(Received for publication, January 31, 1997, and in revised form, June 3, 1997)
From the School of Biochemistry and Molecular
Genetics, University of New South Wales, Sydney NSW 2052, Australia,
the § Institut für Biochemie und Molekulare
Zellbiologie der Universität Wien and Ludwig
Boltzmann-Forschungsstelle für Biochemie, Vienna Biocenter, Dr
Bohrgasse 9, A-1030 Wien, Austria, the ¶ Biocenter Oulu,
Department of Biochemistry, University of Oulu, FIN-90570 Oulu,
Finland, and the
Institut für Tumorbiologie-Krebsforschung
der Universität Wien, Borschkegasse 8a, A-1090
Wien, Austria
-Oxidation is compartmentalized in mammals
into both mitochondria and peroxisomes. Fatty acids with double bonds
at even-numbered positions require for their degradation the auxiliary
enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two
mitochondrial and one peroxisomal, exist in the rat. The
Saccharomyces cerevisiae Sps19p is 34% similar to the
human and rat mitochondrial reductases, and an SPS19
deleted strain was unable to utilize petroselineate (cis-C18:1(6)) as the sole carbon source, but
remained viable on oleate (cis-C18:1(9)).
Sps19p was purified to homogeneity from oleate-induced cells and the
homodimeric enzyme (native molecular weight 69,000) converted
2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an
NADPH-dependent manner and therefore contained
2,4-dienoyl-CoA reductase activity. Antibodies raised against Sps19p
decorated the peroxisomal matrix of oleate-induced cells.
SPS19 shares with the sporulation-specific
SPS18 a common promoter region that contains an oleate
response element. This element unidirectionally regulates transcription
of the reductase and is sufficient for oleate induction of a
promoterless CYC1-lacZ reporter gene. SPS19 is
dispensable for growth and sporulation on solid acetate and oleate
media, but is essential for these processes to occur on
petroselineate.
The -oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase (EC
1.3.1.34) participates in the degradation of unsaturated fatty acids
with double bonds at even-numbered (1, 2) and possibly also at
odd-numbered positions (3, 4). It catalyzes an
NADPH-dependent reduction of
trans-2,cis/trans-4-dienoyl-CoA into
trans-3-enoyl-CoA in eukaryotes (1) and into
trans-2-enoyl-CoA in bacteria (5, 6). Reductases have
previously been purified from Escherichia coli (7), from the
yeasts Candida lipolytica (8) and Candida
tropicalis (9), and from rat and bovine liver (10, 11). Mammals
possess at least two mitochondrial reductases (molecular weights
120,000 and 60,000) and a third peroxisomal one (12, 13), and although
the rat and human cDNAs for the Mr 120,000 mitochondrial isoform have been cloned (14, 15), the peroxisomal
isoform has remained uncharacterized at the molecular level. A
reductase-deficient E. coli mutant unable to grow on
petroselineate (cis-C18:1(6)) as the sole carbon
source (16) further underscored the essential requirement for
this enzyme, and a lethal inborn 2,4-dienoyl-CoA reductase deficiency in a human patient has been reported (17).
In Saccharomyces cerevisiae -oxidation is solely confined
to the peroxisomes (18). In cells grown on oleate,
-oxidation enzymes are induced and peroxisomes dramatically increase in number and
size (19). The induction of a number of other S. cerevisiae genes encoding peroxisomal proteins such as catalase A
(CTA1) and thiolase (FOX3) is mediated via a
positive gene control sequence called the oleate response element
(ORE1; 20, 21). OREs have
recently been shown to act as the binding target for the products of
PIP2 (20) and OAF1 (21), and strains deleted at
either locus were unable to grow on oleate
(cis-C18:1(9)) as the sole carbon source.
SPS19 was cloned together with the S. cerevisiae
sporulation-specific gene SPS18, that was in turn isolated
from a genomic yeast DNA library fused to a promoterless E. coli
lacZ gene following a screen for sporulation-specific expression
(22). Homozygous sps18/19 deletants demonstrated a 4-fold
reduction in sporulation, and the viable spores that did form failed to
become resistant to ether and were more sensitive to lytic enzymes.
