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J. Biol. Chem., Vol. 277, Issue 13, 11481-11488, March 29, 2002
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From the
Received for publication, November 30, 2001, and in revised form, January 8, 2001
A number of Saccharomyces cerevisiae
membrane-bound oxidoreductases were examined for potential roles in
microsomal fatty acid elongation, by assaying heterologous elongating
activities in individual deletion mutants. One yeast gene, YBR159w, was
identified as being required for activity of both the
Caenorhabditis elegans elongase PEA1 (F56H11.4) and the
Arabidopsis thaliana elongase FAE1. Ybr159p shows some
limited homology to human steroid dehydrogenases and is a member of the
short-chain alcohol dehydrogenase superfamily. Disruption of YBR159w is
not lethal, in contrast to previous reports, although the mutants are
slow growing and display high temperature sensitivity. Both Ybr159p and
an Arabidopsis homologue were shown to restore heterologous
elongase activities when expressed in ybr159 Unsaturated fatty acids are essential cellular constituents,
serving not only as structural components of membranes but also as
bioactive metabolites. One important class of these lipids is
collectively known as polyunsaturated fatty acids
(PUFAs)1; these are
defined as fatty acids of 18 carbons or more (C18+), which
contain two or more double bonds (1). In mammals, 20 carbon
(C20) PUFAs have been shown to be the biological precursors of a group of molecules called the eicosanoids, which includes the
prostaglandins, leukotrienes, and thromboxanes (2). The eicosanoids
have roles in inflammation responses as well as cardiac and
reproductive function. This obvious importance of PUFAs has resulted in
considerable interest in the characterization of their biosynthetic
pathway (3), and we have identified a new class of fatty acid
desaturases required for PUFA biosynthesis, which contain an
N-terminally fused cytochrome b5 domain (4).
More recently, we have also identified a component of the PUFA fatty
acyl-chain elongation system, which when heterologously expressed in
yeast, directs the C2 elongation of the C18
PUFA The biosynthesis of C20+ fatty acids has also been observed
in higher plants, although the elongated fatty acids are quite distinct
to those observed in mammals. Although C18 PUFAs such as
Fatty acid elongation is carried out by a microsomal "elongase,"
which consists of four distinct enzymatic reactions (11). In sequential
order, these are a condensation reaction between a CoA-esterified fatty
acyl substrate and malonyl-CoA, We have hypothesized that these heterologous elongating activities
function by "hi-jacking" endogenous microsomal elongases, which in
the case of yeast, are primarily synthesizing the C20-26 saturated fatty acid components of sphingolipids (5). This results in
the presumptive redirection of the three endogenous components
(reductases and dehydratase) toward non-native substrates. Until very
recently, little was known about the identity of these other three
enzyme activities and their precise contribution(s) to the activity and
specificity of microsomal fatty acid elongases. Kohlwein et
al. (12) characterized the TSC13 gene, which is one of
a number of genes identified in a screen for temperature-sensitive (ts)
mutants with defects in sphingolipid synthesis (13). Mutants with ts
alleles of TSC13 displayed phenotypes similar to the
elo2 As part of our studies on microsomal fatty acid elongation, we have
sought to identify other enzymatic components of the fatty acid
elongase. To that end, we screened yeast knockout mutants for
loss-of-function of our heterologous PUFA elongating activity PEA1 (5).
Using this approach we identified a previously functionally uncharacterized gene required for the reconstitution of heterologous elongase activity.
Cloning of Putative Functional Characterization in Yeast--
ORFs encoding
putative Fatty Acid Analysis--
Total fatty acids extracted from yeast
cultures were analyzed by gas chromatography (GC) of methyl ester
derivatives. Lipids were extracted and transmethylated with methanolic
HCl. Fatty acid methyl esters were analyzed as described before
(19).
GC-MS Analysis--
Induced peaks were characterized using GC-MS
(Kratos Analytical Instruments MS80RFA) operating at an ionization
voltage of 70 eV with a scan range 40-500 Da and as described before
(19).
