Peroxisomal Degradation of trans-Unsaturated Fatty
Acids in the Yeast Saccharomyces cerevisiae*
Aner
Gurvitz
,
Barbara
Hamilton,
Helmut
Ruis, and
Andreas
Hartig
From the Institut für Biochemie und Molekulare Zellbiologie
der Universität Wien and Ludwig Boltzmann-Forschungsstelle
für Biochemie, Vienna Biocenter, Dr Bohrgasse
9, A-1030 Vienna, Austria
Received for publication, April 18, 2000, and in revised form, October 4, 2000
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ABSTRACT |
Degradation of trans-unsaturated
fatty acids was studied in the yeast Saccharomyces
cerevisiae. Propagation of yeast cells on trans-9
elaidic acid medium resulted in transcriptional up-regulation of the
SPS19 gene, whose promoter contains an oleate response element. This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent
peroxisomes. Utilization of trans fatty acids as a single
carbon and energy source was evaluated by monitoring the formation of
clear zones around cell growth on turbid media containing fatty acids
dispersed with Tween 80. For metabolizing odd-numbered
trans double bonds, cells required the
-oxidation
auxiliary enzyme
3-
2-enoyl-CoA isomerase
Eci1p. Metabolism of the corresponding even-numbered double bonds
proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and
Dci1p (
3,5-
2,4-dienoyl-CoA isomerase).
trans-2,trans-4-Dienoyl-CoAs could enter
-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and
D-specific 3-hydroxyacyl-CoA dehydrogenase) without the
involvement of Sps19p, whereas
trans-2,cis-4-dienoyl-CoAs could not. This
reductase-independent metabolism of
trans-2,trans-4-dienoyl-CoAs resembled the
situation postulated for mammalian mitochondria in which oleic acid is
degraded through a di-isomerase-dependent pathway. In
this hypothetical process,
trans-2,trans-4-dienoyl-CoA metabolites are
generated by
3-
2-enoyl-CoA
isomerase and
3,5-
2,4-dienoyl-CoA
isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of
2,4-dienoyl-CoA reductase. Growth of a yeast fox2sps19
mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on
trans-9,trans-12 linolelaidic acid medium gave
credence to this theory. We propose an amendment to the current scheme
of the carbon flux through
-oxidation taking into account the
dispensability of
-oxidation auxiliary enzymes for metabolizing
trans double bonds at even-numbered positions.
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INTRODUCTION |
Double bonds in naturally occurring unsaturated fatty acids are
mostly in the cis configuration. However, certain foods such as beef, milk, and margarine, also contain fatty acids in the trans configuration due to their enzymatic synthesis in
ruminants by gastrointestinal microorganisms or their chemical
synthesis during partial hydrogenation (hardening) of fats and
vegetable oils (1). Diets high in trans-unsaturated fatty
acids have previously been shown in healthy humans to lead to increased
levels of serum lipoproteins of the type that can increase the risk of coronary heart disease (2). For this reason, it is important to study
the metabolism of trans fatty acids.
Previous work using rat hepatocytes demonstrated that
trans-unsaturated fatty acids are oxidized preferentially in
peroxisomes (3). Peroxisomes are single membrane-limited organelles
that occur in all eukaryotic cells examined. In the yeast
Saccharomyces cerevisiae peroxisomes represent the sole site
for fatty acid
-oxidation (4). The yeast
-oxidation process is
catalyzed by the enzymes acyl-CoA oxidase (Pox1p), 2-enoyl-CoA
hydratase 2, D-specific 3-hydroxyacyl-CoA dehydrogenase
(Fox2p; multi-functional enzyme type 2;
MFE1 type 2), and
3-ketoacyl-CoA thiolase (Pot1p/Fox3p) as shown in Scheme
1A (5-8). To break down
odd-numbered cis double bonds in unsaturated fatty acids
such as oleic acid (cis-C18:1(9)), yeast
cells rely on peroxisomal Eci1p (9, 10) representing the auxiliary
enzyme
3-
2-enoyl-CoA isomerase
(3,2-isomerase; Scheme 1B). Eci1p is also crucial for
metabolizing even-numbered cis double bonds in fatty acids
such as petroselinic acid (cis-C18:1(6)) in a
process that additionally requires Sps19p (11), which corresponds to
the auxiliary enzyme 2,4-dienoyl-CoA reductase (2,4-reductase;
Scheme 1C).

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Scheme 1.
