From the Department of Physiological Chemistry, Ruhr-Universität Bochum, 44780 Bochum, Germany
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ABSTRACT |
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In Saccharomyces cerevisiae the
metabolic degradation of saturated fatty acids is exclusively confined
to peroxisomes. In addition to a functional -oxidation system, the
degradation of unsaturated fatty acids requires auxiliary enzymes,
including a
2,
3-enoyl-CoA isomerase and an
NADPH-dependent 2,4-dienoyl-CoA reductase. We found both
enzymes to be present in yeast peroxisomes. The impermeability of the
peroxisomal membrane for pyrimidine nucleotides led to the question of
how the NADPH needed by the reductase is regenerated in the peroxisomal
lumen. We report the identification and functional analysis of the
IDP3 gene product, which is a yeast peroxisomal
NADP-dependent isocitrate dehydrogenase. The newly
identified peroxisomal protein is homologous to the mitochondrial Idp1p
and cytosolic Idp2p, which both are yeast NADP-dependent
isocitrate dehydrogenases. Yeast cells lacking Idp3p grow normally on
saturated fatty acids, but growth is impaired on unsaturated fatty
acids, indicating that the peroxisomal Idp3p is involved in their
metabolic utilization. The data presented are consistent with the
assumption that peroxisomes of S. cerevisiae contain
the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate
dehydrogenase, a putative constituent of a peroxisomal
NADPH-regenerating redox system.
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INTRODUCTION |
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Peroxisomes harbor variable metabolic pathways that differ depending on cell type, developmental stage, and food supply (1, 2). In reference to the multiplicity of cellular functions and to the ability of cells to adjust the enzymatic equipment as well as the size and number of these organelles in response to the cellular demand, peroxisomes are appropriately called multipurpose organelles (3). The importance of peroxisomes for cellular function is especially emphasized by a number of inherited diseases in humans that are caused by peroxisomal dysfunction and usually have profound clinical consequences (4).
A typical metabolic pathway of peroxisomes is the -oxidation of
fatty acids (5, 6). In fact, whereas the presence of a mitochondrial
-oxidation system is restricted to mammalian cells and a few
protists (7), the fatty acid oxidation in peroxisomes is nearly
ubiquitous among eukaryotic cells (7, 8). The peroxisomal and the
mitochondrial degradation of fatty acids is performed by functionally
comparable but genetically distinct proteins (8, 9). In fungi and
plants, the degradation of fatty acids exclusively takes place in
peroxisomes, and growth on fatty acids results in proliferation of
peroxisomes accompanied by a massive induction of peroxisomal proteins
including the
-oxidation enzymes (7, 10).
In addition to the chain shortening -oxidation system, the oxidation
of unsaturated fatty acids requires auxiliary enzymes for the
elimination of the double bonds (8). Degradation of unsaturated fatty
acids with odd-numbered double bonds requires a
2,
3-enoyl-CoA
isomerase (Fig. 1B) (11). For the degradation of unsaturated
fatty acid with even-numbered double bonds, an NADPH-dependent 2,4-dienoyl-CoA reductase is needed in
addition to the isomerase (Fig. 1A) (12). Recently, a novel
pathway for the degradation of unsaturated fatty acids with double
bonds at odd-numbered carbon atoms has been described that also
requires the NADPH-dependent reductase described above
(Fig. 1C) (13, 14). The
successive reduction and isomerization of double bonds by these
auxiliary enzymes results in the formation of intermediates that can
reenter the
-oxidation spiral (8). The presence of both the
2,
3-enoyl-CoA isomerase and the NADPH-dependent
2,4-dienoyl-CoA reductase has been demonstrated in all peroxisomes
studied so far (8). As the peroxisomal membrane has been suggested to be impermeable for small solutes (15), the requirement of the peroxisomal enoyl-CoA reductase for NADPH raises the question of the
existence of an NADPH regenerating system in peroxisomes.
