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J Biol Chem, Vol. 275, Issue 1, 235-240, January 7, 2000
1-Acyldihydroxyacetone-phosphate Reductase (Ayr1p) of the Yeast
Saccharomyces cerevisiae Encoded by the Open Reading Frame
YIL124w Is a Major Component of Lipid Particles*
Karin
Athenstaedt and
Günther
Daum
From the Institut für Biochemie und Lebensmittelchemie,
Technische Universität and Spezialforschungsbereich Biomembrane
Research Center, Petersgasse 12/2, A-8010 Graz, Austria
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ABSTRACT |
Biosynthesis of phosphatidic acid through the
dihydroxyacetone phosphate pathway requires NADPH-dependent
reduction of the intermediate 1-acyldihydroxyacetone phosphate before
the second step of acylation. Studies with isolated subcellular
fractions of the yeast Saccharomyces cerevisiae revealed
that lipid particles and the endoplasmic reticulum harbor
1-acyldihydroxyacetone-phosphate reductase (ADR) activity. Deletion of
the open reading frame YIL124w (in the following named
AYR1) abolished reduction of 1-acyldihydroxyacetone phosphate in lipid particles, whereas ADR activity in microsomes of the
deletion strain was decreased approximately 3-fold as compared with the
wild-type level. This result indicates that (i) both lipid particles
and microsomes harbor Ayr1p, which was confirmed by immunological
detection of the protein in these two cellular compartments, and (ii)
microsomes contain at least one additional ADR activity. As a
consequence of this redundancy, deletion of AYR1 neither
results in an obvious growth phenotype nor affects the lipid
composition of a haploid deletion strain. When a heterozygous AYR1+/ayr1 diploid strain
was subjected to sporulation; however, spores bearing the ayr1
defect failed to germinate, suggesting that Ayr1p plays an
essential role at this stage. Overexpression of Ayr1p at a 5- to
10-fold level of wild type caused growth arrest. Heterologous expression of Ayr1p in Escherichia coli resulted in gain of
ADR activity in the prokaryote, confirming that YIL124w is the
structural gene of the enzyme and does not encode a regulatory or
auxiliary component required for reduction of 1-acyldihydroxyacetone
phosphate. Taken together, these results identified Ayr1p of the yeast
as the first ADR from any organism at the molecular level.
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INTRODUCTION |
Phosphatidic acid (PA)1
is a key intermediate in the formation of glycerophospholipids and
triacylglycerols. Two different pathways of PA biosynthesis are known:
(i) the glycerol 3-phosphate (Gly-3-P) pathway and (ii) the
dihydroxyacetone phosphate (DHAP) pathway (1). In the former pathway,
Gly-3-P is first acylated to 1-acylglycerol 3-phosphate
(lysophosphatidic acid, LPA) and further converted to PA by a second
acyltransferase. In the DHAP pathway, the precursor DHAP is acylated to
1-acyl-DHAP. This intermediate has to be reduced to LPA in an
NADPH-dependent reaction before its conversion to PA (Fig.
1). Whereas in plants and bacteria, PA
formation occurs only through the Gly-3-P pathway (2, 3), both pathways
are active in mammals (2, 4, 5) and yeast (6-8).

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Fig. 1.
Pathways of phosphatidic acid biosynthesis in
the yeast. Metabolites: Gly-3-P (G-3-P), DHAP,
1-acyl-Gly-3-P (1-acyl-G-3-P), 1-acyl-DHAP, PA. Enzymes:
glycerol-3-phosphate acyltransferase (GAT),
dihydroxyacetone-phosphate acyltransferase (DHAPAT), ADR,
and 1-acylglycerol-3-phosphate acyltransferase (AGAT).
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In mammalian cells, enzymes catalyzing PA formation were reported to be
localized to mitochondria, the microsomal fraction, and peroxisomes (2,
4, 5). Whereas in mitochondria only Gly-3-P and, in peroxisomes, only
DHAP serve as precursors, both substrates can be acylated in the
microsomal compartment (9). In the yeast Saccharomyces
cerevisiae, the highest specific activity of glycerol-3-phosphate
acyltransferase was detected in lipid particles (10-12). The second
site of glycerol-3-phosphate acyltransferase activity in the yeast is
the endoplasmic reticulum (ER), whereas other organelles,
e.g. mitochondria or vacuoles, are largely devoid of enzymes
forming LPA from the precursor Gly-3-P (11, 12). The highest specific
activity of DHAP acylation was found in lipid particles followed by
microsomes. Surprisingly, 1-acyl-DHAP is also formed with mitochondria
as the enzyme source although at a reduced specific activity (8). It
was shown that the same enzyme, the hypothetical Gat1p, catalyzes
acylation of both precursors, Gly-3-P and DHAP, in lipid particles (8).
