Previous Article | Next Article 
Journal of Bacteriology, July 2000, p. 3655-3660, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Elo1p-Dependent Carboxy-Terminal Elongation of
C14:1
9 to C16:111 Fatty Acids in
Saccharomyces cerevisiae
Roger
Schneiter,*
Verena
Tatzer,
Gabriela
Gogg,
Erich
Leitner, and
Sepp
Dieter
Kohlwein
Institut für Biochemie und
Lebensmittelchemie, Technische Universität Graz, A-8010 Graz,
Austria
Received 20 January 2000/Accepted 18 April 2000
 |
ABSTRACT |
Saccharomyces cerevisiae medium-chain acyl elongase
(ELO1) mutants have previously been isolated in screens for
fatty acid synthetase (FAS) mutants that fail to grow on myristic acid
(C14:0)-supplemented media. Here we report that wild-type cells
cultivated in myristoleic acid (C14:1
9)-supplemented
media synthesized a novel unsaturated fatty acid that was identified as
C16:111 fatty acid by gas chromatography-mass
spectroscopy. Synthesis of C16:111 was dependent on a
functional ELO1 gene, indicating that Elo1p catalyzes
carboxy-terminal elongation of unsaturated fatty acids (
-elongation). In wild-type cells, the C16:111
elongation product accounted for approximately 12% of the total fatty
acids. This increased to 18% in cells that lacked a functional acyl
chain desaturase (ole1 mutants) and hence were fully
dependent on uptake and elongation of C14:1. The observation that
ole1 mutant cells grew almost like wild type on medium
supplemented with C14:1 indicated that uptake and elongation of
unsaturated fatty acids were efficient. Interestingly,
wild-type cells supplemented with either C14:1 or C16:1 fatty acids
displayed dramatic alterations in their phospholipid composition,
suggesting that the availability of acyl chains is a dominant
determinant of the phospholipid class composition of cellular
membranes. In particular, the relative content of the two major
phospholipid classes, phosphatidylethanolamine and phosphatidylcholine,
was strongly dependent on the chain length of the supplemented fatty
acid. Moreover, analysis of the acyl chain composition of individual
phospholipid classes in cells supplemented with C14:1 revealed that the
relative degree of acyl chain saturation characteristic for each
phospholipid class appeared to be conserved, despite the gross
alteration in the cellular acyl chain pool. Comparison of the
distribution of fatty acids that were taken up and elongated
(C16:111) to those that were endogenously synthesized by
fatty acid synthetase and then desaturated by Ole1p
(C16:19) in individual phospholipid classes finally
suggested the presence of two different pools of diacylglycerol
species. These results will be discussed in terms of biosynthesis of
different phospholipid classes via either the de novo or the Kennedy pathway.
| |
INTRODUCTION |
The fatty acid composition of
phospholipids is an important determinant of the biophysical properties
of cellular membranes and as such must be subject to dynamic exchange
to ensure membrane homeostasis under varying environmental conditions
(10, 15). The biochemical control of fatty acid composition
of different lipid classes, i.e., the regulation of the synthesis of
defined lipid molecular species, however, is not well understood.
Saccharomyces cerevisiae appears to be particularly amenable
to study of this process, as both lipid and fatty acid biosynthesis are
well characterized, mutants blocked in most steps of the lipid
biosynthetic pathways are readily available (6, 12, 21), and
the acyl chain composition of the major lipid species has recently been
determined by mass spectroscopy (25).
Long-chain saturated fatty acids of 16 to 18 carbon atoms are the
product of de novo synthesis by the fatty acid synthetase (FAS)
(26, 35). These are desaturated between the carbon atoms in
positions 9 and 10 by the single essential desaturase, Ole1p, to
generate cis-monounsaturated C16:1 and C18:1 long-chain
fatty acids (29, 30). Unsaturated fatty acids typically
comprise 70 to 80% of the total fatty acids of this organism
(9). Their relative composition, however, may vary
considerably, depending on the strain background (13) and
the carbon source (33). Very-long-chain C24 and C26 fatty
acids, on the other hand, are synthesized by a
membrane-bound elongase of which two components, Elo2p and Elo3p,
have recently been characterized (14, 24). Even though
very-long-chain fatty acids comprise only ~1% of the cellular fatty
acid pool (36), their synthesis is essential as they form
part of the yeast ceramide, found in sphingolipids and in the lipid
moiety of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored membrane proteins (12).