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We applied a reverse genetic approach to identify proteins essential for peroxisome function in Saccharomyces cerevisiae. Here we report the identification, characterization, and functional analysis of a peroxisomal NADP-dependent isocitrate dehydrogenase. Deficiency in this enzyme resulted in an impaired growth of S. cerevisiae on unsaturated fatty acids, whereas growth on saturated fatty acids was not affected. The peroxisomal Idp3p is suggested to be involved in the regeneration of the NADPH needed for the peroxisomal degradation of unsaturated fatty acids.
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EXPERIMENTAL PROCEDURES |
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Strains, Growth Conditions, and General Methods--
The yeast
strains used in this study were S. cerevisiae wild-type
UTL-7A (MATa, ura3-52, trp1, leu2-3,112),
pex7 (MATa, pex7::LEU2, ura3-52,
trp1)) (16), idp3
(MATa, ura3-52, trp,
leu2-3,112, idp3::kanMX4) (this paper),
idp1
(MATa, leu2-3,112, his3-1, trp1-289, idp1::URA3) (17), idp1,idp3
(MATa, leu2-3,112, his3-1, trp1-289, idp1::URA3,
idp3::kanMX4) (this paper),
idp1/idp2
(MATa, leu2-3,112, his3-1,
trp1-289, idp1::URA3, idp2::URA3) (18), and
idp1/idp2/idp3
(MATa, leu2-3,112, his3-1, trp1-289,
idp1::URA3, idp2::URA3,
idp3::kanMX4) (this paper).
Purification and Amino Acid Sequencing of Idp3p-- High salt-extracted peroxisomal membranes were prepared from oleic acid-induced SKQ2N cells. Further separation of the peroxisomal membrane proteins was achieved by reverse-phase HPLC1 according to Erdmann and Blobel (24).
For sequencing of Idp3p, the SDS samples of HPLC fractions containing the protein (fractions 42-46; see Fig. 2) were pooled and separated on a 12% SDS-polyacrylamide gel. Polypeptides were electrophoretically transferred onto a polyvinylidene difluoride membrane and visualized with 0.1% Amido Black in 10% acetic acid. Idp3p was excised, and Lys-C-derived peptides of the protein were separated by HPLC and subjected to sequence analysis on a gas phase sequenator according to Fernandez et al. (25). Protein sequence analysis was provided by the Rockefeller University Protein Sequencing Facility, which is supported in part by National Institutes of Health shared instrumentation grants and by funds provided by the U.S. Army and Navy for the purchase of equipment.Isolation and Sequencing of IDP3--
According to the obtained
internal sequences of Idp3p, degenerated sense
(5-GCGAATTCA(C/T)CCIAT(A/C/T)GT(A/G/C/T)GA(A/G)ATG-3
) and antisense
(5
-TCTAAGCTT(A/G/C/T)GCIAC(C/T)TC(A/G)TC(T/G/A)AT-3
) oligonucleotide primers, distinguishing between the
IDP3 gene and the highly homologous IDP1 and
IDP2 genes were designed. The corresponding genomic region
of the IDP3 gene was amplified by the polymerase chain
reaction with yeast genomic DNA (100 ng; Promega, Madison, WI) as
template. The amplification product was isolated and subcloned into a
derivative pBluescript SK(+) using the Srf1 kit (Stratagene,
La Jolla, CA), resulting in pSRF-IDP3. The authenticity of
the insert was confirmed by DNA sequencing. A
[32P]dATP-labeled probe of 520 base pairs was generated
by PCR with oligonucleotide primers iso6 (5
-GCCACTATAACACCCGATG-3
)
and iso1 (5
-CGTACGTTATTTTTAAAGCCTG-3
) and the plasmid
pSRF-IDP3 as template, using a random-primed labeling kit
(Boehringer, Mannheim, Germany). High stringency hybridization
according to Maniatis et al. (26) was performed to screen a
YEp13-based yeast genomic library (27) that was kindly provided by M. Bolotin-Fukuhara. Two positive clones containing the IDP3
gene were isolated, and sequencing was directly performed on one of the
plasmids, YEp13-IDP3, with an automatic sequencer (model
373A; Applied Biosystems, Weiterstadt, Germany), the DyeDesoxy
terminator cycle sequencing kit (Applied Biosystems), and synthetic
oligonucleotides. Both strands of the IDP3 gene were
sequenced.
