(Received for publication, December 2, 1996, and in revised form, January 2, 1997)
From the ¶ Department of Biochemistry and Molecular Biology,
The Albany Medical College, Albany, New York 12208, the
Department of Biochemistry, University of Tennessee
College of Medicine, Memphis, Tennessee 38163, the
§ Institute of Biochemistry, Odense University, DK-5230
Odense, Denmark, and the
Department of Anatomy and Neurobiology,
University of Tennessee College of Medicine,
Memphis, Tennessee 38163
The yeast Saccharomyces cerevisiae is
able to utilize exogenous fatty acids for a variety of cellular
processes including -oxidation, phospholipid biosynthesis, and
protein modification. The molecular mechanisms that govern the uptake
of these compounds in S. cerevisiae have not been
described. We report the characterization of FAT1, a gene
that encodes a putative membrane-bound long-chain fatty acid transport
protein (Fat1p). Fat1p contains 623 amino acid residues that are 33%
identical and 54% with similar chemical properties as compared with
the fatty acid transport protein FATP described in 3T3-L1 adipocytes
(Schaffer and Lodish (1994) Cell 79, 427-436), suggesting
a similar function. Disruption of FAT1 results in 1) an
impaired growth in YPD medium containing 25 µM cerulenin
and 500 µM fatty acid (myristate (C14:0),
palmitate (C16:0), or oleate (C18:1)); 2) a
marked decrease in the uptake of the fluorescent long-chain fatty acid
analogue boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823);
3) a reduced rate of exogenous oleate incorporation into phospholipids;
and 4) a 2-3-fold decrease in the rates of oleate uptake. These data
support the hypothesis that Fat1p is involved in long-chain fatty acid
uptake and may represent a long-chain fatty acid transport protein.
Exogenous long-chain fatty acids represent an important class of
hydrophobic compounds that serve as substrates for lipid biosynthesis,
protein modification, and -oxidation. While the mechanism that
facilitates the uptake of these compounds into eukaryotic cells is not
completely understood, information gleaned over the past 15 years is
consistent with a facilitated, protein-mediated process. In higher
eukaryotic cells, three general classes of membrane-bound fatty acid
transport proteins have been described. The first, identified by
Abumrad and co-workers (1-4) is fatty acid translocase
(FAT).1 This protein represents the
adipocyte homologue of the glycoprotein CD36 and appears to act in
concert with intracellular fatty acid-binding proteins to mediate fatty
acid uptake (5, 6). The second, identified by Berk and Stremmel is a
membrane-bound fatty acid-binding protein (FABPpm) that is identical to
mitochondrial aspartate aminotransferase (7-12). Trotter et
al. (13) have demonstrated that pretreatment with anti-FABPpm sera
does not block the uptake of long-chain fatty acids in human intestinal
cells that normally express FABPpm. These data raise the question of
whether the mitochondrial aspartate aminotransferase actually
represents a membrane-bound fatty acid transport protein. The third,
described by Schaffer and Lodish (14) is FATP (atty
cid ransport rotein). FATP is
differentially expressed in 3T3-L1 adipocytes and is predicted to
function in the transport of exogenous long-chain fatty acids. The
expression of FATP is apparently negatively regulated by insulin at the
level of transcription in cultured adipocytes (15). Studies in yeast
and in bacteria also demonstrate that long-chain fatty acid transport
is a facilitated process (16-19). While the proteins that mediate
uptake in yeast have not been described, considerable evidence has been
accumulated describing the fatty acid transporter FadL in
Escherichia coli (20-23). Exogenous long-chain fatty acids traverse the cell envelope by a high affinity transport process that
minimally requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane-associated acyl-CoA synthetase. The
fatty acid transporter FadL, while mechanistically similar, is quite
distinct on the basis of amino acid similarities, to the eukaryotic
fatty acid transporters described above.
