From the
Four unlinked fatty acid activation ( FAA) genes
encoding acyl-CoA synthetases have been identified in Saccharomyces
cerevisiae and characterized by noting the phenotypes of isogenic
strains containing all possible combinations of faa null
alleles. None of these genes is required for vegetative growth when
acyl-CoA production by the fatty acid synthetase (Fas) complex is
active. When Fas is inhibited by cerulenin, exponentially growing cells
are not viable on media containing a fermentable carbon source unless
supplemented with fatty acids such as myristate, palmitate, or oleate.
The functionally interchangeable FAA1 and FAA4 genes
are responsible for activation of these imported fatty acids. Analysis
of lysates prepared from isogenic FAA1FAA4 and
faa1
Regulation of myristoyl-CoA pool size in Saccharomyces
cerevisiae has an important role in modulating the activity of
myristoyl-CoA:protein N-myristoyltransferase, an enzyme
essential for vegetative growth (Johnson et al., 1994a). There
are at least two metabolic pathways in S. cerevisiae that
produce myristoyl-CoA: de novo synthesis or activation of free
myristate by acyl-CoA synthetases. The de novo pathway uses
malonyl-CoA, produced by acetyl-CoA carboxylase ( Acc1p)
(Mishina et al., 1980), to generate long chain saturated
acyl-CoAs through the enzymatic activities contained within the
cytosolic
On-line formulae not verified for accuracy S. cerevisiae contains at least five genes encoding
acyl-CoA synthetases (or fatty acid
activation proteins). Four unlinked FAA genes have
been recovered to date (Duronio et al., 1992; Johnson et
al., 1994b, 1994c). Faa1p, Faa2p, and Faa3p have been expressed in
and purified from strains of Escherichia coli that do not
produce FadD, the only acyl-CoA synthetase synthesized by this
bacterium (Knoll and Gordon, 1993; Knoll et al., 1994). Faa1p
and Faa2p can accommodate C7-C18 saturated fatty acids as substrates
in vitro (optimum = C12:0-C16:0 for Faa1p and
C9:0-C13:0 for Faa2p). Faa3p prefers C16 and C18 fatty acids with a
cis-double bond at C9-C10 (Knoll et al.,
1994).
The functional contributions of the
Faas to lipid metabolism have been examined by comparing the growth
characteristics of isogenic strains with (i) NMT1 or a mutant
allele ( nmt1-181) that causes temperature-sensitive
growth arrest and myristic acid auxotrophy due to a reduction in the
enzyme's affinity for myristoyl-CoA, (ii) various combinations of
wild-type and null FAA alleles, and (iii) an active or
cerulenin-inactivated de novo pathway for acyl-CoA synthesis
(Johnson et al., 1994b, 1994c). The results indicate that if
Fas is active and NMT1 cells are grown on rich media
containing a fermentable carbon source ( e.g. dextrose), none
of the FAA genes are needed for vegetative growth. However,
when Fas is inactivated, maintenance of the viability of NMT1 cells requires that Faa1p, and to a lesser extent Faa4p, activate
imported fatty acids (Johnson et al., 1994c). Faa2p appears to
be unable to activate imported fatty acid substrates ( e.g. myristate or palmitate) but is involved in activation of
endogenous fatty acids. None of the four FAAs are exclusively
responsible for targeting imported fatty acids to perixosomal
S.
cerevisiae Fas produces acyl-CoAs, while mammalian FAS contains an
associated thioesterase known as TEI (Pazirandeh et al., 1989;
1991). The acyl-CoA specificity of TEI determines the nature of the
free fatty acids released from FAS. Palmitate is the dominant species
generated by TEI-mediated chain termination, while stearate and
myristate are minor products. These free fatty acids must be
subsequently activated by cellular acyl-CoA synthetases so that they
can be targeted to various lipid biosynthetic and degradative pathways.
The functional significance of mammalian acyl-CoA synthetases has been
assessed using triacsins. All four known triacsins (A-D) (Tanaka
et al., 1982; Omura et al., 1986) contain an
unbranched chain of 15 non-hydrogen atoms that includes several
trans-double bonds and a terminal triazenol group
(Fig. 1). Triacsins A and C are competitive inhibitors of the
oleoyl-CoA synthetase activity associated with rat liver microsomes
(IC
A single long chain human acyl-CoA synthetase cDNA
has been recovered to date (Abe et al., 1992). Two long chain
acyl-CoA synthetases have been identified in rat. Although limited
analysis of their acyl chain specificities revealed no significant
differences (Fujino and Yamamoto, 1992), two observations suggest that
they may have different physiologic functions. First, they
have distinct tissue distributions. Rat liver acyl-CoA synthetase
(RLACS) is expressed primarily in liver and adipose tissue (Suzuki
et al., 1990), while rat brain acyl-CoA synthetase is
expressed primarily in neural tissues (Fujino and Yamamoto, 1992).