The carbon flux through
-oxidation. A, the -oxidation spiral for
breaking down saturated fatty acids. The yeast enzymes acyl-CoA oxidase
(Pox1p), 2-enoyl-CoA hydratase 2 and D-specific
3-hydroxyacyl-CoA dehydrogenase (Fox2p), and 3-ketoacyl-CoA thiolase
(Pot1p/Fox3p) are indicated. Enzymes are noted to the left of the
dashed arrows, and metabolites are written above them
in italics. B, the position of the auxiliary
enzyme 3,2-isomerase (Eci1p) in the degradation pathway of unsaturated
fatty acids with cis double bonds at odd-numbered positions.
The original double bond in the carbon skeleton is underlined.
C, 2,4-reductase (Sps19p) and 3,2-isomerase (Eci1p) are required
to reposition the double bonds in the
trans-2,cis-4-dienoyl-CoA metabolite formed in
the breakdown process of unsaturated fatty acids with cis
double bonds at even-numbered positions before further metabolism by
2-enoyl-CoA hydratase 2 (Fox2p).
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In the present work S. cerevisiae was examined to determine
whether it could serve as a model system for studying the degradation of trans-unsaturated fatty acids. The experimental
advantages of using yeast for this study include the availability of
mutant cells blocked at various steps in the
-oxidation process and the possibility of replacing the requisite genes affected in these mutants using the corresponding rat cDNAs. Yeast cells were
examined for growth on trans-unsaturated fatty acids, and
the requirement for
-oxidation auxiliary enzymes for
breaking down trans double bonds was determined. In
addition, the effect of expressing rat monofunctional ECI and
peroxisomal MFE type 1 was monitored in yeast mutants lacking Eci1p or
both Fox2p and Sps19p that were grown on trans-unsaturated
fatty acids.
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EXPERIMENTAL PROCEDURES |
Strains--
Escherichia coli strain DH10B was used
for all plasmid amplifications and isolations. The S. cerevisiae strains used here were
derived from BJ1991 (12), NKY857, or
UTL-7Afox2 and are listed in Table I. Construction of
strains BJ1991eci1
(9), NKY857eci1
(9),
yAG760 (9), yAG856 (9), yAG826 (9), yAG827 (9),
BJ1991sps19
(11), yAG257 (11), yAG259 (11), yAG456 (11),
BJ1991dci1
(13), BJ1991pex6
(14), yAG485 (14), yAG854 (15), and BJ1991pip2
oaf1
(16)
has been described. Strain BJ1991dci1
sps19
(yAG935) was constructed by disrupting the SPS19 locus in
the BJ1991dci1
strain after transformation with an
sps19
::LEU2 fragment generated by
digesting plasmid pAG129 (11) with ScaI and StuI.
Yeast colonies that had been rendered protrotrophic for leucine were
examined for utilization of oleic acid
(cis-C18:1(9)) and petroselinic acid
(cis-C18:1(6)). Strain yAG935 specifically
failed to utilize petroselinic acid, and Southern analysis of
HindIII-digested genomic DNA probed using a 1.4-kilobase SphI-XbaI SPS18/19 fragment from
pAG454 (11) verified the incorporation of the
sps19
::LEU2 disruption fragment at
the SPS19 locus. The UTL-7Afox2sps19
strain
(yAG1181) was constructed by disrupting the SPS19 locus in
strain UTL-7Afox2 (6) as described above. Mutant
UTL-7Afox2 cells expressing rat peroxisomal MFE type 1 (yAG1206) or yeast Fox2p (yAG1207) were generated by transformation with the plasmids pYE352-rMFE (17) or
pYE352::ScMFE-2 (18), respectively, emulating
published strains. UTL-7Afox2sps19
double mutants
expressing either native Fox2p or rat peroxisomal MFE type 1 were
generated by transforming the strain yAG1181 with the aforementioned
plasmids pYE352::ScMFE-2 or pYE352-rMFE,
respectively, to generate strains yAG1204 and yAG1203. For fluorescence
microscopy, strain BJ1991 and the otherwise isogenic strain
BJ1991pex6
expressing GFP-Eci1p from plasmid
pADH2-GFP-ECI1 (9) were used (strains yAG872 and yAG873,
respectively).
