From the Kennedy Krieger Institute and Departments of
Neurology, ¶ Pediatrics, and
Biological
Chemistry, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
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Activation of fatty acids to their coenzyme A derivatives is necessary for subsequent metabolism. Very long-chain fatty acids, which accumulate in tissues of patients with X-linked adrenoleukodystrophy, are activated by very long-chain acyl-CoA synthetase (VLCS) normally found in peroxisomes and microsomes. We identified a candidate yeast VLCS gene (FAT1), previously identified as encoding a fatty acid transport protein, by its homology to rat liver peroxisomal VLCS. Disruption of this gene decreased, but did not abolish, cellular VLCS activity. Fractionation studies showed that VLCS activity, but not long-chain acyl-CoA synthetase activity, was reduced to about 40% of wild-type level in both 27,000 × g supernatant and pellet fractions. Separation of organelles in the pellet fraction by density gradient centrifugation revealed that VLCS activity was associated with peroxisomes and microsomes but not mitochondria. FAT1 deletion strains exhibited decreased growth on medium containing dextrose, oleic acid, and cerulenin, an inhibitor of fatty acid synthesis. FAT1 deletion strains grown on either dextrose or oleic acid medium accumulated very long-chain fatty acids. Compared with wild-type yeast, C22:0, C24:0, and C26:0 levels were increased approximately 20-, 18-, and 3-fold in deletion strains grown on dextrose, and 2-, 7-, and 5-fold in deletion strains grown on oleate. Long-chain fatty acid levels in wild-type and deletion strains were not significantly different. All biochemical defects in FAT1 deletion strains were restored to normal after functional complementation with the FAT1 gene. The level of VLCS activity measured in both wild-type and deletion yeast strains transformed with FAT1 cDNA paralleled the level of expression of the transgene. The extent of both the decrease in peroxisomal VLCS activity and the very long-chain fatty acid accumulation in the yeast FAT1 deletion model resembles that observed in cells from X-linked adrenoleukodystrophy patients. These studies suggest that the FAT1 gene product has VLCS activity that is essential for normal cellular very long-chain fatty acid homeostasis.
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INTRODUCTION |
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Metabolism of fatty acids by either catabolic or anabolic pathways
requires initial activation to their coenzyme A
(CoA)1 thioesters, a reaction
catalyzed by acyl-CoA synthetase (1). Differences in substrate chain
length specificity and subcellular localization distinguish the various
acyl-CoA synthetases present in cells from all species examined,
including animals, yeast, bacteria, and plants (1). Long-chain fatty
acids (C12-C20) are activated by long-chain acyl-CoA synthetases
(LCSs) found in mitochondria, peroxisomes, and microsomes (1),
reflecting the primary roles of these organelles in fatty acid
-oxidation (mitochondria and peroxisomes) and complex lipid
synthesis (microsomes). In contrast, activation of very long-chain
fatty acids (VLCFA; C22 or longer) by very long-chain acyl-CoA
synthetase (VLCS) takes place only in peroxisomal and microsomal
subcellular fractions of rat (2), human (3), and yeast (4) cells. Lack
of mitochondrial VLCS prevents this organelle from catabolizing VLCFA
via
-oxidation, thus relegating this function to the peroxisome.
VLCFA -oxidation is impaired in cells from patients with the
progressive neurodegenerative disease X-linked adrenoleukodystrophy (XALD), resulting in elevated plasma and tissue concentrations of
saturated VLCFA (5). Biochemical studies revealed that VLCS activity
was decreased in peroxisomal, but not microsomal, subcellular fractions
of fibroblasts from XALD patients (3, 6). However, further
investigation of the molecular defect in XALD revealed that key steps
in peroxisomal VLCFA metabolism are still poorly understood. The
product of the gene defective in XALD (ALDP) is not VLCS, but rather is
a peroxisomal membrane protein whose existence was previously unknown
(7). Studies in many laboratories, including our own (8), clearly
demonstrated that XALD patients have mutations in the ALD gene, and
complementation studies confirmed that transfection of XALD cells with
the ALD gene restores their ability to catabolize VLCFA (9, 10). While
studies of XALD suggest that ALDP is required for VLCFA activation, the
biochemical relationship between ALDP and VLCS has not yet been
elucidated. Thus, to further our understanding of peroxisomal VLCFA
activation, we are investigating this process in yeast.
Yeasts are unique model organisms for the study of peroxisomal fatty
acid -oxidation, since they lack the mitochondrial
-oxidation pathway found in higher animals. They have been valuable for
identification of proteins subsequently shown to be defective in human
disorders of peroxisome biogenesis (11-16). We previously reported
that, as in mammalian tissues, peroxisomes and microsomes isolated from the yeast Pichia pastoris contain VLCS activity (4). The
yeast Saccharomyces cerevisiae has been used by Gordon and
co-workers to identify and characterize four fatty
acid activation (FAA1-4) genes that
encode acyl-CoA synthetases (17, 18). They examined the fatty acid
substrate specificity of the protein products of three of these
FAA genes (Faa1p, Faa2p, and Faa3p) in detail; while Faa3p
weakly activated VLCFA, Faa1p and Faa2p had no detectable activity with
these substrates (17). Faa4p was reported to be functionally
interchangeable with Faa1p and thus is not likely to be a VLCS (18).
