionally high
level of squalene. In the ncpr1 strain, the cellular
level of triacylglycerols is almost twice as high
as in the corresponding wild-type. This increase
may be a consequence of a decreased level of
steryl esters thus avoiding accumulation of free
fatty acids.
A tester strain with a specific defect in triacylglycerol metabolism was not available. Genes and
gene products involved in this pathway of the
yeast Saccharomyces cerevisiae have not yet been
identified. Nevertheless, one strain used for this
Yeast 15, 601–614 (1999)
.   .
606
Table 4.
Sphingolipid analysis of tester strains.
cpm [3H]inositol
incorporated per aliquot
of cells (10 6)
PtdIns
Lyso-PtdIns
IPC/C
MIPC+IPC/D*
M(IP)2C
Others
2·6
4·4
2·6
2·1
4·0
2·3
2·8
3·2
2·5
6·8
70·3
72·0
75·3
81·0
67·8
83·2
74·5
77·3
63·3
70·0
0·3
0·5
1·3
0·4
0·3
0·3
0·6
0·2
0·5
0·6
16·4
11·6
7·0
11·8
13·4
5·2
7·3
10·3
21·4
12·5
5·4
4·4
8·4
6·5
9·3
3·6
7·1
4·9
5·4
8·0
7·2
11·3
7·8
0·3
9·0
7·6
10·4
7·1
3·7
8·8
0·4
0·2
0·2
0·1
0·2
0·2
0·2
0·2
0·7
0·2
JL 20 (w.t)
CPR1-Ä1 (ncpr1)
YJ3C 366 (w.t)
YJ3C 344 (csg2)
JS91.15-23 (w.t)
IKY2 (fas1)
W303-1A (w.t)
HLY3 (cho2)
FY1679
CEN.PK2-1C
Percentage of total label incorporated
PtdIns, phosphatidylinositol; Lyso-PtdIns, lysophosphatidylinositol; IPC, inositolphosphoceramide; MIPC, mannosylinositolphosphorylceramide; M(IP)2C, mannosyl(diinositolphosphoryl)ceramide. *Resolution of these two lipids is insufficient for
individual quantitation.
Table 5.
Fatty acid analysis of tester strains.
Percentage of total fatty acids
JL 20 (w.t)
CPR1-Ä1 (ncpr1)
YJ3C 366 (w.t)
YJ3C 344 (csg2)
JS91.15-23 (w.t)a
IKY2 (fas1)a
W303-1A (w.t)
HLY3 (cho2)
FY1679
CEN.PK2-1C
C 16:0
C 16:1
C 18:0
C 18:1
Odd number
F.A.
Unsat.:sat. FA
(Ratio)
12·0
9·1
6·3
6·0
2·6
0
6·1
5·4
20·8
4·5
42·7
62·5
38·3
37·5
15·3
0
24·6
48·7
45·1
40·0
5·3
2·3
3·6
4·7
2·7
0
5·8
5·7
4·8
2·7
40·0
26·1
51·8
51·8
27·5
0
63·5
40·2
27·1
52·0
n.d
n.d
n.d
n.d
24·3
100
n.d
n.d
n.d
n.d
4·8
7·8
9·1
8·3
N.D
N.D
7·4
8·0
2·8
12·8
a
Medium, YEPD plus 0·05% C 13:0 and C 15:0 fatty acid, each.
n.d, Not detected; N.D, not determined.
analysis, namely the fatty acid auxotroph fas1
deletion strain (Schüller et al., 1992) showed a
dramatically reduced level of triacylglycerols
(Table 3). This strain has to be supplemented with
exogenous fatty acids to maintain growth. As a
result, in fas1 not only the amount of triacylglycerols but also that of ergosteryl esters is decreased.
Since both triacylglycerols and steryl esters appear
to be dispensable for cellular growth, the mutant
may use the fatty acid resources mainly for the
synthesis of the ‘more important’ phospholipids.