Elongase Activity Assays--
Microsomes were prepared
from the wild type or ybr159 In an attempt to identify genes or genes that encode the
A Saccharomyces cerevisiae Gene Required for
Heterologous Fatty Acid Elongase Activity Encodes a Microsomal
-Keto-reductase*
,,¶
Institute of Arable Crops Research-Long
Ashton Research Station, Long Ashton, Bristol BS41 9AF, United
Kingdom and the § Department of Biochemistry, Uniformed
Services University of the Health Sciences, Bethesda, Maryland
20814
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutants.
Biochemical characterization of microsomal preparations from
ybr159 cells revealed a primary perturbation in
-ketoacyl reduction, confirming the assignment of YBR159w as
encoding a component of the microsomal elongase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-linolenic acid (18:3(n-6); GLA) to the
C20 PUFA di-homo--linolenic acid (20:3(n-6))
(5). This component (Caenorhabditis elegans ORF F56H11.4,
designated PEA1 for polyunsaturated fatty acid
elongating activity (5, 6)) shows some limited
homology to the yeast ELO gene family, which have been shown
genetically to be required for the synthesis of saturated medium and
very long-chain fatty acids (7, 8). Although the precise biochemical
function of the C. elegans PUFA-elongating activity PEA1 and
the polypeptides encoded by the ELO genes remain to be
elucidated, it is generally considered that they serve as condensing
enzymes (5, 6). The steps in the biosynthetic pathway of
C20 PUFAs are indicated in a generalized scheme (Fig.
1), with the key step of C2
elongation of C18 substrates highlighted.
View larger version (19K):
[in a new window]
Fig. 1.
Generalized pathway for the
biosynthesis of polyunsaturated fatty acids. A generalized scheme
of the enzymes responsible for aerobic desaturation and elongation of
fatty acids is shown. The reaction directed by the polyunsaturated
fatty acid elongase PEA1, resulting in the production of
C20 PUFAs, is boxed. The yeast S. cerevisiae does not synthesize PUFAs, serving as a
convenient host for "gain-of-function" screening.
-linolenic acid and linoleic acid accumulate to high levels in
plants, the presence of C20 PUFAs has not been observed (1). However, some higher plants, most notably members of the Brassicas
such as oil seed rape and Arabidopsis, synthesize
C20 and C22 monounsaturated fatty acids (MUFAs)
(9). These C20+ fatty acids are the product of (several
cycles of) C2 fatty acyl elongation of a monounsaturated
C18 substrate, oleic acid (18:19). The
biosynthesis of higher plant C20/22 MUFAs has
been shown to require a single gene, FAE1 (fatty
acid elongation), which encodes a putative
condensing enzyme (10). The Fae1p condensing enzyme displays
specificity only for saturated and monounsaturated fatty acids (9, 10).
Importantly, although Fae1p shows some limited homology to analogous
condensing enzymes such as chalcone and stilbene synthases, it displays
no similarity to the Elo protein family; conversely no homologues of
Fae1p are present in yeast.
-keto reduction, dehydration, and a
final enoyl reduction. It has been hypothesized that the specificity of
any particular elongation reaction is conferred through the selectivity
of the first condensation step. Conversely it is believed that the
three other components (two reductases and a dehydratase) are common to
all microsomal fatty acyl elongases and have no particular substrate
specificity (11). This is given credence by the observation that
heterologous expression in yeast of the plant Fae1p condensing enzyme
successfully reconstitutes a functional C20+ MUFA-specific
elongase (9). Similarly, we have also observed effective reconstitution
of the PUFA-specific elongase (via by heterologous expression of the C. elegans Pea1p in yeast) (5), even though yeast has no
endogenous capacity for either of these particular biosynthetic reactions.