Second, their responsiveness to perixosome proliferators is
different. Fenofibrate increases ACS mRNA levels 6-fold in rat liver
(Causeret et al., 1993), while ACS expression in heart and
brain appears to be unresponsive to this drug (Schoonjans et
al., 1993). Alignments of the primary structures of the four yeast
and three mammalian acyl-CoA synthetases indicate that human ACS has
greater similarity to rat liver ACS compared with rat brain ACS and
Faa2p has greater sequence similarity to the mammalian acyl-CoA
synthetases than to any of the other yeast Faas (Johnson et
al., 1994c).
The gene products involved in importing fatty
acids into S. cerevisiae are unknown. In E. coli, long chain fatty acid transport involves an integral outer
membrane protein, FadL, and is coupled to activation by FadD, which is
loosely associated with the cytoplasmic face of the bacteria's
inner membrane (Black, 1991; Black et al., 1992). A mouse,
integral membrane, long chain fatty acid transport protein and an
acyl-CoA synthetase have been isolated recently based on their ability
to augment uptake of long chain fatty acids into COS-7 cells (Schaffer
and Lodish, 1994). The acyl-CoA synthetase has 94% identity with RLACS
and a similar tissue-specific pattern of expression. Presumably, it
represents the mouse ortholog of RLACS. The finding that fatty acid
uptake can be increased by overexpression of either fatty acid
transport protein or mouse ACS suggests that both proteins play a role
in long chain fatty acid import; fatty acid transport protein may
facilitate passage of fatty acids across the plasma membrane, while
esterification of imported fatty acids with a polar CoA moiety may
prevent their efflux out of the cell (Schaffer and Lodish, 1994).
The large number of FAA genes present in S. cerevisiae raises the possiblity that a given mammalian cell lineage contains
multiple acyl-CoA synthetases. This may complicate any assignment of
function based solely on the effects of overexpressing a single ACS
gene product. The phenotypes produced in S. cerevisiae through
deletion of one or more FAA genes provides an opportunity for
describing the potential functions of mammalian acyl-CoA synthetases
based on their ability to complement faa null alleles.
Isogenic FAA1FAA4 and faa1
YPD media consists of 1%
yeast extract, 2% peptone, and 2% dextrose.
Supplemented minimal media without
uracil (SMM-URA) contains 0.67% yeast nitrogen base with
ammonium sulfate (Bio 101, Inc.), 2% dextrose, and 0.08% complete
supplement minus uracil (Bio 101, Inc.). YPD/agar plates, supplemented
with (i) 500 µM fatty acid (NuChek Prep) plus Brij-58
(Sigma; 1% w/v), and/or (ii) 25 µM cerulenin (CER, Sigma),
were prepared as described in Johnson et al. (1994b).
To
determine the growth characteristics of various strains on YPD,
YPD/CER, and YPD/CER/fatty acid plates, equivalent numbers of cells
were replica plated using a multiprong applicator (Replaclone, Skatron
Instruments). The applicator was dipped into a 1:4 dilution (in 1
M sorbitol) of each culture after the culture had achieved an
OD
Rat Liver Acyl-CoA Synthetase Is Functionally Analogous to FAA1
and FAA4
S. cerevisiae strains containing faa1 and faa4 null alleles are not viable at 24-37 °C in YPD media
containing 25 µM cerulenin, even when the media is
supplemented with 500 µM myristate, palmitate, or oleate
(Fig. 2 A plus data not shown). Growth of
faa1
The myristoyl-CoA synthetase activities of
faa1
Previous studies of Saccharomyces uvarum and
Saccharomycopsis lipolytica suggested a saturable mechanism
for fatty acid import (Kohlwein and Paltauf, 1983). Uptake of lauric
acid (C12:0) into S. lipolytica was not affected by
pretreatment with azide even though cellular levels of ATP were reduced
to 2% of untreated cells. These results were interpreted to mean that
fatty acid uptake occurs by an energy-independent, carrier-mediated
process and is not coupled to fatty acid activation (Kohlwein and
Paltauf, 1983).
The availability of S. cerevisiae strains
with wild-type and null FAA1 and FAA4 alleles allowed
us to define the effects of Faa1p, Faa4p, and RLACS on the uptake of
C14:0 and C16:0. PanelsA and B of
Fig. 5
show the time-dependent changes in cell-associated
radioactivity obtained when the FAA1,FAA4 strain containing
the GPD vector was incubated at 30 °C with 10 or 100
µM fatty acid. Uptake was linear for the first 60 s at
both concentrations for both fatty acids. Studies conducted at 4 and 30
°C with 100 µM fatty acid indicated that uptake of
C14:0 and C16:0 was temperature-dependent (Fig. 5 C and
data not shown). The concentration of C14:0 or C16:0 in the assay
mixture was varied from 5 to 100 µM, and uptake was
monitored at 30 °C after a 30-s incubation ( i.e. the
midpoint of the linear phase). The results (Fig. 5 D)
indicate the S. cerevisiae contains a saturable fatty acid
transport system with K
These findings, together with two
other observations described above ( i.e. (i) Faa1p and Faa4p
together account for 99% of the myristoyl-CoA and palmitoyl-CoA
synthetase activities in this yeast and (ii) faa1
The results of these genetic complementation studies suggest a number
of experimental systems that may be useful in the future.