The notion of a membrane-bound fatty acid transporter is not universally accepted. One school of thought is that fatty acids are merely transported by diffusion across the membrane. In support of this, it has been demonstrated that when fatty acids are in the nonionized form, uptake into artificial membrane vesicles and 3T3-L1 cells occurs by simple diffusion, obviating the need for a membrane-bound fatty acid transporter (24-26). Therefore, it appears that in 3T3-L1 cells, long-chain fatty acid transport can occur by both diffusional and protein-mediated processes. The need for a protein-mediated process must lie in the requirement to regulate the entry of these compounds. A long-chain fatty acid transporter may be required for cells that specifically use these compounds for the production of metabolic energy or for the synthesis of triglycerides. These transporters are also required by microbes such as E. coli that have a hydrophilic cell envelope that is refractory to hydrophobic compounds.
In the yeast S. cerevisiae, the gene products involved in
uptake of exogenous long-chain fatty acids are unknown. Kohlwein and
Paltauf (16) demonstrated that fatty acid uptake in Saccharomyces uvarum and Saccharomycopsis lipolytica occurs via a
saturable process. The same observations were made in Candida
tropicalis supporting the hypothesis that fatty acid transport is
a facilitated process (17). Knoll et al. (19) have shown the
uptake of exogenous long-chain fatty acids in S. cerevisiae
is saturable and that uptake and activation to CoA thioesters are
separable. On the basis of these studies, it is reasonable to predict
that a membrane-bound long-chain fatty acid transport protein exists in
yeast. S. cerevisiae requires supplementation of oleic acid
(C18:1) to remain viable under anaerobic growth conditions
due to the suppression of fatty acid desaturation (27). Supplementation
of exogenous long-chain fatty acids is also required for cell viability
when fatty acid synthase is blocked by the antibiotic cerulenin (28,
29). Under both conditions, transport and activation of exogenous
long-chain fatty acids are required to overcome growth inhibition. The
acyl-CoA synthetases encoded by FAA1 and FAA4
(Faa1p and Faa4p, respectively) have been shown to be responsible for
activation of imported fatty acids in S. cerevisiae (30).
The overexpression of either Faa1p or Faa4p in faa1
faa4
cells does not increase the levels of fatty acid uptake.
The finding that long-chain fatty acid uptake in yeast is saturable and
occurs in the absence of Faap activity supports the proposal for a
specific plasma membrane-bound fatty acid transporter.
We report the characterization of FAT1 in S. cerevisiae that encodes a 623-amino acid residue protein that shares 33% amino acid identity and 54% amino acids with similar chemical properties to FATP described in 3T3-L1 adipocytes (14). Disruption of FAT1 results in 1) an impaired growth in YPD medium containing 25 µM cerulenin and 500 µM fatty acid (myristate (C14:0), palmitate (C16:0), or oleate (C18:1)); 2) a decrease in the uptake of the fluorescent long-chain fatty acid analogue BODIPY-3823; 3) a reduced rate of exogenous oleate incorporation into phospholipids; and 4) a 2-3-fold decrease in the rates of oleate uptake. These data support the hypothesis that Fat1p is a long-chain fatty acid transport protein.
Yeast extract, yeast peptone, agar, and yeast nitrogen base were obtained from Difco. Fatty acids and cerulenin were obtained from Sigma. 3H-Labeled fatty acids were from DuPont NEN. BODIPY-3823 was purchased from Molecular Probes. Enzymes required for all DNA manipulations were obtained from Epicenter, Promega, New England Biolabs, U.S. Biochemical Corp., or Boehringer Mannheim. The oligonucleotides for DNA amplification were synthesized on a Gene Assembler Plus (Pharmacia Biotech Inc.). All other chemicals were obtained from standard suppliers and were of reagent grade.
Data Base Analysis and AlignmentsHomology searches were performed using Biological Sequence Comparative Analysis Node (BioSCAN) at the University of North Carolina. The nonredundant data base Genpept (Release 93) was used. Alignments of the primary structures of FATP homologies were generated with BESTFIT (Genetics Computer Group) (31).