Based on growth studies of yeast mutants in which all four
FAA genes were disrupted, Gordon and colleagues concluded
that at least one other FAA gene exists in S. cerevisiae (18).
Recently, Hashimoto and co-workers purified an enzyme with VLCS activity from rat liver peroxisomes (rVLCS) (19) and subsequently cloned and sequenced its cDNA (20). We identified an open reading frame on S. cerevisiae chromosome II (YBR041W; locus FAT1) with homology to the rVLCS amino acid sequence. Both rVLCS and the FAT1 gene product, Fat1p, are homologous to mouse fatty acid transport protein (mFATP), described by Schaffer and Lodish (21). Faergeman et al. (22) independently identified the FAT1 (fatty acid transporter 1) gene by virtue of the homology of Fat1p to mFATP. These investigators found that deletion of FAT1 resulted in decreased rates of cellular uptake of labeled long-chain fatty acid, decreased cellular uptake of fluorescent fatty acids, and decreased uptake and incorporation of labeled oleic acid into cellular phospholipids (22). Although they noted amino acid sequence similarities between Fat1p and acyl-CoA synthetases, these investigators found that deletion of the FAT1 gene did not affect LCS activity (22). However, they did not investigate either the role of Fat1p in VLCFA metabolism or the subcellular localization of this protein. In this report, we present evidence that Fat1p has VLCS activity and is present in both peroxisomes and a 27,000 × g supernatant fraction of S. cerevisiae. Furthermore, deletion of the FAT1 gene resulted in accumulation of VLCFA in yeast cells similar to that observed in plasma and tissues of XALD patients. These findings support the hypothesis that Fat1p is a VLCS whose activity is critical for normal cellular VLCFA homeostasis.
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EXPERIMENTAL PROCEDURES |
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Identification of a Candidate S. Cerevisiae VLCS Gene-- Degenerate oligonucleotide primers (5'-GGYTCRTCYTTYTCNACRTC-3' and 5'-ACNGGNACNCCRTANACRTT-3') corresponding to two peptide fragments (referred to as internal sequences 1 and 2) of the rVLCS (19) were used to amplify by PCR a 348-base pair product using a liver cDNA library from a Gemfibrozil-treated rat as the template. Treatment of rats with the peroxisome proliferator Gemfibrozil was previously found to increase peroxisomal rVLCS activity by about 2-fold.2 The PCR product, containing an open reading frame with 45% amino acid identity to mFATP (21), was labeled with [32P]dCTP and used as a probe to screen the rat liver cDNA library. One clone (0.7 kb) containing an open reading frame of 220 amino acids was obtained; the nucleotide sequence of this clone was subsequently found to be identical to the carboxyl terminus of the rVLCS cDNA sequence later published (20). The 220-amino acid open reading frame was compared with all open reading frames of the complete S. cerevisiae genome (Saccharomyces Genome Data Base) using the BLAST algorithm (23). Only one likely candidate for a homologous yeast protein was identified (smallest sum probability = 9e-24). This gene (open reading frame YBR041W; locus FAT1) is found on chromosome II and is identical to that subsequently described by Faergeman et al. (22). The SWISS-PROT Data base entry for the protein (Fat1p) encoded by the FAT1 gene noted similarity to enzymes that act via an ATP-dependent covalent binding of AMP to their substrate.
Yeast Strains, Media, and Growth Conditions--
S.
cerevisiae strain YPH680 (MAT his3
200 leu2
1
lys2
202 trp1
1 ura3-52) isogenic to YPH681
(MATa), a derivative of S288C (24), was the strain
used for PCR-mediated disruption of the FAT1 gene. Cells
were grown at 30 °C in rich medium containing dextrose as primary
carbon source (YPD, consisting of 10 g/liter yeast extract (Life
Technologies, Inc.), 20 g/liter Bacto-peptone (Difco), and 20 g/liter
dextrose (Sigma)). For induction of peroxisomes, yeasts were grown in
YPD to an A600 of 1. Cells were harvested by
centrifugation at 3000 × g for 5 min, resuspended to
an A600 of 0.5 in medium containing oleic acid
as primary carbon source (YPOLT, where 0.2% (w/v) oleic acid (Sigma)
solubilized with 0.02% (w/v) Tween 40 (Sigma) replaced dextrose), and
grown for 12-14 h at 30 °C (25). Yeast growth characteristics were
assessed on YPD/agar plates supplemented with 500 µM
oleic acid and 0.033% (w/v) Tween 40, with or without 25 µM cerulenin (Sigma); 5 µl of cells diluted to an
A600 of 0.01 were applied to plates and incubated for 16 h at 24 and 30 °C.
