From the Division of Life Sciences, the Bureau of Biological Research, Nelson Laboratories, Rutgers University, Piscataway, New Jersey 08854-808
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ABSTRACT |
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The Saccharomyces cerevisiae
FAT1 gene appears to encode an acyl-CoA synthetase that is
involved in the regulation of very long chain
(C20-C26) fatty acids. Fat1p, has homology to
a rat peroxisomal very long chain fatty acyl-CoA synthetase. Very long chain acyl-CoA synthetase activity is reduced in strains containing a
disrupted FAT1 gene and is increased when FAT1
is expressed in insect cells under control of a baculovirus promoter.
Fat1p accounts for approximately 90% of the C24-specific
acyl-CoA synthetase activity in glucose-grown cells and approximately
66% of the total activity in cells grown under peroxisomal induction
conditions. Localization of functional Fat1p:green fluorescent protein
gene fusions and subcellular fractionation of C24 acyl-CoA
synthetase activities indicate that the majority of Fat1p is located in
internal cellular locations. Disruption of the FAT1 gene
results in the accumulation of very long chain fatty acids in the
sphingolipid and phospholipid fractions. This includes a 10-fold
increase in C24 acids and a 6-fold increase in
C22 acids. These abnormal accumulations are further
increased by perturbation of very long chain fatty acid synthesis.
Overexpression of Elo2p, a component of the fatty acid elongation
system, in fat1 In Saccharomyces cerevisiae, most fatty acids (>95%)
are C12-C18 species that are found in membrane
glycerolipids. These are formed de novo by the soluble
cytoplasmic fatty acid synthetase complex. Very long chain
(C20-C26) fatty acids
(VLCFAs),1 which are
predominantly found in sphingolipids, are formed by a separate, ER
membrane-bound system that elongates C16 and
C18 saturated fatty acids to C26 and
C28 species. Most of the very long chain fatty acids found
in yeast are 26:02 and
hydroxy-26:0. C20-C24 VLCFAs are minor
sphingolipid components that are apparently derived from metabolic
intermediates in the formation of the 26-carbon species. Our laboratory
has recently found two genes, ELO2 and ELO3, that
are involved in very long chain fatty acid synthesis (1). Each gene
apparently encodes a single component of a system that elongates
C16 and C18 acids to
C20-C26 VLCFAs. Elo2p is involved in the
elongation of fatty acids up to 24 carbons. Elo3p apparently has a
broader substrate specificity and is essential for the conversion of
24-carbon acids to 26-carbon species (1).
Debilitating human diseases are associated with the accumulation of
VLCFAs. A characteristic of human X-linked adrenoleukodystrophy is an
absence or reduction of very long chain acyl-CoA synthetase (VLACS)
activity, which leads to the accumulation of saturated VLCFAs. This
appears to be caused by the mutations in the X-linked adrenoleukodystrophy gene, which encodes the ABC transporter in peroxisomal membranes (2, 3). The function of the adrenoleukodystrophy protein is not clearly explained but might be involved in the transport
of VLCFAs into peroxisomes or in the transport of proteins that are
required for the Acyl-CoA synthetase activity is required for the biosynthesis and the
catabolic oxidation of fatty acids. Previous studies have identified
four fatty acyl-CoA synthetases in Saccharomyces that appear
to be involved in the metabolism of long chain
(C12-C18) fatty acids (8, 9). We had
previously determined that the two major synthetase activities, encoded
by the FAA1 and FAA4 genes, were not essential
for VLCFA synthesis and that disruption of either or both genes did not
affect VLCFA composition.
Given the important role of acyl-CoA synthetase activity in the
biosynthesis and metabolism of VLCFAs, we attempted to identify a yeast
VLACS homologue by data base searching. The amino acid sequence of open
reading frame YBR041W was found to be homologous to that of the rat
VLACS. Previously, YBR041W had been designated as FAT1 and
was proposed to function in long chain fatty acid transport (10). In
this report, we show that the protein encoded by the YBR041W sequence
is a functional homolog for the rat VLACS and that disruption of the
gene can cause large accumulations of aberrant,
C20-C24 VLCFAs, suggesting that Fat1p plays an
important role in controlling cellular VLCFA levels. Bulk fatty acid
transport does not appear to be affected by the loss of Fat1p activity; however, given its multiplicity of cellular locations, it could also
play a significant role in the uptake of low levels of nutrient fatty
acids. In examining the role of fatty acyl-CoA synthetases in fatty
acid import, however, we identified two fatty acyl-CoA synthetases,
Faa1p and Faa4p, that are essential for the bulk import of nutrient
fatty acids.
Strains and Growth Medium
Yeast strains constructed for this study and their genotypes are
presented in Table I. Strains containing
faa1 cells causes
C20-C26 levels to rise to approximately 20%
of the total fatty acids. These data suggest that Fat1p is involved in
the maintenance of cellular very long chain fatty acid levels,
apparently by facilitating
-oxidation of excess intermediate length
(C20-C24) species. Although fat1
cells were reported to grow poorly in oleic
acid-supplemented medium when fatty acid synthase activity is
inactivated by cerulenin, fatty acid import is not significantly
affected in cells containing disrupted alleles of FAT1 and
FAS2 (a subunit of fatty acid synthase). These results
suggest that the primary cause of the growth-defective phenotype is a
failure to metabolize the incorporated fatty acid rather than a defect
in fatty acid transport. Certain fatty acyl-CoA synthetase activities,
however, do appear to be essential for bulk fatty acid transport in
Saccharomyces. Simultaneous disruption of FAA1
and FAA4, which encode long chain
(C14-C18) fatty acyl-CoA synthetases,
effectively blocks the import of long chain saturated and unsaturated
fatty acids.
INTRODUCTION
Top
Abstract
Introduction
References
-oxidation of VLCFAs.
-Oxidation of VLCFAs in yeast and mammals is known to occur
primarily in peroxisomes (4, 5). The formation of CoA thioesters from
VLCFAs is an initial step for
-oxidation and is catalyzed by a
VLACS. A VLACS was recently purified from the rat liver peroxisomal membranes and microsomes, leading to the identification of its gene (6,
7).
EXPERIMENTAL PROCEDURES
, faa4
, and faa1
/faa4
gene disruptions were obtained from J. Gordon and were previously
described (11). Plasmids constructed for this study are shown in Table
II. Standard yeast genetics methods were used for construction of strains bearing the appropriate mutations. Yeast cells were grown at 30 °C in YPAD (1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose, and 2 mg/liter adenine), YPADt (YPAD plus 1% tergitol nonionic detergent Nonidet P-40 (to disperse fatty
acids), CM (complete synthetic medium (12)), or CMt (CM plus 1%
tergitol) medium. Tergitol is used in the medium to disperse fatty
acids. Unlike Brij and Tween compounds, which are derived from fatty
alcohols or fatty acids, tergitol is a nonylphenol polyethylene oxide
polymer that is apparently not metabolized. Alternative carbon sources
(2% galactose; 2% raffinose; or 3% glycerol, 0.1% (3.54 mM) oleic acid, 0.25% Tween 40) were used by replacing
glucose in appropriate media. Fatty acids were obtained from Sigma or
Nu-Chek Prep (Elysian, MN). Escherichia coli strain DH5
was obtained from Life Technologies, Inc. 1-14C-Labeled
24:0 was obtained from American Radiolabeled Chemicals, Inc. (St.
Louis, MO).