Sequence analysis of the shared SPS18/19 promoter region
showed that it contained potential OREs, and the deduced amino acid
sequence of SPS19 revealed a carboxyl-terminal SKL
peroxisome targeting signal (23, 24). A search of the data bases for
similarities to the Sps19p sequence indicated that it was 34% similar
to the human and rat mitochondrial 2,4-dienoyl-CoA reductase. Since the
roles of
-oxidation and peroxisomes during eukaryotic development
and particularly during yeast sporulation are poorly understood, we
were prompted to elucidate the properties and regulation of this gene
product.
We show here that Sps19p is a peroxisomally localized 2,4-dienoyl-CoA
reductase and that the sps19 deletant is unable to grow
on petroselineate as the sole carbon source, although it remains viable
on oleate. SPS19 is dispensable for ascosporogenesis on
solid acetate and oleate media but is essential for this process to
occur on solid petroselineate medium. It is regulated by an ORE that is
sufficient for heterologous expression of a promoterless CYC1-lacZ reporter gene under oleate induction
conditions.
The S. cerevisiae strains and plasmids used are listed in Table I. E. coli strain DH10B was used for all plasmid amplifications and isolations.
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For RNA isolations, logarithmic
cultures of the wild-type strain MF24-6x were shifted to YP medium (1%
yeast extract, 2% peptone) containing the indicated carbon sources and
grown for a further 16 h (20). For electron microscopy cells were
propagated as described (25), and for -galactosidase measurements
cells were induced in oleate medium following a modified protocol (26). Stationary-phase haploid wild-type strains from overnight precultures consisting of YP medium and 5% glucose were transferred to 100-ml conical flasks containing 50 ml of YP medium containing both 0.2% oleate and 0.02% Tween 80 (adjusted to pH 7.0 with NaOH), 0.05% glucose, and 75 µg/ml ampicillin to an absorbance of
A600 nm = 0.2. The cultures were grown at
30 °C with vigorous aeration at 170 rpm for the periods indicated.
For 2,4-dienoyl-CoA reductase (Sps19p) purification, cell propagation
was scaled up to 1-liter cultures in 5-liter conical flasks; the
extreme flocculence of the strain used obviated the requirement for
absorbance determination prior to transfer into oleate medium. Plates
containing Tween 80 alone (Sigma), or Tween 80 with oleate (E. Merck
AG, Darmstadt, Germany), or petroselineate (Sigma) (0.67% yeast
nitrogen base with amino acids, 0.1% yeast extract, 0.5% potassium
Pi at pH 6.0, and 2% agar, autoclaved with 0.5% Tween 80 alone or with 0.5% Tween 80 and either 0.125% oleate or 0.125%
petroselineate, respectively) were prepared by pouring a thin layer at
a temperature below 55 °C. These plates were used to assess
utilization of the fatty acids (clear-zone formation) and for
sporulation (by direct microscopic examination of cells) after about
7-day incubation.
A 1.8-kb KpnI-XbaI
SPS18/19 fragment from pJC18-2µ (22) was cloned into
pBluescript® SK(+) (Stratagene, La Jolla CA) to yield pAG113. The
189-base pair ClaI-SphI fragment within
SPS19 was then replaced with a 2-kb
SmaI-SphI fragment containing the LEU2
gene from pJJ250 (27), resulting in the disruption plasmid pAG129. A
2.8-kb fragment produced by digesting pAG129 with ScaI and
StuI was used for transformation (Fig. 1B).
SPS19 ORE:CYC1-lacZ Construct
The SalI-delineated SPS19ORE1 and ORE2 oligonucleotides (Table I) defining the oleate response element within the shared SPS18/19 promoter region were annealed, and the double-stranded fragment was cloned into the XhoI site of the integrative vector pMF6 (28), a derivative of pLG669Z (29), to produce pAG244. Nucleotide sequencing revealed that the orientation of the ORE fragment with respect to the lacZ fusion boundary as being in the SPS18 direction, i.e. in the reverse orientation to SPS19, and integration of StuI-linearized pMF6 and pAG244 into the ura3 locus of BJ1991 resulted in strains yAG257 and yAG259, respectively.
