(Received for publication, March 3, 1997, and in revised form, May 6, 1997)
From the Bureau of Biological Research, Rutgers
University, Nelson Laboratories, Piscataway, New Jersey 08855-1059
and the ¶ Merck Research Laboratories, R80Y-230,
Rahway, New Jersey 07065
ELO2 and ELO3 were identified from the Saccharomyces cerevisiae genome data base as homologues of ELO1, a gene involved in the elongation of the fatty acid 14:0 to 16:0. Mutations in these genes have previously been shown to produce pleiotropic effects involving a number of membrane functions. The simultaneous disruption of ELO2 and ELO3 has also been shown to produce synthetic lethality, indicating that they have related and/or overlapping functions. Gas chromatography and gas chromatography/mass spectroscopy analyses reveal that null mutations of ELO2 and ELO3 produce defects in the formation of very long chain fatty acids. Analysis of the null mutants indicates that these genes encode components of the membrane-bound fatty acid elongation systems that produce the 26-carbon very long chain fatty acids that are precursors for ceramide and sphingolipids. Elo2p appears to be involved in the elongation of fatty acids up to 24 carbons. It appears to have the highest affinity for substrates with chain lengths less than 22 carbons. Elo3p apparently has a broader substrate specificity and is essential for the conversion of 24-carbon acids to 26-carbon species. Disruption of either gene reduces cellular sphingolipid levels and results in the accumulation of the long chain base, phytosphingosine. Null mutations in ELO3 result in accumulation of labeled precursors into inositol phosphoceramide, with little labeling in the more complex mannosylated sphingolipids, whereas disruption of ELO2 results in reduced levels of all sphingolipids.
In the yeast Saccharomyces cerevisiae, sphingolipids comprise approximately 10% of the total membrane lipid species (1). The hydrophobic moiety of these lipids is ceramide, which consists of a long chain base coupled to a very long chain fatty acid that is almost exclusively 26:01 or hydroxy 26:0 (2). Although sphingolipids are relatively minor membrane lipid species, they are highly concentrated on the plasma membrane and appear to be essential for a number of critical membrane and cellular functions (3-5). Inhibition of sphingolipid biosynthesis results in growth inhibition and cell death (6, 7). Ceramide has also been implicated as a component of an essential cell signaling pathways in Saccharomyces (8).
In wild type cells, most fatty acids are 12-18-carbon species that are found in glycerolipids. Those species appear to be formed de novo by the well characterized soluble cytoplasmic fatty acid synthase complex. The very long chain (20+ carbon) fatty acids found in sphingolipids, however, are formed by membrane-bound fatty acid elongation systems that are not well characterized. These enzyme systems extend 14-18-carbon fatty acids by 2-carbon units by a sequence of reactions similar to those catalyzed by fatty acid synthases, with the exception of one reduction step, which in mammalian cells appears to be mediated by cytochrome b5 (9).
We recently identified a gene (ELO1) that encodes a membrane protein involved in the elongation of 14:0 to 16:0 (10). Comparison of the amino acid sequence of that gene with the recently completed Saccharomyces genome data base revealed two additional genes with high identity to ELO1. These are referred to as ELO2 and ELO3 in this paper, based on their function in fatty acid elongation. Both genes have been previously identified as open reading frames of unknown function; mutations in these genes induce pleiotropic phenotypes that appear to play a key role in membrane and cytoskeletal functions (11).
ELO2 was initially cloned by complementation of mutants of
GNS1 (12). Those mutants confer resistance to echinocandins
and have defects in -glucan synthase activities. It was also
reported as FEN1 (11, 13), a gene whose mutants exhibited
bud localization defects and resistance to the sterol isomerase
inhibitor SR 31747. ELO3 was previously cloned and
identified, respectively, as APA1 with mutant alleles that
cause a decrease in the level of the plasma membrane ATPase (14),
SUR4 (15), whose mutants suppress the reduced viability on
starvation mutant phenotype (rvs161), and SRE1
(11) whose mutants suppress the effects of the steroid isomerase
inhibitor, SR 31747. At least two laboratories have reported that
simultaneous disruption of ELO2 and ELO3 produces a lethal phenotype (11, 13) which indicates that their encoded proteins
have related and overlapping functions. Similar studies in this
laboratory also support the synthetic lethality of the double
disruptions.