S. cerevisiae strains used in this study
Plasmids used in this study
Cloning and Disruption of FAT1, FAT2, and FAA2
Gene-disrupted cells were constructed by standard yeast methods using cloned gene sequences. Strains containing disrupted genes are shown in Table I. All gene disruptions were verified by PCR using genomic DNA from the disrupted cells as a template.
Disruption of FAT1--
A 2.3-kb DNA fragment containing the
FAT1 coding sequence was derived by PCR using DTY10a genomic
DNA as templates and PCR primers FAT1-1 and FAT1-2 in Table
III. Plasmid pCRSK-FAT1 (Table II),
containing the amplified PCR product, was used to create pCRSK-fat1::LEU2 or pCRSKfat1
::HIS3 (Table
II) in which 1.8 kb of the FAT1 coding region was replaced
with the corresponding LEU2 or HIS3 marker genes.
Linear fat1
::LEU2 and
fat1
::HIS3 disruption cassettes were derived
from those plasmids by digestion with BamHI and
HpaI and transformed into strain DTY10a to create the
fat1
deletion strains. A linear
fat1
::LEU2 sequence was also transformed into
the elo3
::HIS3 strain to create
fat1
/elo3
double deletion stains. A linear
fat1
::HIS3 sequence was also transformed into
fas2
::LEU2 strains to create the
fat1
/fas2
double deletion strains.
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Disruption of FAT2--
An 1840-base pair DNA fragment
containing the FAT2 coding sequence was derived by PCR using
DTY10a genomic DNA as templates and the PCR primers FAT2-1 and FAT2-2
in Table III. The pCRSK-FAT2 plasmid (Table II) containing amplified
PCR product was used to create plasmid pCRSKfat2)::HIS3 in
which 0.4 kb of the FAT2 coding part was released and
replaced with the corresponding HIS3 marker gene. Linear
fat2::HIS3 disruption cassettes were derived
from the plasmid by digestion with BstEII and transformed
into strain DTY10a to create the fat2
strains. A linear
fat2)::HIS3 sequence was also transformed into the
fat1
::LEU2 strain to create the fat1
/fat2
double deletion strains.
Disruption of FAA2--
A 2.3-kb DNA fragment containing the
FAA2 coding sequence was derived from PCR using DTY10a
genomic DNA as templates and PCR primers FAA2-1 and FAA2-2 in Table
III. Plasmid pCRSK-FAA2 (Table II) containing the amplified PCR product
was used to create pCRSKfaa)::URA3 in Table II in which 0.5 kb of FAA2 protein coding sequence was released and replaced
with the URA3 gene. A linear
faa2::URA3 disruption cassette was derived from
the plasmid by digestion with ClaI and transformed into
strain DTY10a to create the faa2
deletion strain shown in
Table I. A linear faa2
::URA3 sequence was also
transformed into the fat1
::LEU2 strain and
fat1
::LEU2/fat2
::HIS3 to create
the fat1
/faa2
double disruption stain and the
fat1
/fat2
/faa2
triple disruption strain.
Construction of GAL1-FAT1 Vectors
2.3 kb of DNA sequence containing FAT1 coding
sequence and 3'-untranslated region was released by BamHI
and HpaI digestion of the pCRSK-FAT1 (Table II). This
element was inserted into the linearized and blunt ended YCpGAL1URA
vector by BamHI, HindIIIj, and Klenow enzyme
digestion to create vector pFAT1. That vector was transformed into
fat1::LEU2 strains, and transformants were selected on CM plates without uracil and leucine.
Construction of FAT1-GFP Fusion Vectors
Protein coding elements of the FAT1 gene were fused in frame to the green fluorescent protein (GFP) sequences by insertion into centromeric plasmid vectors pTS395 and pTS408. These contain the GFP sequence linked to the GAL1 promoter and the 3' terminator sequence from the ACT1 gene. Vector pTS395 was used to fuse GFP to the C terminus, and vector pTS495 was used to fuse GFP to the N terminus of Fat1p. To create vector pTS395FAT1, a DNA sequence containing codons 1-669 of the FAT1 coding sequence was derived from PCR using DTY10a genomic DNA as templates and primers 5'-ATAGGATCCATGTCTCCCATACATGTTGTTGTC-3' (forward) and 5'-TTCTAGATAATTTAATTGTTTGTGCATCG-3' (reverse). To create the vector pTS408FAT1 construct, a DNA sequence containing the same codons was derived from DTY10a genomic DNA and primers 5'-ATAGGATCCATGTCTCCCATACATGTTGTTGTC-3' (forward) and 5'-TTCTAGACTATAATTTAATTGTTTGTGCATCG-3' (reverse). The amplified PCR products were cut with BamHI and XbaI and inserted to vectors digested with the same restriction enzymes.
Fluorescence Microscopy of Cells Containing FAT1-GFP Gene Fusions
To induce expression the FAT1-GFP fusion gene under
control of the GAL1 promoter, fat1 strains
harboring GFP fusion vectors were grown overnight in 5 ml of uracil and
leucine drop-out CM containing 2% raffinose. Cells were induced for
4 h by resuspension in the same media containing 2% galactose.
For oleic acid and galactose induction, cells were grown overnight in
uracil and leucine drop-out CM containing 3% glycerol and induced for
4 h in the same medium containing 2% galactose, 0.1% oleic acid,
0.25% Tween 40. One µg/ml 4',6-diamidino-2-phenylindole was added to the media during the last 1.5 h of galactose induction. After induction, cells were washed with 1 ml of phosphate-buffered saline buffer (pH 7.4) three times and then resuspended in 100 µl of phosphate-buffered saline buffer. In a humid chamber, cells were dropped onto a Superfrost Plus slide (25 × 75 × 1 mm; VWR
Scientific) and allowed to settle for 10 min. Excess cells were flushed
from the slides with 100 µl of phosphate-buffered saline buffer and mounted in 0.1% sea plaque agar under a coverslip. All samples were
viewed on a Nikon Diaphot 300 inverted microscope equipped with a
Chroma Endow GFP filter set 41017 (excite HQ470/40; emitter HQ525/50;
beam splitter Q495 LP). Images were captured using the Acquire software program.
Lipid Extraction and Fatty Acid Analysis
Fatty acid methyl esters were prepared by HCl methanolysis as described previously (13). Gas chromatography was performed on a Varian 3400CX chromatograph using a Supelcowax TM10 30 m × 0.32 mm column (Supelco) temperature programmed from 70 to 240 °C at 40 °C/min. Data were collected and analyzed using the Class-VP Chromatography Data System version 4.1 (Shimadzu Scientific Instruments) software.
Sphingolipid and glycerolipid fatty acids were fractionated from logarithmic phase cells as described by Pinto et al. (14). 5% trichloroacetic acid-washed cell pellets were subjected to mild alkaline hydrolysis. The methanolic-KOH extract, which contains saponifiable fatty acids from glycerolipids, was acidified and extracted with petroleum ether as described previously (15). Extracted fatty acids were dried under nitrogen, and methyl esters were prepared by HCl methanolysis. Sphingolipids were extracted from the saponified cell pellets with an ethanol/water/diethylether/pyridine/NH4OH (15:15:5:1:0.018 by volume) solvent. After drying under nitrogen, sphingolipid fatty acid methyl esters were prepared by HCl methanolysis.