and elo3 mutants, including the
accumulation of high levels of long-chain bases, the accumulation of
ceramides with chain lengths of less than 26 carbons, and a deficiency
in very long chain fatty acid synthesis (12). Microsomal fatty
acid elongation assays revealed that the tsc13 mutation
caused the accumulation of trans-2,3- and 3-hydroxy-acyl
intermediates; these observations, taken together with the homology
between Tsc13p and steroid-5--reductase, are consistent with the
TSC13 gene encoding the enoyl-reductase component of the
microsomal elongase. Furthermore, epitope-tagged Tsc13p was shown to
co-immunoprecipitate with the presumptive condensing enzymes Elo2p and
Elo3p (12). Interestingly, a precise ER location for Tsc13p, enriched
at sites of vacuole-nuclear envelope interaction, was observed.
TSC13 is essential, as is expected for a gene encoding a
non-redundant fatty acid elongating activity (12).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Ketoacyl Reductases in a Yeast Expression
Vector--
The Saccharomyces cerevisiae ORF encoded by
YBR159w was cloned by PCR into the pESC-TRP vector (Stratagene). A
crude DNA extract was prepared using the reference strain W303-1A. 1 ml
of an overnight culture in rich medium (YPD) was centrifuged at
3000 × g for 5 min at room temperature, and the cells
were resuspended in 0.5 ml of TE buffer (10 mM Tris, pH
8.0, mM EDTA). This suspension was boiled for 10 min, and the extract was centrifuged at 10,000 × g for
5 min. 1 µl of the supernatant was used for PCR amplification using primers YBR159.for
(5'-GCGGGATCCACCATGACTTTTATGCAACAGC-3') and
YBR159.rev (5'-GCGGGTACCCTATTCCTTTTTAACCGTCT TGC-3').
The amplified sequence was then restricted using BamHI
and KpnI (underlined in the forward and reverse primers,
respectively), purified using the Qiagen PCR purification kit, and
ligated into BamHI/KpnI-cut pESC-TRP plasmid
vector (Stratagene). An Arabidopsis thaliana expressed
sequence tag (GenBankTM accession number: AA10E08; kindly
provided by Prof. H. J. Bohnert, University of Arizona) derived
from gene F12A21.31 was used as template DNA for PCR amplification
using primers At159.for
(5'-GCGGGATCCACCATGGAGATCTGCACTTACTTC-3') and At159.rev
(5'-GCGCTCGAGTCATTCTTTCTTCATGGAGTC-3'). The amplified sequence was then restricted using BamHI and XhoI
(underlined in the forward and reverse primers, respectively) and
cloned into BamHI/XhoI-cut pESC-TRP as described above.
-ketoacyl-reductase activities and elongase or desaturase
constructs were introduced in Saccharomyces cerevisiae using
a lithium acetate-based method (14). Expression of the transgenes was
induced by addition of 2% (w/v) galactose in the presence or absence
of exogenously supplied fatty acid substrates as described previously
(15). The mutant strain used in this study was CEN.RO16; Mat a/;
ura3-52/ura3-52; his31/his31; leu2-3_112/leu2-3_112;
trp1-289/trp1-289; ybr159::HIS3/YBR159w, obtained from European Saccharomyces
cerevisiae Archive for
Functional Analysis (EUROSCARF) (available at
www.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). This strain was
transformed with a pYES2 construct containing the C. elegans
PUFA elongating activity PEA1 (F56H11.4) and sporulated in SPM liquid
media as described previously (16). After sporulation, asci were
digested with -glucuronidase (Sigma Chemical Co.), and the tetrads
were dissected using light microscopy and a micromanipulator as
described (17). Separated ascospores were grown on YPD for up to 2 weeks at 22 °C, and the mating type of each haploid colony tested
using two yeast strains, a sst1 and sst2
(18). "Wild type" (Mat a or ; ura3-52; his31;
leu2-3_112; trp1-289) and mutant spores (Mat a or ; ura3-52;
his31; leu2-3_112; trp1-289; ybr159::HIS3)
were identified by replica plating on synthetic dextrose medium
lacking histidine. This was also confirmed by PCR using primers
159_prom.for (5'-CGGATTTGGAAGTCCTTTATAG-3') and 5'_his3.rev
(5'-CGCTTTACTAGGGCTTTCTGC-3'). Wild type and mutant spore colonies
containing the PUFAs-elongase construct in pYES2 were selected by
replica plating on synthetic dextrose medium lacking uracil.
