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INTRODUCTION |
Fatty acids serve a number of essential and regulatory functions.
Many cells, although capable of de novo fatty acid
synthesis, scavenge fatty acids from the extracellular milieu thereby
bypassing the energy-expensive synthetic reactions. Cells such as
adipocytes export fatty acids for use by other cells and tissues. The
mechanisms governing fatty acid transport across the plasma membrane in
these cases are poorly understood. Two fundamental mechanisms have been suggested to contribute to this process. (i) Fatty acid transport is
independent of membrane-bound fatty acid transporters and occurs by
simple diffusion. In this case, transport is viewed as driven by a
concentration gradient generated by intracellular utilization. (ii)
Membrane-bound and membrane-associated proteins mediate fatty acid
import and/or export. The challenges we are faced with are to define
the individual contributions of diffusion and membrane-bound and
membrane-associated proteins to promote specificity and selectivity of
the fatty acid transport process. Our laboratory investigates the
process of fatty acid transport using two genetically tractable model
systems, the prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae (reviewed in Ref. 1). Each organism can synthesize endogenous fatty acids or acquire fatty acids from the
extracellular environment for use as a carbon and energy source for
macromolecular synthesis and growth. Yeast in particular is of interest
as a model eukaryote since it shares many features in common with
mammalian systems. Fatty acid synthesis is catalyzed by a type I
(associated) fatty acid synthase (2). Imported fatty acids can be used
for 
-oxidation to supply energy as in muscle cells or for storage in
a lipid body as in adipocytes (3). Fatty acids supplied in the growth
medium repress the transcription of genes encoding proteins required
for fatty acid synthesis (4, 5) and activate the transcription of genes
encoding proteins required for fatty acid degradation (6, 7).
Additionally, as in some mammalian tissues, fatty acids induce the
proliferation of yeast peroxisomes and, therefore, are critical
signaling molecules in organelle biogenesis and development
(8-10).
Import of fatty acids into yeast is an essential function when cells
are auxotrophic for fatty acids. This occurs in the natural environment
when oxygen is limiting due to inhibition of the
O2-requiring 
9-acyl-CoA desaturase (encoded
by OLE1) (11). In addition, a conditional auxotrophy is
imposed when cells are cultured in medium containing the fatty acid
synthase inhibitor cerulenin. In yeast, fatty acid import is saturable
and dependent upon Fat1p, a homologue of the murine fatty acid
transport protein, FATP (12, 13). Our laboratory has hypothesized that
Fat1p is one member of a multi-component fatty acid import apparatus
(12). In the well characterized E. coli model system, fatty
acid import is not only dependent upon the outer membrane-bound
transporter, FadL, but also requires the fatty acyl-CoA synthetase,
FadD (14). In this regard, long-chain fatty acid transport is described
as vectorial acylation. Since in eukaryotic systems, imported fatty
acids must, likewise, be activated by esterification to coenzyme A
prior to metabolic utilization, we hypothesize a similar coupling of
fatty acid import and activation is one mechanism that drives this
process. In the present study we have evaluated the role of the fatty
acyl-CoA synthetases Faa1p and Faa4p of S. cerevisiae in
fatty acid import, activation, and intracellular trafficking.
In S. cerevisiae, four fatty acyl-CoA synthetases encoded by
separate genes have been identified: Faa1p, Faa2p, Faa3p, and Faa4p
(15, 16). Cells carrying deletions in each of the four FAA
genes are viable, suggesting de novo fatty acid synthesis provides sufficient acyl-CoA for essential cellular functions (15).
Faa1p and Faa4p are the primary enzymes involved in activation of
imported long-chain fatty acids (C12-C18),
whereas Faa2p appears to be involved in activation of medium-chain
fatty acids directed toward peroxisomal -oxidation (15). The
physiological role of Faa3p, which is most active toward fatty acids
>C18, has not yet been defined. More recently,
very-long-chain (C22-C26) acyl-CoA synthetase
activity has also been attributed to Fat1p (17, 18), which plays a
pivotal role in long chain fatty acid import (12, 13).
In previous work, our laboratory has devised methods to assess fatty
acid transport in yeast. We define the process of fatty acid transport
as the net movement of fatty acids from the external milieu across the
cell wall and membrane into the internal milieu. Our data support the
hypothesis that transport is complex and includes both diffusional and
protein mediated components. In previous work, we demonstrated one of
the protein components is Fat1p. In the present work, we provide
evidence that a long chain fatty acyl-CoA synthetase, either Faa1p or
Faa4p, is also required. In addition, we demonstrate deletion of
FAA1 and/or FAA4 alters the endogenous acyl-CoA
pools in the absence of exogenous fatty acid, suggesting these enzymes
also play a role in intracellular acyl-CoA metabolism.
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EXPERIMENTAL PROCEDURES |
Strains, Media, and Plasmids--
The Saccharomyces
cerevisiae strains YB332 (a; ura3-52; leu2-3,
112; his3-200; ade2-101; lys2-801), YB513
(a; ura3-52; leu2-3, 112; his3-200;
ade2-101; lys2-01; faa1::HIS3), YB524
(a; ura3-52; leu2-3, 112; his3-200;
ade2-101; lys2-801, faa4::LYS2), YB525
(a; ura3-52; leu2-3, 112; his3-200; ade2-101; lys2-801; faa1::HIS3;
faa4::LYS2), and YB526 (a; ura3-52; his3200; ade2-10; lys2-80; leu2-3, 112;
faa1::HIS3; faa2::LEU2;
faa3::LEU;2
faa4::LYS2) were obtained from Jeffrey I. Gordon (Washington University of Medicine, St. Louis, MO) and used in
all experiments described. YPDA consisted of 1% yeast extract, 2%
peptone, 2% dextrose, and 20 mg/liter adenine-hemisulfate. Yeast
supplemented minimal medium contained 0.67% yeast nitrogen base (YNB),
2% dextrose, adenine (20 mg/liter), uracil (20 mg/liter), and amino
acids as required (arginine, tryptophan, methionine, histidine, and
tyrosine (20 mg/liter); lysine (30 mg/liter); and leucine (100 mg/liter)). To assess growth when fatty acid synthase was inhibited,
cells were grown on YNBD plates supplemented with 45 µM
cerulenin and 100 µM oleic acid (YNB-CER-OLE). To assess growth under hypoxic conditions cells were grown on YNBD plates supplemented with 20 µg/ml ergosterol, 100 µM oleic
acid, and amino acids and uracil as required (YNB-EG-OLE). Growth in
liquid culture and on plates was at 30 °C.