Preparation of Protein Extracts for VLACS Assay
Glucose-grown cells were cultured in 200 ml of YPAD culture to 3 × 107 cells/ml. For oleic acid induced cells, cultures were pregrown in 50 ml of YPAD medium to 3 × 107 cells/ml and washed with YP medium containing 3% glycerol. Cells were resuspended in 200 ml of YP medium containing 3% glycerol, 0.1% oleic acid, and 0.25% Tween 40 at a density of 4 × 106 cell/ml and grown for 18 h to 3 × 107 cells/ml. Formation of spheroplasts, homogenization, and differential centrifugation were performed according to Thieringer et al. (16) with the following modifications.
Cells were washed twice with ice-cold water and incubated in Zymolase buffer (50 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 1 M sorbitol, 30 mM dithiothreitol) for 20 min at room temperature. The cells were then resuspended in Zymolase buffer and incubated with Zymolase (0.1 mg/g of cells (wet weight) for glucose-grown cells and 2 mg/g for oleic acid-induced cells) for 60 min at 30 °C with shaking at 50 rpm.
Spheroplasts were washed by resuspension of centrifuged pellets in 2 volumes of ice-cold Zymolase buffer three times, followed by
resuspension in 2 volumes of lysis buffer (5 mM MES (pH
6.0), 0.6 M sorbitol, 1 mM KCl, 0.5 mM EDTA (pH 8.0), 0.1% ethanol (v/v), 1 mM
phenylmethylsulfonyl fluoride, 4 mM benzamidine, 5 µg/ml leupeptin, 5 µg/ml pepstatin). Spheroplasts were disrupted at 4 °C
in a Potter-Elvehjem homogenizer with 50 strokes. Homogenates were
centrifuged at 3,500 rpm (1,500 × g) for 5 min in a
Sorvall SS-34 rotor to remove nuclei and cell debris. The pellet was
resuspended in lysis buffer, rehomogenized, centrifuged, and combined
with the postnuclear supernatant. Aliquots consisting of 20% of the total volume were quickly frozen to 80 °C. The remaining fraction was centrifuged for 15 min at 25,000 × g. The
supernatants (25,000 × g supernatant) were frozen and
saved. The resulting pellet (25,000 × g pellets) was
gently resuspended in lysis buffer and centrifuged at 600 × g for 5 min to remove aggregated material prior to enzyme assay. Previously frozen supernatant fractions were centrifuged at
80,0000 rpm (256,000 × g) for 40 min in a Beckman
TL100 microultracentrifuge to fractionate microsomes. Protein
concentrations in cell fractions were measured by the Bradford assay
method (17).
Very Long Chain Fatty Acyl-CoA Synthetase Assay
Preparation of the fatty acid substrate and assay of VLACS
activity followed the method of Wanders et al. (18). The
fatty acid substrate [1-14C]lignocerate was prepared as a
100 µM stock solution. Solubilized substrate was obtained
by dissolving the dried fatty acid in 100 mM Tris-HCl (pH
8.5) containing 10 mg/ml -cyclodextrin and incubating for 30 min in
a sonicating water bath at room temperature.
Reactions were started by adding 20 µl of yeast extract to the assay
solution (50 mM Tris-HCl (pH 8.5), 150 µM
coenzyme A, 300 µM dithiothreitol, 10 mM ATP,
10 mM MgCl2, 0.01% (v/v) Triton X-100, 10 µM [1-14C]lignocerate dissolved in
-cyclodextrin) to the final volume of 200 µl at protein
concentrations of 50-100 µg/ml. After 30 min at 37 °C, reactions
were terminated by transferring 150 µl of the incubation mixture to a
glass tube containing 750 µl of Dole's reagent (isopropyl alcohol,
heptane, 1 M H2SO4; 40:10:1 by
volume). After vigorous mixing, the protein was removed by centrifugation, and 650 µl of the supernatant was extracted with 350 µl of heptane plus 190 µl of a solution containing 400 nM Mops-NaOH (pH 6.5). The lower (aqueous) layer was washed
three times with 400 µl of heptane, and the radioactivity in that
fraction was measured by scintillation counting. The radioactivity
measured from the boiled protein control extracts was subtracted from
the corresponding assay to determine the amount of acyl-CoA product.
Fatty Acid Transport Assays
Cells were pregrown in 40 ml of CM to midlogarithmic phase, collected by centrifugation, and reinoculated at a concentration that would yield 2-3 × 107 ml at the end of the experiment in 40 ml of fresh CMt containing 0.5 or 1.0 mM fatty acids. Following incubation with fatty acids, cells were washed with 1% tergitol solution three times and distilled water three times. Aliquots of cells subjected to this procedure were plated on YPD medium and found to show no significant changes in viability compared with unwashed control cells.
To measure fatty acid incorporation in FAT1-overexpressing cells, strains were grown in CMt (2% raffinose) medium, reinoculated in fresh CMt galactose medium, and grown for 4 h to induce the GAL1 promoter. 1 mM 18:2 was then added to these cultures for 1 h.
To compare uptake over short times in fatty acid synthesis-defective
strains, fas2 and fat1
/fas2
cells were
grown in CMt supplemented with a mixture of odd numbered fatty acids
(13:0, 15:0, and 17:0; 0.2, 0.4, and 0.2 mM, respectively).
After 72 h of growth, the native, even chain length, fatty acids
are replaced with odd chain species (Table VII), permitting detection
of imported even numbered species (18:1, 14:0, and 16:0) that would
normally be masked by their endogenous counterparts. Import was
monitored by the addition of 0.5 mM even chain fatty acids
to the growth medium.
For incorporation assays in faa strains, aliquots of
cells grown to late log phase in CMt glucose medium were diluted to 6 × 105 cells/ml in 40 ml of fresh medium and grown
to 2 × 107 cells/ml prior to the addition of the test
fatty acid. In experiments of more than 30-min duration, the initial
cell concentration was modified so that the final culture density would
be 2 × 107 cells/ml.
RNA Isolation and Northern Blot Analysis
Total yeast RNA was isolated as described previously (19). Equal amounts (10 µg) of total RNA from each time point of an experiment were analyzed by Northern blots using 1% formaldehyde gels. RNA from the gels was transferred to Zeta ProbeTM membranes (Bio-Rad) using a vacuum blotter (Bio-Rad). Northern blots were quantified using a PhosphorImager (Molecular Dynamics).
Heterologous Expression of the FAT1 Gene in Sf9 Insect Cells
Cloning FAT1 Gene into a Baculovirus Transfer Vector-- A 2.3-kb DNA fragment containing the FAT1 protein coding sequence and 3'-untranslated region was released by BamHI and NotI digestion of vector pCRSK-FAT1 (Table II). The element was inserted into the pVL1393 vector (Pharmingen) that was linearized by BamHI and NotI digestion, placing the FAT1 sequence under control of the baculovirus polyhedrin promoter. Two vectors (pVLFAT1-1 and pVLFAT1-2) derived from independent E. coli transformation were used for transfection.
