From the Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208
Received for publication, October 15, 2002, and in revised form, February 21, 2003
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ABSTRACT |
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In Saccharomyces
cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are
hypothesized to couple import and activation of exogenous fatty acids
by a process called vectorial acylation. Molecular genetic and
biochemical studies were used to define further the functional and
physical interactions between these proteins. Multicopy extragenic
suppressors were selected in strains carrying deletions in
FAA1 and FAA4 or FAA1 and
FAT1. Each strain is unable to grow under synthetic lethal
conditions when exogenous long-chain fatty acids are required, and
neither strain accumulates the fluorescent long-chain fatty acid
C1-BODIPY-C12 indicating a fatty acid transport
defect. By using these phenotypes as selective screens, plasmids were
identified encoding FAA1, FAT1, and
FAA4 in the faa1 Biological membranes are complex in both their protein and lipid
compositions. This complexity is essential and contributes to the
barrier function of the membrane and to selectively regulated transport
of molecules into and out of the cell. Unlike hydrophilic molecules
such as sugars and amino acids, hydrophobic fatty acids are able to
dissolve in the membrane, and as a consequence, the processes governing
their regulated movement across membranes are likely to be quite
distinct. Recent investigations into the problem of fatty acid
transport have intensified due to findings that exogenous fatty acids
influence a number of important cellular functions, including signal
transduction and transcriptional control. To date, several distinct
membrane-bound and membrane-associated proteins have been identified as
components of fatty acid import systems in eukaryotic cells. Most
notable among these are fatty acid translocase
(FAT,1 the murine homologue
to CD36) (1, 2), fatty acid transport protein (FATP) (3), and fatty
acyl-CoA synthetase (3-6). FAT was identified following protein
modification using sulfo-N-succinimidyl oleate (7), whereas
FATP and fatty acyl-CoA synthetase were both identified using
expression cloning (3). Both FAT and FATP have been claimed to be fatty
acid transport proteins (1, 8, 9). Despite these claims, there is
controversy surrounding the classification of FAT/CD36 and FATP as
bona fide integral membrane-bound fatty acid transporters
(10). Indeed, there are gnawing questions as to whether these proteins
actually function as components of a fatty acid delivery system
(i.e. FAT/DC36) or as components of a utilization driven
fatty acid import system (i.e. FATP), which also includes
fatty acyl-CoA synthetase (2, 4, 8, 10, 11). In this regard, proteins
identified as required for fatty acid transport may function not as
transport proteins per se but in an alternative manner,
perhaps by promoting selectivity and specificity of fatty acid delivery
to downstream metabolic events.
The best characterized fatty acid transport system is that found in
Escherichia coli (4). In this case, the specific integral outer membrane protein, FadL, is required for long-chain fatty acid
binding and transport across that membrane. The fatty acid ligands must
then traverse the bacterial periplasmic space and the inner membrane.
No inner membrane proteins have been identified that are required for
this process. On the basis of studies defining the energetics of fatty
acid transport, we suggested protonated fatty acids flip across the
inner membrane and are subsequently abstracted from the inner membrane
concomitant with activation by fatty acyl-CoA synthetase (12). In this
manner, exogenous fatty acids are metabolically trapped as CoA
thioesters upon transport, which in turn generates a concentration
gradient further driving the system. Overath and colleagues (5) coined
the term "vectorial acylation" to describe this process at the time
they identified the structural gene for the E. coli fatty
acyl-CoA synthetase (fadD). This postulate was initially
expanded by Frerman and Bennett (6) and subsequently by our laboratory
(4, 12) as the underlying mechanism driving long-chain fatty acid
transport in bacteria. Although at the time the model of vectorial
acylation was proposed the bacterial fatty acid transporter FadL had
not been identified, our subsequent studies have clearly shown that both FadL and fatty acyl-CoA synthetase are required for fatty acid
transport in E. coli.
By using the yeast Saccharomyces cerevisiae as a model
eukaryotic system, we have recently shown the fatty acyl-CoA
synthetases Faa1p or Faa4p function in the fatty acid transport system
presumably by activating exogenous fatty acids concomitant with
transport (11). This finding presents somewhat of a conundrum as we
have also shown that long-chain fatty acid import in yeast requires Fat1p, the yeast orthologue of the murine FATP1 (13). One of the
central questions we are now faced with is to determine the mechanisms
by which Fat1p and fatty acyl-CoA synthetase (Faa1p and/or Faa4p) work
in concert to promote fatty acid import. A similar situation appears to
be operational in murine adipocytes, where there are data supporting a
functional association of mmFATP1 with fatty acyl-CoA synthetase (3,
15). We suggest vectorial acylation is one general mechanism of fatty
acid import, which functions to promote the regulated import and
metabolic trapping of exogenous long-chain fatty acids.
In our prior investigations into fatty acid import in yeast, we used
reverse genetic approaches to demonstrate this process requires the
yeast orthologue of mmFATP (Fat1p) and fatty acyl-CoA synthetase (Faa1p
or Faa4p) (11, 13, 14). Despite the information gleaned from these
studies, there are no data demonstrating these proteins function
cooperatively in a physical complex, and there is no information as to
whether there are additional proteins involved in mediating the
regulated import of exogenous long-chain fatty acids. In the present
work, we sought to identify additional components required for fatty
acid transport and to confirm the importance of Fat1p and fatty
acyl-CoA synthetase (Faa1p and Faa4p) by using a genetic approach. A
valuable molecular-genetic method for the identification of
participants in multicomponent cellular processes is the selection of
plasmid-encoded multicopy extragenic suppressors (16). The rationale
behind this approach is that the altered phenotype resulting from a
deficiency in one participant can be suppressed by overexpression of
another participant required for the same process (16). In this manner,
we sought to identify plasmid-encoded multicopy extragenic suppressors
of the deficiency in fatty acid import caused by deletion of
FAT1 and/or FAA1 and FAA4. We report
that plasmids encoding Fat1p, Faa1p, and Faa4p were identified in a
screen for multicopy extragenic suppressors of the transport and
activation deficiency of a faa1 Strains, Media, and Materials--
The S. cerevisiae
strains used in this study are listed in Table
I. The fat1
YPDA consisted of 1% yeast extract, 2% peptone, 2% dextrose, and 20 mg/liter adenine hemisulfate. Yeast-supplemented minimal media
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 or YPDA
plates supplemented with 45 µM cerulenin and 100 µM oleic acid unless otherwise indicated. Growth in
liquid culture and on plates was at 30 °C.