mutant cells as has been
previously described (20). Total elongase activity was measured in a
volume of 200 µl containing 50 mM Tris, pH 7.5, 1 mM MgCl2, 150 µM Triton X-100, 1 mM NADPH, 1 mM NADH, 10 mM
-mercaptoethanol, 40 µM palmitoyl-CoA, 60 µM 2[14C]malonyl-CoA (0.05 µCi/ml) at
37 °C. The reaction was initiated by the addition of 0.4 mg of
microsomal protein. Protein concentrations were determined using the
Bio-Rad protein assay reagent (Bio-Rad Laboratories). For assays of
only the condensing activity, the NADPH and NADH were omitted. At
various times (0.2, 1, or 5 min) the reaction was terminated by adding
200 µl of 5 M KOH/10% MeOH and heating at 80 °C for
1 h. Following addition of 200 µl of 10 N
H2SO4, fatty acids were recovered by two 1.5-ml
extractions into hexane. The extracted fatty acids were resolved by
silica gel TLC using hexane:diethyl ether:acetic acid (30:70:1) as the developing solvent and detected and quantified using a PhosphorImager SI (Molecular Dynamics, Inc.).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-keto-reductase of the microsomal elongase, we searched the complete genome sequence of the yeast S. cerevisiae (predicted to
encode between 5000 and 6000 polypeptides) (21) for protein sequences likely to encode oxidoreductases. This was carried out either on a
homology basis to known oxidoreductases or via the presence of the
diagnostic NADH binding motif (22). Because this generated over 200 candidates, the search was refined to exclude proteins already clearly
functionally characterized, as well as cytosolic oxidoreductases. By
excluding predicted ORFs, which lacked a canonical dilysine ER
retention motif, the search was further refined to identify predicted
oxidoreductases likely to have a transmembrane topology and to be
located in the ER. This approach allowed us to consider a small number
(~10) of genes for functional characterization as potential
microsomal fatty acyl elongase components (listed in Table
I). A number of these selected
oxidoreductases ORFs had previously been shown by high throughput
deletion analysis to be non-essential for viability. Yeast mutants in
which the non-essential candidate genes were disrupted were assayed for any loss of ability to carry out heterologous PUFA elongation. Thus, a
galactose-inducible, URA3-marked plasmid carrying the C. elegans PEA1 PUFA elongating
activity (5) was transformed into these mutant strains, and
the transformed yeast cells were assayed for their ability to direct
the C2 elongation of GLA, forming the basis of a
"loss-of-function" screen.
Yeast oxidoreductases identified as candidate components of the
microsomal elongase
mutant cells
are actually viable. The activity of the heterologous elongating
activity PEA1 in deletion mutant strains is indicated.
Characterization for loss-of-function in haploid knockout mutants of
the eight non-essential oxidoreductases (Table I) revealed no
alteration in their ability to reconstitute the heterologous PUFA
elongase and redirect the elongation of C18 PUFAs. Although the synthesis of very long-chain fatty acids is essential for yeast
cell viability (8), previous directed ethyl methanesulfonate (EMS) mutant screens for defects in fatty acid elongation only identified the ELO1 gene as a potential elongase component,
implying that other elongase genes are either essential or display
functional redundancy (7, 8, 23). To address the possibility that the
(annotated as) essential gene YBR159w encoded an oxidoreductase component of the microsomal elongase, a diploid yeast strain
heterozygous for disruption of YBR159w was transformed with a plasmid
containing the PEA1 ORF. Following sporulation and tetrad dissection,
the spores were allowed to germinate on YPD media. Using this approach, we observed that the ybr159 haploid deletion was in fact
viable, although PCR confirmed the insertional disruption of this
oxidoreductase ORF in mutant haploid cells (data not shown). When
compared with wild type spores isolated from the same tetrad, spores
containing the disrupted gene showed a very reduced growth rate, with
small colonies appearing usually only after 10 days of incubation at 22 °C.