Other Reporter GenesThe plasmids YIp357-19 and YIp357-19M1
were kindly donated by P. Yeoh (University of New South Wales, Sydney,
Australia). Briefly, a 1.4-kb XbaI-SphI fragment
containing the intergenic region and part of the coding regions of both
SPS18 and SPS19 was excised from pUC18-KXC (23)
and inserted into the corresponding sites in YIp357 (30) to create
YIp357-19 (pAG454; SPS19-lacZ) as well as into M13mp19 for
the substitution of the 3 ORE half-site sequence 5
-ACGCCGTGAG-3
with
a unique XhoI site using site-directed mutagenesis. The
substituted DNA was verified by nucleotide sequencing and cloned into
YIp357 to create YIp357-19M1 (pAG23). Integration of the
StuI-linearized plasmids containing the native and
substituted DNA into the ura3 locus of BJ1991 resulted in
strains yAG456 and yAG295, respectively. Cloning of the reverse
orientation by inserting the 1.4-kb XbaI-SphI
fragment isolated from pAG454 into the appropriate sites of YIp356R
resulted in pAG534 (SPS18-lacZ). Similar integration of this
plasmid yielded strain yAG561.
2,4-Dienoyl-CoA reductase was assayed
spectrophotometrically at 23 °C as described (1). The assay mixture
consisted of 0.1 M potassium Pi (pH 7.4), 125 µM NADPH, and 60 µM 2,4-hexadienoyl-CoA (synthesized from trans-2,trans-4-hexadienoic
acid via the mixed anhydride system; (31)) as the substrate. Reductase
activity was expressed as micromoles of substrate metabolized per min. -Galactosidase activity was assayed in crude extracts prepared by
breakage of cells with glass beads (32) and the values reported here,
expressed as nanomoles of
o-nitrophenyl-
-D-galactopyranoside hydrolyzed
per min/mg of protein, were the average of three experiments.
To purify Sps19p S. cerevisiae cells yAG162 harboring pJC18 (22) were grown in oleate
medium for 3 days. The cells were collected by centrifugation, washed
in 2 volumes of cold distilled water, and frozen at 70 °C until
required. All subsequent work was performed at 4 °C. A cell pellet
of 15 g wet weight was thawed, suspended in 150 ml of breakage
buffer (50 mM sodium Pi at pH 7.0, 0.4 M KCl, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and
a crude extract of cells disrupted with glass beads was prepared (33).
A volume of 100 ml of the 6 × 106 × g × min supernatant was diluted to 130 mM
KCl, loaded onto a phosphocellulose P-11 column (2.5 × 15 cm;
Whatman) in equilibrium with 20 mM potassium Pi
(pH 7.0). After washing the column with 55 ml of the same buffer, the
reductase activity was eluted with 20 mM potassium
Pi (pH 7.0) containing 0.5 M NaCl. Following
the adjustment of the salt concentration to 0.7 M NaCl, the
eluted reductase-containing fraction was loaded onto a Matrex gel red A
column (1.5 x.10 cm; Amicon Corp, Lexington, MA) in equilibrium with 20 mM potassium Pi (pH 7.0) containing 0.3 M NaCl. The column was washed with 55 ml of 20 mM potassium Pi (pH 7.0) containing 10%
glycerol and 0.7 M NaCl, and the reductase was eluted with 2.5 M NaCl in the same buffer. The buffer exchange to 50 mM sodium Pi (pH 7.6) containing 10% glycerol
and 1 mM dithiothreitol was carried out with a HiTrapTM
desalting column (Pharmacia Biotech Inc.). The purification of Sps19p
was completed by applying the sample to a SMARTTM-linked ResourceTM S
column (Pharmacia Biotech Inc.) in equilibrium with the buffer
mentioned above, and the bound proteins were eluted with a linear
gradient (20 ml from 0-500 mM NaCl in the equilibration
buffer).
An aliquot
containing 5 µg of ResourceTM S-purified protein was incubated with
810 µl of a mixture that consisted of 0.01 M potassium
Pi (pH 7.5), 125 µM NADPH, and 60 µM 2,4-hexadienoyl-CoA. After all the ester was
metabolized, the reductase was removed by ultrafiltration
(Ultrafree®-MC 10,000; Millipore, Bedford, MA) and 200 µl of the
filtrate was added to 800 µl of buffer containing 40 µmol of
Tris-HCl (pH 9.0), 40 µg of bovine serum albumin, 40 µmol of KCl,
25 µmol of Mg2+, 1 µmol of pyruvate, 1 µmol of
NAD+, and 10 µg of lactate dehydrogenase (EC 1.1.1.27;
from rabbit muscle, Boehringer Mannheim GmbH, Mannheim, Germany). The
following enzymes were sequentially added: 20 µg of
L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35; Sigma), 0.4 µg of 2-enoyl-CoA hydratase I (EC 4.2.1.17; Ref. 34), and 1 µg of
3,
2-enoyl-CoA isomerase (EC 5.3.3.8; Ref.