The plasmids pGALFAA1 and pGALFAA4
(FAA1 and FAA4 under the control of the
GAL7 promoter, respectively) were obtained from InvitroGen.
Transformation of the faa1 faa4 strain was
accomplished using the high efficiency lithium acetate protocol
essentially as described (19).
Qualitative Fatty Acid Uptake Measurements--
Fatty acid
transport was assessed using confocal laser scanning microscopy
(CLSM)1 and the fluorescent
long-chain fatty acid analogue
4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12) as described previously
(12, 13). Cells were grown in YNBD to A600 of
0.8-1.0, harvested by centrifugation, washed twice in
phosphate-buffered saline (PBS), and finally resuspended in 0.1 volume
of PBS. Cells (faa1 faa4) harboring either
pGALFAA1 or pGALFAA4 were grown from an overnight
culture in YNB containing 4% sucrose to an A600
of 0.4, at which time cultures were split, 2% glucose added to one,
2% galactose added to the other. Growth was continued until the cell
density reached an A600 of 0.8. Cells were
harvested, washed with PBS, and resuspended in 0.1 volume of PBS as
detailed above. All steps were performed at room temperature. Washed
cells were incubated with 10 µM
C1-BODIPY-C12 for 60 s, washed in PBS
containing 50 µM fatty acid-free BSA (two times), PBS,
resuspended in PBS, and visualized on an NORAN-OZ CLSM, interfaced with
a Nikon Diaphot 200 inverted microscope equipped with a PlanApo 60×,
1.4 numeric aperture oil immersion objective lens. No efflux of fatty
acid was found during the time course used during these experimental
conditions. The use of C1-BODIPY-C12 at a final
concentration of 10 µM was chosen, as this allowed for
the fluorescent signal to be readily visualized using CLSM. The
instrument settings for brightness, contrast, laser power, and slit
size were optimized for the brightest sample to assure that the CLSM
was set for its full dynamic range. The same settings were used for all
subsequent image collections.
Quantitative Fatty Acid Uptake Measurements--
Rates of fatty
acid transport were also determined using the filtration assay
described previously (13). Cells were grown in YNBD containing the
appropriate supplements at 30 °C to mid-log phase, collected by
centrifugation, washed once in PBS, and resuspended in 0.1 original
volume in PBS. 500 µl of cells (1 × 108 cells) were
preincubated for 10 min at 30 °C in PBS, and the assay was initiated
by the addition of [9, 10-3H]oleate at the fatty acid:BSA
ratios indicated in the table legend. At the defined time points (0, 2, 4, and 6 min), 100-µl duplicate samples were diluted into 5 ml of PBS
containing 0.5% Brij 58 (PBS-Brij) and then were immediately filtered
through a Whatman Gf/B filter. The filters were washed three times with
PBS-Brij. Transport rates were defined within the linear range. No
fatty acid efflux was observed over the 6-min period using this
experimental approach. All wash steps were carried out at room
temperature. Filters were air-dried and radioactivity determined by
scintillation counting. The final data were expressed in picomoles of
oleate transported/min/1 × 108 cells and subjected to
analysis of variance using PRIZM software (GraphPad Software, Inc.).
All data presented represent the mean (± stand error of the mean) from
two independent experiments performed in duplicate.
Fatty Acid and Fatty Acyl-CoA Analyses--
Total fatty acid
profiles were determined in cells grown in YNBD to an
A600 of 0.8. Cell growth and metabolism were
stopped by addition of 0.1 volume of 6.6 M perchloric acid.
Cells were harvested by centrifugation for 10 min at 5,000 × g (4 °C). The cell pellets were resuspended in water and
100 µg of heptadecanoic acid (in hexane) added as an internal
standard. The lipids were extracted using a modification of the
technique described by Bligh and Dyer (20) using glass beads (2 g/50 ml
cultures, 425-600 µm) to break the cells. Following extraction, the
lipids were dried under a stream of N2, resuspended in 0.4 ml of NaOH in methanol (20 g/liter), and incubated overnight at
100 °C. boron trifluoride (0.5 ml; 20%) was added and
incubation continued for 1 h at 100 °C. The methyl esters were
extracted with hexane, dried, resuspended in 1 ml of methyl acetate,
and analyzed by gas chromatography using an HP 225 column. The data are
presented in mol % fatty acid and are the mean of at least three
independent experiments performed in duplicate.
For fatty acyl-CoA determinations, cells were grown overnight in YPDA
and diluted to an A600 of 0.1 in 100 ml YNBD.
When the cell density reached an A600 of 0.8, oleic acid was added to a final concentration of 100 µM
and grown for the times indicated in the figure legend. Cell growth and
metabolism were stopped by addition of 0.1 volume of 6.6 M
perchloric acid. Cells were harvested by centrifugation for 10 min at
5,000 × g (4 °C). Acyl-CoA extraction,
quantification, and identification were performed as described by
Schjerling et al. (21).