An initial study described deletion of YBR159w (in strain ENY.MR17) as
resulting in poor growth (only at low temperatures) with a
pseudo-hyphal phenotype (24), although in a subsequent study it was
reported that YBR159w was an essential gene in the CEN.PK2
background (25). In our current study, we used the same parental
diploid heterozygote as used in the latter study (CEN.PK2 strain
background), although after sporulation the haploid
ybr159 mutants were viable even at 30 °C. Indeed,
although it took about 10 days for mutant spores to germinate and to
form a colony after meiosis (see above), ybr159 cells are
able to grow at 30 °C in rich medium (in the absence of fatty acid
supplements) but at a slower rate than wild type (Fig.
2). Interestingly, cultivating ybr159 cells in a media supplemented with medium and
long-chain fatty acids did not improve the growth rate (data not
shown). However, we observed that the ybr159 mutant
displayed temperature sensitivity when transferred to 37 °C. We also
examined the phenotypic appearance of the CEN.PK strain
ybr159 mutant cells and observed the previously reported
(for strain ENY.MR17) pseudo-hyphal growth (24). However, genetic
analysis indicates that this phenotype is unlinked to the
ybr159
mutation.2
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The Oxidoreductase Ybr159p Is Specifically Required for
Heterologous Elongation Activity in S. cerevisiae--
The ability of
the heterologous elongating activity Pea1p to function in the
ybr159 deletion was tested by galactose induction as
described above. Wild type haploid colonies displayed PUFA elongating
activity resulting in the conversion of 18:3(n-6) (GLA) into
20:3(n-6) (di-homo--linolenic acid), whereas deletion
mutant haploids completely failed to elongate heterologous
C18 PUFAs (Fig. 3, top
panel). We tested the galactose inducibility of other pYES2-directed enzyme activities in this haploid knockout strain to
assess the possibility of pleiotropic effects on either galactose uptake or lipid metabolism. However, when two other (heterologous) enzymes of the PUFA biosynthetic pathway (borage
6-desaturase (19), C. elegans
3-desaturase (26); see also Fig. 1) were tested in this
mutant background, they displayed unaltered activities when compared with wild type yeast (Table II). Northern
blot analysis of mutant cells expressing the heterologous elongase
revealed no alteration in transcript abundance when compared with wild
type (data not shown). Thus, deletion of YBR159w had a specific effect
on the activity of the heterologous PUFA elongase reconstituted by
expression of PEA1. This was given additional weight by our observation
that YBR159w was also required for the activity of the
Arabidopsis microsomal elongase condensing enzyme FAE1 (10);
expression of Fae1p in ybr159 haploid strains failed to
accumulate C20+ MUFAs (Fig. 3, bottom
panel).
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Several ORFs Related to YBR159w Are Present in S. cerevisiae and in
Other Yeast, Plant, and Animal Genomes--
Examination of the deduced
amino acid sequence encoded by YBR159w indicated that the predicted
polypeptide (of 347 amino acids) showed some similarity to human
estradiol-17-hydroxysteroid dehydrogenase (32% similarity). For
this reason, it has recently been suggested that the enzyme encoded by
YBR159w could be responsible for steroid dehydrogenase activities
observed in vitro in yeast extracts (27). However, no
sequences homologous to YBR159w could be detected by PCR and Northern
hybridization in the mesophilic yeasts Candida tropicalis
and Cryptococcus tsukubaensis even though steroid
dehydrogenase activity was observed. These observations have led the
authors to speculate that the Ybr159p oxidoreductase does not function as a steroid dehydrogenase (27); this is in agreement with our present
study. Presumptive orthologues of Ybr159p are also present in fission
yeast (43% similarity), Drosophila melanogaster (34% similarity), C. elegans (30% similarity), and
Arabidopsis (30% similarity) (Fig.