A third elongase, Elo1p, has been identified in a screen for
fas mutant cells that do not grow on myristic
acid-supplemented media. elo1 mutant cells hence fail to
elongate saturated fatty acids of 14 carbon atoms to long-chain fatty
acids of 16 to 18 carbon atoms (17, 32). The substrate of
Elo1p, however, is only poorly defined, and the mechanism of elongation
has not yet been characterized. In the present study, we thus addressed
the question whether Elo1p elongates unsaturated fatty acids by
carboxy-terminal elongation (-elongation), as known from the
microsomal elongation systems of mammals and plants, which catalyze the
synthesis of polyunsaturated fatty acids (5, 8).
The fatty acid supplementation experiments performed in the course of
this work revealed important new information about the regulation of
the synthesis of lipid molecular species in yeast. Wild-type cells
supplemented with either C14:19 or C16:19
fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a
dominant determinant of the phospholipid class composition of cellular
membranes. The degree of acyl chain saturation characteristic for the
different lipid classes (25, 34), however, appeared to be
conserved, despite the gross alteration of the cellular acyl chain pool
brought about by the fatty acid supplementation. These results will be
discussed in terms of biosynthesis of different phospholipid
classes via the de novo or the Kennedy pathway. In the de novo
pathway, decarboxylation of phosphatidylserine (PtdSer) yields phosphatidylethanolamine (PtdEtn), which is further
methylated to phosphatidylcholine (PtdCho), whereas, in the
Kennedy pathway, PtdEtn and PtdCho can both be synthesized from
diacylglycerol (DAG) and either CDP-ethanolamine or CDP-choline
(12, 21).
| |
MATERIALS AND METHODS |
Yeast strains and culture media.
The wild-type S. cerevisiae strain used was W303a (MATa ade2-1
his3-11,15 leu2-3, 112 trp1-1 ura3-1). The
elo1 ole1 double mutant strain
(MATa elo1::HIS3
ole1::LEU2 ade2 his3 leu2 ura3) was obtained
from crossing elo1 (MAT
elo1::HIS3 ade2-1 can1-100
his3-11,15 leu2-2,112 ura3-1) with
ole1 (MATa ole1::LEU2 ade2 his3 leu2-2 ura3), both kindly provided by C. Martin (Rutgers University, Piscataway, N.J.). Standard yeast genetics methods were
used for mating, sporulation, and construction of the double mutant
strain (27). Cells were grown either in complete (yeast extract-peptone-dextrose) medium containing 1% yeast extract (Difco), 1% Bacto Peptone (Difco), and 2% glucose or in minimal (synthetic dextrose) medium containing 0.67% yeast nitrogen base without amino
acids (Difco) and 2% glucose. Media supplemented with fatty acids
contained 1% Brij 58 and 0.5 mM (either) C14:1 or C16:1 fatty acids
(Sigma). For growth rate determination, optical density at 600 nm was monitored.
Lipid and fatty acid analysis.
Cells were harvested in the
late exponential growth phase, washed two times with 0.2% bovine serum
albumin, and used immediately or stored at 
70°C before extraction
of lipids. Lipids were extracted from cell homogenates by the procedure
of Folch et al. (18). Phospholipid classes were separated by
two-dimensional thin-layer chromatography on silica gel 60 plates
(Merck, Darmstadt, Germany), using chloroform-methanol-25% ammonia
(65:35:5 [vol/vol/vol]) as the first and
chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5
[vol/vol/vol/vol/vol]) as the second developing solvent. Spots
detected after exposure to iodine vapor were scraped off, and lipids
were extracted with chloroform-methanol (1:4 [vol/vol]). Total and
individual phospholipids were quantified as described by Broekhuyse
(4).
Fatty acids were converted to methyl esters by
BF3-catalyzed methanolysis (23) and separated by
gas-liquid chromatography (GLC) using a Hewlett-Packard HP 6890 Series
GC, equipped with an HP Innovax column (15 m by 0.25 mm by 0.50 µm in
film thickness), with a temperature gradient (20 min at 200°C,
10°C/min to 280°C, and 15 min at 280°C). Fatty acids were
identified by comparison to commercially available methyl ester
standards (NuCheck, Inc., Elysian, Minn.).
The double bond position of monounsaturated fatty acids was determined
by GLC-mass spectrometry (GLC-MS) of dimethyl disulfide
adducts of
these derivatives as described previously (
37), on
an HP
5890 Series II Plus GC equipped with electronic pressure
control, the
HP Chemstation software package, and an HP 5972 mass
selective
detector. Injector and interface were kept at 250 and
300°C,
respectively. GLC-MS analysis was performed on a capillary
column,
HP-5MS, 30 m by 0.25 mm by 0.25 µm in film thickness,
programmed
from 150 to 320°C at 20°C/min after a 2-min hold at
150°C.
Finally, the column was kept at 320°C for 10 min. All analyses
were
carried out in the constant-flow mode. Helium was used as
carrier gas
with a linear velocity of 34.1 cm/s. Aliquots of 1
µl of the
samples were injected with an HP 7673 autosampler in
splitless mode.