Strains and MediaThe isogenic S. cerevisiae
strains W303a (leu2, ura3, trp1, ade2, his3) and
W303a-fat1-1 (leu2, ura3, trp1, ade2, his3,
fat1
::HIS3) were used in all of the experiments
described. YPD media consisted of 1% yeast extract, 2% peptone, and
2% dextrose. Supplemented minimal media (SMM) contained 0.67% yeast
nitrogen base, 2% dextrose adenine (20 mg/liter), uracil (20 mg/liter)
and amino acids as required for either the gene replacement experiments
or the complementation experiments (arginine, tryptophan, histidine,
and tyrosine (20 mg/liter); lysine (30 mg/liter); and leucine (100 mg/liter)). YPD/agar plates containing 50 µM fatty acid
(myristate, palmitate, or oleate) also contained 0.5% Brij 58, 0.7%
KH2PO4 with or without 25 µM
cerulenin as required (30). Growth characteristics of various strains
on YPD, YPD/CER, and YPD/CER/fatty acid plates were performed according
to Johnson et al. (30). Plates were then incubated at 24, 30, or 37 °C for 72 h. Growth in liquid YPD with or without
cerulenin and fatty acid was monitored over a 40-h period. All
experiments were repeated at least three times.
Yeast genomic DNA was purified from
strain W303a as detailed in Kaiser et al. (32). For
disruption, the FAT1 gene was amplified by thermocycling
from genomic DNA using sets of specific primers; 5-ACCCAGAAATCCTGGGTTATCT-3
(upstream) and
5
-CAACTCTACTTCAGTAGTGGAAAC-3
(downstream), both
containing BamHI sites (underlined). The HIS3 gene was amplified from pHB3 (obtained from David Nelson, University of
Tennessee, Memphis) using the upstream primer
5
-AGCGCCTTTTAAACCACGACGCTTTGTC-3
and the
downstream primer
5
-TACGCACTTGCCACCTATCACCACAA-3
, both
containing EcoRI sites (underlined). The 1549-bp fragment containing the coding sequence of FAT1 was cloned into the
BamHI-site of pACYC177 to generate pNJF1. The 480-bp
EcoRI fragment within FAT1 was replaced by the
1346-bp EcoRI-fragment containing the HIS3 gene to generate
pNJF2. The strain W303a was rendered competent using lithium acetate
using standard procedures and transformed with linearized pNJF2
according to Kaiser et al. (32). Clones were selected on
minimal plates lacking histidine supplementation. His+
isolates were selected and colony-purified on minimal places without
histidine. Several isolates were obtained. The disruption of the
FAT1 gene with HIS3 was confirmed using DNA
amplification of genomic DNA. One such isolate shown to contain a
disruption in FAT1, designated W303a-fat1
-1
(fat1
) was selected for all further studies.
The expression of FAT1 was evaluated in strain W303a grown in YPD containing 500 µM oleate. Total RNA was purified using the RNeasy kit as recommended by the supplier (Qiagen). Reverse transcriptase amplification was performed on 1 µg of total RNA using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase as described by the manufacturer (Boehringer-Mannheim). Controls received no Moloney murine leukemia virus reverse transcriptase. First-strand cDNA was amplified using primers derived from the sequences corresponding to the C termini of Fat1p or histone H4 (used as an internal control).
Fatty Acid TransportFatty acid transport was performed
essentially as described by Knoll et al. (19). Cells (W303a
and W303a-fat1-1) were grown in SMM at 24 or 30 °C to
midlog phase (A600 = 1.0), collected by
centrifugation, washed once in phosphate-buffered saline (PBS), and
resuspended in 1/10 of the original volume in PBS. 200 µl of
cells (1 × 108 cells) were preincubated for 10 min at
24 or 30 °C in SMM, and the assay was initiated by the addition of
[9,10-3H]myristate, [9-10-3H]palmitate, or
[9-10-3H]oleate at the concentrations indicated. All
fatty acids were prepared as ethanolic stocks. At the defined time
points, the reactions were terminated by the addition of 10 ml of
ice-cold PBS. The cells were immediately filtered through a Whatman
Gf/B-filter, washed three times with ice-cold PBS, and air-dried. The
amount of cell-associated radioactivity was determined by scintillation counting. Background counts were subtracted from the experimental samples by evaluating the amount of radioactivity on control filters with no cells. The final data were expressed in nmol of cell-associated fatty acid/min/1 × 108 cells. All transport data were
analyzed using EnzymeKinetics software (version 1.0.4; Trinity
Software). The data presented represents the mean from at least three
independent experiments.