4A). Not only do all these
sequences contain the diagnostic NADH binding motif (22), they also
share a number of conserved residues, in particular the catalytically
essential (for estradiol-17-hydroxysteroid dehydrogenase) motif
Y-X3-K (28). There are also canonical dilysine
ER retention motifs present in both Ybr159p and the
Arabidopsis orthologue, consistent with these proteins'
predicted multiple (presumptively endoplasmic reticulum)
membrane-spanning topology. When the polypeptide sequence of Ybr159p
was used to search the complete yeast genome sequence, several related,
but distinct, ORFs were detected. These included Ymr226p (which shows
some homology to insect short-chain alcohol dehydrogenase; 33%
similar), Ayr1p (1-acyl-dihydroxyacetone-phosphate reductase (29); 24%
similar), and Yir036p (which shows homology to 7-hydroxysteroid
dehydrogenase; 23%) (Fig. 4B). Individual deletion analysis
of these three ORFs had previously indicated that none of these genes
encode essential proteins. More importantly, individual deletion of any
of these ORFs (YMR226c, AYR1/YIL124w, YIR036c) did not alter the
activity of the heterologous PUFA elongase PEA1 as determined by our
loss-of-function assay (data not shown; see also Table I for
summary).
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ybr159 Mutants Can Be Complemented by a Homologous ORF from A. thaliana--
Further confirmation of the role of Ybr159p in
heterologous microsomal elongation activity was obtained by rescue of
either Pea1p or Fae1p (PUFA or MUFA, respectively) elongating
activities by episomal co-expression of either the wild type YBR159w
ORF from S. cerevisiae or a presumptive A. thaliana homologue ORF (F12A21.31). In ybr159
cells, the galactose-induced co-expression of YBR159w with PEA1
resulted in restoration of 72% of C18 PUFA elongating
activity, respectively, when compared with the activity of the same
enzymes in wild type cells (Table III).
When the presumptive Arabidopsis orthologue F12A21.31 was
co-expressed instead of YBR159w, this resulted in a very similar
restoration of PUFA elongation (Table III); for that reason we
hereafter refer to F12A21.31 as At-YBR159. Similarly, co-expression of
YBR159w with FAE1 in ybr159 cells resulted in a 52%
restoration of C18 MUFA elongation, whereas co-expression
with At-YBR159 resulted in restoration of 42% of FAE1 activity,
compared with wild type cells (Table
IV).
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Interestingly, episomal galactose-induced overexpression of YBR159w in
wild type spore colonies (derived from the same tetrads that had
yielded the ybr159 haploids) resulted in moderate
increases in both PEA1 (PUFA) and FAE1 (MUFA) heterologous elongation
activities, compared with the expression of these elongating activities
in wild type cells (Tables III and IV). This suggests that, in contrast with native elongase systems, the condensation reaction may not be the
only rate-limiting activity for heterologous elongation in yeast.
Co-expression of the same elongating activities with At-YBR159 in wild
type yeast resulted in almost equivalent results, again demonstrating
the functional equivalence of the Arabidopsis protein.
ybr159 Mutant Cells Have Reduced Endogenous Fatty Acid
Elongation Activity--
ybr159p clearly plays a role in the fatty
acid elongation mediated by the heterologously expressed Fae1p and
Pea1p activities in yeast. Based on its homology to the
oxidoreductases, it was considered a good candidate for a
-keto-reductase, reducing the 3-keto intermediate formed by the
condensing activity during each cycle of fatty acid elongation. To
address how elongation is affected in the ybr159 mutant
cells, microsomes were prepared and assayed for elongase activity
in vitro. The elongation cycle initiates with the
condensation of malonyl-CoA with an acyl-CoA (e.g.
palmitoyl-CoA) to form a 3-ketoacyl-CoA intermediate. Omitting pyridine
nucleotide from the assay mix prevents the reduction of the
3-ketoacyl-CoA intermediate and thereby allows the first step of
elongation to be measured (12). The condensation activity measured over
a time course of 5 min was very similar whether wild type or
ybr159 microsomes were used for the assay (Fig.