Acyl-CoA Synthetase Activity--
Cells were grown from
overnight cultures in YPDA and grown to an A600
of 1.0. Following growth, cells were harvested by centrifugation, washed twice with PBS, and resuspended to a density of 1.2 × 109 cells/ml in 200 mM Tris-HCl, pH 8.0, 4 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol,
0.01% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride,
4 µM pepstatin A, and 8 µM leupeptin. The
cells were lysed by vigorously vortexing the cell suspension containing
glass beads for 1 min, 5 times at 0 °C. Samples were clarified by
centrifugation (1,500 × g, 5 min), and supernatants
were used to assess acyl-CoA activities. Acyl-CoA synthetase activities
were determined in cell extracts as described (22). The reaction
mixtures contained 200 mM Tris HCl, pH 7.5, 2.5 mM ATP, 8 mM MgCl2, 2 mM EDTA, 20 mM NaF, 0.01% Triton X-100, fatty
acid dissolved in 10 mg/ml 
-dextrin (final concentrations of fatty
acids were 50 µM), 0.5 mM coenzyme A, and
cell extract in a total volume of 0.5 ml. The reactions were initiated
by the addition of coenzyme A, incubated at 30 °C for 20 min, and
terminated by the addition of 2.5 ml of
isopropanol:n-heptane:1 M
H2SO4 (40:10:1). The radioactive fatty acid was
removed by organic extraction using n-heptane. Acyl-CoA
formed during the reaction remained in the aqueous fraction and was
quantified by scintillation counting. Protein concentrations in the
enzyme extracts and purified enzyme samples were determined using the
Bradford assay and bovine serum albumin as a standard (23). The values
presented represent the average from at least three independent
experiments. All experiments were analyzed by analysis of variance
using PRIZM software.
In Vivo -Oxidation--
Cells were grown and peroxisomes
induced in YP medium (0.3% yeast extract, 0.5% peptone, 0.5%
potassium phosphate, pH 6.0, 3% glycerol) containing 0.2% oleate and
1% Brij58 (YPO) as described by Rottensteiner et al. (24).
Following induction, cells were harvested by centrifugation, washed
once in PBS-Brij58, twice in PBS, and finally resuspended to an
A600 of 2.5 in PBS. For assay, aliquots of 200 µl of cell suspension were added to a 25-ml reaction vessel fitted
with a center well (Kontes) in a total volume of 2 ml of PBS. To
initiate the reaction, [1-14C]oleic acid was added from
an ethanolic stock to a final concentration of 10 µM.
Reactions were continued for 30 min at 30 °C and were terminated by
the addition of H2SO4 to 1 N added
directly to the cell sample. Radiolabeled CO2 was trapped
for 60 min in 50% ethanolamine in ethanol in the center well.
Radioactivity trapped in the well was quantified by liquid
scintillation counting. The final data were expressed in
picomoles/min/mg of protein and analyzed using PRIZM. Data presented
represent the mean (± stand error of the mean) from at least three
independent experiments.
RNA Isolation and Northern Blot Analyses--
Cells were grown
from an overnight culture into YPDA to an A600
of 0.1 and grown to an A600 of 0.6. Cells were
harvested, washed twice in YP medium (1% yeast extract, 2% peptone)
and diluted to an A600 of 0.05 in YP medium
containing 2% glucose, 3% glycerol, or 3% glycerol 0.2% oleate, and
1% Brij58 58 and grown to an A600 of 0.6. Cells
were harvested by centrifugation at 0 °C, washed, RNA extracted, and
analyzed by Northern blotting (25). The hybridization probes
(OLE1, POX1, FAA2, and
ACT1) used were PCR-amplified from yeast genomic DNA,
gel-purified, and 32P-labeled using
[-32P]dCTP and the Prime-a-Gene kit from Promega.
Materials--
Yeast extract, yeast peptone, and yeast nitrogen
base were obtained from Difco. Oleic acid was obtained from Sigma.
3H- or 14C-labeled fatty acids were from
PerkinElmer Life Sciences, American Radiochemicals, or Sigma.
C1-BODIPY-C12 was purchased from Molecular Probes. Enzymes required for all DNA manipulations were from Promega, New England Biolabs, United States Biochemical Corp., or Roche Molecular Biochemicals.
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RESULTS |
FAA1 and FAA4 Are Required for Growth of Fatty Acid Auxotrophs Due
to Deficiencies in Both Fatty Acid Import and Activation--
The
Saccharomyces cerevisiae fatty acid-activating enzyme Faa1p
was first identified as a gene required for growth when the fatty acid
synthesis inhibitor cerulenin was included in the growth media
containing the long-chain saturated fatty acids myristate or palmitate
(26). Subsequently, three additional genes encoding the fatty acyl-CoA
synthetases Faa2p, Faa3p, and Faa4p were identified by sequence
comparisons and reverse genetics (27). Further characterization of the
cloned genes and enzymes indicated Faa4p had overlapping functions with
Faa1p, and either one was sufficient to support growth under these
synthetic lethal conditions (15). In the natural environment, yeast are
auxotrophic for long-chain unsaturated fatty acids when growing under
hypoxic conditions since the O2-requiring 9-acyl-CoA desaturase necessary for de novo
unsaturated fatty acid synthesis is inactive (11). Deletion of both
FAA1 and FAA4 was required to completely
eliminate growth under these culture conditions (Fig.
1A). This result was
comparable to that observed when fatty acid synthase was inhibited with
cerulenin (Fig. 1B). Growth of the faa1
faa4 strain was restored under both conditionally lethal conditions by the introduction of expression plasmids containing either
the FAA1 or FAA4 genes under the control of a
galactose inducible promoter; as expected, growth was dependent upon
inclusion of galactose in the culture media (Fig. 1). Thus, Faa1p and
Faa4p can each activate saturated and unsaturated fatty acids imported from the environment to provide essential fatty acids required for
growth.
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Fig. 1.