Plasmids, Transformations, Media, Growth Conditions, and General
Methods--
Plasmids and fatty acids used are listed in Tables I and
II, respectively. Transformations of
yeast strains (19) and general nucleic acid manipulations (20) were
done according to standard protocols. Stock liquid elaidic acid
(trans-C18:1(9)) medium consisted of 0.67%
(w/v) yeast nitrogen base with amino acids added, 0.1% (w/v) yeast
extract, 0.5% (w/v) potassium phosphate buffer at pH 6.0, 0.5% (w/v)
Tween 80, and 0.125% (w/v) elaidic acid. Stock Tween 80 medium was
prepared the same way but without fatty acids. For induction of cells,
media were mixed with an equal volume of YP medium consisting of 1%
(w/v) yeast extract and 2% (w/v) meat peptone to which 0.05% (w/v)
D-glucose was added, and cells were grown as described
(11). Liquid oleic acid or ethanol media consisted of YP to which 0.2%
(w/v) oleic acid and 0.02% (w/v) Tween 80 adjusted to pH 7.0 with NaOH
or 2% (v/v) ethanol were added. Plates used to assess utilization of
fatty acids were prepared as described (11) and contained either
0.125% (w/v) oleic acid (cis-C18:1(9)), 0.125%
(w/v) oleic acid and 2% (v/v) ethanol, 0.125% (w/v) linoleic acid
(cis,cis-C18:2(9,12)), or
linolelaidic acid
(trans,trans-C18:2(9,12)) and 0.5%
(w/v) Tween 80. For assays using culture drops, cells were grown in
rich-glucose medium (YP and 2% w/v D-glucose) overnight to
late log phase or in liquid elaidic acid medium, serially diluted, and
applied to solid media. Preparation of protein extracts and measurement
of
-galactosidase activity were described previously (21).
Electrophoretic mobility shift assays were performed with crude protein
extracts as described (21). The FOX3 oleic acid-response
element (ORE) was excised from plasmid pSKFOX3ORE (22) after digestion
with XhoI and EcoRI and 2.5% (w/v) agarose gel
electrophoresis. The DNA fragment was purified using QIAEX II (Qiagen
Inc., Valencia, CA), labeled with [
-32P]dATP using
Klenow enzyme, and purified again with QIAEX II before incubation with
protein extracts. Fluorescence microscopy of living yeast cells
expressing green fluorescent protein was done according to published
methods (9).
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RESULTS |
Tween 80 as a Dispersing Agent in Trans Fatty Acid Media--
The
methods employed here to address the issue of how yeast cells degrade
trans fatty acids include the use of wild-type yeast cells
and otherwise isogenic pex5
or pex6
mutants
that are unable to degrade fatty acids due to defective peroxisomes.
Yeast is routinely examined for utilization of fatty acids as a single carbon and energy source, but these must be dispersed in media using
agents such as Tween 80 (polyoxyethylenesorbitan monooleate; Sigma).
Since yeast might hydrolyze Tween 80 to release oleic acid, this could
provide abundant cis fatty acids in the medium, thereby
complicating the interpretation of the growth data. To underscore the
appropriateness of using Tween 80 for dispersing the fatty acids used
in this work, drops of serially diluted yeast cultures were applied on
solid media containing either 0.125% (w/v) oleic acid dispersed with
0.5% (w/v) Tween 80 or the latter two combined with 2% (v/v) ethanol
(Fig. 1).

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Fig. 1.
Tween 80 does not represent an alternative
carbon source when used as a dispersing agent in fatty acid
medium. Serially diluted cultures of BJ1991 wild-type cells and
otherwise isogenic mutants devoid of Pex5p or Pex6p were applied to
solid media as indicated. Oleic acid dispersed with Tween 80 could not
be internalized by the pex mutants (upper
panel). However, the addition of 2% (v/v) ethanol to Tween
80-dispersed oleic acid restored the ability of the mutants to take up
the fatty acid, as indicated by the zones clearing surrounding the
colonies (lower panel).
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The results showed that on the opaque medium consisting of oleic
acid/Tween 80 (upper panel; Fig. 1) the wild-type strain produced the characteristic clear zone, whereas the two pex
mutants failed to do so. The clear zone produced by the wild type is
indicative of utilization of the fatty acid as a sole carbon and energy
source. On the other hand, all three strains produced clear zones on
medium containing oleic acid/Tween 80 plus ethanol (lower
panel; Fig. 1). This demonstrated that in the presence of
alternative carbon sources such as ethanol that do not require
peroxisomes for their degradation, mutant yeast cells could internalize
the fatty acids present in the medium. However, Tween 80-dispersed
fatty acids alone could not be internalized by pex mutants,
and therefore, unlike ethanol, Tween 80 combined with fatty acids did
not represent a good alternative carbon source. This plate assay
therefore served to underscore the difference between
-oxidized
fatty acids and those internalized into cells for assimilation into
membranes or lipid droplets.