Generating Recombinant Baculoviruses by Co-transfection-- All reagents, plasmid vectors, and insect cells for baculovirus expression were purchased from Pharmingen, and experiments were done according to the manufacturer's protocols. Uninfected Spodoptera frugiperda (Sf9) cells were maintained by subculturing 1:3 dilutions into 15 ml of TNM-FH medium upon confluency. Cells were incubated at 26-28 °C. For co-transfection, a mixture of 0.5 µg of BaculoGold DNA (consisting of Autographa californica nuclear polyhedrosis virus DNA (Pharmingen, catalog no. 21100D)) and 5 µg of either the recombinant pVLFAT1-1 or -2 vector or the pVL1393 control vector were mixed by gentle vortexing and allowed to sit for 5 min before adding 1 ml of transfection buffer B (25 mM Hepes, pH 7.1, 125 mM CaCl2, 140 mM NaCl). Three drops of healthy Sf9 cells were dropped onto each 60-mm tissue culture plate containing 3 ml of fresh TNM-FH medium. The plates were allowed to sit for 5 min to allow cells to attach. The medium was then aspirated and replaced with 1 ml of transfection buffer A (Grace's medium supplemented with 10% fetal bovine serum). One ml of transfection buffer B/DNA solution was added drop by drop into each of the remaining plates for co-transfection. Plates were incubated at 27 °C for 4 h, washed with 3 ml of fresh TNM-FH medium, and then replenished with 3 ml of fresh TNM-FH medium. The plates were incubated at 27 °C for 4-5 days.
Amplification of Baculovirus Vectors by Serial Infection-- After 5 days of transfection, 0.5 ml of the supernatants were inoculated to the plates containing healthy insect cells growing in 3 ml of fresh TNM-FH medium. Cells infected with viral stocks were grown for 5 days. 0.8 ml of recombinant viral supernatant from the infection plate was transferred to healthy Sf9 cells in 10 ml of TNM-FH medium. If necessary, detached Sf9 cells from the infection plate were cleared from the supernatant by microcentrifugation before transfer.
Preparation of Insect Cellular Extract for the Very Long Chain
Acyl-CoA Synthetase Assay--
Cells grown for 48 h after
infection were transferred to sterile 15-ml centrifuge tubes and
collected by centrifugation at 2,500 × g for 5 min.
Cell pellets were resuspended in 1.5 ml of phosphate-buffered saline
solution by gentle pipetting and washed three times with the same
solution. Cells were resuspended in lysis buffer (5 mM MES
(pH 6.0), 0.6 M sorbitol, 1 mM KCl, 0.5 mM EDTA (pH 8.0), 0.1% ethanol (v/v), 1 mM
phenylmethylsulfonyl fluoride, 4 mM benzamidine, 5 µg/ml
leupeptin, 5 µg/ml pepstatin) and disrupted by three 10-s sonications
with 1 min intervals on ice. Cell debris was removed by centrifugation
at 1,400 × g for 5 min. The supernatant was assayed
for very long chain acyl-CoA synthetase activity using the procedure
described for the analysis of yeast membrane fractions.
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RESULTS |
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The Amino Acid Sequence of the S. cerevisiae FAT1 Gene Is Highly Homologous to Those of Mammalian Very Long Chain Fatty Acyl-CoA Synthetases-- A homology search revealed that FAT1 is related to a rat gene that encodes a very long chain fatty acyl-CoA synthetase. After PCR cloning and DNA sequencing, we determined that the GenBankTM sequences YBR041W that describe the FAT1 sequence contained an error in codon 619. Correction of the sequence yielded an open reading frame encoding a 669-amino acid polypeptide with a significant improvement in homology to the rat protein (Fig. 1). A revised version of the FAT1 gene sequence noting the same error was provided to the sequence data base (GenBankTM number AF065148) by another laboratory while this work was in preparation. The amino acid homology and identity between two proteins are 50 and 33% over 570 amino acids, respectively. By comparison, a human homologue in the data base (GenBankTM number D88308) is 93% identical to the rat protein and 33% identical to Fat1p. In addition to the sequence similarity, Fat1p and the mammalian VLACS share several common characteristics. Both proteins are very close in amino acid length (669 and 620 residues), in theoretical pI (8.54 and 8.82), and in calculated molecular mass (77141 and 70694 daltons). Analysis by the Psort method, which predicts protein localization sites, indicates that Fat1p might be located in the ER membrane, peroxisomes, or plasma membrane. The rat VLACS protein is known to be localized to peroxisomes and microsomes. Both proteins contain an AMP-binding motif that is commonly found in acyl-CoA synthetases, and hydropathy analysis using the Kyte and Doolittle algorithm revealed that the two proteins have four hydrophobic potential membrane-spanning domains located in similar positions.
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Disruption of FAT1 and Measurement of VLACS Activity in Deletion
Strains--
Disruption of the FAT1 gene by homologous
recombination produced viable haploid cells with normal growth
characteristics, indicating that the gene is not essential under normal
culture conditions. To verify that Fat1p functions as a very long chain acyl-CoA synthetase, VLACS activity was measured in cell extracts of
the fat1 and wild type strains using 24:0 as a substrate
(Table IV). The specific enzyme activity
in the postnuclear extract was 2-fold greater in wild type cells that
were grown on glycerol and oleic acid (used to induce peroxisome
maturation) than on cells grown with glucose as a carbon source. Total
enzyme activity toward the 24:0 substrate was reduced to
wild type levels in glucose-grown fat1
cells and to
the wild type levels in oleic acid-grown cells.
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Heterologous Expression of the Yeast FAT1 Gene in Sf9 Cells-- To further verify that the FAT1 gene encodes a very long chain acyl-CoA synthetase, the gene was expressed in a heterologous insect cell system. Results presented in Fig. 2 show that, using a 24-carbon fatty acid substrate, the acyl-CoA synthetase activity in Sf9 cells infected with the FAT1-containing baculovirus was about 4-fold greater than the activity in uninfected cells and 5-fold greater in cells infected with wild type virus. Taken together, the data strongly support the hypothesis that the FAT1 gene encodes a very long chain acyl-CoA synthetase enzyme.
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Intracellular Location of Fa1p-- Reports of subcellular fractionation of the yeast Pichia pastoris and mammalian cells has shown that VLACS activities are primarily found in peroxisomal and microsome fractions (5). VLACS activities in S. cerevisiae were found in similar locations (Table IV). In oleic acid-induced cultures, the total activity was distributed between the 25,000 × g organelle pellet (approximately 30%), and its supernatant fractions (approximately 70%). Fractionation of the 25,000 × g supernatant into a microsomal pellet and 250,000 × g supernatant fraction revealed that approximately 25% of that activity was associated with the microsomes and 75% remained in the supernatant fraction. The latter contains high levels of a low density protein fraction that is apparently associated with oil bodies produced under the oleic acid induced culture conditions. No significant low density fraction was seen in the 250,000 × g supernatant fraction of extracts from glucose grown cells. These results indicate that under oleic acid-induced conditions, the Fat1p-dependent VLACS activity is not peroxisome-specific but is distributed in several cytoplasmic and organellar fractions.
Fat1p Directs Green Fluorescent Protein Chimeras to ER and
Peroxisome-like Structures--
To further determine the cellular
location of Fat1p, the GFP coding sequence was fused to either the C
terminus or the N terminus of FAT1 protein coding sequence
under the control of the GAL1 promoter. Both types of fusion
proteins were functional and restored the wild type very long chain
fatty acid composition in galactose-induced fat1 cells.
In cells grown only on galactose, most of the GFP fluorescence
surrounded the 4',6-diamidino-2-phenylindole-stained nucleus (Fig.