Faa1p or Faa4p are essential for the import
of the long-chain fatty acid oleate (C18:1) to support
growth under hypoxic conditions (A) and in the
presence of the fatty acid synthase inhibitor cerulenin
(B). Cells were spread on YNB agar plates
containing ergosterol (YNB-EG), ergosterol and 100 µM
oleate (YNB-EG-OLE) or 45 µM cerulenin and 100 µM oleate (YNB-CER-OLE) supplemented with 4% sucrose and
either 2% glucose or 2% galactose. YNB-EG and YNB-EG-OLE plates were
incubated at 30 °C for 96 h under anaerobic conditions and
YNB-CER-OLE plates were incubated at 30 °C for 48 h under
aerobic conditions. (1) Wild-type (YB332a); (2) faa1
faa4/YpGALFAA1; (3)
faa1 faa4/YpGALFAA4;
(4) faa1 faa4/YEp24 (vector
control); (5) faa4; and (6) faa1.
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These data did not distinguish whether these fatty acyl-CoA synthetases
were required only for metabolic utilization of imported fatty acids or
whether they were are also required for the transport process in a
manner analogous to that which occurs in Gram-negative bacteria. In an
effort to address whether there is a link between fatty acid import and
activation in S. cerevisiae, studies were conducted to
monitor the intracellular accumulation of the fluorescent fatty acid
analogue C1-BODIPY-C12 using CLSM. Wild-type
cells accumulate exogenous C1-BODIPY-C12
quickly (within 30 s) by an essentially irreversible process,
suggesting this compound becomes metabolically trapped (12, 13). No
efflux of C1-BODIPY-C12 was noted using this
experimental approach to monitor fatty acid import (13). The
accumulation of exogenous C1-BODIPY-C12 was basically unchanged in the faa4 strain but considerably
reduced in the faa1 strain (Fig.
2A). Deletion of both
FAA1 and FAA4 reduced the accumulation of
C1-BODIPY-C12 further (Fig. 2A).
These data are consistent with the notion that Faa1p plays a more
prominent role in fatty acid import. Centromeric clones
(pGALFAA1 and pGALFAA4) encoding these enzymes
transformed into the faa1 faa4 strain led
to a large increase in C1-BODIPY-C12
accumulation when cells were grown in the presence of galactose to
induce the expression of Faa1p or Faa4p (Fig. 2B). These
findings attest to the importance of these two enzymes in the fatty
acid import process. We hypothesize the irreversible nature of import
is due to the metabolic trapping of
C1-BODIPY-C12, presumably as a CoA thioester as
a consequence of either Faa1p or Faa4p.
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Fig. 2.
Fatty acid transport monitored by confocal
laser scanning microscopy using the fluorescent long-chain fatty acid
C1-BODIPY-C12. A. The strains noted were
grown in YNBD and intracellular fatty acid accumulation monitored
following incubation of cells with 10 µM
C1-BODIPY-C12 as detailed in Experimental
Methods. B. faa1 faa4 cells
transformed with plasmids containing FAA1 or FAA4
under the control of a galactose-inducible promoter
(pGalFAA1 and pGalFAA4 respectively) grown in YNB
containing 2% sucrose and either 2% glucose or 2% galactose; fatty
acid accumulation was monitored following incubation with 10 µM C1-BODIPY-C12.
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The results presented in Fig. 2 demonstrated the accumulation of the
fluorescent fatty acid C1-BODIPY-C12 was
compromised in the faa1 faa4 strain and
that expression of either FAA1 or FAA4 from a
galactose inducible promoter on a plasmid following transformation in
essence restored the wild type activity. These data lend support to the
hypothesis that fatty acid import is indeed linked to activation in
yeast. In an effort to quantify these results at the level of fatty
acid import, we used the filtration method (13, 14) to monitor the
import of [3H]oleate in wild type and fatty acyl-CoA
deficient strains as detailed under "Experimental Procedures"
(Table I). As with the experimental
approach detailed above using C1-BODIPY-C12,
there was no measurable efflux of the labeled fatty acid following
transport (13, 14). These results revealed that a deletion of
FAA1 decreased oleate import nearly 2-fold, whereas a
deletion of FAA4 basically had no effect. Although the
reduction in import in the faa1 strain was the trend at
oleate:BSA ratios of 0.5 and 1.0 (p 
0.1), the reduction in import noted at the higher concentration of oleate was
significant (oleate:BSA = 2.0; p < 0.1). The
results acquired for the wild-type, faa1, and
faa4 strains using the filtration method essentially
mirrored those obtained using the
C1-BODIPY-C12. The unexpected result came from
measuring the transport of [3H]oleate using the
filtration method on the faa1 faa4 strain, which had apparent levels of fatty acid import that were nearly 10-fold
higher than wild type. The fatty acid import measurements in the
faa1 faa4 strain were complicated by a
flocculent phenotype, suggesting the cell surface was altered in some
manner, which resulted in cells becoming clumped during growth in
liquid medium. This flocculent phenotype was not observed in the
faa4 strain. The high density of cells required for the
filtration assay appears to exacerbate this problem and resulted in
what we interpret as high levels of nonspecific fatty acid binding. We
predicted the faa1 faa4 stain would have
fatty acid import values comparable to or lower than the
fat1 strain, which is defective in the import of
[3H]oleate (13). The levels of [3H]oleate
import in the fat1 strain, by comparison to the wild-type strain, were reduced nearly 5-fold at all three fatty acid
concentrations tested (13). The most informative observation derived
from these data using the filtration method was the depressed levels of
oleate transport in the faa1 strain. The phenotype
associated with this strain on the fatty acid, cerulenin-containing
plates and under anaerobic conditions suggests Faa1p plays a more
predominant role in activating exogenous long-chain fatty acids, when
compared with Faa4p. The fatty acid import data obtained using both
C1-BODIPY-C12 and [3H]oleate seem
to corroborate these findings.