Electron impact ionization with 70-eV ionization
energy was used for
mass spectroscopy. Data were collected by
scanning from 150 to 600 atomic mass units at 1.6 scans/s.
| |
RESULTS AND DISCUSSION |
Growth of ole1 mutant cells on C14:1-supplemented
media requires a functional elongase.
In our analysis of the fatty
acid requirements of an ole1 mutant strain, we observed
that cells lacking the desaturase displayed significant growth on
C14:1-supplemented media (data not shown). Since pathways for the
modification of supplemented C14:1 fatty acid have not yet been
described, growth of ole1 mutant cells on C14:1 would
indicate that these cells survived by incorporating C14:1 as sole
unsaturated fatty acid in their membrane lipids, a possibility that we
considered unlikely. We thus investigated whether the exogenously
supplemented C14:1 was metabolically converted to a longer-chain
unsaturated fatty acid that, when esterified to lipid, permitted growth
of an ole1 mutant strain on myristoleic acid.
ELO1 has previously been shown to be required for the
elongation of saturated medium-chain fatty acids of 14 carbon atoms
in
length to saturated long-chain fatty acids of 16 to 18 carbon
atoms
(
17,
32). We thus investigated whether Elo1p is required
for
ole1 mutant cells to grow on myristoleic
acid-supplemented
media. To address this question, an
elo1 ole1 double mutant
strain was
generated and tested for growth on media supplemented
with various
unsaturated fatty acids. In contrast to the two parental
strains, the
elo1 ole1 double mutant did not grow on
C14:1-supplemented
media, suggesting that in an
ole1
mutant strain the supplemented
C14:1 fatty acid is elongated to
longer-chain unsaturated fatty
acids and that this elongation is
dependent on Elo1p. On C16:1-supplemented
media, on the other hand,
Elo1p function appeared to be dispensable
for growth of the
ole1 mutant strain, as indicated by the vigorous
growth
of the
elo1 ole1 double mutant strain on
palmitoleic
acid-supplemented media (data not
shown).
Identification of an Elo1p-dependent elongation product of
unsaturated fatty acids in wild-type cells.
To determine the
chemical nature and relative abundance of the predicted Elo1p-dependent
product of medium-chain unsaturated fatty acid elongation, wild-type
and ole1 and elo1 mutant strains were
cultivated in C14:1-supplemented media and their fatty acid profiles
were analyzed. As shown in Fig. 1, a
novel peak, eluting in the range of C16 fatty acid methyl esters, was
detected in wild-type and ole1 mutant cells. This peak
was absent from the fatty acid profile of the elo1
mutant, indicating that synthesis of this compound was directly or
indirectly dependent on Elo1p. In wild-type cells, this elongation
product comprised 12.3% of all the fatty acids. In ole1
mutant cells, uptake and elongation of C14:1 were only slightly
increased, with the elongation product accounting for 18.1% of the
total fatty acids (Table 1). Under these
conditions, elo1 mutant cells had approximately
4.5-fold-more unsaturated than saturated fatty acids and a roughly
equal ratio of acyl chains with 16 carbon atoms to those with 18 carbon
atoms. In comparison, the ole1 mutant strain displayed a
ratio of unsaturated to saturated fatty acids that was intermediate
between those observed in wild-type cells and those observed for
elo1 mutant cells. The ratio of C16 to C18 fatty acids,
however, was grossly perturbed in the desaturase mutant (Table 1).
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 1.
Fatty acid profiles of wild-type and elo1
and ole1 mutant cells cultivated in
C14:19-supplemented media. Wild-type and
ole1 and elo1 mutant cells were cultivated
in C14:19-supplemented minimal media to late exponential
growth phase, cells were harvested, lipids were extracted, and fatty
acid methyl esters were analyzed by gas chromatography as described in
Materials and Methods. Positions of saturated and unsaturated fatty
acids in the chromatograms are indicated. C16:1?
indicates the ELO1-dependent product.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Comparison of fatty acid composition of total lipids from
wild-type and ole1 and elo1 mutant cells
cultivated in C14:19- and
C16:19-supplemented mediaa
|
|
Comparison of growth rates of wild-type and
elo1 and
ole1 mutant cells in media supplemented with either C14:1
or C16:1
indicated that elongation of C14:1 was not growth limiting for
ole1 mutant cells, suggesting that uptake and elongation
of the
supplemented C14:1 are rapid and efficient.
ole1
mutant cells
required 207 min for doubling in C14:1-supplemented media,
compared
to 198 min in C16:1-supplemented media (data not
shown).
Elo1p is required for carboxy-terminal elongation of unsaturated
fatty acids.