First, the dependence of the faa1
We thank Tokuo Yamamoto (Tohoku University Gene
Research Center) for generously supplying the cDNA for rat liver
acyl-CoA synthetase and George Gokel for insights about the structural
similarities between triacsins and cerulenin.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
faa4
strains indicated that Faa1p and Faa4p
together account for 99% of total cellular myristoyl-CoA and
palmitoyl-CoA synthetase activities. Genetic complementation studies
revealed that rat liver acyl-CoA synthetase (RLACS) rescues the
viability of faa1
faa4
cells in media containing a
fermentable carbon source, myristate or palmitate, plus cerulenin.
Rescue is greater at 37 °C compared with 24 °C, paralleling the
temperature-dependent changes in RLACS activity in vitro as
well as the enzyme's ability to direct incorporation of tritiated
myristate and palmitate into cellular phospholipids in vivo.
Complementation by RLACS is blocked by treatment of cells with triacsin
C
(1-hydroxy-3-( E, E, E,2`,4`,7`-undecatrienylidine)triazene).
Even though Faa1p, Faa4p, and RLACS are all able to activate imported
myristate and palmitate in S. cerevisiae, the sensitivity of
Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests
that the rat liver enzyme is functionally more analogous to Faa4p than
to Faa1p. Finally, an assessment of myristate and palmitate import into
FAA1FAA4 and faa1
faa4
strains, with or
without episomes that direct overexpression of Faa1p, Faa4p or RLACS,
indicated that fatty acid uptake is not coupled to activation in S.
cerevisiae.
fatty acid synthetase
(Fas)
(
)
complex (Paltauf et al., 1992).
Palmitoyl-CoA and stearoyl-CoA are the principal products of Fas, while
myristoyl-CoA represents 3-5% of the total acyl-CoAs synthesized
(Hori et al., 1987). Cerulenin, a 12-carbon amide
((2 R,3 S)-2,3-epoxy-4-oxo-7,10- trans,trans-dodecanienamide;
see Fig. 1), is a specific inhibitor of Fas (Nomura et
al., 1972; Vance, 1972; Funabashi et al., 1989). When Fas
is inhibited with cerulenin, wild-type strains of S. cerevisiae require supplementation of media with fatty acids such as
myristate, palmitate, or oleate to remain viable. These fatty acids are
imported and then activated by cellular acyl-CoA synthetases (Duronio
et al., 1992; Johnson et al., 1994b, 1994c).
Figure 1:
Comparison of the structures
of cerulenin
((2 R,3 S)-2,3-epoxy-4-oxo-7,10- trans,trans-dodecanienamide),
triacsin C
(1-hydroxy-3-( E,E,E,2`,4`,7`-undecatrienylidine)triazene), and
myristic acid (tetradecanoic acid). The overall length of cerulenin is
equivalent to lauramide
(CH(CH
)
CO-NH
).
The trans- or E-double bonds present at the 7,8 and
10,11 positions of cerulenin are also present in triacsin C: i.e. they share a pentadiene,
-CH=CH-CH
-CH=CH-. The
alignment of cerulenin and triacsin C also reveals that polar groups
are present at the right hand terminus of both molecules: there are
three contiguous sp
or sp
-like (the epoxide
carbon) atoms in cerulenin while triacsin C has an
N-hydroxytriazene moiety essential for its inhibitory activity
(Tomoda et al., 1987).
The overall reaction catalyzed by an acyl-CoA synthetase is as
follows (Berg, 1956, 1958; Bar-Tana et al., 1973a, 1973b).
(
)
-oxidation pathways (Johnson et al., 1994c). Moreover, a
NMT1 strain with deletions of all four FAA genes is
still viable at 30 °C on media containing myristate or oleate as
the sole carbon source, providing evidence that S. cerevisiae contains one or more other FAAs, which direct fatty acids
to
-oxidation pathways (Johnson et al., 1994c).
= 18 and 9 µM, respectively)
(Tomoda et al., 1987). Proliferation of Raji cells in culture
is inhibited in a dose-dependent fashion by triacsins (order = C
> A > D > B). The degree of inhibition of growth by the
various triacsins can be correlated with their ability to inhibit Raji
cell membrane-associated long chain acyl-CoA synthetase activity in
vitro and by their capacity to inhibit incorporation of oleate
into cellular phospholipids and triacylglycerol (Tomoda et
al., 1991).
faa4
strains also
provide an opportunity to explore the general role of acyl-CoA
synthetases in mediating fatty acid import. We have explored these
issues in this report.
Strains and Media
The isogenic S. cerevisiae strains YB332 ( MATa NMT1 ura3 his3200 ade2 lys2-801
leu2 FAA1 FAA2 FAA3 FAA4) and YB525 ( MATa NMT1 ura3
his3
200 ade2 lys2-801 leu2 faa1
::1.9::HIS3 FAA2 FAA3
faa4
0.3::LYS2) are described in Johnson et al. (1994b and 1994c, respectively).
of 1. Duplicate plates were then incubated at
24-37 °C for 3-4 days. All experiments were repeated at
least 4 times.