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Table I
Long-chain fatty acid transport profiles in wild-type and acyl-CoA
synthetase-deficient strains of S. cerevisiae determined using the
filtration assay
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In Vivo Activation of Imported Fatty Acids and Maintenance of
Endogenous Acyl-CoA Pools--
Yeast strains harboring deletions in
both FAA1 and FAA4 had levels of fluorescent
fatty acid accumulation appreciably reduced when compared with the
isogenic wild type parent attesting to the functional role of fatty
acyl-CoA synthetase in this process. In order to investigate the
linkage between import, activation and subsequent trafficking, we
measured intracellular oleoyl-CoA pools in wild-type,
faa1, faa4, and faa1
faa4 strains following the addition of oleate (final
concentration, 100 µM) to actively growing cells cultured
in YNBD medium. We chose to use 100 µM oleate for these
experiments, as our phenotypic data demonstrated the restrictive
concentrations for fatty acid supplementation when fatty acid
synthetase is inhibited was between 50 and 75 µM oleate.
Aliquots of cells were removed at various time points (t = 10, 30, 60, and 120 min) after oleate addition and
oleoyl-CoA levels quantified as detailed under "Experimental
Procedures." As a base line, endogenous oleoyl-CoA levels were
defined prior to the addition of exogenous oleate. Within 10 min after
the addition of C18:1, oleoyl-CoA levels increased nearly
6-fold in wild-type cells (Fig. 3). By
comparison, the oleoyl-CoA levels for the faa1, faa4, and faa1 faa4 strains
were either reduced or not responsive to added oleate when compared
with the wild type strain. For the faa4 cells, the
measured oleoyl-CoA levels derived from exogenous oleate were between
45 and 55% of wild type. Deletion of FAA1, in contrast,
resulted in a severe reduction of oleoyl-CoA at all time points
evaluated (<10% of wild-type levels). There was not a significant
difference in oleoyl-CoA levels between cells from the strain deleted
for FAA1 alone compared with those with deletions in both
FAA1 and FAA4 or all four FAA genes
(Fig. 3). It should be noted that these are not kinetic measurements
per se, but rather measurements, which established the
linkage between exogenous oleate and intracellular oleoyl CoA pools.
These observations confirmed that Faa1p is the principal acyl-CoA
synthetase responsible for the activation of exogenous fatty acids.
Additional significant findings from these experiments came from the
base-line measurements of endogenous acyl-CoA pools in the
faa1, faa4, and faa1
faa4 strains.
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Fig. 3.
Activation of exogenous oleate occurs
primarily through Faa1p. Oleoyl-CoA levels were determined in the
strains noted prior to and at four time points following the addition
of oleate to a final concentration of 100 µM in mid-log
cultures.
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Strains harboring deletions in FAA1 and/or FAA4
had endogenous oleoyl-CoA pools, which were only ~20% those measured
for the wild type strain. This observation led us to measure endogenous levels of other long chain acyl-CoA species in cells grown in the
absence of exogenous fatty acids (Table
II). Total long chain acyl-CoA levels
were reduced to 74 and 39% of wild-type levels in the
faa1 and faa1 faa4 strains,
respectively. Although the total long-chain acyl-CoA profile in the
faa4 strain was equivalent to the wild type strain, there
were notable differences, particularly in C16:0-CoA levels, which were
elevated 2-fold over the wild type. The reduction in the acyl-CoA pools
in the faa1, faa4, and faa1
faa4 strains was particularly pronounced for the
unsaturated acyl-CoA esters (C16:1 and C18:1).
The levels of saturated long-chain fatty acyl-CoA esters
(C14:0 and C16:0) were increased in the faa4 strain by ~33%, which was due to the spike in
C16:0 CoA levels as noted above. Deletion of both the FAA1
and FAA4 genes reduced the total acyl-CoA ester levels to
39% of wild type levels. In this strain the levels of saturated and
unsaturated long-chain acyl-CoAs were 53 and 19% of wild type,
respectively, indicating a cooperative role between Faa1p and Faa4 in
the maintenance of intracellular acyl-CoA pools.
The data presented above documented unique biochemical phenotypes of
the faa1 and faa4 strains, indicating that
Faa1p and Faa4p are not completely functionally redundant, and indeed
further support the hypothesis that Faa1p is the predominant acyl-CoA synthetase involved in the fatty acid import and activation processes. Previously, acyl-CoA synthetase activities were measured in wild type
and faa1 faa4 strains using saturated fatty
acid substrates (27). It was expected that the severe reduction of
oleoyl-CoA found for the faa1 strain by comparison with
the faa4 strain should be reflected in reduced enzyme
activity. Therefore, we measured oleoyl-CoA synthetase activities in
total cellular extracts of each strain (Table
III). We found the oleoyl-CoA synthetase profiles measured in these experiments mirrored the data obtained on
the oleoyl-CoA levels in the wild type and different mutant strains. Of
particular note was the finding that deletion of FAA1 resulted in oleoyl-CoA synthetase levels that were only 7% of those
measured in the wild-type cells. By contrast, the faa4 strain had oleoyl-CoA synthetase levels that were nearly 3-fold higher
than the faa1 strain, but only 22% of wild type levels. Deletion of both FAA1 and FAA4 or all four
FAA genes reduced acyl-CoA synthetase levels further (to 5%
wild type), but did not eliminate all activity.
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Table III
Fatty acyl-CoA synthetase activities in cell extracts using oleate
(C18:1) as the fatty acid substrate in wild type and strains
defective in one or more fatty acyl-CoA synthetase
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The reductions in the intracellular acyl-CoA pools were particularly
notable in the faa1 and faa1
faa4 strains. To address whether these reduced acyl-CoA
levels also resulted in altering the cellular fatty acid levels, we
identified and quantified total cellular fatty acids and showed that
the fatty acid profiles in the faa1 and
faa1 faa4 strains were essentially
unchanged compared with wild type (Table
IV). This is in contrast to the fatty
acid profiles of a fat1 strain (defective in fatty acid
import and lignoceryl CoA synthetase activity), where marked
differences in the very long-chain fatty acid pools relative to the
wild type are found (13, 17, 18). These data indicate de
novo fatty acid synthesis provides sufficient long-chain saturated
and unsaturated fatty acyl-CoAs to maintain normal levels required for
higher lipid synthesis and other process essential to cell growth.