The position of the double bond in the elongation
product was determined by GLC-MS analysis of fatty acid dimethyl
disulfide adducts, as previously described (37).
Fragmentation of C16:1? into two main products of
m/z 117 and 245 allowed unequivocal identification of the
Elo1p-dependent products as being a C16:111 fatty acid,
indicating that Elo1p is required for -elongation of
C14:19 to C16:111 (Fig.
2). Furthermore, fragmentation of the
Elo1p-dependent product was identical to that of a chemically
synthesized C16:111 fatty acid. In contrast,
GLC-MS analysis of an authentic C16:19 fatty acid methyl
ester yielded major products at m/z 217 and 145, consistent
with the predicted fragmentation properties of C16:19
fatty acid (data not shown).
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 2.
Characterization of the double bond position present in
the ELO1-dependent product of C14:19,
C16:111. Dimethyl disulfide adducts of fatty acid methyl
esters were prepared and analyzed as described in Materials and
Methods.
|
|
To determine the substrate specificity of the elongation process that
is operating on unsaturated fatty acids, wild-type and
ole1 and
elo1 mutant cells were cultivated
in C16:1
9-supplemented media and their fatty acid
profiles were analyzed.
A novel peak in the C18:1 region of the
chromatogram was apparent
in lipid extracts from wild-type cells. This
peak was not detected
in lipid extracts from
elo1 mutant
cells, suggesting that its
synthesis depends on the presence of a
functional elongase. Determination
of the double bond position in this
elongation product by GLC-MS
analysis of fatty acid dimethyl disulfide
adducts allowed unequivocal
identification of the product as
C18:1
11 (data not shown). As shown in Table
1,
elongation efficiency
of C16:1 in vivo is approximately 12-fold lower
than that of C14:1.
Only 1% of C18:1
11 was detected in
wild-type cells grown in the presence of C16:1
9,
compared to 12.3% of C16:1
11 that was synthesized by
the elongation of C14:1 (Table
1). The
notion that C16:1 is a poorer
substrate for Elo1p-dependent elongation
is also apparent from the fact
that only 0.5% of C18:1
13 was detected in cells
supplemented with C14:1, in which case
elongation of
C16:1
11 by a second cycle generates a
C18:1
13 fatty
acid.
The fatty acid profile of cells supplemented with C16:1
9
(Table
1) furthermore revealed that all three strains appeared to
maintain a ratio of unsaturated to saturated fatty acids of between
2 and 3. The ratio of C16 to C18 fatty acids, however, was grossly
disturbed in the two mutants. Under these conditions, the
endogenous
synthesis of C18:1
9 appeared to be strongly
repressed in the
elo1 mutant but not
in the wild type,
suggesting that Ole1p activity is tightly downregulated
by
C16:1
9 (
3), but not by
C14:1
9, and that this repression is somewhat relieved by
the presence
of functional
Elo1p.
Effects of altered fatty acid profile on the phospholipid
composition.
Next, we wished to determine whether an
altered fatty acid composition, generated by cultivation of the
yeast strains in the presence of 9-unsaturated fatty
acids, affected the phospholipid composition of these cells. For this
purpose, phospholipids of wild-type and ole1 and
elo1 mutant cells cultivated in either
C14:19- or C16:19-supplemented media were
analyzed. As shown in Table 2,
phospholipid compositions of wild-type and mutant cells deviated
greatly from each other and were strongly dependent on the chain length
of the unsaturated fatty acid supplied. The most notable observations were as follows. (i) In wild-type cells, the relative content of
the two major phospholipid classes, PtdEtn and PtdCho, was strongly dependent on the type of unsaturated fatty acid supplemented. While PtdEtn was the most abundant phospholipid (74.5%) in wild-type cells supplemented with C14:1, PtdEtn levels were reduced to 38.9% when cells were supplemented with C16:1. Concomitantly, PtdCho increased from 17.9% in C14:1-supplemented cells to 48.7% in cells supplemented with C16:1. This tendency toward an increased content of
PtdEtn at the expense of PtdCho in cells supplemented with C14:1 was
also observed for the ole1 mutant. (ii) Furthermore, in
ole1 mutant cells grown in C14:1-supplemented media, the
relative content of phosphatidic acid (PtdOH) was greatly
elevated (~10-fold compared to the wild type), suggesting that the
unfavorable fatty acid composition of these cells results in
increased steady-state levels of PtdOH, possibly as a result of an
altered phospholipid turnover. (iii) In all three strains, the relative
content of PtdIns was lower in C14:1-supplemented cells than in
C16:1-supplemented cells. This chain-length-dependent decrease of
PtdIns was most pronounced (eight-fold) in the elo1
mutant. (iv) PtdSer levels appeared to be least affected by chain
length alterations or mutations in ELO1 and OLE1.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Comparison of the phospholipid composition of wild-type
and ole1 and elo1 mutant cells cultivated
in C14:19- and
C16:19-supplemented mediaa
|
|
On one hand, this analysis illustrates the flexibility of the cellular
phospholipid composition; on the other hand, it demonstrates
that the
phospholipid composition is greatly affected by the availability
of
acyl chains. These data suggest, in fact, that acyl chain supply
is a
dominant determinant of the phospholipid composition of a
cellular
membrane, suggesting that the lipid head group composition
is more
modulatory. This notion is supported by biophysical data
showing that
the introduction of a single double bond decreases
the melting
temperature of dipalmitoyl-PtdCho by 46°C. Head group
exchange, on
the other hand, appears to affect the phase behavior
in a degree that
is more comparable to that exerted by a two-carbon
increase in acyl
chain length (
11). While lipid phase properties
are
certainly not the sole parameters in vivo, additional variables
such as
charge, size, and polarity of the lipid head group; the
intrinsic
curvature of a lipid species; and even superlattice
formation may all
significantly affect the head group composition
of cellular lipid
bilayers (
2,
28,
31).