Construction of a Plasmid for Expressing Rat Liver
Acyl-CoA Synthetase in S. cerevisiae
pBB358 is a low copy
YCp-based plasmid containing the glyceraldehyde-3-phosphate
dehydrogenase ( GPD) promoter (Johnson et al., 1994c).
pBB360 ( GPD-RLACS) was produced by subcloning a 2.3-kb
EcoRI- PstI fragment from pRACS15 (Suzuki et
al., 1990) into EcoRI- PstI-digested pBB358.
Metabolic Labeling of Cellular Lipids
Strain YB332
( FAA1FAA4), transformed with pBB358 (YCp- GPD expression vector without insert), and strain YB525
( faa1faa4
) transformed with plasmids pBB358, pBB330
( GPD-FAA1, Johnson et al., 1994a;b), pBB365
( GPD-FAA4, Johnson et al. (1994c)), or pBB360
( GPD-RLACS), were grown in SMM-URA medium at 24 or 37 °C
to an OD
0.8. Cultures were transferred to tubes
containing either [9,10-
H]myristate, or
[9,10-
H]palmitate (specific activity = 16
Ci/mmol, 100 µCi/ml culture) and shaken for 1 h at 24 or 37 °C.
Cellular lipids were extracted according to Bligh and Dyer (1959),
except that 425-600-µm glass beads (Sigma) were added in the
initial extraction step. Lipids were resuspended in chloroform/methanol
(1:1). Aliquots of 100,000 dpm from each strain were spotted onto
silica gel 60 high performance thin layer chromatography (HPTLC) plates
(Merck), and separated in a single dimension using methyl acetate,
isopropyl alcohol, chloroform, methanol, and 0.25% KCl (25:25:28:10:7).
Lipid standards (Sigma) were visualized using iodine vapors.
Radiolabeled lipids were detected by spraying the plates with
EN
HANCE and performing fluorography at -80 °C.
Measurement of Cellular Acyl-CoA Synthetase Activity in
Vitro
The myristoyl-CoA and palmitoyl-CoA synthetase activities
of cellular lysates from the strains described above were evaluated in
an in vitro assay system. Twenty-ml cultures were grown in
SMM-URA medium to an OD = 1. Cells were harvested
by centrifugation and lysed by vortexing with glass beads in 500 µl
of yeast lysis buffer (200 mM Tris, pH 8, 4 mM EDTA,
4 µM pepstatin A, 8 µM leupeptin, 1
mM Pefabloc (Boehringer Mannheim), 4 mM benzamidine,
5 mM
-mercaptoethanol, 10% glycerol, and 0.1% Brij 35).
The 100-µl acyl-CoA synthetase assays contained 150 mM
MOPS, pH 7.1, 2 mM CoA, 2 mM ATP, 10 mM
MgCl
, 1 mM dithiothreitol, 0.05% Triton X-100, 100
µM EGTA, 50 µM either
[9,10-
H]myristate or
[9,10-
H]palmitate (1 Ci/mmol), and cell lysates
(concentration, 10-1000 µg of total protein/ml). Following a
15-min incubation at 24 or 37 °C, the reactions were quenched with
an equal volume of 5% trichloroacetic acid (prepared in methanol).
[
H]Fatty acyl-CoA and
H-labeled fatty
acids were resolved by C4 reverse phase HPLC using an isocratic
gradiate of 60% 20 mM potassium phosphate buffer, pH 5.5, and
40% acetonitrile (Knoll et al., 1994). Tritiated products were
quantitated using an in-line scintillation counter (model CR;
Radiomatic Instruments). Each assay was performed in duplicate and all
experiments were repeated at least 3 times.
The Effects of Triacsin C and Cerulenin on Acyl-CoA
Synthetase Activities in Vitro
The abilities of triacsin C
(Kameda Co.) and cerulenin (Sigma) to inhibit acyl-CoA synthetase
activities were evaluated using the in vitro assay system
described above. Cellular lysates were prepared from strain YB525
( faa1faa4
) transformed with pBB330
( GPD-FAA1), pBB365 ( GPD-FAA4), or pBB360
( GPD-RLACS). Faa1p and Faa2p, each with a carboxyl-terminal
tag of 6 histidine residues, were expressed in a
fadD
strain of E. coli (LS6928
fadR
fadD
27-zea::Tn 10)
(Nunn et al., 1986) and purified to apparent homogeneity using
Ni-nitrilotriacetic acid affinity chromatography (Knoll et
al., 1994). The 100-µl acyl-CoA synthetase assays contained
cell lysate (concentration, 10-1000 µg of protein/ml), or
purified Faa1p-6xHis or Faa2p-6xHis (1 µg/ml),
[
H]myristate (50 µM), plus either
1-100 µM triacsin C (reference control = equivalent concentration of dimethyl sulfoxide (Me
SO)) or
100 µM cerulenin (reference control = equivalent
amount of ethanol). After a 15-min incubation at 37 °C, the
reactions were quenched with an equal volume of 5% trichloroacetic
acid/methanol, and products and reactants were resolved by C4 reverse
phase high performance liquid chromatography. Each assay was performed
in duplicate, and all experiments were repeated 3 times.