Therefore, although deficiency in the long-chain fatty acid-activating
enzymes eliminates incorporation of exogenous fatty acids into higher lipids (27), there was no detectable effect on fatty acid incorporation from endogenous pools into higher lipids under the growth conditions used for these studies.
Intracellular Trafficking of Exogenously Derived acyl-CoA--
The
data presented above demonstrated fatty acid import and activation are
severely compromised in the faa1 faa4
strain, fully supporting our hypothesis that one or both enzymes
function as components of a fatty acid import system. In addition, the endogenous acyl-CoA pools were markedly reduced in the
faa1 faa4 strain, supporting the notion
that these enzymes are also involved in endogenous acyl-CoA metabolism.
To further investigate the linkage between import, activation, and
trafficking, we defined 1) the levels of -oxidation of exogenous
fatty acids (to monitor utilization) and 2) differential expression of
POX1 and FAA2 (encoding the peroxisomal genes
acyl-CoA oxidase and medium-chain acyl-CoA synthetase, respectively; to
monitor intracellular signaling) in response to exogenous fatty acids.
In order to assess the role of fatty acid-activating enzymes on the
-oxidation pathway, we analyzed the ability of yeast cells
containing deletions in the FAA genes to degrade exogenously supplied oleate in vivo. For these studies, cells were
cultured under conditions required for the induction of peroxisomes as detailed under "Experimental Procedures." -Oxidation levels of exogenous oleate in strains containing deletions of FAA1 or
FAA4 were reduced to 70 and 85% of wild-type levels,
respectively. Deletion of both genes reduced -oxidation levels to
25% of wild type (Fig. 4). In this case,
each activating enzyme Faa1p and Faa4p appears to contribute to the
acyl-CoA substrate pool. Deletion of both genes had a more substantial
effect on the overall rate of -oxidation than either enzyme alone.
This is in striking contrast to the dominant role of Faa1p over Faa4p
noted above. We suggest this may be due to the differences in growth
conditions used for each assay. For the measurement of acyl-CoA and
fatty acid pools, cells were incubated 0-120 min in minimal medium
with glucose as the carbon source and limiting concentrations
(µM) of oleate, whereas, for -oxidation assays, cells
were grown overnight in YP medium containing glycerol and 4 mM oleate prior to assaying in a reaction mixture
containing 10 µM [14C]oleate.
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Fig. 4.
Maximal levels of
-oxidation of exogenous oleate requires Faa1p or
Faa4p. Cells were grown and incubated under conditions for
peroxisome proliferation and levels of -oxidation measured as
detailed in Experimental Methods. The error
bars represent the standard error of the mean from 3 independent experiments. WT, wild-type.
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As a second measure of the roles of Faa1p and Faa4p in intracellular
fatty acid trafficking, we monitored the expression of genes activated
by fatty acids (POX1 and FAA2) using Northern analyses of RNA prepared from the wild type strain or strains defective
in the fatty acid-activating genes. For comparison and as a control, we
monitored the repression of OLE1 mRNA (encoding the
9 acyl-CoA desaturase) (Fig.
5). It has been demonstrated previously that oleate-mediated repression of OLE1 requires
FAA1 or FAA4 (28). As expected, OLE1
expression was high in wild-type cells cultured in glucose or in
glycerol. The addition of oleic acid essentially eliminated
OLE1 expression and repression of OLE1 was
dependent on Faa1p or Faa4p. In the case of POX1 and
FAA2, there was also a dependence of Faa1p or Faa4p in
linking increased expression to exogenous fatty acid. This is the first
work that demonstrates either Faa1p or Faa4p must be active to induce
the expression of POX1 and FAA2. Unlike
OLE1, however, the expression of patterns of POX1
and FAA2 were more complex. First, both FAA2 and
POX1 were subject to catabolite repression mediated by
glucose. Second, the expression of both genes was detectable when cells were cultured in medium containing glycerol (non-catabolite-repressing conditions). Third, in wild type, faa1 and
faa4 strains, the expression of both FAA2 and
POX1 was high when cells were cultured in oleate-containing
medium (Fig. 5). Deletion of both FAA1 and FAA4 eliminated the increased expression of both genes
in response to oleate in the culture medium. We noted the expression of
FAA2 and POX1 in the faa1
faa4 strain was elevated when cells were grown in medium
containing glycerol when compared with the wild type,
faa1, and faa4 strains. These results are
similar to those observed in strains carrying a deletion in the gene
encoding the transcription factor Pip2p (29).
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Fig. 5.
Northern analyses illustrate the fatty
acid-mediated regulation of expression of FAA2,
POX1, and OLE1 require either Faa1p
or Faa4p. RNA was prepared from the strains noted and resolved as
detailed under "Experimental Procedures." Blots were probed with
32P-labeled fragments corresponding to FAA2,
POX1, and OLE1; ACT1 is included as
the loading control. D, cells grown in YP glucose (2%);
G, cells grown in YP glycerol (3%); O, cells
grown in YP glycerol (3%) and oleate (0.2%) in 1% Brij 58
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On the basis of these data, we conclude the fatty acyl-CoA synthetases
Faa1p and Faa4p function as components of a fatty acid transport and
activation system linking import and trafficking of exogenous fatty
acids to sites of utilization (-oxidation) and intracellular
signaling (differential expression of genes involved in fatty acid metabolism).