Disruption of the Genomic IDP3 Gene--
To construct a
idp3 null mutant, the entire IDP3 open reading
frame was replaced by the kanMX4 marker gene (28). The
PCR-derived construct for disruption comprised the kanMX4
gene flanked by short (40-base pair) homology regions to the
IDP3 3- and 5
-noncoding region. PCR primers were
5
-CACAAGCAACACTTTAGAGATAGTTGTCCAAGTTAAAATGCGTACGCTGCAGGTCGAC-3
(KU179) and
5
-GGCCAGACTTGTCTTTTCAAATGAATGGCGGATTGGTTTAATCGATGAATTCGAGCTCG-3
(KU180), and plasmid pFA6a-kanMX4 served as template.
The resulting amplification construct was introduced into S. cerevisiae wild-type UTL-7A, the idp1
mutant (17),
and the idp1
/idp2
double mutant (18).
Geneticin-resistant clones were selected by growth on YPD plates
containing 200 mg/liter G418 (28).
Antibodies and Immunoblots--
For generating antibodies
against Idp3p, the IDP3-fragment IDP3* (144-210
amino acids) was amplified by PCR using GenBankTM plasmid
YEp13-IDP3 and oligonucleotides KU188
(5-CCGGAATTCCGGGATCCGATCAAGATTAAAAAAGCA-3
) and KU189
(5
-TGCTCTAGACTGCAGCTACTCGAGTGTAAAGAATAACGGTAG-3
). The PCR product
was digested with EcoRI and XbaI and inserted into pBluescript SK(+) (Stratagene, La Jolla, CA) to create
pSKIDP3*. The BamHI/HindIII fragment
of pSKIDP3* was subcloned into pET21b (Novagen), leading to
plasmid pET-IDP3*. The plasmid was transformed into
Escherichia coli BL21-DE3, resulting in an
isopropyl-1-thio-
-D-galactopyranoside-inducible expression of HIS6-tagged Idp3p*. The tagged Idp3p* was
purified by affinity chromatography on a nickel-nitrilotriacetic acid
resin (Quiagen, Hilden, Germany) according to the manufacturer's
protocol. Rabbit polyclonal antibodies to HIS6-tagged
Idp3p* were produced by Eurogentec (Seraing, Belgium) according to
standard methods (29).
Construction and Expression of Idp3pCKL--
To construct a
Idp3p
CKL, the IDP3 gene was amplified by PCR using
plasmid YEp13-IDP3 (see above) and oligonucleotides KU308 5
-CCGCTCGAGGGCTGGTGAAAAGACAGT-3
and KU311
5
-CGCGGATCCTTACATACCTTTCTTGTCTTCAT-3
. The amplification product
was isolated and subcloned (BamHI/XhoI) into the
vector pRSterm, a pRS316 derivative (35) that contained the
HincII/KpnI-prepared CYC1 terminator
of pTerm1 (36). The resulting plasmid pRS-IDP3
CKL was
transformed into idp3
, resulting in an expression of
Idp3p
CKL under the control of its own promoter.
Cell Fractionation-- Spheroplasting of yeast cells, homogenization, and differential centrifugation at 25,000 × g of homogenates were performed as described previously (19).
For subfractionation by isopycnic sucrose density gradient centrifugation, cell lysates or organellar pellets were loaded onto linear 20-53% sucrose density gradients (34). Centrifugation, fractionation of gradients, and preparation of the samples for SDS-PAGE were carried out as described (24). The suborganellar localization of proteins was determined by extraction of 25,000 × g organellar pellets with low salt (10 mM Tris/HCl, pH 8.0; 1 mM PMSF), high salt (10 mM Tris/HCl, pH 8.0; 500 mM KCl; 1 mM PMSF), or pH 11 buffer (0.1 M Na2CO3, 1 mM PMSF) as described by Erdmann and Blobel (37).Protease Protection-- Peroxisomal peak fractions from a sucrose density gradient were pooled and diluted five times in gradient buffer (34). Peroxisomes were sedimented at 25,000 × g for 30 min and subsequently resuspended in homogenization buffer (19) without protease inhibitors but supplemented with 50 mM KCl. Equal amounts of isolated peroxisomes were incubated with increasing amounts of proteinase K for 10 min on ice. Protease was inactivated immediately after the incubation with 4 mM PMSF, proteins were precipitated with trichloroacetic acid, and samples were processed for SDS-PAGE.