W303a and
W303a-fat1-1 cells were grown in SMM at 30 °C to
midlog phase (A600 = 1.0). One ml of cells were
transferred to a tube containing 50 µM
[9,10-3H]oleate (specific activity of 1 Ci/mmol, 50 µCi/ml). At the time points indicated, 1 volume of 10% ice-cold
trichloroacetic acid was added to stop yeast fatty acid metabolism. The
cells were immediately collected by centrifugation (15,000 × g) and resuspended in 100 mM Tris-HCl, pH 7.5. After neutralization with 1 M KOH, cells were washed four
times in 100 mM Tris-HCl (pH 7.5) containing 100 µM fatty acid-free bovine serum albumin. Lipids were
extracted according to Bligh and Dyer (33). Samples were analyzed using high performance thin layer chromatography as described by Knoll et al. (19).
Yeast cells
(FAT1 and fat1) as noted for transport,
harvested by centrifugation, washed twice with yeast nitrogen base,
resuspended to a density of 1.2 × 109 cells/ml in 10 mM Tris-HCl, pH 7.5, and lysed by three cycles of
sonication at 0 °C. Acyl-CoA synthetase activities were determined in sonicated cell extracts as described by Kameda and Nunn (34). The
reaction mixtures contained 200 mM Tris-HCl, pH 7.5, 2.5 mM ATP, 8 mM MgCl2, 2 mM EDTA, 20 mM NaF, 0.1% Triton X-100, 10 µM [3H]oleate, [3H]palmitate,
or [3H]myristate, 0.5 mM coenzyme A, and cell
extract in a total volume of 0.5 ml. The reactions were initiated with
the addition of coenzyme A, incubated at 35 °C for 10 min, and
terminated by the addition of 2.5 ml of isopropyl
alcohol:n-heptane:1MH2SO4 (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 (35). The values presented represent the average
from at least three independent experiments. All experiments were
analyzed using analysis variance (StatView; Abacus Concepts, Inc.)
Cells were grown in SMM at 24 °C and prepared as described above for fatty acid transport. The fluorescent long-chain fatty acid analogue BODIPY-3823 was added from a 5 mM ethanolic stock solution to 1 ml of cells to a final concentration of 50 µM, and cells were incubated for 60 s. Cells were centrifuged and washed three times with 50 µM fatty acid-free bovine serum albumin in PBS. Finally, cells were resuspended in 1 ml of PBS, and the labeled cells were analyzed in a Olympus BH2 microscope using a Zeiss 63 × oil planapo objective, and in an MRC 1024 laser Sharp confocal microscope (Bio-Rad) on an Olympus BX50 with a × 60 objective.
While the processes that mediate fatty
acid uptake in yeast are not defined, it was reasonable to predict that
a homologue of one or more of the three putative fatty acid transport
proteins from higher eukaryotic cells may be required. We compared the sequence of FAT, mitochondrial aspartate aminotransferase (plasma membrane FABPpm), and FATP to open reading frames within the
Saccharomyces cerevisiae data base. We were unable to find
open reading frames within the yeast data base that had significant
homology to FAT. While seven different reading frames on five different
chromosomes were found to have homology to mitochondrial aspartate
aminotransferase, it was difficult to assess which if any of these open
reading frames represented the FABPpm homologue in yeast. Of these
comparisons, the FATP identified in 3T3-L1 adipocytes appeared to be
the most promising candidate. We identified an open reading frame on
yeast chromosome II encoding a 623-amino acid protein with 33%
sequence identity and 54% similarity to FATP (Fig.
1A). We have designated this open reading
frame Fat1p and the structural gene encoding this protein as
FAT1.
The sequence similarities and identities exist throughout the lengths of Fat1p and FATP and thus suggest they may have similar structures and functions. Three segments of these two proteins are nearly identical. The first segment, between amino acids 255 and 268 of Fat1p, contains a consensus sequence (LIYSGTTGLPK) common to members of the AMP-binding protein family including the family of acyl-CoA synthetases (36, 37). The second and third regions of high sequence similarity include amino acid residues 324-399 and 491-544, respectively, and are restricted to Fat1p and FATP. Using the algorithms of Kyte and Doolittle (38), we compared the hydropathy profiles of Fat1p and FATP (Fig. 1B). These analyses demonstrated that both proteins have comparable profiles and in particular predicted that Fat1p, like FATP, contains at least four potential membrane-spanning segments. Fat1p also has four potential N-linked glycosylation sites. One of these glycosylation sites (at amino acid residue 534) is identical to that predicted for FATP. On the basis of these comparisons, we propose that Fat1p represents the yeast homologue of the murine long-chain fatty acid transport protein FATP.