The characterization of the genes described in this paper suggests that they encode proteins required for the production of very long chain fatty acids. Each gene apparently encodes a single enzyme component of one or more systems that elongate C16 and C18 acids to C20-C26 very long chain fatty acids. Disruption of either gene causes either the reduction or loss of 26:0, the end product of the elongation pathway, with a concomitant reduction in ceramide synthesis and striking changes in sphingolipid composition.
The strains used in this study
and their genotypes are presented in Table I. Plasmids
constructed for this study are shown in Table II.
Standard yeast genetics methods were used for construction of strains
bearing the appropriate mutations (16). Cell growth conditions and
growth medium have been previously described (17). Escherichia
coli strain DH5 was obtained from Life Technologies, Inc.
Saccharomyces cerevisiae cells were cultured as described previously (10).
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Fatty acid analysis of long chain and very
long chain fatty acid methyl esters was performed by HCl-methanolysis
of whole cell lipids as described previously (10). Methyl esters were prepared from washed cells grown to a density of 2-3 × 107 ml in 50 ml of CM or CM (URA) medium containing
either 2% glucose or 2% galactose. Gas chromatography temperature
programming was modified to optimize analysis of very long chain fatty
acids on a 0.32 mm × 30 m SupelcoWaxTM10 column.
Two-ml cultures were grown to 2 × 106 cells/ml in CM medium at 30 °C and then labeled with either 20 µCi/ml [3H]serine for 6 h or 1 µCi/ml [3H]dihydrosphingosine (10 µM) for 30 min. Cultures were chilled on ice with an additional 0.5 ml of unlabeled stationary phase cells and centrifuged at 2800 × g for 10 min at 4 °C. Cells were washed 2 times with 5 ml of cold H2O and treated with 5% trichloroacetic acid at 4 °C for 20 min. Lipids were extracted twice in 1 ml of ethanol/water/diethyl ether/pyridine/NH4OH (15:15:5:1:0.018) at 60 °C as described (18). The [3H]serine-labeled extract was subjected to mild alkaline methanolysis by one treatment of 0.5 ml of monomethylamine reagent as prepared by Clarke and Dawson (19) for 30 min at 52 °C. The alkali-stable [3H]serine-labeled lipids were dried under N2, resuspended in 0.1 ml of chloroform/methanol/H2O (16:16:5), applied to Whatman Linear K6D silica gel TLC plates (25 µl/spot), and resolved in CHCl3/methanol/ 4.2 N NH4OH (9:7:2). The [3H]dihydrosphingosine-labeled total lipid extracts were dried under N2, resuspended in 0.2 µl, and subjected to TLC as described (7). Radioactive bands were quantified on a Molecular Dynamics PhosphorImager using a tritium screen and visualized by x-ray film after treatment with EN3HANCE (NEN Life Science Products).
Construction of the ELO2A
1.2-kb2 fragment containing the coding
sequence for ELO2 was derived by PCR using strain DTY10A
genomic DNA as a template and primers ELO2A and ELO2B in
Table III. The amplified product was ligated into the
pCRII cloning vector (Invitrogen), resulting in formation of pCRELO2.
This plasmid was digested with HpaI/MfeI to
remove most part of the ELO2 coding region. A 1.2-kb DNA
fragment of the Saccharomyces HIS3 gene was inserted into
blunt-ended sites of the vector. The resulting plasmid (pCRelo2HIS)
contains the HIS3 gene coding sequence in an orientation
opposite to that of ELO2. A 2.2-kb
elo2::HIS3 linear disruption
cassette was released from that plasmid by digestion with
SalI and XbaI. This DNA fragment was
electroporated into strain DTY10A to creating strain DTY004. Interruption of the ELO2 gene by that fragment was confirmed
by PCR, using primers that flank the ELO2 open reading frame
sequence.