Analysis of the distribution of the elongation product in
individual phospholipid classes.
Having analyzed the dependency of
the phospholipid class composition on the acyl chain length of the
supplemented unsaturated fatty acid, we tested whether incorporation of
the C16:111 elongation product displayed a preference
for certain phospholipid classes. Therefore, the fatty acid composition
of individual phospholipid classes, isolated from wild-type and
ole1 and elo1 mutant cells cultivated in
media supplemented with C14:19, was determined. As shown
in Table 3, this analysis revealed that,
in ole1 mutant cells, the C16:111
elongation product was incorporated in all of the phospholipid classes,
with the highest relative content in PtdCho (18.8%) and PtdEtn
(15.8%), compared to PtdSer (8.7%) and PdtIns (6.1%). This enrichment of unsaturated C16:111 in order from
PtdIns and PtdSer via PtdEtn to PtdCho parallels the general increase in the content of unsaturated acyl chains from
PtdIns via PtdSer and PtdEtn to PtdCho observed
both in C14:1-supplemented cells and in wild-type cells grown in rich
medium (25, 34), an observation which suggests that the
relative degree of acyl chain saturation of the different phospholipid
classes is maintained even under conditions where the endogenous acyl
coenzyme A pool is grossly altered.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Fatty acid composition of individual phospholipid classes
from wild-type and ole1 and elo1 mutant
cells cultivated in
C14:19-supplemented mediaa
|
|
The ratio of C16:1
9 to C16:1
11 in the
different phospholipid classes, however, varied markedly. In
wild-type cells, PtdSer
and PtdEtn contained
approximately fourfold-more C16:1
9 than
C16:1
11. In PtdCho and PtdIns, on the other
hand, the ratio of C16:1
9 to C16:1
11 was
close to 1. This apparent acyl chain preference of different
phospholipid classes is particularly noteworthy in light of the
fact
that both PtdSer and PtdIns are synthesized from a
common
precursor, CDP-DAG. The observation that PtdEtn has a
C16:1
9-to-C16:1
11 ratio similar to that
of PtdSer suggests that the bulk of PtdEtn
is synthesized by
decarboxylation of PtdSer (i.e., the de novo
pathway). In
contrast, the similarity in the
C16:1
9-to-C16:1
11 ratio of
PtdCho and PtdIns suggests that, in this case, the bulk
of
PtdCho is synthesized through the Kennedy (CDP-choline) pathway,
possibly by reutilizing molecular species of DAG that are produced
from
the turnover of PtdIns. Turnover of PtdIns is
required for
the maturation of sphingolipids, in which the synthesis of
inositolphosphorylceramide
from ceramide and that of
mannosyl-diinositolphosphorylceramide
from
mannosylinositol-phosphorylceramide each consumes
PtdIns
and yields DAG (
1). Interestingly, a recent
study of the molecular
species of PtdCho synthesized in hepatocytes
either through the
de novo methylation pathway or through
the Kennedy pathway revealed
that different molecular species of
PtdCho are produced by the
two pathways, with saturated species
predominating in the Kennedy
pathway and arachidonic-acid-containing
species predominating
in the methylation pathway (
16).
Remarkably, these two classes
of PtdCho species appear to be
functionally distinct, since overexpression
of the PtdEtn
N-methyltransferase-2 in conditional mutants of
the
CDP-choline pathway does not rescue the temperature-sensitive
growth
defect of these cells (
19). A functional distinction
of
PtdCho synthesized by the de novo pathway from that formed
by the
Kennedy pathway is also evident in yeast, where attenuation
of PtdCho
synthesis via the Kennedy pathway, but not via the methylation
pathway,
can rescue the temperature-sensitive growth phenotype
of
sec14 mutants (
7,
20,
22).