Heterologous Expression, Purification, and Characterization of
Idp3p--
The IDP3-orf was amplified by PCR using plasmid
YEp13-IDP3 as template and oligonucleotides KU235
(5-CGGAATTCCCATATGAGTAAAATTAAAGTTGTT-3
) and KU236
(5
-CCCTCGAGTAGTTTGCACATACCTTTC-3
). The PCR product was digested with
EcoRI and XhoI and subcloned into pET21b
(Novagen), leading to plasmid pET-IDP3. The plasmid was
introduced into E. coli BL21-DE3, resulting in an
isopropyl-1-thio-
-D-galactopyranoside-inducible expression of HIS6-tagged Idp3p. The tagged Idp3p was
purified by affinity chromatography on a nickel-nitrilotriacetic acid
resin (Quiagen, Hilden, Germany) according to the manufacturer's
protocol.
Analytical Procedures--
NADP-specific isocitrate
dehydrogenase activity was determined as described by Loftus et
al. (18). Catalase and fumarase were assayed as described by
Moreno de la Garca et al. (38). 3,
2-Enoyl-CoA
isomerase was assayed spectrophotometrically at 340 nm in a coupled
assay (39) with 3-trans-decanoyl-CoA as substrate.
2,4-Dienoyl-CoA reductase activity was determined
spectrophotometrically at 340 nm with
2-trans-4-trans-decanoyl-CoA as substrate
according to Kunau and Dommes (12). Total protein was measured by the BCA method (Pierce) using bovine serum albumin as a standard.
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RESULTS |
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Isolation and Identification of Idp3p-- Peroxisomes were isolated from oleic acid-induced S. cerevisiae cells and successively extracted by low and high salt treatments. The proteins of high salt extracted peroxisomes were solubilized with SDS and separated by HPLC and SDS-PAGE (Fig. 2). Lys-C-derived internal fragments of the 45-kDa protein marked in Fig. 2 were subjected to amino-terminal protein sequencing in preparation for DNA cloning and sequencing of the corresponding gene (see "Experimental Procedures"). The open reading frame of the isolated DNA fragment encoded a new protein with a calculated molecular mass of 48 kDa (Fig. 3) that later on also appeared in the yeast genome data base as open reading frame YNL009w. A search of the GenBankTM data base with the predicted amino acid sequence of IDP3 revealed the yeast genes IDP1 (17) and IDP2 (18) as close relatives of the newly identified gene, hereafter referred to as IDP3. The overall identity of Idp3p with Idp1p and Idp2p is 68 and 70%, respectively (Fig. 4). Idp1p and Idp2p represent the two NADP-dependent isocitrate dehydrogenases reported for S. cerevisiae to date. Idp2p is localized in the yeast cytosol, and Idp1p is a mitochondrial isoenzyme that differs from the other two proteins by an N-terminal extension, which functions as a mitochondrial targeting signal (Fig. 4) (18, 40). Idp3p lacks the mitochondrial targeting signal and instead is characterized by an additional nine amino acids at the extreme C terminus. These terminal amino acids of Idp3p comprise the tripeptide cysteine-lysine-leucine (CKL; Fig. 3), a putative peroxisomal targeting signal 1 (PTS1) for S. cerevisiae (41, 42). The prominent presence in the HPLC profile of peroxisomal proteins (Fig. 2), the sequence similarity to Idp1p and to Idp2p (Fig. 4), and the presence of the putative peroxisomal targeting signal (PTS1; Fig. 3) suggested that Idp3p might be a peroxisomal NADP-dependent isocitrate dehydrogenase.