Cloning of FAT1 cDNA--
S. cerevisiae genomic
DNA was isolated (26) and used as a template for PCR amplification of
the FAT1 gene. PCR amplification was performed using the
Expand Long Template PCR System (Boehringer Mannheim) with forward
primer 5'-TACTGTCACCTTGATGAGCGAC-3' and reverse primer
5'-TCGATGGCTTCCCAATCAG-3'. Thirty-five cycles of 94 °C (20 s),
57 °C (60 s), and 68 °C (150 s) were performed. The resulting
2.2-kb amplicon containing the FAT1 gene (from 196 to
+1990, where +1 is the first nucleotide of the translation initiation
codon) was TA-cloned into pCR2.1 (Invitrogen) to produce pJFL3301. A
HindIII/XbaI fragment from pJFL3301 was then
cloned into the low copy number (CEN) vector pRS416 (27), producing pJFL3302. DNA sequencing of pJFL3302 was performed at the Johns Hopkins
University Genetics Core Facility using the fluorescent dideoxy
terminator method of cycle sequencing on an Applied Biosystems Inc.
373a automated DNA sequencer, following ABI protocols. Comparison of
the sequence of pJFL3302 with that of FAT1 as contained in the Saccharomyces Genome Data base revealed that the data
base sequence contained a CpC dinucleotide insertion at bp 1857-1858. Four subsequent attempts to amplify an 896-bp fragment containing this
region using forward primer 5'-GGTGAAGGTTGCTTATGGTAACGG-3' and the
reverse primer described above and PCR kits from three different
manufacturers (Boehringer Mannheim, Promega, and Life Technologies,
Inc.) yielded products with sequence identical to that contained in
pJFL3302, ruling out random PCR-generated error. These findings
suggested that the data base sequence may be in error. To verify this
possibility, reverse transcription-PCR of wild-type yeast RNA was
carried out as described below. RNA was treated with DNase I prior to
cDNA synthesis. A 595-bp fragment of the FAT1 gene
containing the putative CpC deletion region was amplified using the
forward primer 5'-TTTCCTTGATAGAATGGGTG-3' and reverse primer
5'-CCTTTCCATATGTTATTAGG-3'. Sequencing of this fragment confirmed that
the FAT1 transcript also lacked the CpC dinucleotide. Thus,
we concluded that the data base sequence was in error, and this has
been communicated to the curator of the Saccharomyces Genome
Data base. Computer-assisted translation of our corrected
FAT1 sequence yielded an open reading frame of 669 amino
acids ending in -IKL, a yeast peroxisome targeting signal (28). The
correct nucleotide sequence of FAT1 and the predicted amino
acid sequence of Fat1p are shown in Fig. 1.
Construction of FAT1 Gene Deletion Strains-- PCR-mediated gene disruption of the FAT1 gene was performed by the method of Lorenz et al. (29). A forward primer consisting of 40 nucleotides of FAT1 sequence upstream of the initiation ATG and 20 left primer sequence-specific nucleotides for pRS plasmids (5'-TCTATATCGTTGAACTTTTAATAGGCTGCGAATACCGACTCTGTGCGGTATTTCACACCG-3') and a reverse primer consisting of 40 nucleotides of FAT1 sequence downstream of the stop codon (as contained in the Saccharomyces Genome Data base; nucleotides 1871-1910) plus 20 right primer sequence-specific nucleotides for pRS plasmids (5'-TCCAAACCCTTTGGTAATTTTTGCTCTCTATAAACCTTCTAGATTGTACTGAGAGTGCAC-3') were used to amplify pRS303 using a two-step program (24). Seven HIS prototrophs were selected following transformation of YPH680 with this PCR product (26). PCR using the forward and reverse primers used to clone FAT1 (described above) and genomic DNA prepared from wild-type and candidate gene disruption strains was performed to confirm disruption of FAT1. PCR products were separated by electrophoresis on 1% agarose gels. Four of the seven strains contained a PCR product of the expected size (1231 bp) for FAT1 disruption. The expected 2186-bp PCR product was observed in wild-type strains.