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
and American Radiochemicals. C1-BODIPY-C12 was
purchased from Molecular Probes. Enzymes required for all DNA
manipulations were from Promega, Invitrogen, New England Biolabs,
U. S. Biochemical Corp., or Roche Molecular Biochemicals. Anti-V5
antibody and anti-T7 antibodies were purchased from Invitrogen and
Novagen, respectively. Anti-Pma1p was the gift of Dr. Günther
Daum (Technische Universität Graz, Graz, Austria).
Complementation of faa1 Assessment of Fatty Acid Import Capacity--
Fatty acid import
was assessed using confocal laser scanning microscopy to detect
accumulation of 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
(13). Following growth under selective conditions, cells were
harvested, washed with phosphate-buffered saline (PBS) and resuspended
in 0.1 volume of PBS. All steps were performed at room
temperature. Washed cells were incubated with 10 µM
C1-BODIPY-C12 for 60 s, washed with PBS
containing 50 µM fatty acid-free bovine serum albumin
(two times), PBS, resuspended in PBS, and visualized on an NORAN-OZ confocal laser scanning microscopy, interfaced with a Nikon Diaphot 200 inverted microscope equipped with a PlanApo ×60, 1.4 NA oil-immersion objective lens. The instrument settings for brightness, contrast, laser
power, and slit size were optimized for the brightest sample to ensure
that the confocal laser scanning microscopy was set for its full
dynamic range. The same settings were used for all subsequent image collections.
Quantification of Fatty Acyl-CoA Synthetase
Activities--
Cells were grown from overnight cultures in YNBD (with
appropriate supplements) and grown to
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, 4 °C), and supernatants were
used to assess fatty acyl-CoA synthetase activities as described (20).
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 Negative Dominance of Mutant Fat1p Over Fatty Acyl-CoA
Synthetase--
The sequence encoding the C-terminal 125 amino
acids (residues 545-669) of Fat1p was cloned in-frame to the T7
epitope tag of the yeast expression vector YEpGALSET983 to generate
YEpDB213. The resulting T7Fat1p125C fusion was
expressed under the control of the GAL10 promoter. To test
for negative dominance, YEpDB213 was transformed into YB332. Cells
transformed with the vector (YEpGALSET983) served as a control. The
cells were pre-grown in YNBD (without leucine) overnight. The culture
was harvested by centrifugation and resuspended to a cell density of
0.1 A600 in 50 ml of YNB containing 2%
galactose and 2% raffinose to induce expression of
T7Fat1p125C. When the density reached 1.0 A600, cells were harvested by centrifugation, washed once in PBS, and resuspended in 1 ml of breaking buffer (200 mM Tris, pH 8.0, 4 mM EDTA, 10% glycerol, 5 mM Two-hybrid Analysis of Fat1p and Faa1p--
The yeast two-hybrid
system was used to test Faa1p-Fat1p interaction (22). The bait plasmid
vector used was pEG202; the trap plasmid vector was pJG4-5, and the
reporter plasmid was pSH18-34T. To generate the full-length Faa1p-bait
fusion protein, the coding sequence of FAA1 was amplified
using the upstream primer 5'-AGACCCATGGATGGTTGCTCAATATACCG-3' and the downstream primer
5'-AAATGTTGGCGGCCGCAGACGAACTATAAACGGC-3'. The amplified DNA
fragment was cleaved with NcoI and NotI and ligated into pEG202 cleaved with the same enzymes. For the trap plasmids, a single primer was used to amplify DNA at the 3' end of the
gene including the termination codon encoding amino acid 669, 5'-GAACATCCTCGAGTAATTTAATTGTTTGTGC-3', whereas unique primers were used to amplify DNA at the 5' ends. These included
5'-TTTTTAGCGCGCAATACTAAAGGCACTCCG-3' to generate a peptide from
amino acids 169 to 669 of Fat1p (Fat1p500C) and
5'-GAAGATGAATTCACGGCCAGTAACAAAGAAC-3' to generate a peptide from
amino acids 544 to 669 of Fat1p (Fat1p125C). The amplified
DNA fragments were digested with the appropriate restriction enzymes
and ligated into pJG4-5.
To test interaction, the Faa1p bait plasmid and the target trap plasmid
of interest were transformed into yeast strain W303B carrying the
reporter plasmid pSH18-34T. The reporter plasmid pSH18-34T contains the
lacZ gene encoding Co-immunoprecipitation of Fat1p and Faa1p or Faa4p--
To
identify a protein complex containing Fat1p and Faa1p or Faa4p,
plasmids were constructed expressing each protein fused to a peptide
tag, which is recognized by a commercially available antibody.
Full-length Fat1p tagged with a T7 epitope was constructed in the
vector YEpGALSET983 to generate plasmid YEpDB204. The coding sequence
of FAT1 was amplified using the upstream primer
5'-GCGGAGCTCATGTCTCCCATACAGGTTGTTG-3' and the downstream primer
5'-CGCGGTACCATGCTCTAATGGAAAGGTAC-3'. The amplified DNA fragment was
cleaved with SacI and KpnI and ligated into
YEpGALSET983 cleaved with the same restriction enzymes. Expression
clones encoding full-length Faa1p or Faa4p tagged at the C terminus
with a V5 epitope were obtained from Invitrogen (GeneStormTM clones pYES2/YOR317w and pYES2/YMR246w, respectively).