Examination of the DNA flanking the Fat1p coding sequence identified
two potential TATA boxes. The first is found 301 bases upstream from
the presumptive translational initiation codon, and the second is 119 bases upstream. Of the two, the first (TATATAA) is predicted to be a
very strong binding site for TFIID (39). There were also two potential
polyadenylation sites, AATAAAN14/22CA (14-22 nucleotides
without consensus features), found 286 and 294 bases 3 relative to the
presumptive translational termination codon. Examination of the 5
upstream region indicated that expression of the gene may be controlled
by at least three regulators. There are three potential CCAAT elements
located beginning at
846,
882, and
982 and two potential
overcoming glucose repression elements (consensus (A/C)(A/G)GAAT)
beginning at
130 and +88 bp from the predicted start of translation
(40). Since our prediction is that Fat1p is involved in fatty acid
transport and metabolism, we searched for potential regulatory sites
identified particularly in genes encoding other yeast proteins required
for these functions. There were no matches to the consensus sequence
for UASINO, which is CATGTGAAAT (41). These elements are
found in many genes whose product is required for phospholipid and
fatty acid biosynthesis. Several genes encoding yeast peroxisomal
proteins and the
9-acyl-CoA desaturase are activated or repressed,
respectively, after growth in media containing oleate (42). Several
laboratories have identified DNA elements important for response to
oleate (43-45). Each contains the minimal consensus sequence
CGGN15/18CCG. There were no elements of this type
identified in FAT1. A second oleate response element was
identified in the peroxisomal trifunctional fatty acid oxidizing enzyme
gene of C. tropicalis when expressed in S. cerevisiae (40). The element has the consensus sequence
YGTTRTT(A/C/G). We identified four regions upstream of FAT1
at
369,
520,
727, and
835, which conform to this consensus sequence. It is not known at this time whether any of these DNA segments contribute to the expression or regulation of
FAT1.
On the basis of the sequence similarities noted above,
we predicted that Fat1p, like FATP, was involved in the uptake of
long-chain fatty acids. To determine the function of Fat1p in this
process, we disrupted FAT1 in S. cerevisiae by
the replacement of a 480-bp internal EcoRI fragment (within
the open reading frame of FAT1) with a 1.346-kilobase pair
fragment encoding the yeast HIS3 gene. This construction was
crossed onto the chromosome in the yeast strain W303a (his3)
as detailed under "Experimental Procedures," and several
transformants complementing the his3 mutation were identified. One such isolate, designated W303a-fat1-1,
was selected and shown to contain a disruption in FAT1 by
DNA amplification using thermocycling. Genomic DNA purified from both
the FAT1 and fat1
strains were amplified using
oligonucleotides specific to the 3
- and 5
-ends of the open reading
frame within FAT1. The DNA amplification product from the
fat1
strain was approximately 0.9 kilobase pair larger
than the product obtained from the parental strain, confirming that the
FAT1::HIS3 fragment was integrated into the
chromosome. Reverse transcription and amplification of total mRNA
isolated from the FAT1 strain W303a grown in YPD containing 500 µM oleate demonstrated that this gene is expressed
(data not shown).
We initially tested whether the fat1 strain
was phenotypically distinct from the parental FAT1 strain.
FAT1 and fat1
cells were grown to midlog phase
and diluted in yeast nitrogen base, and 2 × 103 cells
were plated on cerulenin-containing media supplemented with oleate,
palmitate, or myristate. The cultures were incubated at 24, 30, and
37 °C for 72 h. As predicted, both strains were unable to grow
on YPD plates containing 25 µM cerulenin (YPD/CER) due to
the inhibition of fatty acid synthesis. Growth of the FAT1 strain could be rescued at all temperatures by the addition of 500 µM myristate, palmitate, or oleate to the YPD/CER plates
(YPD/CER/MYR, YPD/CER/PAL, or YPD/CER/OLE, respectively). The
fat1
strain was viable on both YPD/CER/MYR and
YPD/CER/PAL, although there was an apparent decrease in growth when
compared with the wild type. The growth of the fat1
strain on YPD/CER/OLE was reduced dramatically when grown at 24, 30, and 37 °C when compared with the wild type.