Gat1p is also present in the ER, but this compartment contains at least
one additional set of acyltransferases involved in PA formation. DHAP
acyltransferase of mitochondria, however, appears to be an enzyme
distinct from Gat1p (8, 12). Collectively, different organelles of the yeast contribute to PA formation and may interact during this process.
1-Acyl-DHAP formed during PA synthesis through the DHAP pathway is
reduced to LPA by 1-acyl-DHAP reductase (ADR) (13). In animal cells,
ADR is a key enzyme for the formation of ether lipids and
acylglycerolipids via the DHAP pathway (14). Localization studies
demonstrated that acyl/alkyl-DHAP reductase of guinea pig liver cells,
like other enzymes of the DHAP pathway, is mainly present in
peroxisomes, although some activity of this enzyme was also detected in
the ER (14). Amino acid analysis of acyl/alkyl-DHAP reductase purified
from guinea pig liver peroxisomes revealed that hydrophobic amino acids
composed 27% of the molecule; the amino acid sequence of this protein,
however, was not determined. In the yeast S. cerevisiae ADR
has been detected by its enzymatic activity (13), but neither the gene
nor the gene product was characterized at a molecular level. Yeast ADR
activity is present in lipid particles and the ER (30,000 × g microsomes), whereas mitochondria appear to be devoid of
this enzyme (8).
The present paper describes identification of AYR1, the
structural gene encoding the major ADR of yeast. Dual localization of
Ayr1p in lipid particles and the ER and the existence of at least one
additional ADR activity in the ER are demonstrated. Phenotypic
consequences of an ayr1 deletion are discussed.
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EXPERIMENTAL PROCEDURES |
Strains and Culture Conditions--
The wild-type yeast strains
S. cerevisiae DBY746 (MAT , his3- 1, leu2-3,
leu2-112, ura3-52, trp1-289) and FY1679 (Mat
a/ , ura 3-52/ura 3-52, leu2 1/LEU,
his3 200/HIS3, trp1 63/TRP1, GAL2/GAL2) and mutants deleted of
YIL124w (MAT , his3- 1, leu2-3, leu2-112, ura3-52,
trp1-289, ayr1::kanMX4), YIR036c (MATa,
his3- 1, leu2-3, leu2-112, ura3-52, trp1-289,
yir036c::kanMX4), and YMR226c (MATa
his3- 1, leu2-3, leu2-112, ura3-52, trp1-289,
ymr226c::kanMX4) in the DBY746 background were used
throughout this study.
Cells were grown aerobically in 2-liter Erlenmeyer flasks to the late
logarithmic phase at 30 °C in YPD medium, pH 5.5, containing 1%
yeast extract (Oxoid), 2% peptone (Oxoid), and 2% glucose (Merck). Five hundred ml of culture medium were inoculated with 0.5 ml of a
preculture grown aerobically for 48 h in YPD medium.
For the heterologous expression of yeast 1-acyl-DHAP reductase, the
Escherichia coli strain TOP10F'
F'(lacIqTn10(TetR)) mcrA
(mrr-hsdRMS-mcrBC)
80lacZ M15 lacX74 deoR
recA araD139 (ara-leu)7697
galK rpsL endA1 nupG was
used. Wild-type E. coli and transformants were grown at
37 °C in LB medium containing 10 g of tryptone, 5 g of
NaCl, and 5 g of yeast extract/liter.
Deletion, Overexpression, and Heterologous Expression of
YIL124w--
A dominant resistance marker module, kanMX4, containing
the kanr gene of the E. coli
transposon Tn903, which encodes aminoglycoside phosphotransferase (15), was included in vector pFA6a (16) and used to
replace yeast ORFs. Aminoglycoside phosphotransferase activity renders
S. cerevisiae resistant to the drug geneticin (G418;
Calbiochem) (17). A replacement strategy making use of short flanking
homology regions to the target locus (Fig.