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The
plasmid pCRELO2 was digested with BamHI and SalI.
The released 1.2-kb DNA fragment was ligated into plasmid YCpGAL1URA downstream of the GAL1 promoter sequences. This construct
(YCpGALELO2(U)) was analyzed by diagnostic restriction enzyme digest to
confirm the correct orientation of the ELO2 fragment in
respect to the GAL1 promoter. That plasmid was
electroporated into strain CSY3H for over-expression studies of the
ELO2 gene in the elo3
background.
A 2.9-kb
fragment containing the coding sequence for ELO3 was derived
by PCR using primers ELO3A and ELO3B (Table III) and strain DTY10A
genomic DNA as a template. The PCR product was digested with
KpnI and SalI and ligated into plasmid YEp352 in
which the HindIII site had been destroyed to produce
YEpElo3. This plasmid was digested with HindIII followed by
Klenow fill-in to remove the ELO3 coding region. A 1.2-kb
DNA blunt-ended SalI-XhoI fragment containing the
S. cerevisiae HIS3 gene was inserted into the vector creating plasmid YEpelo3HIS. The resulting plasmid (YEpelo3HIS) contains the HIS3 gene coding sequence in an orientation
opposite to that of ELO3. A 3.4-kb
elo3::HIS3 linear disruption
cassette was released from that plasmid by
KpnI/SalI restriction digest. This DNA fragment
was electroporated into strain DTY10A to disrupt the chromosomal copy
of ELO3. Interruption of the ELO3 gene was confirmed by PCR, using primers that flank the ELO3 open
reading frame sequence.
A DNA
fragment containing the GAL1 promoter derived from vector
YCpGAL1 by EcoRI digestion was inserted upstream of the
ELO3 mRNA coding sequences in plasmid YEpElo3 at a
SacI restriction enzyme site. The 5-untranslated region of
ELO3, including its presumptive basal promoter elements
(TATA), was removed from the plasmid by partial HindIII
digestion. A 2.0-kb DNA fragment, containing the GAL1
promoter and adjacent ELO3 protein coding sequences, was
released from the resulting plasmid by EcoRI digestion. The recovered fragment was ligated into complementary EcoRI
sites on plasmid YCpGAL1 which contains the URA3 gene as a
selectable marker. These constructs YCpGALELO3(U) were analyzed by
diagnostic restriction enzyme digest to confirm the correct orientation
of the EL03 fragment in respect to the GAL1
promoter. These constructs were electroporated into strain DTY004 for
over-expression studies of the ELO3 gene in an
elo2
background.
Fatty acid methyl esters were extracted from washed yeast cell pellets as described previously (17). These were generated by boiling the cell pellets derived from 50-ml cultures containing 1-4 × 107 cells/ml in a mixture of 3 N methanolic-HCl. The samples were then extracted with hexane/anhydrous ethyl ether (1:1). Fatty acid methyl esters were analyzed using a Varian 3400 CX gas chromatograph and with a 30 m × 0.32 mm SupelcoWaxTM-10 column with a film thickness of 0.32 µm using helium as the carrier gas. The injector temperature (splitting ratio 50:1) for gas chromatograph was 240 °C and the oven temperature was increased from 70 to 240 at 20 °C/min. Gas chromatography data was collected and quantified using the Shimadzu EZChrom data system.
Mass SpectroscopyMass spectra data were collected and quantified on a Finnigan Mat 8230 mass spectrograph using the Finnigan Mat SS300 data system. The same gas chromatography column and temperature programming parameters were used to fractionate fatty acids prior to ionization. The ion source temperature for mass spectra was 250 °C, and the filament emission current was 1 mA. Mass spectra were recorded at an ionization voltage of 70 eV. Ions from 35 to 600 amu were scanned at 1 s/decade and interscanned at 0.85 s/decade.