To evaluate these observations further, growth of the FAT1
and fat1 strains was monitored at 30 °C in liquid YPD
with and without cerulenin and fatty acid (Fig. 2).
These data were in agreement with the phenotypic data noted above. The
growth rate of the FAT1 strain while depressed with the
addition of cerulenin was able to be rescued by the addition of fatty
acid (myristate, palmitate, or oleate). The growth of the
fat1
strain was reduced even when supplemented with fatty
acid when compared with the isogenic FAT1 strain. This
reduction was particularly notable with the oleate supplementation,
which paralleled the observations made on YPD agar plates containing
oleate and cerulenin.
Use of the Fluorescent Fatty Acid BODIPY-3823 to Monitor Fatty Acid Uptake
To visualize in a more direct way that fatty acid uptake
was reduced in the fat1 strain of S. cerevisiae, we evaluated the uptake of a fluorophore-labeled
long-chain fatty acid analog BODIPY-3823, employing confocal laser
scanning microscopy. Pagano et al. (46) demonstrated that
the spectral properties of several BODIPY-labeled ceramide analogues
are highly dependent upon the concentration of the probe in lipid
vesicles as characterized by a shift in the emission maximum from green
(515 nm) to red (620 nm) with increasing concentrations. Typical
confocal scanning micrographs of FAT1 and fat1
cells labeled with BODIPY-3823 are shown in Fig. 3. When
the cells were observed in the green channel only, the wild-type strain
appeared highly fluorescent, whereas the disrupted strain showed
limited fluorescent labeling (Fig. 3, A and D,
respectively). When the same cells were viewed using the red channel
(Fig. 3, B and E), the difference between the FAT1 and fat1
strains was even more dramatic.
When labeled cells were viewed in the red and green channels (
520
nm), wild-type cells were yellow-orange in color, in contrast to the
fat1
cells, which appeared pale yellow-green (Fig. 3,
C and F). These results are consistent with the
conclusion that a disruption of FAT1 reduces the cell's
ability to take up the fluorescent long-chain fatty acid
BODIPY-3823.
Incorporation of Exogenous Fatty Acids into Phospholipids in the fat1
To distinguish fatty acid transport from metabolic
utilization, we evaluated the distribution of fatty acids when supplied exogenously to the intracellular fatty acid and phospholipid pools. We
predicted that overall rate of uptake and incorporation of exogenous
long-chain fatty acids into the phospholipid pool in the
fat1 strain would be reduced while the distribution of
incorporated long-chain fatty acids among the phospholipid classes
would be the same as the wild-type. To test these predictions, we
monitored the time-dependent incorporation of exogenous
[3H]oleate into cellular lipids using one-dimensional
high performance TLC as detailed under "Experimental Procedures."
The initial uptake and incorporation of [3H]oleate into
total lipids was notably reduced in the fat1
strain (Fig.
4). Furthermore, the level of free fatty acid was
markedly decreased when compared with the wild-type (Fig.
4A). At the later time points, the differences in
incorporation of exogenous oleate were still striking. We noted that
while oleate incorporation was reduced in the mutant, the pattern of
incorporation into the various classes of phospholipids remained the
same, arguing that the enzymatic machinery required for lipid
biosynthesis was still intact. These data imply that Fat1p must
necessarily operate prior to the incorporation of exogenous oleate into
the phospholipid pool. We interpret these data to suggest a defect in
the uptake of oleate prior to metabolic utilization.