The Effect of Triacsin C on the Growth of S. cerevisiae
Strains Containing Mammalian Acyl-CoA Synthetases
Strain YB332
( FAA1FAA4), transformed with the expression vector without
insert, and strain YB525 ( faa1faa4
), transformed
with the vector minus insert, GPD-FAA1, GPD-FAA4, or
GPD-RLACS episomes, were grown at 30 °C in SMM-URA to an
OD
1. Aliquots (200 µl) were withdrawn from each
culture and then added to 4 ml of SMM-URA media containing 0, 1, or 10
µM triacsin C (prepared in Me
SO) or
Me
SO alone, with or without cerulenin (25 µM).
Growth rates at 37 °C were monitored every 2 h over the next 12 h
by measuring optical density at 550 nm with a Thermo-Max reader
(Molecular Devices). Two independent experiments were performed, each
in duplicate.
Assay for Fatty Acid Uptake
This assay was adapted
from protocols described by Trigatti et al. (1992) for the
uptake of oleate in Candida tropicalis and Lai and McGraw
(1994) for the uptake of inositol in S. cerevisiae. Uptake was
compared in the wild-type strain containing the parental vector and in
the isogenic faa1faa4
strain transformed with
pBB358, pBB330 ( GPD-FAA1), pBB365 ( GPD-FAA4), and
pBB360 ( GPD-RLACS). Cells were grown in SMM-URA media at 30
°C to an OD
between 0.5 and 1.0, harvested by
centrifugation, and washed once with phosphate-buffered saline, pH 7.4.
Cells were resuspended in phosphate-buffered saline to an OD
= 10, a 200-µl aliquot of the suspension was withdrawn
(
10
cells) and added to glass test tubes, and the
mixture was prewarmed at 30 °C or chilled to 4 °C for 10 min.
Assays were initiated by adding [9,10-
H]myristate
or [9,10-
H]palmitate (dissolved in absolute
ethanol; specific activity = 50 mCi/mmol). The final
concentration of fatty acid ranged from 5 to 100 µM. The
final concentration of ethanol was 0.5% in all reactions. The mixtures
were incubated for 0, 30, 60, 120, 300, or 600 s at 4 °C or 30
°C, 10 ml of ice-cold phosphate-buffered saline were added, and
cells were collected by passing the suspension through a GF/A glass
fiber filter (Whatman). The filters were washed 3 times with 10 ml of
ice-cold phosphate-buffered saline, placed in scintillant (Econo-Safe,
RPI), and counted. Each time point was assayed in duplicate.
Experiments were repeated 3 times.
faa4
cells can be restored by introducing an
episome containing FAA1 or FAA4 under the control of
the strong GPD promoter (Fig. 2 A). A
GPD-rat liver acyl-CoA synthetase episome was also able to
sustain growth of faa1
faa4
cells on YPD/fatty acid
plates when Fas was inhibited by cerulenin (Fig. 2 A).
Rescue on YPD/CER/myristate or YPD/CER/palmitate was better at 37
°C than at 24 °C (Fig. 2 A), consistent with the
expected temperature optimum of a mammalian enzyme. Complementation by
RLACS was also evident when cells were grown in liquid selective media
(SMM-URA) containing glucose, myristate, plus cerulenin
(Fig. 2 B).
Figure 2:
Comparison of the growth rate of a
faa1faa4
strain with and without
GPD-acyl-CoA synthetase episomes. PanelA,
an equal number of cells from YB332 ( FAA1FAA4), transformed
with the parental YCp- GPD vector (pBB358), or YB525
( faa1
faa4
), transformed with either the parental
vector, GPD-FAA1 (pBB330), GPD-FAA4 (pBB365), or
GPD-RLACS (pBB360) were plated onto YPD media supplemented
with 25 µM CER, with or without 500 µM
myristate ( MYR) or palmitate ( PALM). Plates were
incubated for 3 days at 24, 30, or 37 °C. Panel B,
analysis of the ability of the GPD-acyl-CoA synthetase
episomes to rescue growth of faa
1faa4
cells in
SMM-URA media supplemented with 500 µM myristate and 25
µM cerulenin. Cells were innoculated, during the mid-log
phase of their growth at 30 °C, into SMM-URA media or into
SMM-URA/MYR/CER and incubated for 12 h at 37 °C. Aliquots were
withdrawn at various times after innoculation, and the optical density
was determined at 550 nm. The mean ± 1 S.D. is plotted at each
time point.