The Response of S. cerevisiae Cells to Elaidic Acid Depends on the
Pip2p Transcription Factor--
S. cerevisiae cells grown
on cis-unsaturated fatty acids such as oleic acid as the
sole carbon source dramatically increase their peroxisomal compartment
(23). This expansion is tightly associated with an induced synthesis of
peroxisomal matrix enzymes (21). From the outset of this study, it was
not clear whether yeast could also utilize trans-unsaturated
fatty acids as a single carbon and energy source. The following two
sections are concerned with whether (i) trans-unsaturated
fatty acids mediate transcriptional up-regulation of genes encoding
-oxidation enzymes, and (ii) growth on these fatty acids requires an
expandable peroxisomal compartment.
To determine whether trans-unsaturated fatty acids emulate
the transcriptional effect seen using the corresponding cis
isomers, levels of transcription of the oleic acid-responsive gene
SPS19 were monitored in cells containing an
SPS19-lacZ reporter gene. Wild-type cells (yAG456) as well
as a pip2
(21) mutant strain (yAG485) in which the
response to oleic acid is abolished (11) were grown to late log phase
on rich-glucose (YP and 2% w/v D-glucose) medium and
transferred to elaidic acid medium for 18 h. The results of three
independent
-galactosidase activity measurements (first two rows of
Table III) demonstrated that expression
of the SPS19-lacZ reporter gene in the wild-type strain was
30-fold greater after subjecting cells to elaidic acid medium
conditions compared with the initial time point. This induction was
abolished in the pip2
mutant strain (first two rows of
Table III). Hence, elaidic acid was efficient in inducing the
transcription of the SPS19-lacZ reporter, which, like
in the situation with oleic acid (11), depended on Pip2p (21).
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Table III
Reporter gene expression after cell propagation on media consisting of
either elaidic acid/Tween 80, Tween 80, or ethanol
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To recapitulate that up-regulation of SPS19 was due to
its ORE (24, 25), the ability of the SPS19 ORE to confer
transcriptional activation on a basal CYC1-lacZ reporter
gene was examined under elaidic acid-medium conditions (third and
fourth rows of Table III). Strains yAG259 and yAG257 harboring an
SPS19 ORE::CYC1- lacZ reporter or the
plasmid vector, respectively, were propagated similarly to the
aforementioned strains yAG456 and yAG485. The results showed that
transfer of late log-phase cells to liquid elaidic acid medium for
18 h resulted in a 10-fold increase in reporter-gene activity due
to the SPS19 ORE, since
-galactosidase activity levels
were not elevated in the control strain carrying the plasmid vector
(third and fourth rows of Table III). Finally, to exclude Tween 80 as
the agent responsible for this ORE-dependent transcriptional up-regulation, the yAG259 and yAG257 strains were propagated on Tween 80 or ethanol media for 18 h. The results demonstrated that Tween 80-dependent levels of
-galactosidase activity were at the lower detection limit of the
assay used (fifth and sixth rows of Table III). These Tween 80 values
were similar to those obtained using ethanol as a sole carbon source
and energy source (last two rows of Table III). Hence, like the
situation with ethanol, Tween 80 alone was not sufficient to trigger
ORE-dependent transcription.
Oleic acid-induced transcription of SPS19, FOX3,
and other similarly regulated genes is instigated by binding of the
Pip2p-Oaf1p transcription factor (16, 21, 26, 27) to the ORE in their promoters. To underscore that transcriptional induction under elaidic
acid-medium conditions was coincidental with the formation of a complex
between Pip2p-Oaf1p and OREs, an electrophoretic mobility shift assay
was performed using the FOX3 ORE. Soluble protein extracts
were obtained from wild-type cells grown in media containing either
Tween 80 or elaidic acid/Tween 80. Extracts were incubated with labeled
DNA, and free and bound DNA were resolved on a 5% (w/v) polyacrylamide
gel (Fig. 2). The results showed that the
intensity of the Pip2p-Oaf1p complex formed under Tween 80 medium
conditions (lane 2; Fig. 2) was significantly reduced compared with that generated using the corresponding elaidic acid/Tween 80 extract (lane 3; Fig. 2). Hence, despite consisting of a
fatty acid derivative, Tween 80 alone represented a poor carbon source for inducing the formation of a Pip2p-Oaf1p complex with
FOX3 ORE. The situation seen here with respect to the
difference in Pip2p-Oaf1p complex intensities between elaidic
acid/Tween 80- and Tween 80-derived protein extracts closely resembled
that known to occur when comparing protein extracts from oleic
acid-grown cells with those grown on ethanol (21).