35), and the reaction was monitored spectrophotometrically at
A303 nm.
The following procedures were performed
according to published methods: nucleic acid manipulations,
formaldehyde gel electrophoresis, blotting and hybridization (36), DNA
fragment isolation (37), yeast transformation (38), verification of
single plasmid integration (39), yeast RNA preparation for Northern
analysis (40), determination of protein concentration (41), sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (42),
separation of organelles using homogenized spheroplasts generated from
oleate-induced cells (43), and nucleotide sequencing of pAG244 using
the CYC1 oligonucleotide (44). Double labeling of the antigens Fox3p
and Sps19p by repeating the antigen-antibody complex incubation
sequence twice in tandem with the two antibodies raised in rabbits was
performed as outlined (45, 46). The use of FOX3 and
ACT1 as probes has been described previously (20), and the
0.9-kb SspI-XbaI SPS18 and 0.5-kb
SspI-SphI SPS19 fragments were
obtained from pAG454. The [-32P]dATP-labeled probes
were generated with a random primer labeling kit (Prime-a-GeneTM,
Promega) according to the manufacturer's instructions. To remove any
remaining minor contaminants, the purified reductase was applied onto a
µRPCTM reverse phase column (Pharmacia Biotech Inc.), and the eluted
polypeptide was used for immunization of rabbits (47). The IgG fraction
was purified from the antiserum using a protein A-Sepharose column
(Pharmacia Biotech Inc.).
Analysis of the amino acid sequence of Sps19p revealed
an overall 28% identity to the human mitochondrial 2,4-dienoyl-CoA reductase and 24% identity to the corresponding rat enzyme (Fig. 1A). Comparison of the
NH2-terminal residues showed that unlike its
mitochondrially targeted mammalian homologues, Sps19p lacked the
appropriate leader sequence and instead contained a carboxyl-terminal SKL peroxisomal targeting signal (24). This suggested that Sps19p may
be an enzyme involved in -oxidation (23), since in yeast this
process is restricted to the peroxisomes (18). The known 2,4-dienoyl-CoA reductases are NADPH-dependent, and the
NAD(P)+-binding site includes a highly conserved
1
2 fold (48). The respective Sps19p
sequence (Ala30 to Glu62) that came closest to
this nucleotide-binding motif (Fig. 1A; Ref. 49) deviated
from the consensus sequence both at the second and third glycine
residues. Sps19p vaguely resembled a reductase, and to obtain a
clearer indication for its physiological requirement for the breakdown
of unsaturated fatty acids, an appropriately deleted yeast strain
was constructed (Fig. 1B).
To elucidate the potential participation of Sps19p
in the metabolism of unsaturated lipids, wild-type and
sps19 strains were grown on plates containing fatty acids
with double bonds at odd-numbered (oleate) and even-numbered
(petroselineate) positions. In these solid media Tween 80 was added to
help form the emulsion in the plate, but also to act as a relatively
poor carbon source. Hence the strains could all grow on these plates
(Fig. 2) but zones of clearing indicate
utilization of the additional fatty acid substrate (oleate or
petroselineate).
We found that the deleted strain was unable to form clear zones and to
sporulate at all on petroselineate, but did so on oleate (Fig. 2). This
indicated that Sps19p was involved in the removal of double bonds at
even-numbered positions. Transformation of the sps19
strain with the multicopy plasmid pJC18 containing the intact
SPS19 (22) complemented the mutant phenotype, since it
restored its ability to utilize petroselineate.
Pilot-scale
production of Sps19p using pJC18 transformed cells yielded crude
extracts that contained a high 2,4-dienoyl-CoA reductase activity (53 nmol × min1 × mg
1 protein). Since
reductase activity in the extracts from similarly propagated parental
strains (without pJC18) was below the detection limit of the used
assay, Sps19p could be identified throughout the ensuing purification
process by enzymatic monitoring. Reductase activity was
chromatographically purified from homogenized oleate-induced cultures
using phosphocellulose P-11 (cation exchanger), Matrex gel red A
(general dye ligand), and ResourceTM S (cation exchanger) columns (Fig.