It is noteworthy that some of the so-called
wild-type strains used in this study exhibit rather
Copyright 1999 John Wiley & Sons, Ltd.
different levels of sterols and triacylglycerols. As
an example, sterol levels of FY1679 are low as
compared to the other strains (see Table 3) but a
significant amount of the sterol precursor lanosterol accumulates. Triacylglycerol levels are also
very low in FY1679. As another example,
CEN.PK2-1C produces an excess of steryl esters
and triacylglycerols. This accumulation may be
caused by an imbalanced ‘overproduction’ of fatty
acids which are stored in the form of steryl esters
and triacylglycerols. Similar to FY1679, the
CEN.PK2-1C strain contains an elevated level of
lanosterol.
Yeast 15, 601–614 (1999)
      
Sphingolipid analysis was carried out by labelling cells with [3H]inositol, because all known yeast
sphingolipids contain inositol. Quantitative chemical analysis of yeast sphingolipids (Hechtberger
et al., 1994) is complicated and cannot be used as a
routine method. The labelling method used in this
study, however, confirmed that a strain with an
established defect in sphingolipid biosynthesis,
csg2 (Beeler et al., 1997), shows an abnormal
pattern of sphingolipids (Table 4). In this deletion
strain, the last product in the cascade of the yeast
sphingolipid biosynthetic pathway, mannosyl(diinositolphosphoryl) ceramide [M(IP)2C], is almost
missing. As a consequence, the precursor inositolphosphorylceramide (IPC) accumulated. Among
the other strains tested, fas1 exhibited a modified
sphingolipid pattern. Levels of all sphingolipids
were reduced in this mutant, especially those of
inositolphosphorylceramide (IPC) and mannosylinositolphosphorylceramide (MIPC). This may be
result of the limited availability of fatty acids that
are also required for the biosynthesis of sphingolipids. The ncpr1 mutant contained an enhanced
amount of M(IP)2C. The FY1679 strain has a
sphingolipid pattern different from other wildtypes. FY1679 showed a reduced incorporation
of [3H]inositol into M(IP)2C but accumulated label
in IPC.
Finally, fatty acids of tester strains were analysed (Table 5). The most dramatic effect was seen
with fas1. To overcome the fatty acid auxotrophy
of this strain odd numbered fatty acids were added
to the medium. This experimental trick was used to
distinguish between endogenously formed and
exogenously added fatty acids. As can be seen
from Table 5, fas1 relies completely on the fatty
acid supply from the medium because all fatty
acids detected in the lipid extract were oddnumbered. The corresponding wild-type, JS91.1523, which was also grown in the presence of
exogenous fatty acids, contained only 24·3% odd
numbered fatty acids. Less dramatic, but nevertheless significant, changes of the fatty acid composition were observed with strains that are not
primarily affected in fatty acid metabolism. The
ncpr1 and cho2 strains exhibited a significantly
altered fatty acid pattern as compared to the
corresponding wild-types. In both mutants, the
amount of C 18:1 was decreased, and the level of
C 16:1 was increased. Furthermore, the ratio of
unsaturated to saturated fatty acids was significantly increased in ncpr1. It is well known that
membrane properties can be modulated by an
Copyright 1999 John Wiley & Sons, Ltd.
607
altered fatty acid composition of complex lipids,
especially phospholipids. Thus, changes in the
fatty acid patterns of ncpr1 and cho2 may be a
consequence of disturbed sterol and phospholipid
metabolism, respectively.
In contrast to other wild-type strains, FY1679
has an unusual fatty acid composition (Table 5).
The amount of palmitic acid (C 16:0) in this strain
is much higher, and that of oleic acid (C 18:1)
much lower than in other wild-types. The ratio of
unsaturated to saturated fatty acids in FY1679 is
extremely low, namely 2·8, as compared to other
wild-type strains (ratio 4·8–12·8). CEN.PK2-1C,
as the other extreme, contains more than 90%
unsaturated fatty acids.
As mentioned above, designing of novel screens
for defects in yeast lipid metabolism is difficult,
since phenotypes can barely be predicted. Nevertheless, we measured growth of tester strains at
different temperatures, in the presence of 2 
NaCl, 0·2 ìg/ml cycloheximide, 0·25 ìg/ml calcofluor, or 0·5% Brij 58, respectively. Under all of
these conditions the tester strains with known
defects in lipid biosynthesis grew like their respective parental strains (data not shown). The only
exceptions were drug sensitivity tests using fluconazole, nystatin and Amphotericin B (Kerridge,
1986). Sensitivity against fluconazole and resistance to Amphotericin B correlated with defects in
the sterol biosynthetic pathway (Figure 1). It has
to be mentioned, however, that certain wild-type
strains, e.g. JS91.15-23 or CEN.PK2-1C, are less
sensitive to Amphotericin B than other wild-type
strains. Nystatin resistance was observed with the
ncpr1 strain as a consequence of the defect in the
sterol biosynthetic pathway. Slightly reduced sensitivity to nystatin was also found with csg2, which
lacks mannosylated sphingolipids (Figure 1). This
result is in line with the previous finding that a
mutant defective in the last step of yeast sphingolipid biosynthesis, the formation of M(IP)2C from
MIPC (Leber et al., 1997), was nystatin-resistant.