2A) was used to construct
deletion cassettes by polymerase chain reaction (PCR) (16, 18).
Deletion cassettes contained the ATG codon of the ORF to be deleted,
the kanMX4 gene, and the stop codon of the ORF, thus
eliminating 100% of the target ORF. All deletions were made in the
DBY746 or FY1679 background.

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Fig. 2.
A, deletion strategy and choice of
primers for the replacement of ORFs by PCR. The short flanking homology
(SFH) strategy (16, 18) used for gene replacement is
described under "Experimental Procedures." B, primers
used for the replacement of the respective ORFs by kanMX4. The
underlined sequence is homologous to kanMX4. C,
primers used for the insertion of YIL124w into the plasmid pYES2 (see
"Experimental Procedures").
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To generate marker DNA flanked by short homology regions, a pair of
oligonucleotide primers with 70 nucleotides homologous to the target
locus at the 5' end followed by 18-19 nucleotides homologous to
pFA6a-kanMX4 multiple cloning sites at the 3' end of the primer were
chosen (Fig. 2B). A 1.65-kilobase PCR fragment was generated
with Pwo polymerase (Roche Molecular Biochemicals) using approximately
50 ng of gel-purified NotI-digested pFA6a-kanMX4 plasmid or
150 ng of plasmid as a template in a standard PCR mixture. The PCR
standard mixture contained the PCR buffer (10 mM
Tris/Cl , pH 8.85, 25 mM KCl, 5 mM
(NH4)2SO4) with 2 mM
MgSO4, 0.2 mM deoxynucleoside triphosphates
(each), and 1 µM primers in a total volume of 25 µl.
After a denaturation step for 5 min at 94 °C, fragments were
amplified during 10 cycles for 30 s at 94 °C, 30 s at
54 °C, 105 s at 72 °C, and 20 cycles for 30 s at
94 °C, 30 s at 65 °C, 105 s at 72 °C followed by a
final elongation step for 12 min at 72 °C. PCR fragments were
ethanol-precipitated, and 400-700 ng were used for transformations.
Yeast cells were transformed using the high efficiency lithium acetate
transformation protocol (19). Transformants were grown in liquid YPD at
30 °C overnight and then spread on YPD plates containing 200 mg/l
G418 (Calbiochem). After incubation for 2-3 days, large colonies were
transferred to fresh YPD-G418 plates. Only those clones that yielded
colonies were considered as positive transformants and further checked
for correct integration of the respective deletion cassette.
Correct replacement of the respective ORFs by the kanMX4 module in
G418-resistant transformants was verified by analytical PCR using
Dynazyme polymerase with whole yeast cell extracts (20). Oligonucleotides were designed to bind outside the target locus, within
the target locus and within the marker module.
Diploid yeast transformants were sporulated in liquid medium containing
0.3% potassium acetate and 0.02% raffinose with or without addition
of 1% sucrose for 3 to 5 days at room temperature. Tetrad dissection
was performed on YPD plates.
For overexpression and heterologous expression YIL124w was inserted
into the plasmid pYES2 (Invitrogen). The ORF was amplified by PCR with
genomic DNA derived from a wild-type yeast strain as template. Primers
used for amplification are shown in Fig. 2C. S1 contained a
BamHI, and S2 contained an EcoRI cleavage site. PCR was performed using the same standard reaction mixture as described
above with approximately 150 ng of genomic DNA as template. After
denaturation for 5 min at 94 °C, the fragment was amplified during
10 cycles for 30 s at 94 °C, 30 s at 53 °C, 80 s
at 72 °C, and 20 cycles for 30 s at 94 °C, 30 s at
65 °C, 80 s at 72 °C followed by a final elongation step for
12 min at 72 °C. The PCR products were purified with a QIAquick
purification kit (Qiagen), and DNA fragments and plasmids were cleaved
with EcoRI and BamHI. The DNA fragment was
inserted into the multiple cloning site of the plasmid, which was used
for transformation of yeast and E. coli Top10 (21).
Isolation and Characterization of Subcellular
Fractions--
Lipid particles were obtained at high purity by the
method of Leber et al. (22) from yeast cells grown to the
late logarithmic phase. Microsomal fractions used in this study were
prepared as described by Zinser et al. (11). Relative
enrichment of markers and cross-contamination were as described by
Zinser and Daum (23).