The plasmid pair encoding the proteins of interest (e.g.
T7Fat1p and V5Faa1p or V5Faa4p) was
transformed into the fat1 Identification of Multicopy Suppressors of Synthetic Lethality
Imposed by Cerulenin on faa1
In an attempt to identify genes that could functionally replace
FAT1 or FAA1 and FAA4, we screened a
yeast genomic multicopy library for clones, which suppressed the
cerulenin-induced lethality of an faa1
We noted that several plasmids isolated from the colonies listed in the
"other" category in Table II did not confer the suppressor phenotype upon re-transformation. Therefore, we presume the phenotype was associated with an undefined chromosomally encoded suppressor.
The Multicopy Suppressors Alleviate Fatty Acid Import
Defects--
In an effort to determine whether fatty acid import was
restored by the plasmid-encoded suppressors, we monitored the
accumulation of the fluorescent fatty acid analogue
C1-BODIPY-C12 using confocal laser scanning
microscopy in wild-type strains and transformants of the
faa1 Deficiencies in Long-chain Acyl-CoA Synthetase Activity in faa1
Our laboratory and others (14, 24) have shown that Fat1p has intrinsic
very long-chain (C22-C26) fatty acyl-CoA
synthetase activity. Indeed, when we first characterized the
FAT1 gene, we noted Fat1p shared similarities to the
adenylate-forming family of enzymes, which includes the fatty acyl-CoA
synthetases (13). We reasoned that when expressed from a high copy
number plasmid, FAT1 would result in sufficient long-chain
fatty acyl-CoA synthetase activity to promote growth of the
faa1
As noted above, Fat1p has been shown to confer very long-chain fatty
acyl-CoA synthetase activity. Therefore, we also measured fatty
acyl-CoA synthetase activities in the same cell extracts from above
using the very long-chain fatty acid, lignocerate (C24:0) as substrate (Table IV). As expected, expression of FAT1
from YEpDB17 increased these activities just over 4-fold, whereas
expression of FAA1 from YEpDB02 or FAA4 from
YEpDB133 had no significant effect on total cellular very long-chain
fatty acyl-CoA synthetase activities. Similar results were obtained for
the fat1 Fat1p and Fatty Acyl-CoA Synthetase Form a Physical
Complex--
In previous work, we provided independent evidence that
Fat1p (13) and Faa1p or Faa4p (11) are each required for fatty acid
import in yeast. The results of the multicopy suppressor analyses
detailed above extended these results to include a functional dependence of fatty acid transport on both Fat1p and fatty acyl-CoA synthetase (Faa1p or Faa4p). Indeed, these data provided evidence suggesting Fat1p and Faa1p or Fat1p and Faa4p interact to coordinately facilitate fatty acid transport. The results from the multicopy suppressor screen are consistent with the notion that, at least in
yeast, no other proteins participate in this process. Yet this experimental approach did not address whether Fat1p and fatty acyl-CoA
synthetase form a physical complex. To address this question, we
employed three different experimental strategies as follows: 1)
negative dominance of mutant Fat1p over fatty acyl-CoA synthetase; 2)
yeast two-hybrid analyses to investigate the hypothesized physical linkage between Fat1p and Faa1p or Faa4p; and 3) co-immunoprecipitation of Fat1p and a cognate fatty acyl-CoA synthetase.
Often when two proteins physically interact to form a functional
complex, inactivation of one protein due to a mutation will result in a
reduction in activity for the partner protein. This phenomenon is
called negative dominance. Long-chain acyl-CoA synthetase activity in
yeast is primarily contributed by Faa1p (
Another method, which has become standard to evaluate
protein-protein interactions, is the yeast two-hybrid system. These experiments employed plasmids encoding a bait protein, which consisted of a fusion between full-length Faa1p and the DNA binding domain of
bacterial LexA and several trap proteins, which consisted of protein
fusions containing full-length Fat1p and peptides derived from Fatp1
and the Gal-driven activation domain. In this system a third reporter
plasmid contains the DNA-binding site of LexA in the promoter region
driving expression of lacZ (encoding
Additional evidence for specific protein-protein interactions
between Fat1p and Faa1p or Faa4p was obtained using
co-immunoprecipitation. As detailed under "Experimental
Procedures," Fat1p was tagged with a T7 epitope
(T7Fat1p), and the fatty acyl-CoA synthetases were tagged
with a V5 epitope (V5Faa1p and V5Faa4p).
Following growth, extracts were prepared from cells expressing T7Fat1p and V5Faa1p or V5Faa4p and
immunoprecipitated using anti-T7 or anti-V5 antibodies. The presence of
the second protein in the complex was detected by Western blot analyses
using the reciprocal antibody. The data presented in Fig.
4 showed that V5Faa1p and
V5Faa4p are co-immunoprecipitated with T7Fat1p
whether the precipitating antibody was anti-T7 directed against Fat1p
or anti-V5 directed against one of the Faa proteins. In our control
experiments using protein A-Sepharose beads alone (Fig. 4) or using an
unrelated antibody (c-Myc) (not shown), we did not pull down the
Fat1p-fatty acyl-CoA synthetase complex. Additionally, to test for
nonspecific protein-protein interactions, we probed the immunocomplex
using an antibody against Pma1p, an unrelated plasma membrane protein
(25). No co-immunoprecipitation of Pma1p with Fat1p, Faa1p, or Faa4p
was detected (Fig. 4). These data are in agreement with the results of
the multicopy suppressor analysis, negative dominance, and yeast
two-hybrid data presented above and fully support the notion that Fat1p
and Faa1p or Faa4p form a physical complex, which we suggest is crucial
to the process of vectorial esterification of exogenous long-chain
fatty acids.