Altered sensitivity to nystatin is believed to be an
indicator of altered properties of the plasma membrane. Unfortunately, screening for nystatin resistance often yields ambiguous results, since the drug
is unstable. Thus, tests with nystatin were omitted
from further routine screenings.
Analysis of EUROFAN deletion strains
A homology search carried out in collaboration
with MIPS revealed that the yeast genome
Yeast 15, 601–614 (1999)
608
.   .
Figure 1. Drug resistance of yeast strains with established defects in lipid metabolism. Strains were spotted to plates containing
drugs at concentrations as indicated at dilutions of 1: 102:104:106 and incubated at 30C for 2 days.
contains approximately 170–200 open reading
frames with proven or putative roles in lipid
metabolism. Functional categories of genes
involved in phospholipid, fatty acid and sterol
metabolism, degradation and utilization of lipids,
regulation of lipid biosynthesis, transport and
binding of complex lipids and fatty acids,
â-oxidation of fatty acids and protein modification
by lipids were established. Several candidate
strains bearing a deletion of an open reading frame
hypothetically involved in yeast lipid metabolism
were constructed during the EUROFAN programme. Eight of these deletion mutants were
available during the early phase of EUROFAN I
and selected for detailed investigations (Table 6).
At this stage we had to decide which alterations
in the lipid composition should be considered
significant. As demonstrated by studies with tester
strains (see above) changes in the lipid composition are sometimes only secondary effects. The
underlying primary effects need not even be linked
to lipid metabolism, but may concern general
cellular metabolism, cell structure, organelle bioCopyright 1999 John Wiley & Sons, Ltd.
synthesis, etc. Thus, we considered changes of
50% of the control levels of major lipids as
a significant alteration. Since concentrations of
minor components, such as degradation products
and lipid intermediates, can vary to a large extent
for various reasons, they were not considered.
Among the eight deletion strains tested, only
one showed significant changes of the phospholipid composition (Table 7). The strain with a
deletion of YDL019c contained significantly more
cardiolipin than wild-type and other mutants. This
mutant also contained reduced amounts of ergosterol and ergosteryl esters (Table 8). The gene
product of YDL019c is homologous to oxysterolbinding proteins from other cell types and may
fulfil a similar function in yeast, thus affecting the
cellular level of sterols. Similar to the ncpr1 tester
strain (see Table 3), the YDL019c deletion strain
contains elevated amounts of triacylglycerols,
probably as a compensatory means of fatty acid
storage instead of forming steryl esters. Sterol
depletion and increase of the level of triacylglycerols was also observed with a strain deleted of
Yeast 15, 601–614 (1999)
EUROFAN deletion strains analysed for lipid composition.