3, A and B),
indicating that the fusion proteins are mainly localized to ER. In many
cells, GFP fluorescence was also associated with one or two small,
membrane structures in close proximity to the plasma membrane. These
are similar in size and location to microbodies (peroxisomes)
previously identified by serial electron microscope sections and
immunofluorescence-stained cells of Saccharomyces (20, 21)
grown in fatty acid-deficient medium conditions. In cells induced with
galactose plus oleic acid, the fluorescence is associated with more
numerous and larger intracellular bodies that are more irregularly
shaped than those in galactose-grown cells. This is also consistent
with EM and fluorescence observations of oleic acid-induced cells,
which undergo peroxisomal proliferation and the formation of large oil
bodies (20, 21). The location of the fluorescence in cells grown under
both conditions was distinct from the
4',6-diamidino-2-phenylindole-stained mitochondria.
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FAA2, Not FAT2, Contributes Most of the Residual VLACS Activity in
fat1 Cells--
The increased levels of residual VLACS enzyme
activity detected in fat1
strains grown in oleic
acid-containing medium suggest that an additional VLACS enzyme is
induced by the fatty acid. Two genes that encode oleic acid-induced
peroxisomal AMP-binding proteins were identified that might be the
source of the inducible residual activity. PcsP60/FAT2 (21)
and FAA2 (22) were previously identified as oleic
acid-inducible genes that encode peroxisomal proteins. Although its
function is not clearly understood, Fat2p contains an
ATP-dependent AMP binding site and shows a high degree of
similarity to the E. coli long chain acyl-CoA synthetase.
FAA2 has been previously shown to be a fatty acyl-CoA synthetase (9). Faa2p is reported to be a peroxisomal fatty acyl-CoA synthetase that is
localized to the matrix side of peroxisomal membrane, where it is
involved in the conversion of medium and long chain fatty acids to
their CoA derivatives (22).
To examine the roles of Faa2p and Fat2p in VLCFA activation, we
constructed strains containing combinations of disrupted
FAT1, FAT2, and FAA2 genes. VLACS
activity levels in fat1/fat2
cells grown on
glycerol-oleic acid medium did not differ from that observed in the
fat1
disruption strain (Table IV), indicating that Fat2p is not the source of the residual activity. VLACS activity in the oleic
acid-induced fat1
/faa2
strain, however, was reduced in
the postnuclear supernatant fraction by 80% compared with the fat1
strain. Those cells contained less than 5% of the
total VLACS activity found in wild type. As expected, due to the
peroxisomal matrix location of Faa2p, the loss of residual activity was
primarily associated with the organelle pellet fraction (Table IV).
These data indicate that Fat1p and Faa2p constitute the major VLACS activities in yeast, with Fat1p being the primary activity. Although previous reports suggest that Faa2p is involved in medium and long
chain fatty acid activation (22), these data indicate that it also
exhibits activity toward very long chain fatty acid substrates. This is
consistent with reports on the rat enzymes, which showed that the long
chain acyl-CoA synthetase in rat peroxisome exhibits VLACS activity,
although its activity was much stronger (359-fold) toward long chain
fatty acid 16:0 than toward 24:0. By contrast, the purified rat
peroxisomal VLACS enzyme was only 1.5-fold more active toward the 16:0
substrate than the 24:0 substrate (6).
FAT1 Expression Is Not Induced by Fatty Acids-- The FAA2 gene has been shown to contain oleate-responsive elements in its promoter and to be strongly induced in cells grown on glycerol-oleate medium (22) in a manner similar to that of peroxisomal protein genes that are repressed by glucose, de-repressed by glycerol, and activated by fatty acids (23, 24). Examination of the FAT1 promoter region did not reveal sequences that were homologous to oleate-responsive elements. To see if the FAT1 gene is regulated by similar conditions, quantitative measurements of its mRNA levels were performed by Northern blot analysis using the constitutively expressed ACT1 (actin) gene as an internal standard.
No differences were observed in FAT1 mRNA levels in wild type cells grown in glucose medium with or without fatty acids (data not shown). FAT1 mRNA levels are approximately 2 times higher in glucose grown cells than in glycerol-grown cells that are cultured in fatty acid-deficient medium (Fig. 4a, lanes 1 and 2). The addition of 0.5 mM 16:0 to cells grown in glycerol medium elevates FAT1 mRNA levels 2-fold to the same level as in glucose-grown cells (Fig. 4a, lanes 4, 6, and 8). Identical results were observed when the fatty acid 18:2 was added to the growth medium. Under similar induction conditions, expression of peroxisomal genes is increased 10-250-fold (16, 23, 25). To test the response of FAT1 when cells were dependent on the import of nutrient fatty acids, fatty acid synthetase activity was inhibited by cerulenin (Fig. 4a, lanes 3, 5, 7, and 9). No significant changes in FAT1 expression were found under those conditions. Overexpression of Elo2p, a condition that we found to cause an increase in VLCFAs (Fig. 4a), also did not increase the expression of FAT1 (Fig. 4b). These data indicate that FAT1 does not follow the same pattern of expression seen with peroxisomal proteins that are induced by exogenous fatty acids. This may be linked to a requirement for FAT1p function in nonperoxisomal membrane fractions that are insensitive to fatty acid induction.
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FAT1 Deletion Causes an Accumulation of Intermediate Chain Length
Saturated VLCFAs--
A characteristic of a human X-linked
adrenoleukodystrophy is a reduction of VLACS activity that leads to the
accumulation of saturated VLCFAs. We suspected that FAT1
deletion might cause a similar accumulation of VLCFAs. This was
confirmed by quantitative gas chromatography of total fatty acids
isolated from wild type and fat1 cells grown on minimal
glucose medium.
No significant differences were seen between the fatty acid
compositions of wild type and fat1 cells with respect to
the major, C14-C18, long chain fatty acid
species. The FAT1 deletion, however, caused striking changes
in very long chain fatty acid levels (Table
V). Wild type cells usually contain less
than 5% very long chain fatty acids. Within that class, 26:0 and
hydroxy-26:0 are the most abundant species, and intermediate length
(C20-C24) VLCFAs usually comprise less than
10% of the total VLCFAs. Disruption of FAT1 dramatically
increased the levels of C22-C26 saturated VLCFAs but did not change in the levels of hydroxy-26:0 species (Table
V). In logarithmic phase cells, there was a 2-fold increase in 20:0, a
6-fold increase in 22:0, a 10-fold increase in 24:0, and 2-fold
increase 26:0. Accumulation of VLCFAs in fat1
strains reached even higher levels as the cells approached stationary phase
(data not shown). Under those conditions, 24:0 comprises 3.7% of the
total fatty acids compared with 0.13% in wild type, and 26:0 is
elevated to 6% from 2.6% in wild type strains.
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The abnormal accumulation of intermediate chain length fatty acids was
found in both the sphingolipid and phospholipid fractions (Table
VI). In sphingolipids of the
fat1 strain, 22:0, 24:0, hydroxy-24:0, and 26:0 were
increased 24-, 53-, 5-, and 2.4-fold, respectively. By contrast, 22:0,
24:0, and 26:0 levels in the phospholipid fraction were increased 5.5-, 6.8-, and 1.6-fold. No increase in hydroxy-VLCFAs was observed in the
phospholipid fraction, which is consistent with our previous report
that Fah1p, a sphingolipid fatty acid hydroxylase, adds an
-hydroxyl
group to the fatty acid after it is acylated with sphingosine to form a
ceramide (15). A small increase in free fatty acid levels was observed
in the fat1
strain compared with wild type cells (5.4 versus 3.0%, respectively). The increase was not caused by disproportionate accumulations in very long chain fatty acid species, however, and consisted almost exclusively of increases in
C12-C18 fatty acids. To verify that VLCFA
accumulations were caused by the absence of Fat1p activity, a plasmid
containing a copy of the FAT1 gene under control of the
GAL1 promoter was transformed into fat1
cells.