Interestingly, the supplemented C14:1 was not detected in
PtdSer species of wild-type and
elo1 mutant
cells but was incorporated
to 14.6% in PtdSer of
ole1 cells (Table
3). This apparent exclusion
of C14:1
from PtdSer in desaturation-competent cells is remarkable
and
may reflect the preference and/or restricted accessibility
of
PtdSer synthase for/to certain molecular species of CDP-DAG
(
12). In
ole1 cells, on the other hand, this
species preference
appears to have been overcome, possibly due to an
acute requirement
of the cell to increase the degree of lipid
unsaturation (Table
1).
Conclusions.
Taken together, the present study
provides evidence that the yeast ELO1 gene is
required for carboxy-terminal elongation of unsaturated fatty acids.
Since the two very- long-chain elongases, Elo2p and Elo3p, are closely
related to Elo1p, with more than 52% identity and 72% homology
(24), it appears reasonable to suggest that these operate by
a similar mechanism. The second important observation made in this
study is that the exogenous addition of unsaturated fatty acids results
in a dramatic alteration in the phospholipid composition, suggesting
that acyl chain supply is a dominant parameter of the lipid
composition. Altering the cellular acyl chain pool, however, appears
not to affect the degree of acyl chain saturation characteristic of
each phospholipid class (25, 34). The third and possibly
most interesting observation is that the
C16:19-to-C16:111 ratio of
individual phospholipid classes may be used to trace the biosynthetic
origin of the corresponding lipid species. Experiments to validate this
approach utilizing mutants in the de novo pathway and those defective
in the Kennedy pathway are currently being performed.
| |
ACKNOWLEDGMENTS |
We thank G. Daum for liberal support and valuable comments on the
manuscript, C. Martin for generously providing the elo1 and ole1 mutant strains used throughout this study, and
D. Ribitsch for synthesizing the C16:111 fatty acid
employed as a standard for the GLC-MS analysis.
This work was supported by grants from the Fonds zur Förderung
der wissenschaftlichen Forschung in Österreich P11731 (to S.D.K.)
and M00304 and 13767 (to R.S.) and the Swiss National Science
Foundation (823A-046702 to R.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry, Technical University Graz, Petersgasse 12, A-8010 Graz, Austria. Phone: 43-316-873-6955. Fax: 43-316-873-6952. E-mail: f548roge{at}mbox.tu-graz.ac.at.
| |
REFERENCES |
| 1.
|
Becker, G. W., and R. L. Lester.
1980.
Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae.
J. Bacteriol.
142:747-754[Abstract/Free Full Text].
|
| 2.
|
Bloom, M.,
E. Evans, and O. G. Mouritsen.
1991.
Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective.
Q. Rev. Biophys.
24:293-397[Medline].
|
| 3.
|
Bossie, M. A., and C. E. Martin.
1989.
Nutritional regulation of yeast delta-9 fatty acid desaturase activity.
J. Bacteriol.
171:6409-6413[Abstract/Free Full Text].
|
| 4.
|
Broekhuyse, R. M.
1968.
Phospholipids in tissues of the eye. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl-ether phospholipids.
Biochim. Biophys. Acta
260:449-459.
|
| 5.
|
Browse, J., and C. Somerville.
1991.
Glycerolipid synthesis: biochemistry and regulation.
Annu. Rev. Plant Physiol. Plant Mol. Biol.
42:467-506[CrossRef].
|
| 6.
|
Carman, G. M., and S. A. Henry.
1999.
Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes.
Prog. Lipid Res.
38:361-399[CrossRef][Medline].
|
| 7.
|
Cleves, A. E.,
T. P. McGee,
E. A. Whitters,
K. M. Champion,
J. R. Aitken,
W. Dowhan,
M. Goebl, and V. A. Bankaitis.
1991.
Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein.
Cell
64:789-800[CrossRef][Medline].
|
| 8.
|
Cook, H. W.
1996.
Fatty acid desaturation and chain elongation in eukaryotes.
New Compr. Biochem.
31:129-152.
|
| 9.
|
Cottrell, M.,
B. C. Viljoen,
J. L. F. Kock, and P. M. Lategan.
1986.
The long-chain fatty acid compositions of species representing the genera Saccharomyces, Schanniomyces and Lipomyces.
J. Gen. Microbiol.
132:2401-2403.
|
| 10.
|
Cullis, P. R., and B. de Kruijff.
1979.
Lipid polymorphism and the functional roles of lipids in biological membranes.