Strain
ORF/gene deleted
Genotype
Characterization
FSCA009 (A)
YBR041w/FAT1
FDAM004 (A)
YDL019c
MATa ura3-52 LEU2 trp1Ä63 HIS3
YBR041w::KanMX4
MATa ura3-52 LEU2 trp1Ä63 HIS3
YDL019c::KanMX4
FDAM002 (A)
YDL109c
Similarity to M. musculus fatty acid
transport protein
Similarity to Osh1p; has
oxysterol-binding protein family
signature
Protein with lipase serine active site
FMZN002 (AL)
YGL144c
FDHN0023 (A)
YJL132w
FPIN040 (A)
YNL040w
FHBT011 (A)
YNL045w
FVKT004 (A)
YNR008w
MATa ura3-52 LEU2 TRP1 HIS3
YDL109c::KanMX4
MATá ura3-52 leu2Ä1 trp1Ä63
HIS3 YGL144c::KanMX4
MATa ura3-52 LEU2 TRP1
his3Ä200 YJL132w::KanMX4
MATa ura3-52 leu2Ä1TRP1 HIS3
YNL040c::KanMX4
MATa ura3-52 leu2Ä1TRP1
his3Ä200 YNL045w::KanMX4
MATa ura3-52 leu2Ä1 trp1Ä63
HIS3 YNR008w::KanMX4
Similarity to YDL109c; putative
protein with serine active lipase site
Protein with similarity to
phospholipase D
Has phospholipase A2 active site
signature
Strong similarity to human
leukotriene-A4 hydrolase
Protein with similarity to human
phosphatidylcholine-sterol
O-acyltransferase precursor
Constructed by
I. Sa-Correia, Portugal
G. Daum, Austria
G. Daum, Austria
M. J. Mazon, Spain
J. Dohmen, Germany
      
Copyright 1999 John Wiley & Sons, Ltd.
Table 6.
P. Philippsen, Switzerland
C. Herbert, France
G. Volckaert, Belgium
All strains were grown on YPD medium. The corresponding wild-type for all strains is FY1679.
609
Yeast 15, 601–614 (1999)
610
Copyright 1999 John Wiley & Sons, Ltd.
Table 7.
Phospholipid analysis of EUROFAN deletion strains.
Percentage of total phospholipids
Strain
FY1679
FSCA009 (A)
FDAM004 (A)
FDAM002 (A)
FMZN002 (AL)
FDHN0023 (A)
FPIN040 (A)
FHBT011 (A)
FVKT004 (A)
ORF
deleted
mg PL/g
CDW
PA
PtdSer
PtdEtn
PtdCho
PtdIns
CL
DMPtdEtn
LYSOPL
LPtdEtn
Others
YBR041w
YDL019c
YDL109c
YGL144c
YJL132w
YNL040w
YNL045w
YNR008w
31·5
34·5
22·5
25·0
28·8
28·3
30·0
30·8
30·0
2·0
0·8
1·3
0·8
1·3
1·0
2·0
1·4
1·7
9·2
8·3
5·8
8·0
7·9
8·5
7·0
5·4
7·3
20·4
21·0
22·0
22·6
24·4
21·4
23·8
21·3
23·5
40·0
42·3
45·8
46·1
41·8
44·7
42·9
44·4
45·4
19·1
20·0
14·5
17·4
14·9
16·7
14·5
21·0
14·4
2·9
2·2
5·1
4·2
2·5
3·3
2·0
2·7
2·0
4·0
4·9
2·5
2·3
5·3
2·9
5·8
3·3
4·9
1·0
0·6
n.d
0·5
1·8
1·2
0·9
n.d
0·3
1·0
n.d
n.d
n.d
n.d
0·3
0·1
n.d
n.d
0·2
n.d
1·2
1·0
0·2
0·1
0·8
0·6
0·4
n.d, Not detected. For abbreviations, see Table 2.
Table 8.
Sterol and triacylglycerol analysis of EUROFAN deletion strains.
Strain
Ergosterol
(mg/g CDW)
Erg-ester
(mg/g CDW)
Ergosterol+
Erg-ester
(mg/g CDW)
Ergosterol
(% of total
sterol)
Lanosterol
(% of total
sterol)
Squalene
(% of total
sterol)
Triacylglycerols
(mg/g CDW)
YBR041w
YDL019c
YDL109c
YGL144c
YJL132w
YNL040w
YNL045w
YNR008w
6·0
4·7
1·9
5·1
4·4
3·7
5·3
3·2
3·9
2·6
2·5
1·2
2·2
2·0
2·0
4·3
0·8
2·5
8·5
7·2
3·1
7·3
6·4
5·7
9·7
4·0
6·4
75·3
81·9
81·7
79·3
86·1
86·0
79·4
77·6
83·4
17·0
14·8
11·9
11·2
10·3
11·8
15·2
14·2
13·7
7·7
3·3
6·4
9·5
3·6
2·2
5·4
8·3
2·9
2·4
2·0
3·9
6·0
1·3
1·9
2·0
4·0
1·5
.   .