Polymerase Chain ReactionThe polymerase chain reaction
(PCR) was performed according to standard protocols (20). All PCR
reactions were performed in a "OmniGene" thermal cycler (Hybaid,
Ltd.) using a heat-stable recombinant "Vent" DNA polymerase with
5-3
- and 3
-5
-exonuclease activity. This allowed for greater
fidelity of PCR products with an error rate of approximately 1 in
105 base pairs of DNA. All PCR reactions used either a 50 or 55 °C annealing temperature. Extension times at 72 °C were
typically between 2.0 and 4.0 min or were adjusted to the size of the
expected product. PCR reactions were routinely run for a total of 30 cycles. A list of all PCR primers used in this work appears in Table
III.
Two genes were identified from the S. cerevisiae genome data base that had high identity to ELO1, a gene involved in the fatty acid synthase-independent elongation of 14:0 to 16:0 (10). ELO2 is located on yeast chromosome III at the locus designated YCR34W. ELO3 is located on chromosome XII at the locus designated YLR372W.
Fig. 1 shows regions of homology between
ELO1, ELO2, and ELO3 protein coding
sequences. Elo2p and Elo3p are, respectively, 76 and 72% similar and
56 and 52% identical to that of Elo1p. The three genes contain
multiple regions of contiguous identical residues throughout the
protein sequence. Hydropathy analyses of ELO1p, ELO2p, and Elo3p by the
TMpredict algorithm (Fig. 2) suggest that the three
proteins contain five membrane-spanning regions. The identified core
regions of these sequences align identically with previously predicted
transmembrane regions of Elo1p (10). The regions of highest identity in
all three genes lie between presumptive transmembrane-spanning regions
II and III (Fig. 2). That region has 16 identical amino acid residues located between residues 185 and 200 of Elo3p (from the amino terminus)
and contains a cluster of four consecutive basic residues followed by
an HXXHH motif, which has been previously identified with
fatty acid desaturase, ribonucleotide reductase, hemerithrin, and other
iron-containing proteins (9). Elo2p also has a hydrophobic stretch in
that region that contains several polar residues, suggesting that it
might serve as a hydrophobic cleft associated with an active site of
the enzyme. The amino- and carboxyl-terminal regions of Elo2p and Elo3p
are most dissimilar to Elo1p. In all three proteins the C-terminal
sequences that follow transmembrane segment V contain unusually high
numbers of basic residues, suggesting that those domains have some
homologous function. Both ELO2 and ELO3 peptide
sequences lack a GSA motif near the carboxyl terminus of
ELO1 that is proposed to be an NADPH binding site for a
number of lipid biosynthetic enzymes (10).
Disruption of ELO2 or ELO3 Produces Changes in Long Chain (14-18-Carbon) Fatty Acids
Analysis of total fatty acids from
elo2 and elo3
strains showed only minor
changes in the composition of C14-C18 fatty
acids (Table IV). Those species comprise approximately
95% of the total cellular fatty acids. Compared with wild type,
elo2
C14 fatty acids were reduced by 45%,
16:1 was reduced approximately 8%, and 18:1 was increased by about
10%. The elo3
strain showed complementary changes with
increased 16:1 (~30%) and decreased 18:1 (~30%).
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The very long chain fatty acid composition of wild type,
elo2, and elo3
strains is shown in Fig.