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Fig. 2.
Eletrophoretic mobility shift assay using
protein extracts from cells propagated on elaidic acid/Tween 80 or
Tween 80 media. Labeled FOX3 ORE was incubated with
protein extracts as indicated, and protein-bound DNA was resolved from
unbound DNA using a 5% (w/v) polyacrylamide gel. The intensity of the
signal representing the Pip2p-Oaf1p complex in soluble protein extracts
from cells grown on elaidic acid/Tween 80 (lane 3) was
compared with that in extracts from cells grown on Tween 80 alone
(lane 2). The Pip2p-Oaf1p complex was missing from cells
devoid of Pip2p and Oaf1p (lane 1) or from the lane
containing only labeled DNA (lane 4). WT, wild
type.
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Yeast Growth on Elaidic Acid Medium Requires Functional
Peroxisomes--
A milestone in the sequence of events leading to the
ability of yeast to utilize fatty acids is the expansion of the
peroxisomal compartment (23), which represents the sole site of
-oxidation in this organism (4). The state of this compartment could
be studied under trans fatty acid medium conditions, using a
peroxisomally targeted green fluorescent protein (GFP-Eci1p).
Expression of this reporter in wild-type cells resulted in punctate
fluorescence, whereas in pex6
or pex8
cells
devoid of functional peroxisomes, the reporter protein remained
cytosolic (9, 10).
To view yeast peroxisomes under trans-unsaturated fatty
acid-medium conditions, a wild-type strain expressing the GFP reporter was propagated in liquid elaidic acid medium. As a control,
GFP-expressing pex6
cells devoid of normal peroxisomes
(29) were also examined. The results demonstrated that like the
situation with oleic acid, under elaidic acid-medium conditions, cells
exhibited a fluorescence pattern that consisted of closely bunched
points (Fig. 3). On the other hand, the
punctate pattern was significantly less dense under both Tween 80 or
ethanol medium conditions (Fig. 3). Expression of the reporter protein
in pex6
cells resulted in diffuse fluorescence (Fig. 3).
Hence, unlike the situation with Tween 80-grown cells, under elaidic
acid-medium conditions, GFP-Eci1p was compartmentalized into
peroxisomes that were both numerous and bunched.

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Fig. 3.
Fluorescence microscopy of living cells
expressing GFP-Eci1p on elaidic acid/Tween 80 or Tween 80 media.
Expression of GFP-Eci1p in wild-type cells grown on
(trans-9) elaidic acid or (cis-9) oleic acid
resulted in bright, bunched fluorescent points. Under Tween 80 or
ethanol medium conditions, the punctate fluorescent pattern was more
dispersed. Green fluorescence was diffuse in mutant pex6
cells devoid of functional peroxisomes.
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Finally, to determine whether yeast cells could actually grow on
trans-unsaturated fatty acids and whether this growth
depended on peroxisomes, wild-type cells were compared with otherwise
isogenic pex5
and pex6
mutants lacking a
functional peroxisomal compartment (30) for growth on elaidic acid
medium. Late log phase cells were transferred to elaidic acid medium at
an A600 = 0.2, and cell growth was monitored by
a vital count after 4 days of incubation. The results demonstrated that
on elaidic acid/Tween 80 medium conditions wild-type cells gave rise to
cell culture concentrations that were at least 4-fold more dense than
those produced by the two pex mutants used
(Table IV). On Tween 80, no difference
was detected between the three strains. To reiterate the difference in
the number of wild-type cells compared with the pex6
mutant after growth on the two media, 2.5-µl culture aliquots from
the serial dilutions used for the vital counts were applied to
His
glucose medium (Fig.
4). The results demonstrated an order of magnitude more wild-type cells than pex6
mutants after
propagation on elaidic acid/Tween 80 medium. This growth difference was
not manifested after propagation in medium containing Tween 80 alone (Fig. 4). This indicated that, like the situation with other fatty acid
media, growth on elaidic acid medium was facilitated by functional peroxisomes.

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Fig. 4.
Yeast growth on elaidic acid is facilitated
by functional peroxisomes. The indicated strains were grown on
elaidic acid/Tween 80 medium or on Tween 80 alone. After 4 days of
incubation, culture aliquots were serially diluted and applied to
His glucose medium.