Biochim. Biophys. Acta
559:399-420[Medline].
|
| 11.
|
Cullis, P. R.,
D. B. Fenske, and M. J. Hope.
1996.
Physical properties and functional roles of lipids in membranes.
New Compr. Biochem.
31:1-33.
|
| 12.
|
Daum, G.,
N. D. Lees,
M. Bard, and R. Dickson.
1998.
Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae.
Yeast
14:1471-1510[CrossRef][Medline].
|
| 13.
|
Daum, G.,
G. Tuller,
T. Nemec,
C. Hrastnik,
G. Balliano,
L. Cattel,
P. Milla,
F. Rocco,
A. Conzelmann,
C. Vionnet,
D. E. Kelly,
S. Kelly,
E. Schweizer,
H. J. Schuller,
U. Hojad,
E. Greiner, and K. Finger.
1999.
Systematic analysis of yeast strains with possible defects in lipid metabolism.
Yeast
15:601-614[CrossRef][Medline].
|
| 14.
|
David, D.,
S. Sundarababu, and J. E. Gerst.
1998.
Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast.
J. Cell Biol.
143:1167-1182[Abstract/Free Full Text].
|
| 15.
|
de Kruijff, B.
1997.
Lipid polymorphism and biomembrane function.
Curr. Opin. Chem. Biol.
1:564-569[CrossRef][Medline].
|
| 16.
|
DeLong, C. J.,
Y. J. Shen,
M. J. Thomas, and Z. Cui.
1999.
Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.
J. Biol. Chem.
274:29683-29688[Abstract/Free Full Text].
|
| 17.
|
Dittrich, F.,
D. Zajonc,
K. Hühne,
U. Hoja,
A. Ekici,
E. Greiner,
H. Klein,
J. Hofmann,
J. J. Bessoule,
P. Sperling, and E. Schweizer.
1998.
Fatty acid elongation in yeast biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.
Eur. J. Biochem.
252:477-485[Medline].
|
| 18.
|
Folch, J.,
M. Lees, and G. H. Sloane-Stanley.
1957.
A simple method for the isolation and purification of total lipids from animal tissues.
J. Biol. Chem.
226:497-509[Free Full Text].
|
| 19.
|
Houweling, M.,
Z. Cui, and D. E. Vance.
1995.
Expression of phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an impaired CDP-choline pathway in mutant Chinese hamster ovary cells.
J. Biol. Chem.
270:16277-16282[Abstract/Free Full Text].
|
| 20.
|
Kearns, B. G.,
J. G. Alb, and V. A. Bankaitis.
1998.
Phosphatidylinositol transfer proteins: the long and winding road to physiological function.
Trends Cell Biol.
8:276-282[CrossRef][Medline].
|
| 21.
|
Kent, C.
1995.
Eukaryotic phospholipid biosynthesis.
Annu. Rev. Biochem.
64:315-343[CrossRef][Medline].
|
| 22.
|
Kent, C., and G. M. Carman.
1999.
Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus.
Trends Biochem. Sci.
24:146-150[CrossRef][Medline].
|
| 23.
|
Morrison, W. R., and L. M. Smith.
1964.
Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.
J. Lipid Res.
5:600-608[Abstract].
|
| 24.
|
Oh, C. S.,
D. A. Toke,
S. Mandala, and C. E. Martin.
1997.
ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation.
J. Biol. Chem.
272:17376-17384[Abstract/Free Full Text].
|
| 25.
|
Schneiter, R.,
B. Brugger,
R. Sandhoff,
G. Zellnig,
A. Leber,
M. Lampl,
K. Athenstaedt,
C. Hrastnik,
S. Eder,
G. Daum,
F. Paltauf,
F. T. Wieland, and S. D. Kohlwein.
1999.
Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.
J. Cell Biol.
146:741-754[Abstract/Free Full Text].
|
| 26.
|
Schweizer, E.
1984.
Genetics of fatty acid biosynthesis in yeast.
New Compr. Biochem.
7:59-83.
|
| 27.
|
Sherman, F.,
G. R. Fink, and J. B. N. Hicks.
1986.
Methods in yeast genetics: a laboratory course manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 28.
|
Somerharju, P.,
J. A. Virtanen, and K. H. Cheng.
1999.
Lateral organisation of membrane lipids. The superlattice view.
Biochim. Biophys. Acta
1440:32-48[Medline].
|
| 29.
|
Stukey, J. E.,
V. M. McDonough, and C. E. Martin.
1989.
Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae.
J. Biol. Chem.
264:16537-16544[Abstract/Free Full Text].
|
| 30.
|
Stukey, J. E.,
V. M. McDonough, and C. E. Martin.
1990.
The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene.