As noted above, Fat1p shares amino acid sequence similarities with the
acyl-CoA synthetases; we therefore evaluated acyl-CoA synthetase
profiles in the FAT1 and fat1 strains using
oleate, palmitate, and myristate as substrates (Table
I). Acyl-CoA synthetase activities using all three fatty
acid substrates were comparable, although the fat1
strain
had higher levels of oleoyl-CoA synthetase activity. On the basis of
these data, we conclude that the observed decrease in the uptake of
BODIPY-3823 and the incorporation of exogenous oleate into the
phospholipid pool observed for the mutant strain were not the
consequence of decreased acyl-CoA synthetase activity.
|
The
transport of fatty acid (oleate, palmitate, and myristate) was
evaluated in FAT1 and fat1 cells following
growth in SMM. The data gleaned from these types of assays must be
evaluated with caution, since they measure both fatty acid transport
and subsequent metabolic utilization. As transport precedes
utilization, we routinely measured levels of cell-associated fatty acid
for 90 s following the initiation of the reaction and thus
interpret the data in terms of uptake. We found that the uptake of
oleate was linear for the first 90 s although reduced in the
fat1
strain when compared with the wild type. Fig.
5 illustrates the substrate-dependent uptake
of oleate at 30 and 24 °C in both the FAT1 and
fat1
strains. Using the program EnzymeKinetics (Trinity
Software), the calculated maximal transport rates at 30 °C from
these data demonstrated that the fat1
strain transported
oleate at 64% of wild-type levels (8.36 nmol/min/108 cells
versus 12.99 nmol/min/108 cells). The
differences were most pronounced at 24 °C (6.54 nmol/min/108 cells for the FAT1 strain
versus 2.74 nmol/min/108 cells for the
fat1
strain). From these analyses, the apparent Kt values for oleate transport at 24 °C were
similar for both the fat1
and FAT1 strains
(2.3 and 13.7 µM, respectively). Likewise at 30 °C,
both strains had comparable apparent Kt values (63.5 µM for fat1
and 61.5 µM for
FAT1). These calculated Kt values at
30 °C are similar to those previously defined in S. cerevisiae and C. tropicalis (17, 19). These data are consistent with the notion that Fat1p functions to maximize oleate uptake. Our initial data indicated that the growth rates on the fat1
strain on YPD/CER with palmitate and myristate were
also reduced when compared with the isogenic FAT1 parental
strain. We therefore tested the rates of uptake in the FAT1
and fat1
strains using 100 µM palmitate or
myristate as substrate (Table II). These data
demonstrated that the levels of palmitate and myristate uptake were
also decreased in the fat1
strain when compared with the
wild type (20 and 47% decrease, respectively). Although these data
demonstrate that fatty acid uptake is compromised in the
fat1
strain, it is clear that an additional transport component remains operational.
|
The present work describes the identification of the fatty acid transport protein Fat1p in the yeast S. cerevisiae. Disruption of the FAT1 structural gene results in a marked decease in 1) cell growth on YPD containing oleate, palmitate, or myristate and cerulenin, 2) the uptake of the fluorescent long-chain fatty acid BODIPY-3823, 3) the uptake and incorporation of exogenous oleate into the phospholipid pool, and 4) the rates of long-chain fatty acid uptake. We hypothesize that Fat1p represents the yeast homologue of the murine fatty acid transport protein FATP.
Fat1p was identified on the basis of amino acid similarity to the murine fatty acid transport protein FATP and is encoded within a structural gene located on chromosome II. Fat1p and FATP are remarkably similar proteins in that they 1) are of comparable length (623 and 646 amino acid residues, respectively) and calculated molecular mass (71,700 and 71,200 daltons, respectively), 2) have comparable calculated isoelectric points (8.14 and 8.32, respectively), and 3) share 54% amino acid sequence similarity and 33% amino acid sequence identity. It is predicted using the algorithms of Kyte and Doolittle (38) that each protein has four potential membrane-spanning segments. The hydropathy profiles of Fat1p and FATP are remarkably alike, suggesting these two proteins span the membrane in similar ways. Fat1p has four potential glycosylation sites, while FATP has three. Both proteins are members of the family of AMP-binding proteins on the basis of sequences conserved in adenylate-forming enzymes. In addition to Fat1p and FATP, this family of enzymes includes the CoA synthetases from mammals, yeast, and bacteria. On the basis of these conserved sequences, it is tempting to postulate that fatty acid transport proteins FATP and Fat1p and the acyl-CoA synthetase have a common evolutionary lineage.