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IDP3 Is Induced upon Growth on Oleic Acid--
Antibodies were
generated against an internal fragment of Idp3p comprising amino acids
144-210, which displays the lowest similarity of the protein to Idp1p
and Idp2p (Fig. 4). A polypeptide with the predicted molecular mass for
Idp3p (48 kDa) was detected in wild-type but not in idp3
yeast extracts (Fig. 5A).
Although binding of the antibodies to other proteins was observed under low stringency conditions, none of these bands disappeared in mutants
lacking either Idp1p or Idp2p, indicating that the antibodies generated
do not recognize Idp1p or Idp2p but are specific for Idp3p (Fig.
5A).
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Peroxisomes of S. cerevisiae Contain an NADP-dependent
Isocitrate Dehydrogenase--
The subcellular localization of
NADP-dependent isocitrate dehydrogenases in S. cerevisiae
was first analyzed by differential centrifugation of cell homogenates
from oleic acid-induced wild-type yeast cells. More than 60% of the
Idp activity was found in the supernatant fraction, suggesting that the
cytosolic isoform might be responsible for the majority of the
endogenous enzyme activity (data not shown). Sedimented organelles were
further fractionated by sucrose density gradient centrifugation.
NADP-dependent isocitrate dehydrogenase activity was found
in both the mitochondrial and the peroxisomal fractions (Fig.
6). The peroxisomal peak, however, comprised a smaller fraction of the total particular enzyme activity. To exclude the possibility that the activity found in the peroxisomal fraction was due to a mitochondrial contamination, the subcellular localization of the enzymes was also analyzed in a idp1
mutant strain, lacking the mitochondrial NADP-dependent
isocitrate dehydrogenase. After differential centrifugation of cell
homogenates of the idp1
mutant strain, about 15% of the
total NADP-dependent isocitrate dehydrogenase activity was
still localized to the organellar fraction. Subsequent sucrose density
gradient fractionation confirmed the absence of the mitochondrial
enzyme and demonstrated the activity to exclusively co-segregate with
peroxisomal marker enzymes (Fig. 6), consistent with the presence of a
peroxisomal isoenzyme of the NADP-dependent isocitrate
dehydrogenases in S. cerevisiae.
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The Peroxisomal NADP-dependent Isocitrate Dehydrogenase
Activity Is Performed by Idp3p--
As a first step to analyze whether
the peroxisomal NADP-dependent isocitrate activity is due
to Idp3p, we studied the subcellular localization of the protein.
Immunological detection of Idp3p in fractions generated by differential
centrifugation of yeast cell homogenates revealed that the protein is
exclusively found in the organellar pellet (Fig.
7A). Immunoblot analysis of
fractions gained by subsequent sucrose density gradient centrifugation
of the organellar pellets demonstrated the protein to be exclusively localized in the peroxisomal fractions (Fig. 7B). In
agreement with Idp3p being responsible for the peroxisomal
NADP-dependent isocitrate dehydrogenase activity, the
absence of this protein in idp3 mutant cells correlated
with the disappearance of the enzyme activity in the organellar pellet
and in the peroxisomal fractions of sucrose density gradients (Fig.
7).
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Idp3p Is Localized in the Peroxisomal Lumen-- An organellar fraction isolated from spheroplasts of yeast wild-type cells was subjected to extraction by low salt, high salt, and carbonate at pH 11 according to Ref. 37. Idp3p was resistant to low salt extraction but was released by high salt and carbonate treatment of the organelles (Fig. 10A). These extraction properties distinguished Idp3p from two other peroxisomal proteins. Pex3p was resistant to all treatments, consistent with it being an integral membrane protein (34). As expected for a matrix protein, peroxisomal thiolase (Fox3p) (30) was extracted by all treatments. The extractability of Idp3p by carbonate treatment suggested that Idp3p does not span the peroxisomal membrane. Idp3p also does not seem to be tightly associated with the peroxisomal membrane, since part of the protein could be extracted with high salt. The extraction properties of Idp3p are similar to those observed for Pcs60p, a protein of the peroxisomal matrix that is also loosely associated with the peroxisomal membrane (32). To distinguish whether Idp3p is associated with the outer aspects of peroxisomes or whether the protein resides in the peroxisomal lumen, we analyzed the sensitivity of organellar Idp3p to externally added proteases. In the presence of detergents and proteases, all proteins were rapidly degraded. When detergents were present, degradation of proteins was also observed without the addition of protease, presumably due to the liberation of endogenous proteases (data not shown). However, in the absence of detergents, both the intraperoxisomal thiolase (Fox3p) and Idp3p were protected against added proteases (Fig. 10B). Under the same conditions, Pex14p, which is located at the cytosolic face of the peroxisomal membrane (33), was rapidly degraded. Taken together, these results are consistent with an intraperoxisomal localization of Idp3p.