Yeast 15, 601–614 (1999)
FY1679
FSCA009 (A)
FDAM004 (A)
FDAM002 (A)
FMZN002 (AL)
FDHN0023 (A)
FPIN040 (A)
FHBT011 (A)
FVKT004 (A)
ORF
deleted
      
611
Table 9. Incorporation of [2-14C]acetate into non-saponifiable components of EUROFAN deletion strains.
Percentage of label incorporated into total sterols
Strain
FY1679
FSCA009 (A)
FDAM004 (A)
FDAM002 (A)
FMZN002 (AL)
FDHN0023 (A)
FPIN040 (A)
FHBT011 (A)
FVKT004 (A)
ORF deleted
Ergosterol
Lanosterol
Squalene
Others
YBR041w
YDL019c
YDL109c
YGL144c
YJL132w
YNL040w
YNL045w
YNR008w
64·1
64·8
61·2
72·7
71·3
67·9
73·3
70·4
72·0
21·3
18·9
23·1
14·2
13·7
19·9
18·6
13·5
12·7
18·3
16·3
14·8
13·1
15·1
12·0
8·0
16·6
14·0
<1·0
<1·0
<1·0
<1·0
<1·0
<1·0
<1·0
<1·0
<1·0
YNL045w (Table 8). The role of this yeast gene
product which has strong similarity to a human
leukotriene-A4 hydrolase remains obscure. Accumulation of triacylglycerols without an effect on
the sterol level was observed with the mutant
bearing a deletion of the YDL109c gene (Table 8).
This gene contains a sequence motif characteristic
for lipases. It would be most interesting to study
this gene and its product in more detail, because
triacylglycerol lipase(s) of the yeast have not yet
been identified at the molecular level. Two other
yeast genes possibly involved in triacylglycerol
metabolism, namely TGL1 (Abraham et al., 1992)
and TGL2 (van Heusden et al., 1998), were
recently detected. Both gene products, however,
could not be unambiguously identified as
triacylglycerol lipases.
So far, quantitative analysis of lipids was
described as a basis to identify possible defects in
lipid metabolism in yeast deletion strains. As an
alternative, labelling of yeast lipids with appropriate precursors can help to pinpoint certain defects.
This type of analysis not only documents the
amount of certain lipids formed but also reflects
their rate of biosynthesis. As an example, labelling
of yeast cells with [2-C14]acetate was used to obtain
more information about the biosynthesis of sterols
and related unsaponifiable products. As can be
seen from Table 9, however, none of the eight
deletion strains tested exhibited an abnormal
labelling pattern.
Labelling of yeast sphingolipids with [3H]inositol demonstrated again that deletion of YDL019c
affects lipid metabolism (Table 10). Besides other
effects described above, the level of M(IP)2C in
Copyright 1999 John Wiley & Sons, Ltd.
this strain is significantly enhanced. The cellular
concentrations of other sphingolipids, however,
appear to be normal. It is noteworthy that sterols
and sphingolipids are considered as partner lipids
in membrane formation. Both classes of lipids are
highly enriched in the plasma membrane, not only
in higher eukaryotes but also in yeast (Zinser and
Daum, 1995). Sterols and sphingolipids have been
reported to cooperate in forming membrane subdomains. Thus, it is possible that defects in sterol
biosynthesis and/or transport cause a compensatory overproduction of sphingolipids. Such an
effect was also observed with the ncpr1 tester
strain (see Table 4). Enhanced levels of
M(IP)2C were also found in strains deleted of
YGL144c (homology to a lipase) and YNR008w
(homology to a human phosphatidylcholine-sterol
O-acyltransferase). Neither effect can be explained
at present.
Fatty acid patterns of deletion strains tested in
this study are shown in Table 11. The fatty
acid composition of all mutant strains was not
significantly different from that of wild-type.
Screens for resistance to certain drugs has
yielded strains mutated in lipid biosynthesis, as
mentioned above and shown in Figure 1. Sensitivity of yeast strains to fluconazole and resistance to
Amphotericin B are caused by mutations in ergosterol biosynthesis and are thought to be a consequence of an altered permeability of the plasma
membrane. Among the mutants tested in this
study, only the strain deleted of YDL019c exhibited significant resistance against Amphotericin B
but showed resistance to fluconazole like wild-type
(Figure 2). It is noteworthy that deletion of
Yeast 15, 601–614 (1999)
.   .