To study overexpression of Ayr1p in yeast, cells transformed with pYES2
bearing YIL124w under a GAL+ promoter were grown on yeast
extract, peptone, 2% galactose. At different time points, aliquots of
the culture corresponding to an A600 of 3.0 were
harvested by centrifugation. Samples were suspended in 0.5 ml of
culture medium and incubated with 50 µl of 1.85 M NaOH
for 10 min on ice. Then, 50 µl of trichloroacetic acid (50%) was
added for a further incubation of 1 h on ice. The resulting
precipitate was isolated by centrifugation, dissolved in 70 µl of
sample buffer (24), and heated for 30 min at 37 °C. Aliquots were
subjected to SDS-polyacrylamide gel electrophoresis and Western blot
analysis as described below.
Homogenate of E. coli was obtained from cultures grown
overnight at 37 °C. Cells were harvested and disintegrated by glass beading using a Merckenschlager homogenizer (25).
Protein Analysis--
Protein was quantified by the method of
Lowry et al. (26) using bovine serum albumin as a standard.
Proteins were precipitated with trichloroacetic acid at a final
concentration of 10%. The protein pellet was solubilized in 0.1% SDS,
0.1% NaOH. Before protein analysis samples of the lipid particle
fraction were delipidated. Nonpolar lipids were extracted with 2 volumes of diethyl ether, the organic phase was withdrawn, residual
diethyl ether was removed under a stream of nitrogen, and proteins were
precipitated from the aqueous phase as described above.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried
out by the method of Laemmli (24). Samples were dissociated at 37 °C
to avoid hydrolysis of polypeptides, which may occur at higher
temperature. Western blot analysis was performed as described by Haid
and Suissa (27). Immunoreactive proteins were detected by enzyme-linked
immunosorbent assay using rabbit antisera as the first antibody and
goat anti-rabbit IgG linked to peroxidase as the second antibody. For
the generation of antibodies against Ayr1p, the polypeptide was
purified from lipid particles by SDS-polyacrylamide gel electrophoresis
and eluted from gel slices using an Electro Eluter model 422 (Bio-Rad).
This protein solution was used to immunize rabbits by standard procedures.
Amino acid sequence analysis of proteins separated by
SDS-polyacrylamide gel electrophoresis was performed by Protana
(Odense, Denmark) following the procedures of Shevchenko et
al. (28). Briefly, proteins in gel slices were reduced with
dithiothreitol and alkylated with iodoacetamide followed by overnight
digestion with trypsin. Peptides generated by this procedure were
extracted with 50% acetonitrile and 5% formic acid. The mixture of
lyophilized peptides was purified on micro-columns with POROS R2
perfusion chromatography material and analyzed on a Finnigan LCQ ion
trap mass spectrometer (Finnigan, San Jose, CA) equipped with a
nanoelectrospray source. Proteins were identified by querying a
nonredundant sequence data base containing more than 300,000 entries
with partial amino acid sequences (peptide sequence tags) deduced from
mass spectroscopy spectra. The software used for this search was
PepSea, Version 1.0 (Protana A/S, Odense, Denmark).
Molecular data about proteins were obtained from the yeast protein data
base, Saccharomyces genome data base, SwissProt, and Munich
Information Center for Protein Sequences. Homology searches were
performed with BLAST Search (29).