Normal levels of fatty acids were observed when the plasmid-borne gene
was expressed by adding galactose to the growth medium (data not
shown).
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Overexpression of Fatty Acid Elongation Enzymes in a fat1 Strain
Further Elevates VLCFA Levels--
We recently identified two
essential genes, ELO2 and ELO3, that are
components of an ER membrane-bound enzyme system that forms VLCFAs (1).
Elo2p appears to be involved in the formation of
C20-C24 VLCFAs, and Elo3p is involved in the
formation of C20-C26 VLCFAs and is essential
for the formation of 26:0. Disruption of either gene causes reduced
VLCFA levels, the accumulation of intermediate length VLCFAs, and a
concomitant reduction in ceramide synthesis and sphingolipid levels.
Given the accumulation of intermediate length fatty acids in
fat1 strains and the cytoplasmic/microsomal location of a
fraction of Fat1p, we hypothesized that Fat1p might act to remove
intermediate length VLCFAs that accumulate abnormally during the fatty
acid elongation process and thus play a role in maintaining VLCFA
levels. To test this interaction, we perturbed VLCFA levels by
manipulating Elo2p and Elo3p activities.
Elo2p expression under control of the GAL1 promoter was
elevated by induction with galactose for 8 h. In wild type cells, overexpression of Elo2p results in a 6-fold increase of the production of C20-C26 saturated VLCFA species (Fig.
5a). When Elo2p was
overexpressed in fat1 cells, saturated VLCFA levels were
15-fold greater than in wild type cells. The combined effect of Elo2p
overexpression and fat1 deletion was to elevate VLCFA levels
to 20% of total cell fatty acids. The elevated production of VLCFAs,
however, did not affect the levels of hydroxy-VLCFAs, changing the
normal 1:1 ratio of 26:0 to hydroxy-26:0 to about 4.5:1.
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In wild type cells, overexpression of Elo3p in conjunction with
fat1 produced a strikingly different pattern of VLCFA
accumulation. This resulted in a 2-fold increase in 26:0 with no
significant changes in intermediate VLCFAs (Fig. 5b).
Overexpression of Elo3p in the fat1
strains did not
increase the elevated levels of 22:0 and 24:0 but caused a greater than
2-fold further increase in the 26:0 levels elevated by either the
disruption of FAT1 or the overexpression of Elo3p.
An elo3/fat1
Double Disruption Strain Accumulates 22:0, but
Not 24:0 or 26:0, with Severely Retarded Growth--
Oh et
al. (1) showed that elo3
gene-disrupted strains
failed to elongate 24:0 to 26:0. This results in the absence of 26:0
and elevated intermediate VLCFA levels. The most abundant of these are
22:0, hydroxy-16:0, and hydroxy-22:0. Elo3 deletion strains also grow
very slowly, possibly due to the lack of normal sphingolipid
composition. Simultaneous disruption of ELO3 and FAT1 resulted in more severe retardation of growth (data not
shown) and further changes to fatty acid composition that included
elevation of 20:0 and 22:0 and the loss of accumulated hydroxy-22:0,
hydroxy-24:0 and a greater than 2-fold reduction in hydroxy-16:0 (Table
V).
Growth of the fat1/fas2
Strains Is Severely Retarded on
18:1-containing Medium but Is Similar to Its Parental FAT1/fas2
Strain When Grown on Saturated Fatty Acids
Previous reports indicated that FAT1 plays a role in the transport of nutrient fatty
acids (10). Disruption of FAT1 was reported to impair the
uptake of long chain fatty acids and reduce the rate of growth on fatty acid-supplemented growth medium when fatty acid synthetase activity was
inhibited by the antibiotic cerulenin. Although inactivation of fatty
acid synthase causes a cellular requirement for saturated fatty acids,
reduced growth rates were most pronounced on cerulenin-treated fat1
cells that were grown in oleic acid-containing medium.
We examined this effect further to try to discriminate between
fat1-derived fatty acid transport defects and growth
defects that might be caused by the failure to metabolize the
internalized fatty acids. The requirement for exogenous fatty acids in
cerulenin-treated cells does not appear to affect the expression of
FAT1. Our tests indicate that FAT1 mRNA
levels do not differ between cells grown in glucose (data not shown) or
glycerol medium with or without cerulenin and 0.5 mM 16:0
(Fig. 4a). To test the effects of inactivated fatty acid
synthetase on transport, we disrupted the FAT1 gene in a
previously constructed fas2
::LEU2 strain.
FAS2 encodes the
-subunit of fatty acid synthetase, and
its disruption inactivates saturated fatty acid biosynthesis without
affecting other enzyme systems that might also be sensitive to
cerulenin. The resulting fat1
::HIS3/fas2
::LEU2 disruption
strains were isolated on media supplemented with a mixture of 14:0 (0.2 mM), 16:0 (0.4 mM), and 18:0 (0.2 mM).
The fat1/fas2
and fas2
strains were
grown to midlog phase in YPD medium supplemented with a mixture of
saturated fatty acids (14:0, 16:0, and 18:0 (0.2, 0.4, and 0.2 mM, respectively)). Washed cells from that culture were
used to inoculate, at a density of 105 cells/ml, minimal
glucose medium containing no fatty acids, 14:0, 16:0, 18:1, or a
mixture of 14:0, 16:0, and 18:0. Growth was monitored by counting cells
with a hemocytometer (Fig. 6). Both types
of cells failed to grow on medium containing no fatty acids. Active and
identical growth patterns were observed for the fas2
and fat1
/fas2
strains on medium containing 16:0 or a
mixture of 14:0, 16:0, and 18:0. In the 14:0-supplemented culture,
growth of the fat1
/fas2
strain was slightly slowed
compared with that of the FAT1/fas2 strain. By contrast, the
growth rate of the fat1
/fas2
strains in the
18:1-supplemented culture was severely retarded compared with the
FAT1/fas2
parent (Fig. 6d). Under those
conditions, the fat1
/fas2
cells grew only two
generations before growth was arrested.
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To examine the incorporation of fatty acids into these strains, the
fas2/FAT1 and fas2
/fat1
strains were
grown for 72 h in medium containing the odd chain fatty acids,
13:0, 15:0, and 17:0. As we have previously observed with wild type
cells (26), both strains grew on these supplements at a rate similar to
that observed with the 14:0/16:0/18:0-supplemented medium. The imported 13-, 15-, and 17-carbon fatty acids are readily elongated to odd chain
VLCFAs and converted to monounsaturated acids in similar proportions to
those found with endogenous even chain fatty acids in wild type cells.
Under those conditions, the odd chain acids replaced virtually all of
the even chain species. No significant differences between the fatty
acid compositions of the fas2
/FAT1 and
fas2
/fat1
strains were observed (Table
VII). Furthermore, the odd chain fatty
acids 25:0 and hydroxy-25:0 were the most abundant VLCFAs in those
strains, indicating that the imported saturated acids can serve as
substrates for the fatty acid elongation system. Levels of 25:0 were
elevated 9-fold in the fat1
/fas2
strain compared with
the FAT1/fas2
, indicating that Fat1p plays a role
controlling the levels of VLCFAs derived from imported fatty acids as
well as the endogenously derived species.