In the present study, a deletion in the FAT1 gene has been
generated to evaluate the role of Fat1p in the uptake and metabolism of
fatty acids. Strains carrying a deletion of FAT1 are
phenotypically asymptomatic unless the cells are grown on cerulenin,
thereby blocking fatty acid biosynthesis. Under these conditions,
wild-type cells overcome growth inhibition by supplementation with
long-chain fatty acids. However, the fat1 strain was
unable to be rescued by fatty acid supplementation. These results
indicated that deletion of FAT1 causes a block in one of the
steps in fatty acid utilization including fatty acid transport,
activation by acyl-CoA synthetase, or synthesis of essential higher
lipids including phospholipids. There was no decrease in acyl-CoA
synthetase in the fat1
strain compared with the wild-type
strain, and while the overall rate of incorporation of fatty acids into
lipids was reduced, there was no indication that a specific class of
lipid was altered. Therefore, we conclude that fat1
is
specifically deficient in fatty acid transport.
Fatty acid transport is a complex process that contains both
protein-mediated and diffusional components. The protein-mediated component appears to be operational at low or physiological levels of
long-chain fatty acids and thus must play a role in governing the
accessibility of exogenous long-chain fatty acids for metabolic utilization. The murine fatty acid transporter, FATP, is induced during
adipogenesis and, in addition to fat cells, is expressed at high levels
in cardiac and skeletal muscle (14). In this regard, FATP plays a
pivotal role in regulating available long-chain fatty acid substrates
from exogenous sources in tissues undergoing high levels of
-oxidation or triglyceride synthesis. The identification of Fat1p in
S. cerevisiae as the homologue to the murine FATP is
especially significant, since this model system will allow us to
specifically address the mechanism of long-chain fatty acid transport
across the plasma membrane in eukaryotic cells. Like FATP, Fat1p
functions to mediate the uptake of exogenous long-chain fatty acids and
thus may play a pivotal role in regulating accessibility of these
hydrophobic compounds prior to metabolic utilization. We hypothesize
that Fat1p acts to facilitate long-chain fatty acid transport in
S. cerevisiae by a saturable, high affinity process. It is
clear that a second mechanism for the uptake of exogenous long-chain
fatty acids remains operative in fat1
derivatives. The
efficiency of uptake using this alternative pathway is severely compromised. We suggest that, under physiological conditions, Fat1p
functions primarily when long-chain fatty acids are limiting and
required for growth to facilitate the efficient uptake of these
hydrophobic compounds into the cell.
The connection between the uptake and metabolism of long-chain fatty acids in eukaryotic cells is largely unresolved. However, a murine isoform of acyl-CoA synthetase, when expressed in COS7 cells, increases the rate of oleate uptake, suggesting a role for this enzyme in the transport of long-chain fatty acids (14). In E. coli, the acyl-CoA synthetase must necessarily operate in conjunction with the outer membrane-bound long-chain fatty acid transport protein FadL to mediate the efficient uptake of long-chain fatty acids across the bacterial cell envelope (18, 47, 48). In the context of transport, the activity of acyl-CoA synthetase of E. coli is described as "vectorial acylation" (49). Our present data are consistent with the postulate that Fat1p of S. cerevisiae acts to facilitate the uptake of exogenous long-chain fatty acids and is distinct from the acyl-CoA synthetases. The activity of Fat1p must result in an increase the intracellular pool of long-chain fatty acids. The cell, in turn, may respond to this increased pool by activating these compounds to long-chain acyl-CoA thioesters via acyl-CoA synthetase.
In the context of yeast physiology, Fat1p may play a significant role in the uptake of exogenous unsaturated long-chain fatty acids under anaerobic conditions or conditions were oxygen is limiting. Under these conditions, the desaturase involved in the synthesis of unsaturated fatty acids is dysfunctional, resulting in auxotrophy for unsaturated long-chain fatty acids. While the present study has not evaluated the requirement of Fat1p under anaerobic conditions, the use of cerulenin to block fatty acid biosynthesis coupled with long-chain fatty acid rescue provides compelling evidence suggesting an important role of Fat1p in the uptake of these hydrophobic compounds under conditions where fatty acid synthesis is compromised. This work represents a valuable foundation to further explore the problem of long-chain fatty acid transport in eukaryotic cells. The use of S. cerevisiae as a model system is of great utility as transport can be evaluated at the genetic level in addition to the biochemical and physiological levels.
We thank Qing Zhang and Changsong Bu for technical assistance and David Nelson for technical advice.