When long-chain fatty acids are supplied in the growth media,
S. cerevisiae transports these compounds into the cell by a process, which requires Fat1p and the fatty acyl-CoA synthetase Faa1p.
Even though Faa1p and Faa4p have been suggested to be functionally redundant, previous results and those presented here show that Faa1p,
rather than Faa4p, plays a more distinct role in fatty acid import (11,
26). Importantly, the experiments reported here provide substantial
genetic and biochemical evidence that Fat1p and fatty acyl-CoA
synthetase (Faa1p or Faa4p) form a physical complex required to
facilitate fatty acid import. These data are consistent with the
hypothesis that the fundamental mechanism driving the accumulation of
exogenous fatty acids within the cell is vectorial acylation whereby
exogenous fatty acids are metabolically trapped as acyl-CoA thioesters.
Until this time, the physical and functional association of FATP and
fatty acyl-CoA synthetase has been inferential (3, 11, 15). The present
studies indicate that in the natural environment when fatty acids are
limiting as, for example, occurs during hypoxia, Fat1p and Faa1p are
each required for fatty acid import. Whereas each protein fulfills a
separate function, the activities are coordinated and facilitated by a
physical interaction. The former conclusion is based on the observation
that in single copy neither gene can substitute for the other. The
distinct functions for Fat1p and Faa1p were apparent in enzymatic
analyses of acyl-CoA synthetase specificity and activity and in our
fatty acid transport studies. In multicopy, Faa1p can substitute for
Fat1p, and in turn, Fat1p can substitute for Faa1p in potentiating
fatty acid import. Thus the apparent increase in accumulation of
C1-BODIPY-C12 when either of these genes is
overexpressed appears related to the essential role of long-chain fatty
acyl-CoA synthetase activity in import and utilization rather than to a
transport function per se. Thus utilization creates a
diffusional gradient dependent upon the acyl-CoA synthetase Faa1p (and
to a more limited extent Faa4p) but not Fat1p. The role of Fat1p in
fatty acid import appears to be distinct from Faa1p and essential only
at limiting fatty acid concentrations ( There is substantial data showing Fat1p plays a role in long-chain
fatty acid import yet has intrinsic very long-chain
(C22-C26) fatty acyl-CoA synthetase activity
(14, 24). This presents somewhat of a dilemma. Our results are
consistent with the notion that the specificity of the fatty acid
import system in yeast is for long-chain fatty acids as opposed to very
long-chain fatty acids. Addition of very long-chain fatty acids to the
growth media of yeast strains defective in very long-chain fatty acid
synthesis does not alleviate the growth defect, suggesting the very
long-chain fatty acids cannot be trafficked from an exogenous source to
the site of metabolic utilization (27). Yet Fat1p is a central
component of the long-chain fatty acid import system in yeast, being
required both under anaerobic conditions and under cerulenin-induced
conditional lethality, where exogenous long-chain fatty acids are
required for growth (14). We have provided evidence recently (28) that the very long-chain fatty acyl-CoA synthetase activity intrinsic to
Fat1p can be distinguished from fatty acid import in specific mutant
alleles of FAT1 with single amino acid substitutions.
Additionally, the specificity of Fat1p-dependent import is
for long-chain fatty acid substrates,
whereasFat1p-dependent fatty acyl-CoA synthetase activity
is for very long-chain substrates (14, 24). The deletion of
FAA1 encoding the major long-chain fatty acyl-CoA synthetase decreases fatty acid import nearly 3-fold; therefore, we suggest this
enzyme is primarily responsible for activating fatty acids from an
exogenous source and therefore contributes to the specificity of the
import system (11).
The ability of FAT1 encoded within a high copy number
episome to suppress the phenotype on YNBD containing oleate and
cerulenin of a faa1 Although we did not identify new partners in the fatty acid trafficking
pathway by selecting multicopy suppressors, these results are of
particular significance because they confirmed by using a powerful
genetic approach the importance of an interaction between Fat1p and
Faa1p in fatty acid import. Indeed, with one note of caution based on
the suppressors presumed to be chromosomally encoded, these studies
indicate these two proteins may be the only components mediating this
process in yeast. Our present results parallel the previous work of
Schaffer and Lodish (3) that identified independent clones encoding
murine FATP1 and a fatty acyl-CoA synthetase using a functional cloning
strategy. Functional cloning requires, in essence, overexpression of
the protein target in a manner analogous to our studies using multicopy
suppression. The murine FATP1 and fatty acyl-CoA synthetase each were
identified and shown to function to promote the accumulation of
C1-BODIPY-C12 (3). By analogy, we have shown
that Fat1p and Faa1p, when expressed from a 2-µm plasmid also
function to promote the accumulation of
C1-BODIPY-C12. The murine FATP1 also has
intrinsic very long-chain acyl-CoA synthetase activity (29). Likewise,
we and others (14, 24) have shown yeast Fat1p is a very long-chain
acyl-CoA synthetase. Previously, we have shown (14) murine FATP1
complements the biochemical phenotypes associated with the
fat1 The present work demonstrates for the first time a physical interaction
between Fat1p and Faa1p or Faa4p. In each series of experiments
(i.e. negative dominance, two-hybrid analyses, and co-immunoprecipitation), the full-length proteins and C-terminal peptides of Fat1p resulted in positive interactions. One outcome from
these experiments suggests the protein-protein interaction domain of
Fat1p is localized at least in part to the C-terminal 125 residues. At present, we have no data localizing an interaction domain
within Faa1p or Faa4p. Those experiments are currently underway.