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This fatty acid replacement technique was used to further monitor the
transport of 18:1 and related unsaturated fatty acids in the
FAT1/fas2 and fat1
/fas2
strains. No
significant differences were seen in the uptake of 18:1 or 18:2 over
intervals of 10 min (Fig. 7). Similar
rates of import between the two strains were also obtained with the
even chain saturated species 14:0 and 16:0 (data not shown).
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To test for the effects of Fat1p function on long term import,
glucose-grown wild type and fat1 cells were simply
incubated with 18:2, 17:0, 18:1, or 17:1 for 4-6 h prior to analysis
(Fig. 8a). No significant
differences were observed in the levels of any of the fed fatty acids
between the two strains. These tests indicated that the fat1
deletion does not affect the bulk uptake of fatty acids at levels
needed to sustain growth. They further indicate that 18:1 and other
long chain fatty acids are imported at similar rates to the saturated
fatty acids required to sustain growth of the
fat1
/fas2
strain. This suggests that the growth retardation of fat1
/fas2
strains in 18:1-supplemented
medium is caused by the defects in the metabolism of that fatty acid after it is imported.
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As an additional test of the role of Fat1p in fatty acid transport, we
examined the import of fatty acids in cells containing a
FAT1 gene expressed to high levels under the control of the strong GAL1 promoter (Fig. 8b). Cells were tested
for their ability to incorporate 18:2 for 1 h after 4 h of
growth on the 2% galactose inducer. In the wild type strain under
those conditions, levels of the incorporated 18:2 reach approximately
20% of the total cellular fatty acid mass. There were no significant
differences observed in the incorporation of 18:2 between the wild
type, fat1, and the fat1
strain containing
the induced FAT1 gene.
Faa1p and Faa4p Are Required for Bulk Transport of Fatty Acids in
Saccharomyces--
A similar but more pronounced response to oleic
acid has been previously reported for the two fatty acyl-CoA
synthetases, Faa1p and Faa4p. FAA1 and FAA4 are
functionally interchangeable genes that account for 99% of the total
cellular 14:0-CoA and 16:0-CoA synthetase activities (8) and are
responsible for the activation of these imported fatty acids.
Simultaneous disruption of the two genes blocks growth and eventually
leads to cell death when faa1/faa4
cells are grown on
cerulenin. We had also previously observed that fatty acid-specific
repression of the Saccharomyces OLE1 gene was
blocked by simultaneous disruption of the FAA1 and FAA4 genes (11). Previous metabolic radiolabeling studies
had suggested that 14:0 and 16:0 were incorporated into the
faa1
/faa4
cells, and the growth defect was attributed
to the inability of the cells to activate the imported fatty acids
(8).
Given the similarity of this phenotype to the response of
fat1-disrupted cells, we examined the ability of
faa1
/faa4
cells to incorporate sufficient quantities
of fatty acids to sustain growth in the presence of inactivated fatty
acid synthetase activity. Wild type and faa1
/faa4
gene-disrupted cells were incubated with 1 mM 18:2 for up
to 6 h.
Analysis of fatty acid methyl esters derived from the washed cell
pellets showed a rapid incorporation of the 18:2 into the wild type
cells so that by 6 h the supplemented fatty acid comprised approximately 60% of the total cellular fatty acids (Fig.
9a). Fatty acid import into
the faa1/faa4
strain was negligible under the same
conditions. Similar effects were observed in the transport of saturated
fatty acids. Incorporation of 15:0 into wild type cells resulted in its
rapid import and either desaturation to 15:1 or elongation to 17:0 and
subsequent desaturation of that species to 17:1. The total accumulation
of the imported and subsequently modified species in wild type cells
over 6 h was greater than 65% of the total cellular fatty acids
(Fig. 9b). Under the same condition, negligible
incorporation of 15:0 was observed in the faa1
/faa4
strain. Furthermore, no 15:1, 17:0, or 17:1 was observed in the
faa1
/faa4
cells, indicating that the small amounts of bound 15:0 were not accessible to the microsomal desaturase or elongation systems. The overlapping functions of Faa1p and Faa4p were
illustrated by comparison of cells containing single disruptions of
each gene with the double disrupted strain (Fig. 9a). Cells containing the single faa4
disruption imported fatty
acids at slightly higher levels than wild type, whereas
faa1
cells closely paralleled the wild type
incorporation.
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DISCUSSION |
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In this report, we show that the yeast FAT1 gene encodes a very long chain fatty acyl-CoA synthetase. Although Fat1p was previously reported to have homology to a presumptive murine fatty acid transport protein (10), further analysis revealed other homologies to members of the acyl-CoA synthetase enzyme family that include an ATP-dependent AMP binding sequence (27) and elements of a recently identified rat very long fatty acyl-CoA synthetase (7). The correction of an error in the FAT1 DNA sequence from the yeast genome data base significantly improved the homology comparison between Fat1p and the mammalian VLACS genes.
A number of fatty acyl-CoA synthetases have been recently identified in
yeast. Faa1p and Faa4p, which account for 99% of the total acyl-CoA
synthetase activity toward long chain fatty acid substrates, apparently
do not catalyze the activation of very long chain fatty acids (9, 28).
The role of Fat1p in VLCFA metabolism, however, is readily evident.
Disruption of the FAT1 gene causes a severe loss of VLACS
activity and the subsequent accumulation of VLCFAs. In glucose-grown
cultures, Fat1p accounts for about 90% of the total activity toward
C24 fatty acids. In oleic acid-induced cultures, Fat1p is
responsible for approximately 70% of the total VLACS activity.
Experiments described here indicate that most of the remaining activity
toward very long chain substrates in the induced cultures is catalyzed
by Faa2p. FAA2 has recently been shown to be an oleic
acid-inducible gene that encodes a peroxisomal matrix protein involved
in the -oxidation of medium chain and long chain fatty acids (22).
The ability of Faa2p to form 24:0 CoA in these studies indicates that
it has an broader range of substrate specificity than the
C6-C20 activity observed when it is expressed
as a recombinant protein (28). Faa3p does show activity toward
C9-C24 substrates when expressed in E. coli (28), but its activity toward all substrates is very low. It
apparently plays a minor, if detectable, role in vivo.
Fractionation studies of Fat1p activity suggest that it is distributed over multiple cellular locations, particularly under growth conditions that induce peroxisomal proliferation. This is consistent with a previous report of VLACS activities in the yeast P. pastoris (5). The intracellular locations of Fat1p in oleic acid-induced and -uninduced cells is further supported by a fusion of green fluorescent protein to elements of Fat1p. In uninduced and induced cells, the most intense fluorescence is found in areas immediately surrounding the nucleus, which is consistent with an ER location. In many uninduced cells, intense fluorescence is also associated with one or two small, spherical regions that are typically located below the plasma membrane. This is consistent with descriptions of microbodies (peroxisomes) found by electron microscopy of serial sections of Saccharomyces grown in fatty acid-depleted medium (20). The same studies revealed that growth under oleic acid-induced conditions produces multiple microbody profiles and the proliferation of lipid bodies. This is consistent with the pattern of fluorescence produced by the Fat1p-GFP chimera in oleic acid-induced cells.