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A Sole
-Oxidation Auxiliary Enzyme, Eci1p, Is Required for S. cerevisiae Cells to Metabolize Trans-unsaturated Fatty Acids with
Double Bonds at Odd-numbered Positions--
The only double bond that
can be degraded by the classical
-oxidation process is at the
2-position and in the trans configuration
(Scheme 1). Hence, removal of other double bonds necessitates
-oxidation auxiliary enzymes (31). For this reason yeast cells
lacking the auxiliary enzyme Sps19p are unable to degrade
cis-unsaturated fatty acids with double bonds at
even-numbered positions (11). In addition, cells devoid of the
auxiliary enzyme Eci1p, which is essential for repositioning cis double bonds from the
3 to the
2 positions, are blocked in the breakdown process of
cis-unsaturated fatty acids with double bonds at any
position (9, 10). DCI1-deleted yeast cells (32) devoid of
3,5-
2,4-dienoyl-CoA isomerase
(di-isomerase) are not known to be blocked at any step of
-oxidation
of cis-unsaturated fatty acids (13, 33, 34).
To determine whether repositioning of trans double bonds at
odd- or even-numbered positions other than
2 also
requires
-oxidation auxiliary enzymes, wild-type and mutant yeast
cells were streaked on trans-9,trans-12
linolelaidic acid medium (upper panel; Fig.
5). As a control, cells were also applied to medium containing the corresponding cis,cis
isomer linoleic acid (lower panel; Fig. 5). The results
demonstrated that eci1
mutant cells did not form clear
zones in either media (Fig. 5). This indicated that Eci1p was critical
at least for metabolizing odd-numbered trans double bonds
originally at position 9 in the fatty acid substrate and, therefore,
represents a physiological
3-cis/trans-
2-trans-enoyl-CoA
isomerase.

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Fig. 5.
Eci1p is essential for yeast to utilize
unsaturated fatty acids with trans double bonds.
Plate assay for the ability of yeast mutants to form clear zones in
media containing trans-9,trans-12 linolelaidic
acid or cis-9,cis-12 linoleic acid. The
BJ1991-derived strains used are indicated.
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On the other hand, the results in Fig. 5 demonstrated that
sps19
cells, which failed to form a clear zone in the
control cis-9,cis-12 linoleic acid medium due to
their inability to metabolize the
12 cis
double bond, formed a broad clear zone in the corresponding trans-9,trans-12 linolelaidic acid medium. This
showed that although Sps19p was essential for transforming
trans-2,cis-4-dienoyl-CoAs to trans-3
metabolites suitable for acting as substrate for Eci1p (Scheme
1C), metabolism of
trans-2,trans-4-dienoyl-CoAs did not depend on
this enzyme. This implied that either an additional, novel, peroxisomal
2,4-reductase specific for
trans-2,trans-4-dienoyl-CoAs exists or that
even-numbered trans,trans metabolites act
directly as substrate for the second step of classical
-oxidation
represented by Fox2p.
The demonstration in Fig. 5 that dci1
cells formed well
defined clear zones in both media supports our earlier studies
reporting the dispensability of Dci1p for utilizing fatty acids (13). Consonant with this observation, the
sps19
dci1
double mutant in which the
putative reduction pathway for metabolizing odd-numbered cis
double bonds is completely blocked also gave rise to a broad clear zone
in the trans-9,trans-12 linolelaidic acid medium
(upper panel; Fig. 5). The absence of a corresponding clear
zone around the double mutant in the cis-9,cis-12
linoleic acid medium (lower panel; Fig. 5) was due to the
missing Sps19p required for metabolizing the double bond originally at
position
12. Hence, the salient points derived from the
experiments depicted in Fig. 5 were as follows. (i) Yeast Eci1p acted
on trans-3-enoyl-CoA metabolites to support degradation of
unsaturated fatty acids with odd-numbered trans double
bonds, and (ii) to degrade even-numbered trans double bonds,
yeast cells did not rely on Eci1p, Sps19p, or Dci1p.
Expression of Rat Monofunctional 3,2-Isomerase or Rat
Multifunctional Enzyme Type 1 in eci1
Mutant Cells Restores
Utilization of Trans-unsaturated Fatty Acids--
The results depicted
in Fig. 5 demonstrated that eci1
cells did not utilize
trans-unsaturated fatty acids such as linolelaidic acid.
Hence, these mutants could serve as a test organism for examining
whether, in addition to accepting cis-3 metabolites, mammalian Eci1p analogs could also accept trans-3
metabolites as a physiological substrate. Previous work using
cis-unsaturated fatty acids has shown that yeast Eci1p can
be functionally replaced by the two rat proteins peroxisomal MFE type 1 (9) and monofunctional ECI (15). Therefore, to determine the effect on
mutant eci1
cells expressing the respective
heterologous enzymes, transformants were streaked on
trans-9,trans-12 linolelaidic acid and, as
a control, also on the corresponding linoleic acid media (Fig.