Fatty acid transport in S. cerevisiae is tightly coupled to
utilization and is primarily dependent upon the products of two genes,
Fat1p and Faa1p. These proteins function in concert to couple fatty
acid import to fatty acid activation and metabolic utilization, a
process first described in bacteria as vectorial acylation. Due to the
functional conservation of these proteins in higher eukaryotes, yeast
provides a valuable, genetically tractable model system useful to
further elucidate the mechanisms that underpin fatty acid import in
eukaryotic systems.
faa4
strain
and encoding FAA1 and FAT1 in the
faa1
fat1
strain. Multicopy
FAA4 could not suppress the growth defect in the
faa1
fat1
strain indicating some
essential functions of Fat1p cannot be performed by Faa4p.
Chromosomally encoded FAA1 and FAT1 are not
able to suppress the growth deficiencies of the fat1
faa1
and faa1
faa4
strains, respectively, indicating Faa1p and Fat1p play distinct roles
in the fatty acid import process. When expressed from a 2µ
plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity,
which indicates vectorial esterification and metabolic trapping are the
driving forces behind import. Evidence of a physical interaction
between Fat1p and FACS was provided using three independent biochemical
approaches. First, a C-terminal peptide of Fat1p deficient in fatty
acid transport exerted a dominant negative effect against long-chain
acyl-CoA synthetase activity. Second, protein fusions employing Faa1p
as bait and portions of Fat1p as trap were active when tested using the
yeast two-hybrid system. Third, co-expressed, differentially tagged
Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively,
these data support the hypothesis that fatty acid import by vectorial
acylation in yeast requires a multiprotein complex, which consists of
Fat1p and Faa1p or Faa4p.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
faa4
strain, and plasmids encoding only Fat1p and Faa1p were identified as multicopy extragenic suppressors of the transport deficiency of a
faa1
fat1
strain. Additional biochemical
evidence is provided demonstrating Fat1p and acyl-CoA synthetase
interact in a physical complex. This work establishes for the first
time a genetic, physical, and functional linkage between Fat1p and
fatty acyl-CoA synthetase and substantiates the hypothesis that these
proteins, perhaps exclusively, are required for long-chain fatty acid
transport in yeast.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::G418
mutation was introduced by transformation of the strain of interest
with linear DNA generated by amplification of the kanamycin resistance
cassette (resulting in G418 resistance) using oligonucleotides
complementary to both FAT1 and the cassette as described
(17). The oligonucleotide for the coding strand was
5'-CACTGTCAAGAAGGGCAAGAAGGCAGCAGTATGGCTTGGGCATAGGCCACTAGTGGATCTG-3', and the oligonucleotide for the template strand was
5'-CCACTGGATCATTCGTAAGTGATCCTGAAACAAACCATTCAGCAGCTGAAGCTTCGTACGC-3'. Chromosomal replacement of the native gene was confirmed by Southern analysis of chromosomal DNA from the transformants by comparison to DNA
obtained from the parental strain. Yeast strains were transformed by
the lithium acetate method (18).
Yeast strains used in this study
faa4
and faa1
fat1
Using
Multicopy Extragenic Suppression--
Cells of the faa1
faa4
strain or faa1
fat1
strain were rendered competent using lithium acetate as noted above,
transformed with a yeast multicopy library in YEp24, and transformants
selected on YNBD containing the appropriate supplements but lacking
uracil (19). Thirty thousand individual Ura+ transformants
were selected from the library and were screened for growth following
replica plating on YPD plates containing 45 µM cerulenin
and 100 µM oleic acid (YPD-CER-OLE). Transformants that
were able to grow on YPD-CER-OLE were colony-purified on the same media
and phenotypes validated on YPD-CER-OLE. Plasmids were isolated from
those that retained positive growth on all three media and
retransformed into the faa1
faa4
and
faa1
fat1
strains. Additionally, the same
plasmids were propagated in the E. coli strain DH5
,
purified using QiaPrep columns (Qiagen), and sequenced using two
plasmid-specific primers flanking the insert (upstream,
5'-GGAGCCACTATCGACTACGC-3'; downstream, 5'-CCTGTGGCGCCGGTGATG-3') using
an Applied Biosystems automated fluorescence DNA sequencer. The
sequences obtained were compared with the Saccharomyces
genome data base for identification.
-cyclodextrin (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 isopropyl
alcohol, 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
cell extracts were determined using the Bradford assay and bovine serum
albumin as a standard (21). The values presented represent the average
from at least three independent experiments performed in duplicate. All
experiments were subjected to analysis of variance (StatView, SAS
Institute, Inc.).
-mercaptoethanol, 0.01% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 4 µM pepstatin A, and 8 µM leupeptin). The cells were lysed by
vortexing with glass beads and assayed for long-chain acyl-CoA
synthetase activity as detailed above.
-galactosidase driven by a promoter
controlled by eight LexA operators. To maintain each plasmid,
transformants were selected and maintained on YNBD media lacking uracil
(for pSH18-34T), histidine (for the pEG202-derived bait), and
tryptophan (for the pJG4-5-derived traps). Expression of
-galactosidase activity was measured using the liquid assay employing o-nitrophenyl
-D-galactopyranoside
as substrate as described previously (23). For these experiments, cells
were grown overnight in YNBD (without uracil, histidine, or tryptophan) and subcultured to A600 0.02-0.1 in 10 ml of
YNB containing 2% galactose and 2% raffinose. Growth was continued
until the A600 reached 0.5-1.0, at which time
was stopped by placing the cultures on ice. Aliquots of cells (1 ml)
were harvested by centrifugation (14,000 rpm for 3 min). The cell
pellets were resuspended in 200 µl of 0.1 M Tris, pH 7.5, containing 0.05% Triton X-100. The sample was frozen on dry ice and
stored at
80 °C prior to assay. All experiments defining
-galactosidase activities were performed in duplicate at least five
times as described previously (23); the data were analyzed using paired
t-tests against cells containing the bait (Faa1p), the trap
vector (pJG4-5), and the reporter (StatView, SAS Institute, Inc.).