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DISCUSSION |
S. cerevisiae shares many biochemical and regulatory
pathways in common with more complex eukaryotic organisms. Among these is the ability to synthesize fatty acids for cellular growth, to import
fatty acids from the environment when fatty acids are available in the
extracellular milieu, and to respond to fatty acids by altering gene
expression. In the present work, we provide evidence that imported
fatty acids are activated to coenzyme A thioesters concomitant with
transport across the cell membrane. We propose the coupling of import
with activation is an important feature of this process and involves
Fat1p and fatty acyl-CoA synthetase (Faa1p or Faa4p). The deficiency of
fatty acid import and activation noted in the faa1
faa4 strain results in lowering intracellular acyl-CoA
pools, which in turn leads to diminished cellular utilization of
exogenous fatty acids and changes in intracellular signaling to fatty
acid-responsive genes. The predominant fatty acid-activating enzyme
during log phase growth is Faa1p although Faa4p is partially
compensating. These results are consistent with previous data
demonstrating Faa1p plays the principal role in activating exogenous
fatty acids destined for incorporation into complex lipids (16). The
activity of Faa1p is likely to represent the limiting step linking
import of exogenous fatty acids with intracellular utilization. When
FAA1 and FAA4 are both deleted, growth under
conditions in which cells are dependent upon exogenous fatty acids
(i.e. during hypoxia or when fatty acid synthase is
inhibited by cerulenin) is abolished, supporting the hypothesis that
fatty acid import and activation are linked processes.
A central tenet of the present work is that fatty acid transport is
coupled to metabolic activation in yeast. We define the term transport
as the net movement of the fatty acid ligand across a membrane from one
compartment to another. The current experiments limit evaluation of
transport activity to import of fatty acids from the extracellular
environment. Fatty acid import is distinct from those processes
governing the transport of hydrophilic ligands due to the hydrophobic
nature of these compounds. The mechanism of fatty acid transport is
likely to include diffusion of the fatty acid across the membrane,
where there is flip of the uncharged fatty acid from the outer leaflet
to the inner leaflet (30). However, it is clear, from our work and that
by others in the field, that there is a need for a sink to establish a
concentration gradient from the outside to the inside of the cell;
otherwise, the fatty acid would remain trapped in the membrane. Our
data are consistent with the conclusion that formation of the acyl-CoA thioester catalyzed by Faa1p or Faa4p is the sink that governs transport in yeast.
In S. cerevisiae, fatty acid import is restricted in strains
carrying a deletion in FAT1 as well as in strains carrying
deletions in both FAA1 and FAA4. The
fat1 and faa1 faa4 strains
have indistinguishable phenotypes when grown on YPD containing oleate and cerulenin and under anaerobic conditions (Ref. 13 and this work).
This information implies that minimally Fat1p and either Faa1p or Faa4p
are components of a metabolic system linking fatty acid import and
utilization. The data presented in this work support the concept that
Faa1p, rather than Faa4p, is the predominant fatty acyl-CoA synthetase
involved in this process. We base this conclusion on the following
results. First, Faa1p functions as the major fatty acyl-CoA synthetase
within the cell during logarithmic phase growth. Second, fatty acid
import was significantly reduced in Faa1p-deficient cells. Third,
formation of oleoyl-CoA from oleate supplied exogenously was reduced in
the faa1 strains. Fourth, the levels of -oxidation
were more severely depressed in strains containing a deletion in
FAA1 when compared with the wild type and those containing a
deletion in FAA4.
Our interpretation that fatty acid import is linked to activation in
yeast is in discord with a previous report. Knoll et al.
(27) reported that cell-associated fatty acid levels were essentially
identical in wild type and fatty acyl-CoA synthetase-deficient strains
(including faa1 faa4). These investigators
employed a radioactive fatty acid transport filtration assay using
fatty acid concentrations between 10 and 250 µM (in the
absence of either BSA or detergent) and ice-cold wash solutions. We
believe our present data can be reconciled with these previous
observations by comparing the methods of analyses employed. Assay
systems that attempt to measure fatty acid transport present a number
of technical challenges. First, the concentration of free fatty acids
in the assay system and within the membrane must be considered. Second, the nonspecific binding of radioactive fatty acids to the cell membrane
or the filter presents a background problem, which often can be
misinterpreted as transport or cell-associated fatty acids. Biological
membranes have a limited capacity to accept free fatty acids, which can
yield data misinterpreted as showing fatty acid binding occurs by a
protein-dependent, saturable process (30). Third, the use
of cold wash solutions dramatically increases the retention of fatty
acids on filters. To minimize these problems, our quantitative fatty
acid transport assay system employs fatty acid concentrations limited
to the mid nM range. By comparison, the transport of the
long chain fatty acid analogue, C1-BODIPY-C12 visualized using confocal laser scanning microscopy provides a qualitative measure of transport. For either assay, we use non-ionic detergent or fatty acid-free BSA in the wash solutions, and all steps
are conducted (including the washes) at room temperature or 30 °C.
These washing steps are presumed to strip any free fatty acids
dissolved in the membrane from the cells. One possible bias of this
approach is that this measure of fatty acid import is simply a gauge of
activation as part of the process, given that we have not specifically
blocked fatty acid efflux as is routine for mammalian cells. In
mammalian systems where there appear to be two distinct processes
operational in fatty acid import (33), it is essential to eliminate
fatty acid efflux. This is routinely accomplished by using phloretin in
the stop solution (34). Previous work evaluating fatty acid import in
yeast and bacteria has shown there is essentially no efflux of fatty
acid following transport, supporting the idea that transport and
activation are linked processes in these systems (12-14, 35). In
addition we have used phloretin as a stop solution in our filtration
fatty acid transport assays and observed no differences in wild-type
cells when compared with experiments done in the absence of phloretin,
which we interpret to mean the imported fatty acid is rapidly activated
and unable to efflux from the
cell.2
Our fatty acid transport assay systems, like most, do not distinguish
import across the membrane from activation and utilization. Our
conclusions that import and activation are coupled were also based upon
direct measurement of oleoyl CoA pools in cell extracts following the
addition of limiting concentrations of oleate to cultures of actively
growing wild type and acyl-CoA synthetase-deficient strains. These data
showed that strains lacking Faa1p or both Faa1p and Faa4p were
essentially unable to activate exogenous long-chain fatty acids.