The apparent ability of RLACS to activate
imported fatty acids in S. cerevisiae is consistent with the
fact that the mammalian cell lineages, which produce this acyl-CoA
synthetase (hepatocytes and adipocytes) are normally involved in
directing plasma-derived fatty acids to their intracellular lipid
metabolic pathways. The ability of Faa1p, Faa4p, and RLACS to activate
and direct imported fatty acids to phospholipid biosynthetic pathways
was compared directly by analyzing cellular lipids from isogenic
FAA1FAA4 or faa1faa4
strains containing
GPD-FAA1, GPD-FAA4, or GPD-RLACS episomes. Total
cellular lipids were isolated and then fractionated by single dimension
HPTLC. The wild-type strain containing Faa1p and Faa4p incorporates
label from tritiated myristate or palmitate into
phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and
phosphatidylcholine when incubated at 24 or 37 °C. This is not the
case with the faa1
faa4
strain (compare lanes1 and 2 and lanes6 and 7 in Fig. 3, A and B). The failure of the
faa1
faa4
strain to activate imported fatty acids for
phospholipid biosynthesis is associated with a large increase in
cellular free fatty acids. Transformation of the faa1
faa4
strain with either GPD-FAA1 or GPD-FAA4 restores
phospholipid labeling with [
H]myristate and
[
H]palmitate to a level identical to that
observed in the wild-type FAA1FAA4 strain ( lanes3 and 4 and lanes8 and 9 of Fig. 3 , A and B). RLACS is also able to
direct incorporation of [
H]myristate into
phospholipids, but the host strain still has a large pool of free fatty
acids ( lanes5 and 10 in panelsA and B). Labeling of phospholipids with
tritiated palmitate is greater with RLACS at 37 °C
(Fig. 3 B) compared with 24 °C
(Fig. 3 A). At both temperatures, incorporation of label
from [
H]palmitate into phospholipid species is
lower than that observed with myristate (Fig. 3). These
differences in incorporation cannot be ascribed to differences in the
efficiency of import of myristate and palmitate since the extent of
phospholipid labeling is similar when an isogenic control, containing
wild-type FAA1 and FAA4 alleles and the GPD vector without insert, is incubated with either tritiated fatty
acid (compare lanes1 and 6 in Fig. 3,
A and B).
Figure 3:
Metabolic labeling of cellular lipids
produced in S. cerevisiae strains containing
GPD-acyl-CoA synthetase episomes. Strain YB332
( FAA1FAA4), transformed with the parental YCp- GPD vector ( lanes1 and 6) and strain YB525
( faa1faa4
), transformed with the the parental vector
( lanes2 and 7), pBB330 ( GPD-FAA1;
lanes3 and 8), pBB365 ( GPD-FAA4;
lanes4 and 9), or pBB360
( GPD-RLACS; lanes5 and 10), were
grown at 24 °C ( panelA) or 37 °C ( panelB) and labeled with either
[
H]myristate (C14:0; lanes1- 5) or [
H]palmitate
(C16:0, lanes6- 10). (the specific
activities of the tritiated fatty acids were identical). Cellular
lipids (100,000 dpm/lane) were separated by HPTLC in a single
dimension, and the plates were subjected to autoradiography for 15 h.
The position of migration of lipid standards are shown: NL,
neutral lipids; FA, free fatty acids; PE,
phosphatidylethanolamine; PI, phosphatidylinositol;
PS, phosphatidylserine; PC,
phosphatidylcholine.
The results of these metabolic labeling
studies can be correlated with the different growth phenotypes of
faa1faa4
( GPD- episome) cells on
YPD/CER/MYR or YPD/CER/PALM plates at 24 and 37 °C
(Fig. 2 A) and with the myristoyl-CoA and palmitoyl-CoA
synthetase activities measured in lysates prepared from
faa1
faa4
cells containing GPD-FAA1, GPD-FAA4, or GPD-RLACS (). Lysates prepared from
FAA1FAA4 ( GPD-vector) cells during the mid-log phase
of their growth had 70-230-fold higher acyl-CoA synthetase
activity compared with faa1
faa4
lysates when assayed
at 24 and 37 °C using myristate or palmitate as substrates (
and data not shown). Since Faa1p and Faa4p together
represent 99% of cellular myristoyl-CoA and palmitoyl-CoA synthetase
activities in the wild-type strain during exponential growth in
glucose-rich media, the low background of endogenous acyl-CoA
synthetase activity in faa1
faa4
cells provides a
convenient assay system for qualitative and quantitative assessment of
yeast or mammalian acyl-CoA synthetases produced from
GPD-episomes. The myristoyl-CoA and palmitoyl-CoA synthetase
activities produced by GPD-FAA4 in faa1
faa4
cells are 9-18-fold lower than those obtained with
GPD-FAA1. Nonetheless, they are still 40-130-fold
greater than in the faa1
faa4
strain containing the
parental GPD-vector. GPD-RLACS was less effective
than GPD-FAA4 in increasing cellular myristoyl-CoA and
palmitoyl-CoA synthetase activities; values were 3-21-fold higher
than the background levels of faa1
faa4
( GPD-vector) lysates (). While the
myrisotyl-CoA and palmitoyl-CoA synthetase activities of
faa1
faa4
( GPD-RLACS) lysates are
6-8-fold greater when assayed at 37 °C compared with 24
°C, a much more modest temperature effect (2-fold) was evident with
faa1
faa4
( GPD-FAA1) and
faa1
faa4
( GPD-FAA4) lysates
().