5, compare lanes 4-6 and
10-12). However, when the time course of the overall
elongation reaction (with NADH/NADPH included) was conducted and the
products were analyzed by TLC using conditions that resolved the four
intermediates of fatty acid elongation, differences between the wild
type and ybr159 mutant were apparent. The only
intermediate seen in the elongation catalyzed by the wild type
microsomes was a small amount of 3-hydroxy intermediate in the 5-min
time point (Fig. 5, lane 3) under conditions where a large
amount of fully elongated product accumulated. In contrast, a large
amount of the 3-keto intermediate accumulated and the formation of the
fully elongated product were greatly delayed during the elongation
catalyzed by the ybr159 mutant microsomes (Fig. 5,
lanes 8 and 9). Based on the homology of Ybr159p
to oxidoreductases, it may directly catalyze the reduction of the
3-keto intermediate that is formed in each cycle of elongation. However, in addition to the 3-keto intermediate, elevated amounts of
the 3-hydroxy intermediate also accumulated (compare the difference in
the ratio of this intermediate to the fully elongated product in the
mutant to that in the wild type, Fig. 5, lane 3 versus lane 9). Although the accumulation of the
3-keto intermediate of elongation would be consistent with Ybr159p
functioning as a -keto-reductase, it is not clear why the 3-hydroxy
intermediate would also accumulate in the mutant (see
"Discussion" for possible explanations). In addition, although
Ybr159p is not essential for viability, very long chain fatty
acids are. Therefore, if Ybr159p is a -keto-reductase
activity that functions in elongation, there must be functionally
redundant activity. This is consistent with the non-lethality of the
disruption of this gene.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we report the identification of a yeast gene
required for the reconstitution of a heterologous microsomal fatty acid
elongase. This gene, identified as YBR159w is predicted to encode an
integral membrane protein, most likely located in the ER. The
encoded protein shows some limited homology to mammalian steroid dehydrogenases, most notably estradiol-17-hydroxysteroid dehydrogenase, and is a member of the short-chain dehydrogenase superfamily (Sanger Protein Family Data base ID: Pfam00106).
Interestingly, the Arabidopsis homologue of Ybr159p
functionally characterized by us in this study was previous annotated
by Xu et al. (30) as being an orthologue of the maize
Glossy8 gene. Transposon-induced mutations in this maize gene result in
defects in the synthesis of the cuticular waxes deposited on the outer
epidermis of the plant's cells, giving the mutant plant a
characteristic "glossy" appearance (30). The maize
glossy8 locus was cloned via transposon inactivation tagging
and, although not characterized biochemically, was hypothesized (on the
basis of homology to other dehydrogenases) to encode a
-ketoacyl-reductase (30). Homozygous glossy8 maize mutants display decreased levels of C24+ components of wax esters but are viable, indicating either gene redundancy or the presence of distinct elongases for fatty acid and wax synthesis. A
partial-length cDNA clone from Arabidopsis
(GenBankTM accession number U89512) was also identified in
that study as being a likely orthologue of maize Glossy8. That
Arabidopsis transcript is derived from F12A21.31, the gene
identified by us as encoding a functional homologue of the yeast
microsomal -keto-reductase Ybr159p. Based on the data presented in
this paper, we believe it is now possible to assign the function of
-keto-reductase to the maize Glossy8 locus.