Induction of Peroxisomes and Subcellular Fractionation of Yeast-- Peroxisomes were induced in wild-type and gene deletion yeast strains by growth in YPOLT medium as described above. Spheroplasts were prepared, homogenized, and fractionated into 27,000 × g supernatant and organelle pellet as described by Crane et al. (25). In some experiments, the organelle pellet was further fractionated by centrifugation on linear Nycodenz (sold as Accudenz by Accurate Chemical and Scientific) gradients (30). 0.5 ml of Maxidens (sold as Purdenz by Accurate Chemical and Scientific) was placed at the bottom of a 13-ml Beckman Quickseal tube, followed by a 10.5-ml linear gradient of 15% Nycodenz and 0.25 M sucrose (top) to 42.5% Nycodenz and 0.125 M sucrose (bottom) in 5 mM MES, pH 6.0, 1 mM EDTA, 1 mM KCl, 0.1% ethanol. The organelle pellet in homogenization buffer (5 mM MES, pH 6.0, 0.6 M sorbitol, 0.5 mM EDTA, 1 mM KCl, 0.1% ethanol) was layered on top of the Nycodenz, and tubes were centrifuged for 75 min at 174,000 × g in a vertical ultracentrifuge rotor. Fractions of 0.5 ml were collected from the bottom of the tube and assayed for acyl-CoA synthetase activity (below) and marker enzyme activity as described previously (4). Because protein cannot be quantitated accurately in Nycodenz solutions, and because this compound had been shown to inhibit other fatty acid metabolic pathways (31), in some experiments, peak peroxisomal and mitochondrial fractions were pooled and dialyzed against 10 mM MES, pH 7.0, prior to assay.
Assay of Acyl-CoA Synthetase Activity--
Acyl-CoA synthetase
activity was measured essentially as described previously (32). Labeled
fatty acids, [1-14C]lignoceric acid (C24:0; American
Radiolabeled Chemicals) and [1-14C]palmitic acid (C16:0;
NEN Life Science Products) were solubilized in -cyclodextrin (Sigma)
and incubated with enzyme for 20 min at 30 °C in 40 mM
Tris-Cl, pH 7.5, containing 10 mM ATP, 5 mM MgCl2, 0.2 mM CoA, and 0.2 mM
dithiothreitol. Labeled acyl-CoAs were extracted from the reaction
mixture by the method of Dole (33) and quantitated in a Beckman LS 3801 liquid scintillation counter. Protein was determined by the method of
Lowry et al. (34).
Reverse Transcription-PCR of FAT1 cDNA-- Total RNA was isolated from spheroplasts prepared from yeast, grown in either YPD or YPOLT medium, using an RNeasy kit (Qiagen). cDNA synthesis was from 1.0 µg of RNA (pretreated with DNase I; Boehringer Mannheim) from yeast grown under each condition and utilized 20 ng of (dT)15 (Boehringer Mannheim) and 100 ng of hexanucleotides (Sigma) as primers. The reaction was carried out at 42 °C for 1 h with 20 units of Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim), buffer supplied with the enzyme, 20 units of RNasin (Promega), and 125 µM each dNTP. After treatment at 95 °C for 3 min, an equal aliquot of each reverse transcription reaction was used as PCR template with forward primer 5'-TTTCCTTGATAGAATGGGTG-3' and reverse primer 5'-CCAATCAGCAGCGGTCAAGA-3' designed to amplify a 424-bp fragment of FAT1 cDNA. An additional PCR was carried out using forward primer 5'-CCCAGATCTATGAGCAGTCGTGTGTGCTAC-3' and reverse primer 5'-CCCGAGCTCAGTTGTGAAGCACTTTAGTTAC-3' (kindly provided by Dr. M. Geraghty) designed to amplify a 1116-bp fragment containing YOR180C (locus EDH2), a peroxisomal enoyl-CoA hydratase gene with a consensus upstream oleate response element. Thirty-five cycles of 94 °C (45 s), 55 °C (45 s), and 72 °C (60 s) were performed using the Expand High Fidelity PCR system (Boehringer Mannheim).
Fatty Acid Analysis-- Yeast grown in either YPD (250 A600) or YPOLT (60 A600) were harvested by centrifugation, washed three times with deionized water, suspended in 0.5 ml water, and subjected to sonic disruption. After determination of the protein concentration by the method of Miller (35), total lipids were extracted by a modification of the method of Folch et al. (36), and total fatty acids were quantitated by gas chromatography as their methyl esters using the method of Moser and Moser (37).
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RESULTS |
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Identification of FAT1 as a Candidate S. cerevisiae VLCS Gene-- A cDNA library from the liver of a rat treated with the peroxisome proliferator Gemfibrozil was screened, and a 660-bp fragment containing a 220-amino acid open reading frame thought to be the carboxyl terminus of rat VLCS was cloned as described under "Experimental Procedures." The sequence of this fragment was 100% identical to that of authentic rat liver VLCS, which was subsequently published by Uchiyama et al. (20). The 220-amino acid sequence was compared with all open reading frames of the complete S. cerevisiae genome using the BLAST algorithm. Only one likely candidate for a homologous yeast protein was identified (smallest sum probability = 9e-24). The gene encoding this protein was identical to that recently reported by Faergeman et al. (22), which they designated FAT1 by virtue of its homology to mFATP.