(
)
In Vitro and in Vivo Sensitivity to Triascin C Provides
Evidence for the Functional Similarities of Faa4p and RLACS
In Vitro Studies
Triacsin C is a potent competitive
inhibitor of purified Faa2p with a Kof
15 nM using oleic acid (C18:1
) as a substrate
(Faa2p's K
for oleate = 25
µM). In contrast, the oleoyl-CoA synthetase activities of
purified Faa1p and Faa3p are unaffected by triacsin C in vitro (IC
> 500 µM).
(
)
faa4
( GPD-FAA4) and
faa1
faa4
( GPD-RLACS) lysates at 37 °C
were also strongly inhibited by triacsin C (IC
=
4.5 and 6 µM, respectively; ). The
myristoyl-CoA synthetase activity in a control faa1
faa4
( GPD-FAA1) lysate was not reduced by 100 µM
triacsin C ().
In Vivo Studies
The growth rate of the
faa1faa4
strain, with or without the GPD-FAA1,
GPD-FAA4, GPD-RLACS, or YCp- GPD episomes, is
similar to that of the isogenic FAA1FAA4 strain at 37 °C
in SMM-URA media containing dextrose as the fermentable carbon source,
500 µM myristate, but no cerulenin
(Fig. 4 A). Thus, when Fas is active, there are no
obvious growth differences between the two strains in liquid culture,
nor do the episomes produce a deleterious effect. We next defined
whether triacsin C could block the rescue by GPD-RLACS,
GPD-FAA4, or GPD-FAA1 of faa1
faa4
cells at 37 °C in media supplemented with 500 µM
myristate and 25 µM cerulenin. Both GPD-FAA1 and GPD-FAA4 produce complete rescue of the
faa1
faa4
strain throughout the course of a 12-h
incubation in SMM-URA/MYR/CER. GPD-RLACS produces partial
rescue that is first evident at the 6-h time point. The growth
advantage (compared with cells containing YCp- GPD vector
alone) becomes progressively more obvious during the next 6 h
(Fig. 2 B). This finding provides an indication of the
minimum time required to determine if triacsin C affects the activity
of RLACS in vivo. Addition of 1 or 10 µM triacsin
C had no effect on the growth kinetics of the wild-type FAA1FAA4 strain in SMM-URA/MYR/CER (Fig. 4 B). As expected,
faa1
faa4
cells containing the YCp- GPD vector minus insert failed to grow under these conditions
(Fig. 4 B). Introduction of GPD-FAA1 fully
rescued growth in SMM-URA/MYR/CER at 37 °C even in the presence of
10 µM triacsin C (Fig. 4 C). This in
vivo result is consistent with the resistance of Faa1p to triacsin
C inhibition in vitro (see above). In contrast, triacsin C
produces a dose-dependent reduction in growth of faa1
faa4
cells containing GPD-FAA4 (Fig. 4 D) or
GPD-RLACS (Fig. 4 E).
Figure 4:
Effect of triacsin C on the growth of a
S. cerevisiae faa1faa4
strain containing various
GPD-acyl-CoA synthetase episomes. PanelA,
evidence that the episomes have no deleterious effect on the growth of
a faa1
faa4
strain at 37 °C in SMM-URA media
supplemented with 500 µM myristate. Note that cellular Fas
activity has not been inhibited by cerulenin in this experiment.
Panel B, triacsin C at concentrations up to 10 µM
has no effect on growth of a wild-type strain of S. cerevisiae at 37 °C in SMM-URA media containing 500 µM
myristate and 25 µM cerulenin. In contrast, the isogenic
faa1
faa4
strain fails to grow under these
conditions. Note that in panelsA and B,
growth is monitored as a function of time after innoculation into
SMM-URA/CER/MYR. (The innoculum consisted of cells harvested during the
mid-log phase of their growth at 30 °C in SMM-URA media minus MYR
or CER). Panels C-E, comparison of the ability
YCp- GPD episomes encoding Faa1p ( GPD-FAA1; panelC) , Faa4p, ( GPD-FAA4; panelD), or rat liver acyl-CoA synthetase ( GPD-RLACS;
panelE) to rescue growth of a faa1
faa4
strain at 37 °C in SMM-URA supplemented with 500
µM myristate, 25 µM cerulenin, triacsin C (1
and 10 µM), or an equivalent amount of the dimethyl
sulfoxide solvent used to prepare stock solutions of the triacsin (0
µM). Growth is plotted as a function of time after
innoculation of exponentially growing cells into the culture media. The
mean value ± 1 S.D. is shown.
These results establish
that Faa4p and rat liver acyl-CoA synthetase are both inhibited by
triacsin C in vivo. A corollary to this conclusion is that
triacsin C is able to enter S. cerevisiae during exponential
growth and gain access to both of these enzymes. Our data also suggest
that RLACS is functionally more analogous to Faa4p than to Faa1p (at
least based on their ability to activate imported fatty acids and to be inhibited by triacsin C in vivo). This
apparent functional similarity is particularly intriguing given the
fact that Faa1p and Faa4p have similar degrees of sequence identity to
RLACS (23%, cf. Johnson et al. (1994c)). Analysis of Fatty Acid Uptake in FAA Deletion Strains
= 55 and 46 µM for C14:0 and C16:0, respectively. These calculated
K
values are similar to those reported
for oleate in C. tropicalis (56 µM; Trigatti
et al., 1992).