Enzyme Analysis--
Enzyme activity of ADR was measured as
described by Bates and Saggerson (30). Yeast homogenate (2-3 mg of
protein), lipid particles (40-80 µg of protein), or microsomes
(0.3-1.0 mg of protein) were used as the enzyme sources. Samples were
incubated at 30 °C in a final volume of 1 ml of 120 mM
KCl, 50 mM Tris/Cl , pH 7.5, 4 mM
MgCl2, 8 mM NaF, 4 mg of bovine serum albumin, 65 nmol of oleoyl-CoA, 80 mM NADPH, 500 nmol of
[U-14C]fructose 1,6-bisphosphate (0.4 µCi), 0.44 units
of aldolase and 28 units of triose phosphate isomerase. A 600-µl
portion of the reaction mixture was preincubated for 16 min at 30 °C
before the addition of 400 µl of samples containing the respective
enzyme source. Incubations were carried out for 10 min at 30 °C and
terminated by the addition of 3 ml of chloroform/methanol (1:2; v/v)
and 0.7 ml 1% perchloric acid. The organic phase was washed three times with 2 ml of 1% perchloric acid each, and total radioactivity was measured by liquid scintillation counting using a 1500 Tricarb Beckman scintillation counter. For the analysis of individual lipids
the extract was applied to high performance TLC plates (silica gel 60;
Merck), and chromatograms were developed in an ascending manner using
the solvent system chloroform, methanol, acetic acid, 5% sodium
metabisulfate (100:40:12:4; vol.). After chromatographic separation,
the radioactively labeled lipids formed during the assay were detected
by thin layer chromatography scanning using a Tracemaster 20 automatic
TLC linear analyzer (Berthold). In addition, lipids were visualized on
high performance TLC plates by staining with iodine vapor, bands were
scraped off, and radioactivity was measured by liquid scintillation
counting using LSC Safety (Baker, Deventer, The Netherlands) + 5%
water as a scintillation mixture.
For an alternative assay, 1-acyl-DHAP was generated with lipid
particles from the wild-type strain following the procedure described
above but without the addition of NADPH to the assay mixture. The
resulting 1-acyl-DHAP was extracted with chloroform/methanol (1:2; v/v)
and used as substrate for conversion to PA in an assay mixture
containing 120 mM KCl, 50 mM
Tris/Cl , pH 7.5, 4 mM MgCl2, 8 mM NaF, 4 mg of bovine serum albumin, 65 nmol of
oleoyl-CoA, and 80 mM NADPH in a final volume of 350 µl.
After the addition of the enzyme source (40-80 µg of lipid particle
protein or 2-4 mg of E. coli homogenate, respectively), incubations were carried out for 10 min at 30 °C. Termination of the
assay by lipid extraction and analysis of products were carried out as
described above.
Lipid Analysis--
Lipids of whole yeast cells were extracted
after disruption of cells with glass beads by the procedure of Folch
et al. (31). Individual phospholipids were separated by
two-dimensional thin layer chromatography (8) and quantified by the
method of Broekhuyse (32). Triacylglycerols, ergosterol, and ergosteryl
esters were quantified as described by Athenstaedt et al.
(8). Individual sterols were analyzed after alkaline hydrolysis (33) of
the lipid extract by gas liquid chromatography. Gas liquid
chromatography was performed on a Hewlett-Packard 5890 equipped with a
flame ionization detector operated an 320 °C using a capillary
column (Hewlett-Packard 5, 30m × 0.32 mm × 0.25-µm film thickness).
After a 1-min hold at 150 °C, the temperature was increased to
310 °C at 10 °C/min. The final temperature was held for 10 min.
Nitrogen was used as the carrier gas, and 1-µl aliquots of samples
were injected onto the column. Relative retention times of sterols were
similar as described previously (34, 35).
Fatty acids were also analyzed by gas liquid chromatography. Lipids
extracted as described above were subjected to methanolysis using
BF3/methanol (14%) and converted to methyl esters (36). Fatty acid methyl esters were separated by gas liquid chromatography using the same equipment as described above. A temperature program of 2 min at 150 °C, then 10 °C/min to 300 °C was used. Fatty acids were identified by comparison to commercial fatty acid methyl ester
standards (NuCheck, Inc., Elysian, MN).
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RESULTS |
Two lines of evidence led us to identify AYR1, the gene
encoding a 1-acyldihydroxyacetone-phosphate
reductase (ADR) of the yeast S. cerevisiae.
First, enzymatic analysis using isolated subcellular fractions revealed
that ADR activity is present in lipid particles and the ER (30,000 × g microsomes) (8). Second, systematic amino acid sequence
analysis of yeast lipid particle proteins by mass spectrometry resulted
in the identification of 15 ORFs encoding polypeptides associated with
this compartment (37). One of these gene products with an apparent
molecular mass of 33 kDa (Fig. 3), which
is encoded by the ORF YIL124w, exhibits homology to an insect-type
alcohol/ribitol dehydrogenase and was the only putative protein of
yeast lipid particles with an oxidoreductase motif. At the N terminus
of Ayr1p (amino acids 13-37), an NADPH binding site, was identified by
analogy to NADPH-dependent enzymes of other cell types. The
tyrosine residue 157 (Y) of the protein may be part of the active site
of this enzyme.