6).

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Fig. 6.
Yeast eci1
cells expressing 3,2-isomerases on
trans-9, trans-12 linolelaidic acid
or cis-9,cis-12 linoleic acid
media. Plate assay for the ability of yeast Eci1p, rat peroxisomal
MFE type 1, or rat monofunctional 3,2-isomerase to restore degradation
of trans,trans- or
cis,cis-unsaturated fatty acids in
eci1 cells. The eci1 strains used were
yAG854 (expressing rat monofunctional 3,2-isomerase;
eci1 [rat ECI]), yAG826 (expressing yeast 3,2-isomerase
Eci1p; eci1 [Eci1p]), yAG827 (harboring the plasmid
vector; eci1 [vector]), and yAG856 (expressing rat
peroxisomal MFE type 1; eci1 [rat MFE]).
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The results demonstrated that the rat proteins had efficiently
substituted for yeast Eci1p under both medium conditions, since their
expression in the mutant strain gave rise to clear zones that were
indistinguishable from those produced by the self-complemented cells.
Control eci1
mutants harboring the plasmid vector did not
form a clear zone in either medium (Fig. 6). Therefore, rat peroxisomal
MFE type 1 and monofunctional ECI represented proteins with
3-cis/trans-
2-trans-enoyl-CoA
isomerase activities.
Rat Peroxisomal MFE Type 1 Accepts trans-2,trans-4 Metabolites as
Substrate in Vivo--
Current understanding of
-oxidation
stipulates that the second reaction step represented by
trans-2-enoyl-CoA hydratase cannot proceed if a double bond
is present at the
4 position (Scheme 1C). The
results in Fig. 5 (upper panel) demonstrated that for
utilizing trans-2,trans-4-dienoyl-CoA
intermediates formed during the metabolism of the
trans-double bond originally at the
12
position, Sps19p (and therefore also Eci1p) were dispensible. This
raised the issue of whether yeast Fox2p could accept
trans,trans intermediates as substrates. Such
intermediates have been previously shown to be degraded by
trans-2-enoyl-CoA hydratase in the absence of 2,4-reductase
(35). However, these experiments were conducted using an in
vitro mammalian
-oxidation system reconstituted from mitochondrial enzymes. Hence, to examine whether 2,4-reductase might be
dispensable for degrading such trans,trans
metabolites in vivo, a system based on the mammalian enzyme
representing peroxisomal trans-2-enoyl-CoA hydratase was used.
The rat protein corresponding to this enzyme activity, peroxisomal MFE
type 1, has previously been shown to substitute for yeast Fox2p during
utilization of oleic acid (17). To determine whether an exchange
between yeast Fox2p and rat peroxisomal MFE type 1 would also function
in a strain additionally devoid of Sps19p, a fox2sps19
double mutant was generated and transformed with the plasmid expressing
this rat protein. As a control, these mutant cells were transformed
with the native Fox2p. The strains were streaked on
trans-9,trans-12 linolelaidic acid and
cis-9,cis-12 linoleic acid media (Fig.
7). The result demonstrated that Sps19p, which was necessary for utilizing linoleic acid (lower
panel; Fig. 7), was dispensable for generating clear zones in the
trans,trans fatty acid (upper panel;
Fig. 7). This indicated that both yeast Fox2p and rat peroxisomal MFE
type 1 could metabolize
trans-2,trans-4-dienoyl-CoA intermediates without
depending on the auxiliary enzymes 2,4-reductase and 3,2-isomerase.

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Fig. 7.
Yeast fox2sps19
cells expressing rat peroxisomal MFE type 1 or native Fox2p on
trans,trans- or cis,cis-unsaturated
fatty acid media. Plate assay for the dispensability of
2,4-reductase (Sps19p) during the utilization of
trans-9,trans-12 linolelaidic acid. The strains
used were yAG1203 (fox2sps19 cells expressing rat
peroxisomal MFE type 1; fox2sps19 [rat MFE]), yAG1204
(fox2sps19 cells expressing yeast MFE type 2;
fox2sps19 [Fox2p]), yAG1206 (fox2 cells
expressing rat peroxisomal MFE type 1; fox2 [rat MFE]),
and yAG1207 (fox2 cells expressing yeast MFE type 2;
fox2 [Fox2p]).