Figure 5:
Fatty acid uptake in isogenic FAA1FAA4 and faa1faa4
strains. PanelsA and B, time course of uptake of myristate
(A) or palmitate ( B) into FAA1FAA4 cells containing the parental YCp- GPD vector. The y axis on the left side measures cell-associated
radioactivity when the concentration of fatty acid in the assay was 100
µM. The y axis on the right side of
these two panels shows the values obtained when [fatty acid]
= 10 µM. Panel C, uptake of myristate into
FAA1FAA4 cells containing YCp- GPD at 4 and 30 °C.
Panel D, fatty acid uptake into FAA1FAA4-YCp- GPD cells after a 30-s incubation at 30 °C in the presence of 5,
10, 20, 50, or 100 µM C14:0 or C16:0. Panels
E-G, uptake of C14:0 or C16:0 into (i) the FAA1FAA4 strain transformed with the parental YCp- GPD vector and
(ii) the isogenic faa1
faa4
strain transformed with
the parental vector, GPD-FAA1, GPD-FAA4, or
GPD-RLACS. The fatty acid and its final concentration are
described in each panel. The mean ± 1 S.E. is plotted at each
time point for each strain. Note that introduction of GPD-FAA1, but not GPD-FAA4 or GPD-RLACS, reduces import of
C16:0 2-fold. No effect is noted on C14:0 import. The significance of
the GPD-FAA1 effect remains unclear although it is
reproducible ( n = 3 experiments, each done in
duplicate).
Isogenic FAA1FAA4 and
faa1faa4
strains containing YCp- GPD vector,
GPD-FAA1, GPD-FAA4, or GPD-RLACS, were
incubated with C14:0 or C16:0 at 30 °C. Uptake was assayed at two
concentrations of fatty acid: 10 µM (subsaturating) and
100 µM (saturating; defined according to
Fig. 5D). We used two concentrations because we were
concerned that at high levels of fatty acid, the rate of transport may
exceed the catalytic capacity of the acyl-CoA synthetases and therefore
the effects of the latter on transport could be negligible. Conversely,
at lower concentrations, metabolism of the fatty acids may function as
kinetic trap that displaces the transporter from equilibrium and
thereby effectively couples the rate of transport to acyl-CoA
formation. The isogenic FAA1FAA4 (YCp- GPD) and
faa1
faa4
(YCp- GPD) strains exhibit no
significant differences in their ability to transport myristate and
palmitate when fatty acid is present at either 10 or 100
µM (Fig. 5, D- G and data not
shown). Moreover, addition of GPD-FAA1, GPD-FAA4, or
GPD-RLACS to the faa1
faa4
strain produces
no increase in the import of either fatty acid (Fig. 5,
D- G).
faa4
(YCp- GPD) cells accumulate free C14:0 and C16:0
( cf. lanes2 and 8, in
Fig. 3
, A and B)) suggest that import and
activation of myristate and palmitate are not coupled processes
in S. cerevisiae. Prospectus
faa4
strain on rat liver acyl-CoA synthetase for growth in YPD/CER/MYR
or SMM-URA/CER/MYR media represents a biological assay for identifying
inhibitors of this mammalian enzyme. Such inhibitors may have
therapeutic uses. Second, the faa1
faa4
strain and these growth conditions could also be used to isolate
cDNAs encoding acyl-CoA synthetases that are responsible for activating
imported fatty acids in other species ( e.g. human, various
human pathogens). Selective or nonselective inhibitors of these enzymes
could then be identified with the biological assay. Finally,
the functions of other types of mammalian acyl-CoA synthetases as well
as their interactions with fatty acid transport proteins may be more
readily definable, in exponentially growing or stationary phase
(Werner-Washburne et al., 1993) S. cerevisiae than in
their cells of origin. This belief is based on ease of genetic
manipulation of S. cerevisiae plus the availability of
isogenic strains with various combinations of faa
alleles
and well-characterized mutations that affect enzymes and/or organelles
that participate in lipid metabolism ( e.g. peroxisomal
assembly ( PAS) mutants; Erdmann et al., 1989; 1991).
desaturase, accounts for all de novo production of
monoenoic fatty acids from saturated acyl-CoA substrates in S.
cerevisiae (Bossie and Martin, 1989; Stukey et al., 1989,
1990; McDonough et al., 1992).
faa4
strains containing
GPD-FAA4, GPD-FAA1 or GPD-RLACS had no
significant effect on their myristoyl-CoA and palmitoyl-CoA synthetase
activities, providing further evidence that cerulenin's
inhibitory effect on acyl-CoA synthesis occurs via its effects on Fas
rather than on Faa1p, Faa4p, or RLACS.
was chosen because it is a substrate for
purified Faa1p, Faa2p and Faa3p (Knoll et al.,
1994a).)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.