While preparing expression constructs to investigate the biochemical properties of Fat1p, the product of the FAT1 gene, we detected an error in the sequence as contained in the Saccharomyces Genome Data Base. The published sequence contained a CpC dinucleotide insertion at bp 1857 and 1858, yielding an open reading frame of 623 amino acids. The corrected nucleotide sequence of FAT1 and its predicted amino acid sequences are shown in Fig. 1. The FAT1 gene encodes a protein of 669 amino acids with 33.7% identity and 57.2% similarity to rVLCS. Although the signal that targets rVLCS to peroxisomes is not certain, the protein ends in the carboxyl-terminal tripeptide -LKL (20) and may be a functional variant of the well characterized -SKL peroxisome targeting signal 1 (38). Fat1p ends in the carboxyl-terminal tripeptide -IKL, which has been shown to function as a peroxisome targeting signal 1 in yeast (28). To investigate the function of Fat1p, PCR-mediated gene disruption was used to replace the FAT1 gene with the HIS selectable marker as described under "Experimental Procedures."
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VLCS and LCS Activity of FAT1 Deletion Strains--
In order to
identify mutants in which the FAT1 gene had been disrupted
and to ascertain whether Fat1p had VLCS activity, seven HIS
prototrophs were assayed for their ability to activate a VLCFA, lignoceric acid (C24:0), to its CoA derivative. Spheroplasts were prepared from wild-type yeast and these candidate FAA1
deletion strains. Spheroplast homogenates from four strains in which
FAA1 gene disruption was confirmed by PCR (see
"Experimental Procedures") also had decreased ability to activate
lignoceric acid, suggesting that Fat1p has VLCS activity that was
indeed disrupted. In these strains, LCS activity was similar to that of
wild-type. Data from the two strains chosen for further study,
designated fat1-1 and fat1
-2, are shown in
Table I.
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Growth of FAT1 Deletion Strains on Fatty Acids-- Growth of wild-type, FAT1 deletion, and FAT1 deletion complemented with FAT1 cDNA strains on YPD plates supplemented with 500 µM oleic acid was comparable at both 24 and 30 °C; in contrast, growth of the FAT1 deletion strain was markedly reduced at 24 °C and somewhat reduced at 30 °C when cerulenin, an inhibitor of fatty acid synthase (39), was added to the oleic acid growth medium (data not shown). This growth phenotype is similar to that reported previously for FAT1 deletion mutants (22). Transformation of FAT1 deletion strains with FAT1 cDNA restored growth to normal (data not shown).
FAT1 Gene Deletion Strains Accumulate VLCFAs--
In XALD and in
peroxisome biogenesis disorders, failure of peroxisomes to activate
VLCFAs results in their accumulation in tissues (5). To determine
whether deletion of the FAT1 gene affected VLCFA
accumulation in yeast, fatty acid analysis was performed on wild-type,
fat1-1, and fat1
-2 strains grown in both
YPD medium and in YPOLT medium to induce peroxisomes. As shown in Fig.
2, the cellular concentration of
saturated fatty acids with chain lengths greater than or equal to C22
was increased in FAT1 deletion strains grown on YPD. The
concentrations of saturated fatty acids containing 14-20 carbons were
similar in both wild-type and deletion strains (data not shown). When
wild-type yeasts were grown on YPOLT instead of YPD, concentrations of
saturated fatty acids, both VLCFA (Fig. 2) and long-chain fatty acids
(data not shown), were 5-10-fold higher. As in YPD-grown yeast,
FAT1 deletion strains grown on YPOLT had increased
concentrations of VLCFA, particularly C24:0 and C26:0 (Fig. 2),
relative to wild-type yeast grown under the same conditions. Long-chain
fatty acid concentrations were similar in YPOLT-grown wild-type and
FAT1 deletion strains.
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VLCS Activity of Both Organelle Pellet and Supernatant Fractions Is
Reduced upon Deletion of FAT1 and Is Restored by Expression of FAT1
cDNA--
We previously demonstrated that in the yeast P. pastoris, VLCS activity was present in both 27,000 × g microsome-rich supernatant and organellar pellet fractions
(4). When homogenates of S. cerevisiae spheroplasts were
subjected to the same procedure, VLCS activity was also found in both
the peroxisome- and mitochondria-rich organelle pellet and the cytosol-
and microsome-rich supernatant (Table
II). As expected, these fractions also
exhibited high LCS activity. In wild-type yeast, the organelle pellet
fraction contained about 60% of the total cellular LCS activity and
about 30% of the total VLCS activity (data not shown). To determine
whether Fat1p was responsible for VLCFA activation in supernatant,
organelle pellet, or both, fat1-1 and
fat1
-2 strains were also fractionated. In these deletion
strains, the specific activity of VLCS was decreased in both the
organelle pellet and the supernatant fractions. The relative decrease
was greater in the microsome-rich supernatant than in the organelle
pellet. The LCS activity of the fat1
-1 strain was more
variable and slightly lower than in either the wild-type or the
fat1
-2 strain; the cause of this variability is
unknown.