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Fig. 3.
Amino acid sequence of the Ayr1p. The
underlined stretch of amino acids is the NADPH binding site
of the protein, and tyrosine residue 157 (bold and
double-underlined) may be part of the active site of the
enzyme according to the yeast genome data base.
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Fig. 4 shows the hydropathy blot of the
polypeptide encoded by AYR1. This protein does not contain
transmembrane-spanning domains but two hydrophobic stretches
(hydrophobicity index 2) (38), one in the middle of the molecule and
one at the C terminus. Lack of transmembrane domains and the presence
of only few hydrophobic domains had been demonstrated to be a common
feature of lipid particle proteins identified so far (37).
To test whether AYR1 indeed encodes ADR, a mutant deleted of
this ORF was constructed. When the gene deletion was carried out in a
diploid wild-type strain, subsequent tetrad analysis yielded two
colony-forming spores per tetrad, whereas two spores failed to
germinate. The viable colonies lacked the kanMX4 marker (data not
shown), indicating that the germination defect was caused by deletion
of AYR1. When deletion of AYR1 was performed with a haploid wild-type strain, transformants were viable, grew like wild
type on glucose, glycerol, ethanol, and lactate and neither exhibited
temperature nor cold sensitivity. Phospholipid pattern, neutral lipid
composition, fatty acid composition, and sterol pattern of total cell
extracts of the deletion strain were also the same as in the
corresponding wild type (data not shown). To confirm the germination
defect of ayr1, the haploid deletion strain was back-crossed
to wild type. When the heterozygous
(AYR1+/ayr1 ) diploid strain
was sporulated and subjected to tetrad analysis, only those spores were
able to germinate that were bearing the wild-type AYR1
allele. The same result was obtained with the wild-types DBY746 and
FY1679, thus indicating that the observed effect was not
strain-dependent. Thus, AYR1 is essential for
spore germination but not for vegetative growth of yeast cells.
Comparison of the protein patterns of lipid particles isolated from the
ayr1 deletion strain and the corresponding wild type showed
that a protein with an apparent molecular mass of 33 kDa was missing in
the mutant (data not shown). This result was confirmed by Western blot
analysis using a monospecific antibody raised against Ayr1p (Fig.
5). The presence of Ayr1p, however, is
not only restricted to lipid particles, but this protein was also detected in the ER (30,000 × g microsomal fraction) of
the wild type, although at a smaller amount. The enrichment factor of
Ayr1p in the lipid particle fraction was approximately 300 to 500 over the homogenate, but only 1 in the microsomes. Other organelles of wild
type such as mitochondria or vacuoles were found to be devoid of the
polypeptide. In the ayr1 deletion strain, the immunoreactive protein was missing in the homogenate, in lipid particles, and in
30,000 × g microsomes (Fig. 5).

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Fig. 5.
Subcellular localization of Ayr1p.
Homogenates of the wild-type DBY746 (1) and the
ayr1 deletion strain (2), lipid particles of wild
type (3) and ayr1 (4), and 30,000 × g microsomes (endoplasmic reticulum) of wild type
(5) and ayr1 (6) were tested by
Western blot analysis using a monospecific rabbit antiserum against
Ayr1p. The amount of total protein per lane was 10 µg (lanes 1, 2, 5, and 6) or 2 µg (lanes 3 and
4), respectively.
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Enzymatic analysis with wild-type organelles revealed that only lipid
particles and 30,000 × g microsomes contained ADR
activity that is in line with data of the Western blot analysis (see
Fig. 5). Since these two compartments contain also DHAP acyltransferase and 1-acyl-Gly-3-P acyltransferase activities (8), they are able to
synthesize PA from the precursor DHAP. Fig.