faa1
strain to
test Fat1p-Faa1p interaction or the fat1
faa4
strain to test Fat1p-Faa4p interaction. The cells
were pre-grown in YNBD without leucine and uracil to maintain both
plasmids; cells were subsequently subcultured to 0.1 A600 in 75 ml of YNB containing 2% galactose
and 2% raffinose (without leucine and uracil) to induce expression of
the epitope-tagged target proteins. When the cell density reached 1.0 A600, the cells were harvested, washed once with
PBS, and resuspended in 1.5 ml of lysis buffer containing 50 mM Tris, pH 7.5, and 150 mM NaCl. The cells
were lysed by vortexing with glass beads on ice as detailed above. The
glass beads were pelleted by centrifugation (2,000 rpm, 2 min,
4 °C). The supernatant was removed to a new tube, and Triton X-100
was added to a final concentration of 1%, and the mixture was
incubated on ice for 45 min. The sample was clarified by centrifugation
(4,000 rpm, 15 min, 4 °C). The resultant supernatant was split into
three 0.5-ml aliquots (~0.7 mg/ml); 2 µg of anti-T7 or 2 µg of
anti-V5 antibodies was added to the first two, and an equal volume of
lysis buffer was added to the third as a control (protein A-Sepharose
bead control). The samples were incubated with gentle rotation
overnight at 4 °C. Protein A-Sepharose beads (50 µl of 50%
slurry) were added to each sample, which were then incubated for 2 h with gentle rotation at 4 °C. The protein A-Sepharose beads
(containing the antigen-antibody complex) were pelleted by
centrifugation (1,000 rpm, 1 min, 4 °C) and subsequently washed 5 times in 50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Triton X-100. The final pellets containing the protein A-Sepharose
beads/antigen-antibody complex were resuspended in 70 µl of SDS
sample buffer. Samples were boiled 5 min, and the proteins from 5 µl
of the cell lysate or 15 µl of the immunoprecipitated sample were
separated by electrophoresis on a 12.5% SDS-polyacrylamide gel. After
electrophoresis the proteins were transferred to nitrocellulose for
immunoblotting. Tagged proteins were detected using the appropriate
antibody (anti-V5, anti-T7, or anti-Pma1p) as detailed in the figure legends.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
faa4
or faa1
fat1
Strains--
When yeast cells are grown on media containing the
fatty-acid synthase inhibitor cerulenin, they become auxotrophic for
long-chain (C14-C18) fatty acids.
Supplementation of the media with 100 µM oleate is
sufficient to restore growth to wild-type strains. However, cells
carrying deletions in FAT1 or FAA1 and
FAA4 are not viable on media containing cerulenin despite
the addition of fatty acids. For fat1
strains, we have
shown previously (13) this phenotype is due to a defect in the ability
to import fatty acids and not due to depressed levels of long-chain
fatty acyl-CoA synthetase activities. Strains carrying deletions in the
genes encoding the fatty acyl-CoA synthetases Faa1p and Faa4p have a
similar phenotype, which we hypothesize is due to a specific coupling
between Fat1p-mediated fatty acid transport and Faa1p/Faa4p-mediated
fatty acid activation (11). The esterification of the exogenous fatty
acid to coenzyme A is required for all subsequent metabolic processes.
faa4
strain (deficient in long-chain fatty acyl-CoA synthetase activity) and
an faa1
fat1
strain (deficient in fatty acid import and with reduced long-chain fatty acyl-CoA synthetase activity). Primary transformants were selected on YNBD plates lacking
uracil and subsequently were replica-plated to YNBD plates containing
cerulenin and oleate. Plasmids were isolated from colonies that grew on
the selective media, and the individual plasmid encoded suppressors
verified by retransformation. The identities of the inserts were
determined by restriction enzyme analysis and by sequencing using
plasmid-specific primers flanking the site of insertion. In both
screens, multiple isolates of each plasmid-borne suppressor were
identified indicating all possible suppressing clones available in this
genomic library had been identified (Table II). As expected, because both strains
carried a deletion in FAA1, most of the plasmids identified
in either strain encoded the fatty acyl-CoA synthetase Faa1p. A
surprising result was that FAT1 was identified at high
frequency, whereas FAA4 was identified in only two cases in
the screen using the faa1
faa4
strain.
FAA4 was not identified as a multicopy suppressor in the
faa1
fat1
strain. Subsequent analyses of
the faa1
fat1
strain transformed with a
YEp24 plasmid derivative encoding Faa4p (YEpDB133) verified this fatty
acyl-CoA synthetase could not substitute for FAA1 and FAT1 in this strain. Three plasmids identified as multicopy
suppressors using these screens were chosen for further
characterization. They were YEpDB02 encoding Faa1p, YEpDB133 encoding
Faa4p, and YEpDB17 encoding Fat1p (Table II; Fig.
1).
Characterization of plasmids encoding multicopy suppressors of strains
deficient in fatty acid import and long-chain acyl-CoA synthetase
activity
View larger version (71K):
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Fig. 1.
FAA1, FAA4, and
FAT1 are multicopy suppressors of the synthetic
lethality of faa1 faa4
and faa1
fat1
. Cells were streaked YNBD
plates containing 100 µM oleate and 45 µM
cerulenin, and the cultures were incubated for 48 h at 30 °C.