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Overexpression of VLCS Activity in Wild-type and FAT1 Deletion
Strains--
To verify that Fat1p catalyzes VLCS activity, we
overexpressed FAT1 cDNA in yeast strains. pSJW981, a
high copy plasmid containing FAT1 cDNA, was transformed
into both the wild-type and fat1-2 strains. As shown in
Fig. 5, a 4-fold increase over wild-type VLCS activity was observed in the wild-type strain transformed with
pSJW981. The VLCS activity of the pSJW981-transformed deletion strain
was nearly 3-fold higher than wild-type level (Fig. 5). Comparison of
the VLCS activities of both the wild-type strain and
fat1
-2 strain transfected with the low copy CEN plasmid
(Table III) versus the high copy 2µ construct (Fig. 5)
showed a correlation between level of VLCS activity and the level of
expression of the transgene. These results indicate that Fat1p indeed
catalyzes VLCS activity.
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VLCS Is Not Induced by Peroxisome Proliferation--
Treatment of
rats with peroxisome proliferators such as Gemfibrozil was found to
increase the specific activity of peroxisomal VLCS.2 In
yeast, peroxisome proliferation and the synthesis of several peroxisomal -oxidation enzymes are induced by growth on medium containing a fatty acid such as oleic acid as primary carbon source (40, 41). A 21-24-bp oleate response element has been identified in
the 5'-untranslated region of the genes encoding these proteins; however, Faergeman et al. (22) reported that there was no
consensus oleate response element in the 5'-untranslated region of the
FAT1 locus. We wanted to ascertain directly whether
oleate-induced peroxisome proliferation in S. cerevisiae
would increase VLCS activity and the level of FAT1 mRNA.
When wild-type yeast were grown in oleic acid-containing medium, we did
not observe an increase in VLCS activity over that of dextrose-grown
yeast (data not shown). To estimate relative FAT1 mRNA
levels, total RNA was isolated from spheroplasts prepared from yeast
grown in either medium. When equal amounts of RNA were used for reverse
transcriptase-catalyzed cDNA synthesis, no increase in
FAT1 message in oleate-grown cells was detected by PCR (Fig.
6), consistent with the lack of an
upstream oleate response element. In contrast, there was a marked
increase in mRNA for EDH2, a gene encoding a peroxisomal
enoyl-CoA hydratase that contains an oleate response element (Fig.
6).
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VLCS Activity of Fat1p in the Organelle Pellet Fraction Is
Peroxisomal--
Because peroxisome induction by oleate in wild-type
yeast failed to increase either FAT1 message (Fig. 6) or
VLCS activity (data not shown), we questioned whether the VLCS activity
in the organelle pellet was associated with peroxisomes or
mitochondria. Although fatty acid -oxidation does not take place in
mitochondria, VLCFA activation for complex lipid synthesis or
elongation could take place in this organelle. To address this
question, we fractionated the organelle pellet from wild-type and gene
deletion strains by density gradient centrifugation. As shown in Fig.
7, C and D,
peroxisomes and mitochondria were well separated in both wild-type and
FAT1 deletion strains. VLCS activity was associated mainly with peroxisomal gradient fractions, with a small amount of activity found in a low density fraction near the top of gradients (Fig. 7B). Mitochondrial fractions had very little VLCS activity.
In contrast, LCS activity was present in mitochondrial, peroxisomal, and low density fractions (Fig. 7A). It is likely that this
low density fraction contains both enzyme released from peroxisomes during processing and heavier microsomes that sedimented at 27,000 × g. Using this fractionation procedure, VLCS activity in
both peroxisomal and low density fractions of the FAT1
deletion strains was found to be decreased (Fig. 7B).
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DISCUSSION |
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In this report, we provide evidence that the S. cerevisiae
FAT1 gene product, Fat1p, has VLCS activity. While several yeast enzymes with acyl-CoA synthetase have been reported, Fat1p is the first
described that appears to play a significant role in VLCFA metabolism.
Four yeast acyl-CoA synthetases were previously described by Gordon and
colleagues (17, 18). These investigators also predicted the existence
of at least one additional fatty acid activation gene that directs
fatty acids to the -oxidation pathway after they found that strains
in which all four FAA genes were disrupted were viable on
media containing fatty acids as sole carbon source (18). Faa1p, Faa2p,
and Faa4p were very active toward medium- to long-chain fatty acids but
showed no VLCS activity. Faa3p, expressed in bacteria as a
His6 fusion protein, weakly activated fatty acids over a
broad range of chain lengths from 12 to 24 carbons. Although Faa3p was
the only one of this group that demonstrated any activity toward
VLCFAs, the activity of the recombinant enzyme with fatty acids of all
chain lengths was very low. Studies to determine whether Faa3p or any
of the other known yeast acyl-CoA synthetases are responsible for the
residual VLCS activity in FAT1 deletion strains are in
progress.