6 shows relative amounts of products
formed in an in vitro assay with lipid particles or
microsomes of wild type and ayr1 as the enzyme source. Lipid particles of the ayr1 mutant completely lack the ability to
reduce 1-acyl-DHAP, resulting in the accumulation of this intermediate. ADR activity in the ER fraction (30,000 × g
microsomes) of the deletion strain was also markedly reduced, and
1-acyl-DHAP accumulated in this fraction to some extent, indicating
that Ayr1p is also a component of microsomes and contributes
significantly to the DHAP pathway of PA biosynthesis in this
compartment. In contrast to lipid particles, however, microsomes of the
ayr1 strain retained the ability to form a small but
significant amount of PA (30% of wild-type level), indicating that
residual ADR activity was still present in this organelle. Thus,
AYR1 encodes a protein that is the only ADR of lipid
particles but only one isoenzyme with ADR activity of the ER. This
result is reminiscent of the dual localization and the redundancy of
glycerol-3-phosphate acyltransferase (Gat1p) and
1-acylglycerol-3-phosphate acyltransferase (Slc1p) (12). Similar
observations with squalene epoxidase, Erg1p, had led us previously to
speculate about a relationship of these two compartments and the
hypothesis that lipid particles might originate from the ER (39).

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Fig. 6.
Lipid products formed from DHAP in
lipid particles and 30,000 × g microsomes of the
wild-type DBY746 and the ayr1 deletion strain.
Data are the mean values of three independent experiments with a
deviation of ± 7%. AD, 1-acyldihydroxyacetone
phosphate.
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Searches for yeast homologues of Ayr1p, which might catalyze the
residual ADR activity in the ER of the ayr1 deletion strain, identified two ORFs of unknown function, namely YMR226c and YIR036c, as
candidates to encode ADR isoenzymes. YMR226c encodes a hypothetical protein with similarity to ketoreductases and has 35% identity to
YIL124w. YIR036c encodes another hypothetical protein that is a
probable member of the short chain family of alcohol dehydrogenases and
exhibits 28% identity to YIL124w. Deletions of YMR226c and YIR036c,
however, did not affect ADR activity in the microsomal fraction (data not shown). Thus, it is unlikely that YMR226c and YIR036c encode enzymes that significantly contribute to ADR activity of
the yeast. Further studies, however, will be needed to establish whether these genes encode ADR isoforms with minor enzymatic activity.
As an alternative to the occurrence of ADR isoforms in different
cellular compartments, residual ADR activity in microsomes of the
ayr1 deletion strain may be result of a bypass reaction. In
such a pathway 1-acyl-DHAP may be dephosphorylated followed by
reduction of 1-acyldihydroxyacetone to 1-acylglycerol. Then, a kinase
may phosphorylate this intermediate to 1-acylglycerol 3-phosphate
(LPA), which can re-enter the regular pathway of PA formation. The
enzyme activities required for such a bypass are present in animal
cells (40) and may also exist in yeast.
Results presented so far did not prove unambiguously that YIL124w is
the structural gene encoding ADR. To distinguish between the possible
role of Ayr1p as ADR enzyme or effector of ADR activity, the protein
was heterologously expressed in E. coli. As mentioned in the
Introduction, plants and bacteria lack the DHAP pathway for PA
biosynthesis, and DHAP is converted to Gly-3-P in an
NADH-dependent reaction before the two steps of acylation
that yield PA. For this reason radioactively labeled 1-acyl-DHAP had to
be used as a substrate for the enzyme assay with E. coli
homogenate as an enzyme source (see "Experimental Procedures"). As
shown in Fig. 7, yeast Ayr1p expressed in
E. coli can convert 1-acyl-DHAP to 1-acyl-Gly-3-P (LPA)
which is further metabolized to PA by the bacterial acyltransferase.
The positive control with lipid particles of the yeast wild-type strain
demonstrated that assay conditions were appropriate. The negative
control with E. coli bearing the empty plasmid showed that
ADR activity in E. coli was indeed due to the presence of
the AYR1 gene product. In the absence of NADPH, only
background activity was observed in all three assays.

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Fig. 7.
Heterologous expression of Ayr1p in E. coli. Formation of PA from the precursor 1-acyl-DHAP
was measured in a standard assay (see "Experimental Procedures")
with lipid particles of the wild-type DBY746 (A), homogenate
of E. coli transformed with the pYES2 plasmid bearing
YIL124w (B), and homogenate of E. coli
transformed with the empty pYES2 plasmid (C). Assays were
carried out in the presence (black bars) or absence
(gray bars) of NADPH.