The accumulation of C20-C24 VLCFAs in
fat1 strains and their further elevation when VLCFA
synthesis is perturbed appears to be an important clue to the primary
function of Fat1p. Although disruption of FAT1 results in
the major loss of very long chain-specific acyl-CoA synthetase
activity, it appears unlikely that Fat1p is directly involved in VLCFA
biosynthesis. Very long chain fatty acid levels, including the normally
abundant C26 species, are actually increased in
fat1
cells. Taken together, these data suggest that Fat1p
primarily plays a catabolic role in VLCFA metabolism, presumably by
mobilizing excess very long chain species for degradation via
-oxidation. This further indicates that other, unidentified, acyl-CoA synthetase activities are employed in the microsomal fatty
acid elongation system.
The high levels of Fat1p activity suggest that VLCFA biosynthetic rates
may be greater than indicated by steady state levels of cellular very
long chain fatty acids. The production of excess acids might, in fact,
be a distinctive property of the membrane-bound elongation system.
Unlike fatty acid synthetase, which keeps its substrate covalently
linked to the multifunctional enzyme complex until the
C14-C18 product is released, the VLCFA
biosynthetic system elongates fatty acids by a series of reactions that
are catalyzed by separate membrane-bound enzymes (29). If transfer of
substrates between reaction centers is inefficient, significant amounts
of C20-C24 species might be displaced from the
system before they were extended to 26:0. This could require high
levels of Fat1p as a component of a system that "scavenges"
intermediate chain length fatty acids and transfers them to peroxisomes
for -oxidation. The existence of this system might allow cells to control VLCFA levels primarily through fatty acid degradation to
prevent toxic accumulations of VLCFAs. Such accumulations cause lethal
conditions such as X-linked adrenoleukodystrophy and similar lipid-mediated disorders. This regulatory mechanism could act on fatty
acids prematurely released from the elongation system or in concert
with phospholipases that remove excess VLCFAs that have been
erroneously acylated into ceramides, triglycerides, or phospholipids.
This would also be consistent with the multiple cellular locations
observed for Fat1p.
A regulatory role of Fat1p is also consistent with the effects seen in
fat1 cells when fatty acid elongation is perturbed. Our
previous observations (1) indicate that overexpression of
ELO2 or ELO3 produces higher levels of VLCFAs
than in wild type cells. The large elevations of VLCFAs in
fat1
cells when ELO genes are either
overexpressed or deleted support that hypothesis and suggest that
maintenance of optimal VLCFA levels requires a carefully coordinated
balance of control between elongation gene expression and
Fat1p-mediated VLCFA degradation.
The role that FAT1 plays in fatty acid import is difficult to resolve. The level of homology of Fat1p to the presumptive mouse fatty acid transporter (10) is virtually the same as the level of homology to the mammalian peroxisomal acyl-CoA synthetases. Previous reports indicate that disruption of FAT1 can cause a 2-3-fold decrease in the rate of oleate uptake (10). This was based on carefully done kinetic studies using relatively low levels of radiolabeled fatty acids. The cell fractionation and fluorescence localization studies, however, suggest that the majority of Fat1p is located in intracellular membranes and not concentrated at the cell surface.
We attempted to assess whether Fat1p contributes to the functionally
significant import of fatty acids under conditions where cells require
them to sustain growth. Under those conditions, we could see no clear
differences between fat1 and FAT1 strains in
the rate of import or the level of incorporation for either saturated
or unsaturated long chain fatty acids. We cannot rule out, however,
that Fat1p might play some role in importing fatty acids that are
present at very low concentrations in the growth medium. The ability of
fat1
strains to import fatty acids contrasts markedly,
however, with the inability of the faa1
/faa4
strains to transport fatty acids under the same experimental conditions. The
loss of import when these two genes are disrupted are consistent with
previous reports that fatty acyl-CoA synthetases play a functional role
in vectorial transport of fatty acids in E. coli (27, 30) and mammalian cells (31).
Previous reports of fat1 and faa1
/faa4
gene-disrupted strains with inhibited fatty acid synthetase activity
indicate that they exhibit similar growth defect phenotypes when they
are supplied with 18:1 (8, 10). This could be interpreted as caused by defects either in the transport of 18:1 or in its metabolism after it
is internalized. We attempted to replicate these experiments by
specifically inactivating fatty acid synthetase through gene disruption. In the case of the faa1
/faa4
strain,
growth inhibition appears to be caused by a simple failure to import
sufficient 18:1 (or other saturated and unsaturated fatty acids) to
sustain growth. By contrast, the fat1
/fas2
strain
appears to import sufficient saturated and unsaturated fatty acids but
is apparently unable to employ the imported 18:1 in some essential
metabolic function.
The inability of fas2/fat1
strains to metabolize 18:1
suggests that Fat1p has a broad acyl chain length specificity that includes 18-carbon fatty acids and that it plays a role in the metabolism of both long chain and very long chain fatty acids. The
homologous rat liver peroxisomal VLACS activity is also reported to be
active toward both long (C16) and very long chain
(C24) fatty acids (7), indicating that the function of
these enzymes may be to modulate a wide range of fatty acid species.
Experiments described in this paper indicate that a primary role Fat1p
involves the maintenance of very long chain fatty acid levels by a
mechanism that is not clearly understood. One of the most striking
effects caused by inactivation of Fat1p activity is the elevation of
VLCFA levels under conditions in which very long chain fatty acid
biosynthesis rates are perturbed. This suggests that Fat1p-mediated
modulation of VLCFA levels is a component of a regulatory system that
prevents toxic accumulations of those fatty acids. In humans, loss of
control of VLCFA metabolism and accumulation of VLCFAs can be
devastating, resulting in progressive degeneration of neural and organ
tissue and eventual death. The interactions of the
Saccharomyces Fat1p and its VLCFA biosynthetic system may
provide a useful model system for understanding complex genetic
diseases associated with VLCFA regulation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Jeffrey Gordon for providing FAA deletion strains and for valuable discussions on fatty acid transport. We also thank Joanne Barbiaz for technical help on fluorescence microscopy and Dr. Chan-Seok Oh for the pGALELO2 and pGALELO3 vectors. We thank Dr. Jon Huibregtse for assistance and advice with the expression of the FAT1 gene in insect cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM45768.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: Nelson Biological
Laboratories, Bureau of Biological Research, Dept. of Cell Biology and
Neuroscience, Rutgers University, Piscataway, NJ 08854-8082. Tel.:
732-445-3972; Fax: 732-445-0644.
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ABBREVIATIONS |
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The abbreviations used are:
VLCFA, very long
chain fatty acid;
VLACS, very long chain fatty acyl-CoA synthetase;
PCR, polymerase chain reaction;
kb, kilobase pair(s);
ER, endoplasmic
reticulum;
MES, 4-morpholineethanesulfonic acid;
Mops, 4-morpholinepropanesulfonic acid;
GFP, Green fluorescent protein;
FAT1, gene encoding presumptive VLACS;
FAT2, gene
encoding a peroxisomal protein with AMP-binding motif;
FAA2, gene encoding a peroxisomal fatty acyl-CoA synthetase;
FAA1, FAA4 genes encoding fatty acyl-CoA synthetase;
FAS2, gene encoding the -subunit of fatty acid synthetase.
2 Fatty acids are denoted by a standard designation that indicates the number of carbons followed by the number of double bonds (e.g. 26:0, hexacosanoic acid, a 26-carbon fatty acid with no double bonds; 18:2, linoleic acid, an 18-carbon fatty acid with two double bonds).
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REFERENCES |
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