3. The most abundant species in wild type are 26:0 and
- hydroxy 26:0 (2, 21). This was confirmed by GC/MS of total fatty
acid methyl esters and by separate GC/MS analyses of authentic
standards. They comprise approximately 2.2 and 0.9% of the total fatty
acid mass in mid-log cells grown in glucose medium. Minor peaks
representing 22- and 24-carbon-saturated, monounsaturated, and hydroxy
fatty acid species were also identified in the wild type strains. These
combined represent less than 0.6% of the total fatty acids and are
apparently derived from metabolic intermediates in the fatty acid
elongation pathway. GC/MS analysis also identified several prominent
peaks with retention times similar to the long chain species. These are
the free fatty acids 16:0, 16:1, 18:0, and 18:1. They are apparently
equilibrium products of the transmethylation reaction and do not
represent a unique pool of unesterified species in vivo. The
relative levels of the esterified and unesterified forms were combined
for each species in data shown in Table IV.
Disruption of ELO2 or ELO3 Produces Changes in the Very Long Chain Fatty Acid Chain Length Distribution
Quantitative gas
chromatography and GC/MS analysis of fatty acid methyl ester fractions
indicate that the 26:0 and HO-26:0 species are absent in the
elo3 strain. Sharply reduced levels of 26:0
(approximately 20% wild type levels) and HO-26:0 (approximately 40%
of wild type levels) were found in the elo2
strain (Fig. 3, Table V). The reductions in C26 species
were accompanied by increases in minor (and in some cases,
undetectable) wild type species. Lipids from the elo2
strain displayed new peaks with retention times between 9 and 11 min.
GC/MS analysis indicated that they were hydroxy 16- and 18-carbon fatty
acids with fragmentation patterns consistent with the
-hydroxy fatty
acid standards. (In wild type, those species were not detected.) No
C20 fatty acids were detected in the elo2
strain and the total C20-C26 species were 30%
of wild type levels.
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The elo3 strain contained elevated levels of C20
and C22 fatty acids (Table V). The most abundant species was
22:0 which averaged 3.1% of the total fatty acyl mass, a 10-fold
increase over wild type levels. Large increases in the levels of
hydroxy C16-C24 fatty acids were also observed
in this strain. Unlike the elo2
strain, ELO3
disruption resulted in an approximate 20% increase in the total levels
of very long chain species.
The synthetic lethality of elo2 and
elo3
suggests that their encoded proteins have
overlapping functions. To test whether increased activity of Elo2p and
Elo3p can compensate for loss of the other's function, their genes
were each placed under the control of the strong, inducible
GAL1 promoter and transformed into cells with the disrupted
homologue. The very long chain fatty acid distributions of these
strains and wild type are shown in Table V and Fig.
4.
Very long chain fatty acid distributions of wild type,
elo2, and elo3
controls (which did not
contain the plasmid) on the non-fermentable carbon source, galactose,
were similar to those found for these same strains when grown on
glucose medium (Table V). Both gene-disrupted strains contained 16- and
18-carbon-hydroxylated species. The elo2
strain exhibited
reduced levels of 26:0 and HO-26:0 and undetectable levels of
C20 and C22 species, whereas the
elo3
strain showed the characteristic large increase in
C20 and C22 fatty acids and no C26
species. Galactose-induced over-expression of Elo3p in an
elo2
/GAL1-ELO3 strain resulted in a 50-fold decrease in
the hydroxy 16:0 fatty acids and reduced levels of the less abundant
hydroxy 16:1, 18:0, and 18:1 species (Table V). The strain also had
slight, but significant, increases in hydroxy 26:0 (which is elevated
to wild type levels) and 26:0 (which is elevated from 15 to 36% of the
wild type levels).
More dramatic changes were observed on over-expression of Elo2p in the
elo3/GAL1-ELO2 strain. This failed to restore
the missing 26-carbon species and also did not reduce the
characteristic high levels of 22:0. Large increases in C20
and C24 carbon species were observed, however. There was a
44-fold increase in 24:0 and a 5-fold increase in hydroxy 24:0 (Table
V, Fig. 4). Similar increases were observed in 20:1 (2-fold) and 22:1
(5-fold). Induction of Elo2p in this strain also produced high levels
of a 24:1 species that was not detected in the elo3
or
wild type strains. All of the shorter chain hydroxy
16:0,
16:1,
18:0, and
18:1 species present in the elo3
strain
were reduced to undetectable levels in cells containing
over-expressed Elo2p.