J. Biol. Chem.
265:20144-20149[Abstract/Free Full Text].
|
| 31.
|
Tate, M. W.,
E. F. Eikenberry,
D. C. Turner,
E. Shyamsunder, and S. M. Gruner.
1991.
Nonbilayer phase of membrane lipids.
Chem. Phys. Lipids
57:147-164[CrossRef][Medline].
|
| 32.
|
Toke, D. A., and C. E. Martin.
1996.
Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae.
J. Biol. Chem.
271:18413-18422[Abstract/Free Full Text].
|
| 33.
|
Tuller, G.,
T. Nemec,
C. Hrastnik, and G. Daum.
1999.
Lipid composition of subcellular membranes of an FY1679-derived haploid yeast wild-type strain grown on different carbon sources.
Yeast
15:1555-1564[CrossRef][Medline].
|
| 34.
|
Wagner, S., and F. Paltauf.
1994.
Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions.
Yeast
10:1429-1437[CrossRef][Medline].
|
| 35.
|
Wakil, S. J.
1989.
Fatty acid synthase, a proficient multifunctional enzyme.
Biochemistry
28:4523-4530[CrossRef][Medline].
|
| 36.
|
Welch, J. W., and A. L. Burlingame.
1973.
Very long-chain fatty acids in yeast.
J. Bacteriol.
115:464-466[Abstract/Free Full Text].
|
| 37.
|
Yamamoto, K.,
A. Shibahara,
T. Nakayama, and G. Kajimoto.
1991.
Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts.
Chem. Phys. Lipids
60:39-50[CrossRef].
|
Journal of Bacteriology, July 2000, p. 3655-3660, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ejsing, C. S., Sampaio, J. L., Surendranath, V., Duchoslav, E., Ekroos, K., Klemm, R. W., Simons, K., Shevchenko, A.
(2009). From the Cover: Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl. Acad. Sci. USA
106: 2136-2141
[Abstract]
[Full Text]
-
Agbaga, M.-P., Brush, R. S., Mandal, M. N. A., Henry, K., Elliott, M. H., Anderson, R. E.
(2008). Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids. Proc. Natl. Acad. Sci. USA
105: 12843-12848
[Abstract]
[Full Text]
-
Serra, M., Pina, B., Abad, J. L., Camps, F., Fabrias, G.
(2007). A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone. Proc. Natl. Acad. Sci. USA
104: 16444-16449
[Abstract]
[Full Text]
-
Paul, S., Gable, K., Beaudoin, F., Cahoon, E., Jaworski, J., Napier, J. A., Dunn, T. M.
(2006). Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA Synthase Gene Family Substitute for the Elop Proteins of Saccharomyces cerevisiae. J. Biol. Chem.
281: 9018-9029
[Abstract]
[Full Text]
-
Ren, G., Vajjhala, P., Lee, J. S., Winsor, B., Munn, A. L.
(2006). The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy. Microbiol. Mol. Biol. Rev.
70: 37-120
[Abstract]
[Full Text]
-
Boumann, H. A., Gubbens, J., Koorengevel, M. C., Oh, C.-S., Martin, C. E., Heck, A. J.R., Patton-Vogt, J., Henry, S. A., de Kruijff, B., de Kroon, A. I.P.M.
(2006). Depletion of Phosphatidylcholine in Yeast Induces Shortening and Increased Saturation of the Lipid Acyl Chains: Evidence for Regulation of Intrinsic Membrane Curvature in a Eukaryote. Mol. Biol. Cell
17: 1006-1017
[Abstract]
[Full Text]
-
Gaigg, B., Timischl, B., Corbino, L., Schneiter, R.
(2005). Synthesis of Sphingolipids with Very Long Chain Fatty Acids but Not Ergosterol Is Required for Routing of Newly Synthesized Plasma Membrane ATPase to the Cell Surface of Yeast. J. Biol. Chem.
280: 22515-22522
[Abstract]
[Full Text]
-
Han, G., Gable, K., Kohlwein, S. D., Beaudoin, F., Napier, J. A., Dunn, T. M.
(2002). The Saccharomyces cerevisiae YBR159w Gene Encodes the 3-Ketoreductase of the Microsomal Fatty Acid Elongase. J. Biol. Chem.
277: 35440-35449
[Abstract]
[Full Text]
-
Kohlwein, S. D., Eder, S., Oh, C.-S., Martin, C. E., Gable, K., Bacikova, D., Dunn, T.
(2001). Tsc13p Is Required for Fatty Acid Elongation and Localizes to a Novel Structure at the Nuclear-Vacuolar Interface in Saccharomyces cerevisiae. Mol. Cell. Biol.
21: 109-125
[Abstract]
[Full Text]
Top
Abstract
Introduction