Deletion of the FAT1 gene resulted in decreased growth on
rich medium containing both dextrose and oleic acid when an inhibitor of fatty acid synthesis, cerulenin, was present. This result is in
agreement with findings reported by Faergeman et al. (22), who independently identified the FAT1 gene by virtue of the
homology of Fat1p to mFATP, a mouse protein thought to be involved in
fatty acid transport (21). In their study, FAT1 deletion
strains showed evidence of defective fatty acid uptake into cells and
decreased incorporation of labeled oleic acid into cellular
phospholipids (22). However, when we quantitated total fatty acids in
yeast grown on either dextrose- or oleate-containing medium, no
difference in the concentrations of C14:0, C16:0, or C18:0 were found
in wild-type, fat1-1, and fat1
-2 strains
(Fig. 3). FAT1 gene deletion caused yeast to accumulate
VLCFA when grown on either dextrose or oleic acid as primary carbon
source, suggesting that the primary role of Fat1p is in VLCFA
metabolism. Furthermore, it is possible that the biochemical effects in
deletion mutants ascribed to defects in fatty acid transport result
from perturbation of the cell membrane lipid composition by increased
VLCFA concentrations. Alterations in membrane properties by VLCFAs have
been described in model membranes (42), erythrocyte membranes (43), and
adrenocortical cell membranes (44).
Our results clearly indicate that FAT1 deletion strains are
unable to effectively utilize VLCFA. Accumulation of VLCFA could result
from overproduction, failure to incorporate these fatty acids into
complex lipids, failure to degrade excess fatty acids by peroxisomal
-oxidation, or a combination of these processes. When yeast are
grown on rich medium containing oleate, the cellular long-chain fatty
acid content is 5-10-fold higher than when cells are grown on dextrose
medium (data not shown), and VLCFA would be expected to arise primarily
from chain elongation. However, when cells are grown in dextrose
medium, fatty acids (including VLCFA) for biosynthesis of complex
lipids could originate either from de novo synthesis or from
lipids present in the yeast extract component of the medium. In either
case, failure to activate VLCFA, either for incorporation into complex
lipids or for degradation via
-oxidation, could result in
significant accumulation within yeast cells. Furthermore, if Fat1p, in
addition to its role in VLCFA activation also functions to transport
VLCFA across organellar membranes, lack of this protein could similarly
result in cellular accumulation of VLCFA.
Because of the association of defective peroxisomal fatty acid metabolism with human neurodegenerative diseases, we wanted to verify that yeast Fat1p was an acyl-CoA synthetase and to determine its subcellular location. Faergeman et al. (22) reported that deletion of the FAT1 gene did not decrease acyl-CoA synthetase activity in sonicated cell extracts when myristate (C14:0), palmitate (C16:0), or oleate (C18:1) was supplied as substrate. Similarly, we found no decrease in LCS activity in yeast spheroplast homogenates using palmitate as substrate (Table I). In contrast, total yeast VLCS activity as well as that of peroxisomes and a microsome-rich 27,000 × g supernatant fraction was significantly decreased when the FAT1 gene was deleted, indicating that Fat1p has VLCS activity (Tables I and II). The only other yeast acyl-CoA synthetase whose subcellular distribution has been studied is Faa2p, which was found to be almost exclusively associated with peroxisomes (45). Faa2p, which had no VLCS activity, was found to be most active toward medium chain length saturated fatty acids (C10:0-C14:0) (17). The subcellular distribution of Faa1p, Faa3p, and Faa4p is unknown.
Decreased peroxisomal VLCS activity and elevated cellular VLCFA levels
are consistent biochemical features of XALD. As reported here, deletion
of the gene encoding the only S. cerevisiae protein that
shares significant homology with mammalian VLCS produces a similar
biochemical phenotype. However, the gene defective in XALD encodes
ALDP, a protein of the ATP-binding cassette membrane transporter
superfamily, and not VLCS (7). ALDP and at least three other
structurally related proteins of this family are found in mammalian
peroxisomal membranes (46-48). Yeasts contain only two peroxisomal
proteins belonging to this family, Pxa1p (49) and Pxa2p (50) (also
known as Pat2p and Pat1p, respectively (45)), which have been proposed
as functional homologs of ALDP. Deletion of either the PXA1
or PXA2 genes resulted in decreased growth on medium
containing oleate as primary carbon source (45, 49, 50), whereas no
growth phenotype of FAT1 deletion strains was evident under
the same conditions. Shani et al. (50) found that Pxa1p and
Pxa2p function as a heterodimer. Hettema et al. (45)
concluded that these two yeast ATP-binding cassette transporter proteins functioned to transport long-chain fatty acyl-CoAs into peroxisomes for catabolism via -oxidation. Furthermore, they concluded that the acyl-CoAs transported by these proteins were activated by acyl-CoA synthetases located on organelles other than
peroxisomes. They further concluded that Pxa1p and Pxa2p were not
required for transport into peroxisomes of medium chain free fatty
acids, which were subsequently activated inside the peroxisome by
Faa2p. Preliminary data indicate that, in contrast to deletion of
FAT1, deletion of PXA1 or PXA2 did not
increase cellular C22:0 or C24:0 concentrations
significantly.3 Thus, it
appears that the functional relationship between VLCS and ATP-binding
cassette transporters may differ between yeasts and humans and
introduces the possibility that Fat1p both activates and transports
peroxisomal VLCFAs. Further studies to probe the relationship among
Fat1p, Pxa1p, and Pxa2p and to determine whether Fat1p also functions
as a peroxisomal transporter are currently in progress.