The
effects of disrupting ELO1, ELO2, and
ELO3 on sphingolipid biosynthesis are shown in Fig.
5, A and B. The strains were pulse-labeled (30 min) with [3H]dihydrosphingosine
(lanes 1-5) or labeled for 6 h with
[3H]serine (lanes 6-9), and the lipid
extracts were chromatographed by TLC either before or after mild
alkaline methanolysis, respectively. The methanolysis deacylates fatty
acids on phospholipids that are linked to the glycerol-head group
moiety by O-acyl bonds, but leaves the N-acyl
ester bonds of sphingolipids intact. A thin layer chromatogram of those
lipids is shown in Fig. 5A.
The lipids in wild type and elo1 cells had a similar
pattern; most of the label incorporated into sphingolipids was found in
the major inositol-containing species including inositol
phosphoceramide as well as the more complex mannosylated forms,
mannosyl phosphorylceramide and mannosyl
diinositoldiphosphorylceramide. Sphingolipid intermediates were in low
abundance with almost no label incorporation into the long chain bases,
phytosphingosine, or dihydrosphingosine, and only a faint band for
ceramide, a precursor composed of a long chain base and a very long
chain fatty acid linked by an N-acyl bond. The
elo2
and elo3
strains, by comparison,
displayed large accumulations of label in the long chain bases,
primarily phytosphingosine, and the ceramide band was absent. Label
incorporation into the mature sphingolipids was greatly reduced. In
elo3
almost all of the label accumulated into the
inositol phosphoceramide species, with very little labeling of the
mannosylated forms, while in elo2
there were reduced
amounts of all of the sphingolipids. The major serine-labeled
phospholipids, which run as their more polar glycerol-head groups after
the alkaline hydrolysis procedure, were normal in all of the strains.
Pulse labeling with [3H]dihydrosphingosine gave
qualitatively similar results where elo2
and
elo3
strains accumulated phytosphingosine and were very
deficient in ceramide and the inositol-containing sphingolipids compared with wild type and elo1
cells.
Quantitation of the [3H]dihydrosphingosine labeling by
PhosphorImager is shown in Fig. 5B. The majority of the
converted label (81%) was found in the sphingolipid fraction in the
wild type and elo1 strains. In these lipids,
approximately 90% of the label was in the form of inositol
phosphoceramide and mannosyl phosphorylceramide and 10% was in the
form of mannosyl diinositoldiphosphorylceramide. Approximately 2% of
the converted dihydrosphingosine label was in phytosphingosine, 10% in
ceramide, and 8% found in phospholipid fractions. The label in
phospholipids is derived from the degradation of dihydrosphingosine to
an aldehyde intermediate which is then converted to a fatty acid. In
elo2
and elo3
, the amount of converted dihydrosphingosine label was reduced to approximately 30% of wild type
due to reductions in ceramide and sphingolipids. Phytosphingosine was
increased approximately 2-fold in the elo2
strain and by 1.5-fold in elo3
, whereas the label incorporated into the
phospholipid fractions was similar to that observed in wild type. A
similar pattern of dihydrosphingosine labeling was found in wild type cells that were treated with the ceramide synthase inhibitor, australifungin, as seen in Fig. 5A. These data suggest that
disruption of ELO2 or ELO3 has the effect of
reducing the level of ceramide synthesis which results in the
concomitant reduction in cellular sphingolipid levels.
The roles of Elo2p and Elo3p as components of the very long chain fatty acid elongation system are supported by several observations. Hydrophobicity analysis indicates that Elo2p and Elo3p are intrinsic membrane proteins with multiple membrane-spanning regions, which is consistent with the reported tight membrane association of fatty acid elongation activities (9). ELO2 and ELO3 encode polypeptides that have a high degree of homology to the ELO1 gene product which is involved in the highly specific elongation of 14 carbon fatty acids (10). Disruption of either gene alters the composition of very long chain fatty acids and causes the accumulation of intermediate length fatty acid precursors.