The host strain was either YB525 (faa1
faa4
) (A) or LS2086 (faa1
fat1
) (B) carrying the vector, YEp24, or the
plasmids encoding FAA1 (YEpDB02), FAA4
(YEpDB133), or FAT1 (YEpDB17).
faa4
and faa1
fat1
strains. Wild-type cells import C1-BODIPY-C12 quickly (within 30 s) by a
process that is essentially irreversible, which we suspect reflects
metabolic activation (14). Previous work from our laboratory (13, 14)
has shown a deletion within FAT1 severely restricts
C1-BODIPY-C12 accumulation. Deletion of
FAA1 alone appears to decrease, but not completely
eliminate, accumulation of C1-BODIPY-C12.
Deletion of FAA4 has essentially no effect. In contrast,
when both FAA1 and FAA4 are deleted, the accumulation of C1-BODIPY-C12 is restricted in
a manner similar to that observed in FAT1 mutants (11). As
illustrated in Fig. 2, the accumulation
of C1-BODIPY-C12 was restored in the
faa1
faa4
strain harboring FAT1,
FAA1, or FAA4 on a multicopy plasmid. These
results point out that FAT1 is a true multicopy suppressor. Only when expressed from a 2µ plasmid can FAT1
compensate for deletions in FAA1 and FAA4. These
data support the notion that Fat1p and Faa1p or Faa4p form a functional
network facilitating the import and activation of exogenous fatty
acids, and in wild-type cells each functions in a distinct yet
coordinate manner. It is important to note that FAA4 on a
multicopy plasmid (YEpDB133) did not restore in
C1-BODIPY-C12 accumulation in the
faa1
fat1
strain, whereas both
FAA1 and FAT1 did (Fig. 2). These results indicated that Faa1p, Faa4p, and Fat1p have overlapping, yet distinct roles. Of particular importance was the finding that Faa1p and Fat1p
appeared to be functionally linked. Table
III summarizes both the phenotypes and
fatty acid transport profiles of the mutant strains alone and
transformed with the selected multicopy suppressor plasmids. These
findings are consistent with data obtained on the mmFATP1 and fatty
acyl-CoA synthetase, which are proposed to form a functional complex
(15).
View larger version (40K):
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Fig. 2.
Fatty acid import in the
faa1 faa4
strain
(A) and faa1
fat1
(B) containing the
indicated multicopy suppressor plasmids monitored by following the
accumulation of the fluorescent long-chain fatty acid
C1-BODIPY-C12. Shown are the following:
YEp24 (vector control), FAA1 on plasmid YEpDB02,
FAA4 on plasmid YEpDB133, and FAT1 on plasmid
YEpDB17.
Characteristics of yeast strains with mutations in FAT1 and the FAA
genes
faa4
and faa1
fat1
Strains Can Be Compensated by Multicopy
FAT1--
The identification of FAT1 as a multicopy
suppressor in experiments using the faa1
faa4
and faa1
fat1
strains
demonstrated that in high copy FAT1 alone as well
FAA1 alone could compensate for the defects with regard to
importing exogenous long-chain fatty acids. These data imply that under
these conditions Fat1p contributed an enzymatic activity to promote the
unidirectional transport of exogenous long-chain fatty acids.
Previously, we have shown that deletion of FAT1 does not
reduce long-chain fatty acyl-CoA synthetase activities measured using
whole cell extracts, whereas deletion of FAA1 and
FAA4 reduced these activities ~95% (Table
IV) (11, 13). Likewise, when
FAT1 is cloned into a centromeric plasmid (a pRS316
derivative designated pDB102) (14) and transformed into the
faa1
faa4
strain, long-chain fatty acyl-CoA
synthetase activities are not substantially elevated, and no
complementation was observed on YPD-CER-OLE plates (data not
shown).
Fatty acyl-CoA synthetase activities in yeast strains with mutations in
FAA1, FAA4, and/or FAT1 alone and transformed with
multicopy suppressor plasmids
faa4
and faa1
fat1
strains under the synthetic lethal conditions used
in this study. To test this idea, we measured fatty acyl-CoA synthetase
activities in total cell extracts from the parental strain and strains
harboring the multicopy suppressor plasmids using oleate
(C18:1) as a substrate (Table IV). Extracts prepared from
the faa1
faa4
strain harboring YEpDB17
(encoding Fat1p) had ~4-fold higher oleoyl-CoA synthetase activity
compared with the strain carrying the vector YEp24, which was 30% of
the level obtained for the wild-type strain. This modest increase in
oleoyl-CoA synthetase activity correlated with a 3-fold increase in
protein level estimated using Western blot analysis of cellular
extracts employing a Fat1p-specific antibody and analyzed using NIH
Image analysis software. The same strain transformed with YEpDB02 and
YEpDB133 (encoding Faa1p and Faa4p, respectively) had 10- and 2-fold
oleoyl-CoA synthetase activities, respectively, compared with the same
control cells. It is unclear why increased dosage of FAA4
had such a limited impact on total oleoyl-CoA synthetase activity. This
may be due to protein instability as noted for the purified enzyme (26)
or due to regulatory parameters poorly defined at the present time. In
the case of the faa1
fat1
strain, we noted
similar results. Most notable among these was the finding that YEpDB17
(FAT1) resulted in oleoyl-CoA synthetase activities, which
were increased 6-fold over the same strain harboring the plasmid vector
(Table IV).
faa1
and fat1
host
strains (Table IV).
95%). Therefore, we
reasoned that the overexpression of nonfunctional Fat1p would result in
a reduction of long-chain acyl-CoA synthetase activity if the proteins
physically interact to facilitate vectorial acylation. For
these experiments, we expressed a peptide derived from Fat1p made up of
the C-terminal 125 amino acids (residues 545-669;
T7Fat1p125C). This peptide derived from Fat1p
was non-functional in transport and activation, yet when analyzed using
SDS-PAGE it formed a dimer, which was stable to boiling, suggesting it
might contain a protein-protein interaction domain (data not shown).