612
Table 10.
Sphingolipid analysis of EUROFAN deletion strains.
ORF
PtdIns
Lyso-PtdIns
IPC/C
MIPC
+IPC/D
M(IP)2C
Others
YBR041w
YDL019c
YDL109c
YGL144c
YJL132w
YNL040w
YNL045w
YNR008w
6 700
23 800
17 800
20 700
23 100
20 300
25 400
17 100
20 000
63·3
65·5
58·2
68·6
60·2
65·9
64·5
67·1
58·8
0·5
0·9
1·8
0·9
1·0
1·0
0·9
1·2
1·3
21·4
24·0
21·2
19·2
23·8
22·4
23·5
20·9
22·5
5·4
4·8
7·1
5·6
5·2
5·8
4·5
5·3
6·7
3·7
3·3
10·1
4·1
8·2
3·6
5·2
4·2
9·6
0·7
0·5
0·8
0·7
0·6
0·5
0·6
0·4
0·5
Strain
FY1679
FSCA009 (A)
FDAM004 (A)
FDAM002 (A)
FMZN002 (AL)
FDHN0023 (A)
FPIN040 (A)
FHBT011 (A)
FVKT004 (A)
Percentage of total label incorporated
cpm [3H]inositol
incorporated per
aliquot of cells
For abbreviations, see Table 4.
Table 11.
Fatty acid analysis of EUROFAN deletion strains.
Percentage of total fatty acids
Strain
FY1679
FSCA009 (A)
FDAM004 (A)
FDAM002 (A)
FMZN002 (AL)
FDHN0023 (A)
FPIN040 (A)
FHBT011 (A)
FVKT004 (A)
ORF deleted
YBR041w
YDL019c
YDL109c
YGL144c
YJL132w
YNL040w
YNL045w
YNR008w
Cc14
2·2
2·4
1·6
1·2
2·2
1·3
1·8
<1·0
1·8
C 16:0
C 16:1
C 18:0
C 18:1
Other
F.A
20·8
21·0
20·4
18·7
19·8
21·2
19·3
19·1
19·9
45·1
45·8
46·3
47·0
47·6
45·1
48·7
45·9
46·7
4·8
4·2
4·7
4·2
4·6
4·3
3·4
5·7
3·8
27·1
26·6
27·0
28·9
25·8
28·1
26·8
29·3
27·8
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d, Not detected.
YDL019c causes abnormalities of the lipid pattern, as shown in Tables 7 and 8. Thus, a certain
correlation between nystatin resistance and lipid
composition of this mutant may exist. Further
experimental evidence will be required for a more
precise characterization of the defects causing
these properties.
Of course, the screening methods presented here
cannot explain metabolic defects or structural
abnormalities of yeast deletion strains at the
molecular level. Results of quantitative lipid analysis can only give us some hints on how to proceed
during more detailed investigations in the future.
The final goal of functional analysis will be to
explain cellular defects due to deletion of a certain
open reading frame. This is, however, a major
Copyright 1999 John Wiley & Sons, Ltd.
difficulty for studies of yeast lipids because of
multiple effects that may contribute to the overall
phenotype. Local disturbances of the membrane
lipid composition of certain organelles may be
more pronounced than alterations in total lipid
extracts. As a consequence, specific processes
bound to these subcellular compartments would be
affected. In such cases the more detailed analysis of
lipids in each organelle will be required to pinpoint
defects and to study the role of genes involved.
ACKNOWLEDGEMENTS
We would like to thank T. Dunn and S. D.
Kohlwein for providing yeast strains for our investigations; K. Albermann and his colleagues at
Yeast 15, 601–614 (1999)
      
Figure 2.
613
Drug sensitivity of EUROFAN deletion strains. For experimental details see Figure 1.
MIPS for their contribution to establish the functional categories of genes involved in lipid metabolism; and all EUROFAN members who sent us
strains prior to submission to EUROSCARF. This
study was financially supported by the following
grants: EUROFAN project BIO4-CT95-0080;
project 950080 of the Austrian Ministry of Science
and Transportation to G.D.; project 12076 of the
Fonds zur Förderung der wissenschaftlichen
Forschung in O
} sterreich to G.D.; Grant No.
95.0191-3 from the BBW (Switzerland) to A.C.
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