The lethality caused by the simultaneous disruption of the
ELO2 and ELO3 genes indicates that their products
have a high degree of overlapping functions. The different pattern of
accumulation of very long chain fatty acid intermediates in the
elo2 and elo3
strains, however, suggests
that Elo2p and Elo3p may play roles in independent, and to some degree
parallel, metabolic pathways. These may be located in different parts
of the cell. Previous studies of fatty acid elongation enzyme
activities in Saccharomyces suggest, in fact, that there may
be elongation systems both in the endoplasmic reticulum and on the
mitochondrial surface (22).
Clues about the enzymatic characteristics of Elo2p can be seen
from the analysis of the ELO2+,
elo3 strain. The absence of a C26 fatty acid
indicates that Elo2p cannot catalyze the elongation of 24:0 to 26:0.
Elo2p apparently has the highest catalytic specificity for
C20 acyl-CoA, which results in the observed accumulation of
22:0. Experiments in which ELO2 is over-expressed in
elo3
cells indicate that Elo2p can convert 22:0 to 24:0
with less efficiency. This reduced activity toward C22
substrates can be compensated for by over-expression of Elo2p which
shifts in the accumulated species from 22:0 to 24:0.
Elo3p appears to act on a broader range of substrates.
Twenty-six-carbon fatty acids are formed when the masking Elo2p
activity is removed in the ELO3+,
elo2 disruption strain. Taken together with the absence of C26 species in the elo3
strain, this
indicates that conversion of 24:0 to 26:0 is exclusively performed by
the Elo3p-dependent elongation system.
The lower levels of sphingolipids associated with the disruption of either ELO2 or ELO3 genes appear to be the result of reduced ceramide synthesis. Ceramide is the sphingolipid precursor that is formed from the direct condensation of very long chain fatty acids with a long chain base by ceramide synthase. This sensitivity of sphingolipid biosynthesis to changes in the very long chain fatty acid distribution in the disrupted strains could be caused by several factors. Reduced levels of very long chain acids could limit the pool of precursors available to ceramide synthase, producing a rate-limiting step at ceramide formation. Ceramide synthase could also have an optimal substrate specificity for 26:0 CoA and reduced activity in the presence of shorter chain substrates found in the disrupted strains. A third possibility is that the spatial distribution of fatty acid precursors in the cell is altered by ELO2 or ELO3 null mutations, possibly due to different cellular locations of the ELO2 and ELO3 elongation systems. This might result in the inability of ceramide synthase to make contact with available substrates.
The marked changes in cellular sphingolipid fatty acid composition in
elo2 and elo3
strains provide a rational
explanation for the pleiotropic effects reported for mutant alleles of
these genes. Mutations or disruptions of ELO2 and
ELO3 were previously reported as affecting
-glucan
synthase activity (12), the plasma membrane, (H+)-ATPase
(14), bud localization defects (13), and resistance to sterol synthesis
inhibitors (11, 13). All of these functions are apparently produced by
the activities of intrinsic membrane proteins, which could be affected
by changes in the composition of their lipid environment or by the
absence of an essential sphingolipid-membrane protein interaction.
Although Elo2p and Elo3p appear to be essential for the formation of very long chain fatty acids, there are apparently a number of other components that catalyze other steps in the elongation cycle that have yet to be identified. The lack of information about the structure, function, and cellular location of these enzymes gives an unclear picture of how they are organized and how intermediates are transferred from one reaction center to the next. Until now, the intrinsic hydrophobicity of these enzymes has hindered previous attempts to purify and characterize the components of these systems. The identification of ELO2 and ELO3 in this paper provides new tools that can be used to resolve questions concerning the mechanism, organization, and structure of these complex systems.