|
|
To study overexpression of Ayr1p in yeast, an ayr1 deletion
strain was transformed with a multicopy plasmid bearing AYR1
under control of a GAL+ promoter. When these transformants
were shifted from glucose- or raffinose-containing media to inducing
conditions (galactose-containing medium), cells stopped growing within
1 h. During the period of induction, the amount of Ayr1p increased
5- to 10-fold as compared with noninducing conditions and remained
constant for more than 50 h. When transformants were shifted back
from inducing to noninducing conditions at any time point during this
period, cells recovered and formed growing colonies. Thus, either the
increased amount of Ayr1p or accumulation of metabolic intermediates
formed upon the induction caused a reversible growth arrest.
 |
DISCUSSION |
Only a few genes and gene products involved in the biosynthesis of
PA in the yeast have been identified at a molecular level. The two
components characterized so far are Slc1p, a 1-acylglycerol-3-phosphate acyltransferase (12, 24), and Ayr1p, a reductase catalyzing the
conversion of 1-acyl-DHAP to LPA, described in this study. Most
noteworthy, Ayr1p of the yeast is the first enzyme of this type
characterized at a molecular level.
The redundancy of enzymes involved in PA biosynthesis and their
localization to different subcellular compartments (8, 12), namely
lipid particles, the ER, and mitochondria, raises the question as to
the interplay of organelles during synthesis of this key intermediate
of lipid metabolism. As an example, 1-acyl-DHAP formed through
acylation of DHAP in mitochondria cannot be further metabolized in this
compartment, because mitochondria lack ADR. As a consequence,
1-acyl-DHAP has to be transported to a site of ADR activity, namely
lipid particles or the ER, to get converted to LPA. This process may
not require a specific transport mechanism, since 1-acyl-DHAP is
assumed to be largely water-soluble and might reach the site of
reduction by diffusion. As an alternative, translocation of 1-acyl-DHAP
may occur through membrane contact between the ER and mitochondria. A
specific subfraction of the ER that associates with mitochondria (MAM,
mitochondria associated membrane)
of the yeast (41, 42) may be involved in this process.
The obvious reason for a missing growth defect caused by an
ayr1 deletion is the presence of additional ADRs or a bypass
pathway to form PA in the ER. Thus, involvement of Ayr1p in PA
formation through the DHAP pathway is not essential for vegetative
growth of the yeast. The notable property of the ayr1
deletion strain, however, is its germination defect. This result
suggests that a certain level of intermediates of the DHAP pathway may
be required for spores to germinate.
Overexpression of AYR1 in yeast resulted in growth arrest
but left cells viable. Thus, larger quantities of lysophospholipids accumulating in an overexpressing strain may disturb membrane proliferation. As a hypothetical alternative, ADR present in excess may
act as a reductase of other substrates. The respective products might
inhibit cell proliferation or prevent the formation of intermediates that are required for cell growth.
In animal cells, the DHAP pathway for PA formation is not only used for
the synthesis of diacylglycerolipids but is also obligatory for the
formation of ether lipids. In yeast, incorporation of DHAP appears to
be restricted to glycerolipids in vitro (20) and in
vivo (6, 8). The possible occurrence of ether lipids in this
microorganism has been a long-standing matter of dispute. Most recently
it was shown by analysis of isolated organelle membranes that alkyl
ether lipids may be present at trace amounts in the yeast S. cerevisiae (43). A mutant lacking total ADR activity would be
required to demonstrate whether or not yeast is able to synthesize
alkyl ether lipids from DHAP as a precursor. Such a mutant would also
allow the study of the possible physiological role of alkyl ether
lipids and the contribution of the DHAP pathway to overall glycerolipid
synthesis in yeast.
 |
FOOTNOTES |
*
This work was financially supported by the Fonds zur
Förderung der Wissenschaftlichen Forschung in Österreich
(projects 11491 and F706), the Austrian Ministry of Science and
Transportation (project 950080), and EUROFAN project BIO-CT95-0080.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.: 43-316-873-6462;
Fax: 43-316-873-6952; E-mail: f548daum@mbox.tu-graz.ac.at.
 |
ABBREVIATIONS |
The abbreviations used are:
PA, phosphatidic
acid;
Gly-3-P, glycerol 3-phosphate;
DHAP, dihydroxyacetone phosphate;
LPA, lysophosphatidic acid;
ER, endoplasmic reticulum;
ADR, 1-acyldihydroxyacetone-phosphate reductase;
YPD, yeast
extract/peptone/dextrose;
ORF, open reading frame;
PCR, polymerase
chain reaction.
 |
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