Previous assessments of the requirement of YBR159w for yeast viability
yielded slightly contradictory data, with the gene being described as
essential in the CEN.PK2 background but viable at reduced temperatures
in the ENY.MR17 background. One explanation for the discrepancy in
previous viability data may be explained by our observation of a long
"lag" period in the initial growth of newly isolated haploid
mutants. This initial slow growth may be related to some form of
adaptive response to the loss of this microsomal elongase component.
After this "adaptation" the mutant spore colonies formed are viable
in the absence of fatty acid supplement, although growing at a much
slower rate than wild type cells (Fig. 2). In that respect it may be
analogous the adaptation of srp54 null mutants, in which
the loss of a component of the ER protein targeting machinery is
bypassed through an adaptive mechanism (31).
Mutagenic approaches to the identification of genes involved in fatty
acid elongation have previously only identified one component, Elo1p,
that is believed to be a condensing enzyme. The failure to recover
mutants in the other enzymatic reactions that comprise the elongase
system have been interpreted as indicating either functional redundancy
(as exemplified in the case of the ELO2/3 genes) or
lethality upon disruption. The essential nature of the activity of the
microsomal elongase for yeast cell viability is clear from the
synthetic lethality of elo2elo3 mutants, as well as the
requirement for a functional copy of the TSC13 gene, which
encodes the enoyl-reductase. Our observation that YBR159w, a
non-essential gene, is required for microsomal elongase
activity is intriguing, especially because of the lack of (functionally redundant) candidate homologues in the yeast genome. It is also clear
that the lethality (or not) on the loss of YBR159w is
strain-dependent; our current studies in the CEN.PK2
background indicate the viability of ybr159 mutants.
Although there are no obvious homologues of Ybr159p, it is possible
that related, ER co-located oxidoreductases (i.e. paralogues
of Ybr159p) are capable of (inefficiently) metabolizing 3-ketoacyl-CoA
intermediates. This could explain the viability of the
ybr159 mutant as well as the observed pronounced
reduction (but not ablation) in elongase activity.
The experiments presented here suggest that Ybr159p is the
-keto-reductase activity of the microsomal fatty acid elongating system. Consistent with this proposal, the in vitro
elongation assays reveal an accumulation of the 3-keto-fatty acyl
elongation intermediate. The accumulation of 3-hydroxacyl-CoA
intermediates in the flux through the elongase of ybr159
is equally intriguing. This could be due to a perturbation of the
physical interactions between the various distinct enzymatic components
as a result of the loss of Ybr159p. As such, this would give further
credence to the hypothesis that the microsomal elongase is likely to
function as a large multimeric protein complex. In that respect,
proximal physical interactions between other microsomal elongase
components such as Tsc13p and Elo2p/Elo3p have previously been
demonstrated (12).
Conclusions--
In this study we have used a
"loss-of-heterologous-function" screen to genetically identify a
component of the microsomal fatty acid elongase. Biochemical
characterization confirms the identification of the encoded polypeptide
as the -keto-reductase. As such, this is the first report of the
identification of a gene encoding this microsomal enzyme. Moreover,
overexpression of the -keto-reductase can result in enhanced
activity of (heterologous) elongases, challenging the concept that the
condensing enzyme is the rate-limiting step in fatty acyl elongation.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. This work was also supported by a grant (to T. M. D.) from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 44-1275-549-424; Fax: 44-1275-549-225; E-mail: jon.napier@bbsrc.ac.uk.
Published, JBC Papers in Press, January 15, 2002, DOI 10.1074/jbc.M111441200
2 T. Dunn, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
PUFA, polyunsaturated fatty acid;
GLA, -linolenic acid;
MUFA, monounsaturated fatty acid;
ORF, open reading frame;
PEA1, polyunsaturated fatty acid elongating activity;
FAE1, fatty acid
elongation;
ts, temperature-sensitive;
ER, endoplasmic reticulum;
YPD, yeast peptone dextrose media;
GC-MS, gas chromatography-mass
spectrometry;
WT, wild type.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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