|
|
 |
DISCUSSION |
In the present study yeast
-oxidation was exploited for
studying the degradation of unsaturated fatty acids with
trans double bonds. Metabolism of trans double
bonds has previously been studied in rat hepatocytes, where it was
shown that elaidic acid is degraded at a higher rate than oleic acid
(3). In addition, trans-unsaturated fatty acids were
preferentially oxidized by hepatic peroxisomes (3). Pioneering work
aimed at determining whether metabolites of
trans-unsaturated fatty acids could be metabolized directly by mammalian
-oxidation demonstrated that
trans-2,trans-4-decadienoyl-CoA, but not its
trans-2,cis-4 isomer, could be readily degraded
without the requirement for 2,4-reductase (35). Although it was later shown that trans-2,trans-4-octadienoyl-CoA is a
substrate for mitochondrial 2,4-reductase (36), a more recent in
vitro study reported that such trans,trans
metabolites can in fact be degraded by mitochondrial
-oxidation
without first being modified by 2,4-reductase (28). Hence, it was
hitherto not clear whether 2,4-reductase was physiologically critical
for degrading trans-2,trans-4-CoAs.
We demonstrated here for the first time that S. cerevisiae
cells utilize trans double bonds at even-numbered positions
in the absence of the known
-oxidation auxiliary enzymes. The
S. cerevisiae genome does not contain any novel peroxisomal
proteins with obvious homologies to 2,4-reductases or 3,2-isomerases
that could have potentially substituted for Sps19p or Eci1p. In light of these new findings, we propose an amendment to the accepted scheme
of the carbon flux through
-oxidation (Scheme
2). In contrast to the dispensability of
auxiliary enzymes for metabolizing even-numbered trans
double bonds, yeast cells engaged the same
-oxidation auxiliary enzyme Eci1p used for breaking down the corresponding cis
isomers (Scheme 1C). Both rat 3,2-isomerases, peroxisomal
MFE type 1 and monofunctional ECI, were shown here to be able to
execute the requisite
3 to
2
repositioning of trans double bonds for ensuring the carbon
flux through
-oxidation. Due to the dual distribution of ECI (15), at least in rodents this faculty is extended to both the peroxisomal as
well as the mitochondrial
-oxidation compartments.

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Scheme 2.
Schematic representation of the modified
route taken by peroxisomal -oxidation for
degrading trans double bonds at even-numbered
positions. trans-2,trans-4-Dienoyl-CoA
metabolites are direct substrates for the second step of classical
-oxidation and, unlike
trans-2,cis-4-dienoyl-CoA metabolites, do not
require 2,4-reductase (Sps19p) and 3,2-isomerase (Eci1p) for
repositioning of the double bonds. The double arrowheads
represent the two catalytic steps of Fox2p.
|
|
Like the situation in yeast in which Fox2p accepts
trans-2,trans-4-dienoyl-CoAs as substrate,
the second step of mammalian
-oxidation executed by rat peroxisomal
MFE type 1 also accepted this intermediate without requiring the
repositioning of the double bonds by Sps19p. This is the first in
vivo demonstration of the final step in the
di-isomerase-dependent route for degrading fatty acids
(28). In this di-isomerase-dependent route, which
represents an alternative to the reductional pathway, 3,2-isomerase and
di-isomerase are sufficient to establish the carbon flux, since the
combined enzyme activities result in the production of
trans-2,trans-4-dienoyl-CoA intermediates that
feed directly into
-oxidation.
 |
ACKNOWLEDGEMENTS |
We thank Leila Wabnegger (University of
Vienna, Austria) for preparing media and soluble protein
extracts and Professor J. Kalervo Hiltunen (University of Oulu,
Finland) for critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by the Fonds zur Förderung der
wissenschaftlichen Forschung (FWF), Vienna, Austria Grant P12118-MOB (to A. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Biochemie und Molekulare Zellbiologie, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria. Tel.: 43-1-4277 52804; Fax: 43-1-4277 9528;
E-mail: AG@abc.univie.ac.at.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M003305200
 |
ABBREVIATIONS |
The abbreviations used are:
MFE, multifunctional
enzyme;
2, 4-reductase, 2,4-dienoyl-CoA reductase;
3, 2-isomerase,
3-
2-enoyl-CoA isomerase;
di-isomerase,
3,5-
2,4-dienoyl-CoA isomerase;
GFP, green
fluorescent protein;
YP, yeast extract/peptone;
ORE, oleic
acid-response element.
 |
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