3). In the final preparation, an overall
purification of about 50-fold was obtained with an approximate 26%
yield, and this preparation showed a specific activity of 1.79 µmol × min
1 × mg
1 protein for
2,4-hexadienoyl-CoA (Table II).
SDS-polyacrylamide gel electrophoresis revealed a single protein band
with an apparent molecular weight of 34,000, and size exclusion
chromatography on a SuperdexTM 200 HR 10/30 column (Pharmacia Biotech)
yielded a native molecular weight of 69,000, indicating that the
reductase was present as a homodimer. The monomer size so obtained
agreed with the molecular weight of 31,400 calculated from the deduced amino acid sequence (23).
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The amino-terminal residues of the protein were determined by
microsequencing following reverse-phase chromatography and the resulting sequence (N-T-A-N-T-L-D-G-A-F-V-T-) conformed to the deduced
residues 5-15 of Sps19p. Analysis of the SPS19 nucleotide sequence had revealed two potential translational start sites, each of
which was followed by the TTA codon for Asn (23). Comparison of the
data base sequences for Sps19p had shown a number of differences, most
notably at position 2, where Asn (in GenBankTM number M90351) is
replaced by Asp (in GenBankTM number X78898; Fig. 1A). The consensus for the N at position 5 is significant since asparagine is
one of nine most abundant penultimate amino-terminal residues (4% of
all Saccharomyces genes). Moreover, the finding that the Met
at position 4 most likely acted as the signal for initiation of
translation concurred with the consensus sequence:
5-(A/Y)A(A/Y)A(A/Y)AAUG-3
with a strong
preference for A at
3 (50).
In
eukaryotes, the -oxidation of fatty acids with double bonds at
even-numbered positions will result in an unsaturated acyl-CoA intermediate with the double bonds at positions 2 and 4. This intermediate serves as a substrate for 2,4-dienoyl-CoA reductase, which
introduces a double bond at position 3, resulting in 3-enoyl-CoA. This
in turn is the substrate for
3,
2-enoyl-CoA isomerase (Fig.
4A). By generating a
protein-free 2,4-hexadienoyl-CoA-depleted reductase reaction using
purified Sps19p (Fig. 4B), and then adding the enzymes
downstream in the
-oxidation pathway, we were able to identify the
end product of the enzyme (Fig. 4C).
The addition of 2-enoyl-CoA hydratase I to the depleted reductase
filtrate in the presence of NAD+ and
L-3-hydroxyacyl-CoA dehydrogenase failed to initiate the generation of 3-ketohexanoyl-CoA. However, subsequent addition of
3,
2-enoyl-CoA isomerase to the mixture
drove the reaction forward, clearly demonstrating that 3-enoyl-CoA and
not 2-enoyl-CoA was the product of the previous reaction. With the
enzymatic activity and the reaction end product defined, completion of
the characterization of Sps19p required that its location within the
cell be determined biochemically and immunohistochemically.
The protein
A-Sepharose purified IgG fraction from a rabbit injected with the
purified Sps19p as antigen was applied to immunoblots containing crude
extracts of ethanol-propagated and oleate-induced wild-type and
sps19 strains (Fig.
5A). A single band with the molecular weight of 34,000 was detected solely for the oleate-induced wild-type and not in the sps19
strain (for which no band
was detected under any conditions), thereby confirming the antibodies' monospecificity and potential utility in studying the subcellular localization of Sps19p. Subsequent fractionation studies of homogenized yeast spheroplasts demonstrated that Sps19p was exclusive to the organellar pellet (Fig. 5B). We then studied the subcellular
location of Sps19p using immunoelectron microscopy and found that
anti-Sps19p antibodies applied to sections of wild-type cells grown on
oleate medium clearly decorated the peroxisomal matrix (Fig. 5,
C and D).
The SPS18/19 Promoter Region Is Unidirectionally Activated under Oleate Conditions
Oleate response elements, best described as
palindromic CGG triplets spaced by 15-18 base pairs, mediate the
transcriptional regulation of a number of peroxisomal protein-encoding
genes, including POX1 (51), FOX3 (52),
CTA1 (28), and PMP27 (53). The smallest element
capable of relaying the fatty acid signal, a single ORE half-site
(5-CGGNNNTNA-3
), is sufficient for bi-directional induction (28).