VLCSs and mFATP appear to belong to the same protein family. rVLCS shares 40% amino acid identity and 73% similarity with mFATP (20). Faergeman et al. (22) noted that Fat1p and mFATP contain several regions of high sequence similarity, including a consensus sequence (amino acids 255-268 of Fat1p; see Fig. 8, Motif 1) common to all known acyl-CoA synthetases and other proteins (e.g. insect luciferases and plant coumarate CoA ligase) with an AMP-binding domain (51). Another region of sequence similarity between Fat1p and mFATP contains a motif (amino acids 519-540 of Fat1p; Fig. 8, Motif 2) that is highly conserved between these proteins, rat peroxisomal VLCS (20), and human VLCS4 (52). When the comparable regions of rat and human LCSs and yeast Faa1p-4p are aligned, it is clear that proteins in this group are more closely related to each other than to the VLCS/FATP group. An acyl-CoA synthetase "signature motif" described by Black et al. (53) (consisting of eight invariant amino acid residues in a 25-amino acid consensus sequence) is partially contained in motif 2; residues 19-25 of the signature motif are residues 1-7 of motif 2. Conserved residues 4 and 5 of motif 2 (DR) are invariant amino acids of the signature motif. However, only amino acids 1, 7, and 8 (DXXXXXGD) of the signature motif were also present in rVLCS, a protein known to possess acyl-CoA synthetase activity, indicating that the signature motif may require only five invariant residues. Notably, the DXXXXXGD sequence was also present in all enzymes of the VLCS/FATP group (data not shown), suggesting that all of these proteins could have acyl-CoA synthetase activity.
|
Yeasts have proven to be extremely valuable in the study of peroxisomes
and peroxisomal lipid metabolism. This organism has been extensively
used to identify and characterize many proteins required for peroxisome
biogenesis; indeed, protein defects in 6 of the 11 recognized
complementation groups of human peroxisome biogenesis disorders were
elucidated by virtue of their homology to proteins first identified in
yeast (11-16). In this study, we have demonstrated that deletion of
the FAT1 gene results in VLCFA accumulation, a cellular
phenotype resembling that found in XALD. The finding that VLCS activity
was not totally abolished in FAT1 gene deletion strains
suggests that a known (Faa1p-4p) or unknown acyl-CoA synthetase is
responsible for the residual activity. This conclusion is in concert
with the observation that in XALD, peroxisomal -oxidation of VLCFA
is reduced (20-30% of control) but not decreased to the same extent
as seen in the disorders of peroxisome biogenesis (<10% of control)
(54). The nature of the enzyme(s) responsible for residual VLCS
activity in yeast and the elucidation of similar enzyme(s) in humans
are the subject of current investigation.
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ACKNOWLEDGEMENTS |
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We thank Ann Moser for assistance with fatty acid analysis, Susan J. Wang for expert technical assistance, Dr. Michael Geraghty for primers for PCR amplification of YOR180C, Drs. Noam Shani and David Valle for sharing their data on fatty acid levels in PXA1 and PXA2 deletion yeast strains prior to publication, Stephan Kemp for the amino acid sequence of mouse VLCS, and Dr. Hugo Moser for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD10981, HD24061, and NS10533.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: Kennedy Krieger Institute, 707 N. Broadway, Baltimore, MD 21205. Tel.: 410-502-9493; Fax: 410-502-8279; E-mail: watkins{at}kennedykrieger.org.
1 The abbreviations used are: CoA, coenzyme A; VLCFA, very long-chain fatty acid; VLCS, very long-chain acyl-CoA synthetase; rVLCS, rat VLCS; mFATP, mouse fatty acid transport protein; LCS, long-chain acyl-CoA synthetase; FAT1 and Fat1p, S. cerevisiae fatty acid transporter/VLCS gene and protein, respectively; FAA and Faap, S. cerevisiae fatty acid activation gene and protein, respectively; XALD, X-linked adrenoleukodystrophy; ALDP, adrenoleukodystrophy protein; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); MES, 4-morpholineethanesulfonic acid.
2 P. A. Watkins, unpublished observations.
3 N. Shani, J.-F. Lu, and D. Valle, submitted for publication.
4 S. J. Steinberg and P. A. Watkins, submitted for publication.
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