The expression of T7Fat1p125C significantly
reduced oleoyl-CoA synthetase activity (compared with vector control)
(Fig. 3A). The reduction in
activity was correlated with expression of the
T7Fat1p125C peptide, detected using a Western
blot following expression with anti-T7 antibodies (Fig. 3B).
Under these conditions, the T7Fat1p125C peptide
is expressed at levels nearly 10-fold higher when compared with native
Fat1p (data not shown). These data are consistent with the proposal
that Fat1p and fatty acyl-CoA synthetase form a functional complex.
View larger version (18K):
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Fig. 3.
Negative dominance of mutant Fat1p over fatty
acyl-CoA synthetase. Extracts were prepared from wild-type cells
carrying a plasmid encoding T7Fat1p125C under
the control of a galactose-inducible promoter and used to assess
oleoyl-CoA synthetase activity (A) and peptide expression
(B) using a Western blot probed with anti-T7 antibody.
-galactosidase), which is dependent on specific protein-protein interactions between the
bait (bound to the DNA binding site) and the trap (fused to activation
domain, which interacts with yeast RNA polymerase II). As shown in
Table V, positive interactions between
full-length Faa1p and either full-length Fat1p or two peptides carrying
C-terminal fragments of Fat1p (Fat1p500C and
Fat1p125C) were found when compared with the trap vector
control alone. The peptide, which conferred negative dominance to fatty
acyl-CoA synthetase activity (T7Fat1p125C)
detailed above, also results in a positive interaction with Faa1p using
the yeast two-hybrid system.
Yeast two-hybrid analyses of Faa1p-Fat1p interaction
View larger version (49K):
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Fig. 4.
Co-immunoprecipitation of Fat1p and Faa1p or
Faa4p. A, anti-T7 antibody ( -T7) was used
to pull down full-length T7Fat1p in extracts prepared from
cells co-expressing T7Fat1p and V5Faa1p or
V5Faa4p as indicated. The proteins were separated by
SDS-PAGE, and subsequent Western blots were probed with anti-V5
(
-V5) antibody to detect V5Faa1p or
V5Faa4p as shown. B, similarly, anti-V5 was used
as the precipitating antibody to pull down V5Faa1p or
V5Faa4p following co-expression of T7Fat1p and
V5Faa1p or V5Faa4p, and the resultant blot was
probed with anti-T7. IB, antibody used in the immunoblot;
T, total cell extract; IP, samples
immunoprecipitated with the indicated antibody; Beads,
protein A-Sepharose alone without an immunoprecipitating antibody.
Anti-Pma1p was used as a control protein specific to a yeast plasma
membrane protein but unrelated to Fat1p, Faa1p, or Faa4p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 µM) such as
might occur when cells are growing under hypoxic conditions in the
natural environment. We suggest this mechanism of fatty acid transport
by vectorial acylation exemplifies a system common to eukaryotes
including mammalian cells that functions through FATP and a cognate
fatty acyl-CoA synthetase.
faa4
strain and the
corresponding ability of plasmid-encoded Faa1p to suppress the same
phenotype of the faa1
fat1
strain is
consistent with a functional interrelationship between Fat1p and Faa1p
in long-chain fatty acid import. However, multicopy suppression might
also result from alterations in intracellular metabolism and regulation
distinct from the coupled transport/activation process when fatty acid
import or fatty acyl-CoA synthetase activity is highly elevated by
comparison to activities contributed by a single copy of the native
gene. As detailed in these studies, we did not observe oleoyl-CoA
synthetase activities comparable with or exceeding wild-type levels for
either strain expressing FAT1 or FAA4 in high
copy. In the case of the faa1
faa4
strain (wild type for FAT1) transformed with YEpDB17
(FAT1), there was sufficient oleoyl-CoA synthetase activity
(albeit only ~30% wild-type), which appeared to drive the coupled
import/activation process. On the other hand in the faa1
fat1
strain transformed with YEpDB133 (FAA4),
there was detectable oleoyl-CoA synthetase activity (~10% wild
type), but this was not sufficient to overcome the block as a
consequence of a deletion in FAT1. By comparison, the
faa1
fat1
strain transformed with YEpDB02
(FAA1) had robust oleoyl-CoA synthetase activity (~121%
wild type), which was sufficient to overcome the block due to the
fat1
deletion. Therefore, we believe the suppression is
caused by overexpression of one of the partners in the import
process, Fat1p or Faa1p.
strain in yeast indicating that the yeast and the
mouse proteins are functionally equivalent. Collectively, these data
support the notion that the fatty acid import mechanism working through
Fat1p (or FATP) and fatty acyl-CoA synthetase is primarily through the
esterification of the fatty acid with CoA, which results in metabolic
trapping. Our working hypothesis is that Fat1p functions to increase
fatty acid binding to the membrane, which in turn potentiates diffusion across the membrane. The fatty acid is subsequently metabolically activated concomitant with abstraction from the membrane by the Faa1p-Fat1p complex thereby generating a concentration gradient, which
further drives the import process.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the members of the AMC FATTT laboratory for many fruitful discussions during the course of this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM56840 (to P. N. B. and C. C. D.) and by American Heart Association Grant 0151215T (to C. C. D.).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.
Supported by Predoctoral Fellowship Grant 0215328T from the New
York State Affiliate of the American Heart Association.
§ Supported by Postdoctoral Fellowship Grant 99020225T from the New York State Affiliate of the American Heart Association. Present address: Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense, M, DK-5230, Denmark.
¶ 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.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M210557200
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ABBREVIATIONS |
---|
The abbreviations used are: FAT, fatty acid translocase; FATP, fatty acid transport protein; PBS, phosphate-buffered saline; C1-BODIPY-C12, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid.
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REFERENCES |
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