Previous nucleotide analysis of the shared SPS18/19 promoter
region had identified potential ORE half-sites (Fig. 1C),
two of which occurred as an appropriately spaced inverted repeat (23).
We studied the ability of the intragenic region to initiate oleate
induced bi-directional transcription by testing the expression of a
lacZ reporter gene fused to either end of it (Fig.
6A). We noted that
-galactosidase expression by the SPS19-lacZ integrant
under oleate conditions was 27-fold higher compared with that expressed
by the strain carrying SPS18-lacZ. We also observed that
haploids were able to express SPS19 in a sporulation-independent manner.
We investigated the transcription of the two genes under different carbon source conditions (Fig. 6B) and showed that while the profile of SPS19 was similar to that of oleate-inducible FOX3, no signal was detected on the Northern filter when radiolabeled SPS18 was applied. Hence, SPS19 transcription was likely to be mediated by an orientation-governed ORE, and identification of the responsive element, so as to demonstrate its sufficiency for oleate induction, would be facilitated using a reporter gene in which it was mutated.
The SPS19 ORE Is Sufficient for Oleate-dependent Initiation of TranscriptionOf the three potential ORE half-sites
present within the promoter, we chose to test the one that fully
conformed to the 5-CGGNNNTNA-3
consensus (Fig. 1C). A
derivative of the SPS19-lacZ reporter gene with the distal
end of the palindromic ORE replaced by an XhoI site was
tested against the parent construct (Fig. 6C). The loss of
all
-galactosidase activity in the strain carrying the mutated reporter clearly indicated that this was the sequence responsible for
the oleate inducibility of SPS19. We then monitored the
expression of a UAS-less CYC1-lacZ reporter gene into which
the palindromic SPS19 ORE had been inserted in the direction
of SPS18. The single integrant of the recombinant construct
demonstrated a 22-fold increase in
-galactosidase expression
compared with the control (Fig. 6D). Thus, the
SPS19 ORE was sufficient for oleate-dependent initiation of transcription.
This work describes the first molecular characterization of a
peroxisomal 2,4-dienyol-CoA reductase. The S. cerevisiae
enzyme (Sps19p) is compartmentalized differently from its rat and human mitochondrially targeted homologues (14, 15), and its peroxisomal location is in line with the fact that in yeast -oxidation is exclusively a peroxisomal process (18). Although the peroxisomal protein has been enzymatically and immunocytochemically demonstrated in
rat liver (10, 47, 54), it has not yet been cloned. The relevance of
reductase isoforms to human health has been underscored by the finding
that an infant with a lethal 2,4-dienoyl-CoA reductase deficiency
(postulated to be mitochondrial) retained only 40 and 17% of the
normal level of activity in the liver and muscles, respectively. The
residual activity was attributed to a peroxisomal (17) as well as a
second functional mitochondrial isoform (13).
The deduced Sps19p amino acid residues deviate from the conserved NADPH-binding site in reductases, serving to broaden the consensus for this moiety (49). The E. coli 2,4-dienoyl-CoA reductase consists of a single polypeptide chain with a molecular weight of 70,000 (6), and the Candida lipolytica enzyme (33,000 monomer) has been reported to consist of 10-12 identical subunits (8). The mammalian reductases (molecular weight 120,000) act as homotetramers, whereas Sps19p (molecular weight 69,000) is a homodimer. The significant homology of the yeast protein to the mammalian homologues, together with the demonstration of its reductase activity and peroxisomal localization, places Sps19p as a closely related auxiliary enzyme to the one proposed to exist in humans. Work is currently underway to test whether the phenotype of the yeast mutant is complemented by the appropriately targeted human mitochondrial reductase.
The end products of mammalian 2,4-dienoyl-CoA reductases are always
trans-3-enoyl-CoA esters (Fig. 4A), although they
accept both trans-2,cis-4-, and
trans-2,trans-4-dienoyl-CoA as substrates (6,
10). The inability of sps19 strains to grow on
petroselineate plates demonstrated that the S. cerevisiae
reductase is physiologically indispensable for the degradation of the
trans-2,cis-4-dienoyl-CoA intermediate that
arises from the degradation of this fatty acid (cis-6-octadecenoic acid). In our end product accumulation
assay we had used
trans-2,trans-4-hexadienoyl-CoA. Although the
formation of the 3-enoyl-CoA product for the S. cerevisiae
reductase had clearly been demonstrated, there exists some uncertainty
regarding the ester's chirality, and we reason that it is likely to be
the trans isomer as it is in mammals.