Although indirect, collectively the present data support a coupled
import-activation process for exogenous long-chain fatty acids in
yeast. These data are consistent with our earlier studies where oleoyl
CoA pools were monitored in the fat1 strain following the
addition of oleate (13). In this strain, as in the faa1
faa4 strain, the formation of oleoyl CoA was reduced
4-5-fold, further supporting the notion that import and activation are
coupled in yeast. An important distinguishing characteristic of
fat1 from faa1 or faa1
faa4 strains is that fat1 strains do not
have a reduction in oleoyl-CoA synthetase activity.
A subtle, yet important result from the present work was the finding
that endogenous fatty acyl-CoA levels are also dependent upon
FAA1 and FAA4. In contrast, the fatty acid
profiles in the faa1, faa4, and
faa1 faa4 strains were essentially
unaffected, suggesting cellular de novo fatty acid synthesis
provides sufficient substrates for complex lipid synthesis and other
acylation reactions under the growth conditions employed in this study.
In the course of the present work, we did note that strains carrying
deletions in both FAA1 and FAA4 were flocculent
in liquid culture. The cause of the flocculence has not been determined
at this time, but is hypothesized to be due to alterations in cell wall
or membrane composition especially later in the growth cycle since
flocculence was more pronounced in saturated cultures. We conjecture
this may indicate the endogenous acyl-CoA pools maintained by Faa1p and/or Faa4p play an important role in stationary phase survival when
fatty acid synthase has reduced activity and membrane lipids are the
main source of fatty acids for essential functions such as protein
acylation (31). It is also possible the endogenous pools provide a
transitional source of acyl-CoA as cellular metabolism readjusts, for
example, to conditions of hypoxia, starvation, or other types of stresses.
The regulation of gene expression in response to an exogenous
source of fatty acids occurs in many organisms. In S. cerevisiae, the gene OLE1 is negatively regulated by
unsaturated fatty acids and, as confirmed in the present work, this
regulation is dependent upon Faa1p or Faa4p (28). However, if import of
fatty acids is coupled to activation as supported by the present data,
then it becomes difficult to prove unequivocally that fatty acyl-CoAs as opposed to fatty acids (or some other fatty acid derivative) result
in differential expression of genes involved in fatty acid metabolism.
A similar conundrum occurred in E. coli, where import and
activation are tightly coupled processes. However, purification and
extensive characterization of the transcription factor FadR proved
long-chain fatty acyl-CoAs directly regulate DNA binding and
transcription activation or repression mediated by FadR (35). Regulation of OLE1 transcription is sensitive to unsaturated
fatty acids derived from an exogenous source and is presumed to occur subsequent to activation to the fatty acyl-CoA thioester. Recent evidence demonstrated fatty acid-dependent regulation of
OLE1 transcription is mediated by one of two homologous
transcription factors, Spt23p and Mga2p (36, 37). Each of the factors
is synthesized as a membrane-bound precursor (36). Proteolytic processing is required for nuclear translocation and activation of
OLE1 transcription. The regulation of proteolysis is
undefined at this time but is suggested to occur in response to changes in membrane fluidity in a manner analogous to the mammalian steroid receptor element-binding protein family. Thus, one role of acyl-CoA in
the processing of Spt2p and Mga2p may be to provide substrates for
membrane remodeling, thereby altering membrane fluidity to suppress the
proteolytic processing.
The regulation of the fatty acid-inducible genes, POX1 and
FAA2, is different from OLE1 in two respects.
Expression is positively regulated in response to fatty acids and
negatively regulated by glucose-mediated catabolite repression. Two
transcription factors, Oaf1p (Pip1p) and Pip2p (Oaf2p), are
required for fatty acid-mediated increases in gene expression (10, 29).
There is no evidence that either transcription factor has a
membrane-bound precursor form. Oaf1p and Pip2p dimerize, bind an
oleate-response element within the promoters of target genes, and
activate transcription when fatty acids are included in the growth
medium (10, 29). As reported here, deletion of FAA1 and
FAA4 affected both the fatty acid-mediated activation and
glucose-mediated catabolite repression of POX1 and
FAA2. This is the first report demonstrating either
FAA1 or FAA4 are required for this fatty
acid-mediated effect. Derepression in glycerol was enhanced and
induction by fatty acids reduced in the
faa1faa4 strain compared with the isogenic
wild-type strain. The apparent increase in expression of
POX1 in cells grown in glycerol observed in our experiments is similar to results obtained by Karpichev and Small (29) for a strain
carrying a deletion in PIP2. Pip2p has been suggested to be
required both for derepression when cells are grown in non-catabolite repressive medium and fatty acid-mediated induction of gene expression. At the present time, there is no evidence that fatty acids or fatty
acyl-CoA directly interact with Pip2p and/or Oaf1p to effect transcriptional activation. Rather, most evidence supports an indirect
model for fatty acid-mediated regulation of these transcription factors. The present data suggest the fatty acyl-CoA synthetases Faa1p
and Faa4p are involved in this complex regulatory pathway, perhaps by
controlling the activity of an upstream regulator.
Fatty acid import in yeast, as in bacteria and mammals, is a complex
multicomponent process. In the well characterized E. coli
system, fatty acid transport, activation, metabolic utilization, and
gene regulation are tightly coupled processes (35). Some mammalian
systems are believed to operate by similar mechanisms. For example, in
adipocytes, FATP (the mammalian homologue of Fat1p) has been linked to
fatty acyl-CoA synthetase by functional assays, expression patterns,
and cellular localization (39, 40). As in yeast, mammalian ACBP is
believed to play a significant role in intracellular transport and
trafficking of acyl-CoAs (38). The yeast system offers a powerful tool
with which to investigate other components in fatty acid trafficking
due to ability to grow on fatty acids as a sole carbon and energy
source and to natural, conditional auxotrophy for unsaturated fatty
acids when cells grow anaerobically.
§,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Center for
Cardiovascular Sciences, Albany Medical College MC-8, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-6435; Fax: 518-262-8101; E-mail: dirussc@mail.amc.edu.