We also provide here physiological data on the phenotype of the first organism devoid of a 2,4-dienoyl-CoA reductase. Although a reductase-deficient E. coli strain had been described previously, it retained 12% of the wild-type enzymatic activity and expressed this defective protein to apparently normal levels (16). Our observation that the deleted yeast strain was unable to utilize petroselineate as the sole carbon source, and that it remained viable on oleate, agrees with the data presented for the bacterial reductase mutant.
If the 3,5,
2,4-dienoyl-CoA isomerase
pathway as demonstrated in rats is taken into account, then reductases
are also required for the efficient breakdown of fatty acids with
double bonds at odd-numbered positions (3, 4). However, the likelihood
that the observed normal growth of sps19
cells on oleate
may have been due to the action of a second, antigenically related
2,4-dienyol-CoA reductase isoform seems low. Anti-Sps19p antibodies
capable of cross-reacting with the rat homologue from livers of
clofibrate-treated animals (data not shown) did not react with crude
extracts from the oleate-induced deletant. In addition, no isoforms are
known to exist for other
-oxidation enzymes such as Pox1p (55),
Fox2p (56), and Fox3p (57), where the missing activity in the
respective mutants was not replaced by that of other putative isomers.
Therefore, the capability of the sps19
cells to grow on
oleate suggested that dienoyl-CoA isomerase is not obligatory for the
breakdown of unsaturated fatty acids with double bonds at odd-numbered
positions.
The unidirectional transcription of SPS19 is mediated by an ORE within the SPS18/19 intergenic region. We identified the site responsible for oleate-induced expression of the reductase gene and demonstrated its sufficiency for oleate-mediated transcription. The shared SPS18/19 promoter region is capable of versatile regulation, where in diploids under sporulation conditions both genes are activated during meiosis, but SPS18 is induced to a 4-fold greater extent than SPS19. In contrast, under oleate conditions SPS19 is exclusively transcribed, and this induction is independent of ploidy.
Curiously, Sps19p is dispensable for the breakdown of oleate, although it is nonetheless induced by the presence of this fatty acid. We speculate that under physiological conditions yeast cells are unlikely to be confronted with a single lipid species as the sole carbon source, and hence when sensing the fatty acid signal cells will metabolically prime themselves to oxidize carbon double bonds at both odd- as well as at even-numbered positions. A deeper insight into how fatty acids induce Sps19p is required.
The yeast locus for the reductase had previously been isolated in a
screen for sporulation genes, and the acetate medium-derived ascospores
from the poorly sporulating, doubly deleted diploid strain
(sps18/19) were sensitive to diethyl ether treatment and to lytic enzymes in a manner suggesting a spore wall defect (23). We
found no evidence for reductase requirement for solid oleate and
acetate media-driven sporulation, nor for development of ether resistance in spores formed in liquid acetate medium (data not shown).
SPS18 is therefore likely to play a key role in this
developmental process, and its urgent characterization may also shed
light on its potential involvement in lipid utilization.
There is evidence for both SPS19 transcript accumulation as
well as production of reductase activity in cells transferred to
acetate medium (data not shown), although functionally, the reductase
is dispensable for sporulation in this medium type. There is however no
ambiguity regarding the essential requirement of the reductase for
ascosporogenesis using solid petroselineate medium, on which
sps19 mutants failed to grow, to form clear zones, and to
sporulate. It remains unclear whether the deletant's incapacity for
sporulation on this nonfermentable carbon source is due to its
inability to obtain energy for completion of this process or due to the
potential toxicity of the substrate, and further work is required to
elucidate the role of this auxiliary enzyme in particular, and the
roles of
-oxidation and peroxisomes in general, during fungal
sporulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M90351 (S. cerevisiae), L26050 (human), and S11021 (rat).
We thank Hannelore Wrba, Tanja Kokko, Leila Wabnegger, and Walter Stadler for excellent technical assistance and Arnoud J. Kal for his insights from the inception of this work. We especially thank Geoff Kornfeld for facilitating the liaison between the laboratories where this work was conducted. Aner Gurvitz thanks Professor Helmut Ruis for making his stay in Vienna possible.