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Peroxisomal Targeting of Idp3p Depends on the Presence of the Three
C-terminal Amino Acids--
The amino acids CKL at the extreme C
terminus of Idp3p fit the consensus for a yeast PTS1 (41, 42). To
analyze whether this putative PTS1 of Idp3p is functional, we analyzed
the subcellular localization of a mutated Idp3pCKL lacking the last
three amino acids. Idp3p
CKL was expressed in an idp3
strain, and localization of the protein was determined by subcellular
fractionation of whole-cell homogenates on sucrose density gradients.
Idp3p
CKL did not co-segregate with the peroxisomal markers but
instead was exclusively found in the loading zone of the gradient,
suggesting a cytosolic localization of the protein (data not shown).
This result indicated that the last three amino acids of Idp3p are essential for the peroxisomal targeting of the protein. The presence of
a functional PTS1 in Idp3p is in line with the observed protease resistance of the protein (Fig. 10B), since this signal
sequence is known to target proteins to the peroxisomal matrix (41,
45).
Idp3p Is Required for the Peroxisomal Degradation of Unsaturated
Fatty Acids--
In search for the function of Idp3p in peroxisomal
metabolism, we tested the growth abilities of idp3 cells
on different carbon sources. Cells grew normally on medium containing
glucose, glycerol, or stearate as a single carbon source (Fig.
11A). Also, on oleic acid
plates, no significant growth differences between wild-type and
idp3
mutant cells were observed (Fig. 11B). In
liquid oleic acid medium, however, the generation time of
idp3
mutant cells increased from 8 h as determined
for the wild type to 12 h for the mutant (Fig. 11B).
Because the only difference between stearic acid and oleic acid is the
presence of one double bond in position 9, the observed growth defect
suggested that the peroxisomal Idp3p might play a role in the
degradation of unsaturated fatty acids. This assumption was further
supported by the complete inability of cells lacking Idp3p to grow on
petroselinic acid, an unsaturated fatty acid that contains a double
bond at position 6 (Fig. 11C). The observed growth defects
on oleic acid and petroselinic acid medium were complemented upon
transformation of the idp3
mutant with the wild-type
IDP3 gene (Fig. 11, B and C). These
results confirmed that the impaired growth of idp3
mutant
cells on unsaturated fatty acids was indeed caused by the lack of
Idp3p.
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Yeast Peroxisomes Contain Auxiliary Enzymes Needed for the
Degradation of Unsaturated Fatty Acids--
The ability of S. cerevisiae to grow on unsaturated fatty acids as the single carbon
source (Fig. 11), the presence of an NADP-dependent isocitrate dehydrogenase in peroxisomes (Fig. 6), and its suggested role of supplying NADPH for the degradation of unsaturated fatty acids
encouraged us to search for auxiliary enzymes of this pathway. Both the
2,
3-enoyl-CoA isomerase and the NADP-dependent
2,4-dienoyl-CoA reductase activities were detected in whole-cell yeast
lysates (data not shown). For subcellular localization of the
activities, wild-type yeast homogenates were subjected to sucrose
density gradient centrifugation, which did result in a clear separation of peroxisomes and mitochondria as judged by organelle-specific marker
enzymes (Fig. 12). Both the
2,
3-enoyl-CoA isomerase and the NADP-dependent
2,4-dienoyl-CoA reductase activities co-segregated with the peroxisomal
marker catalase, demonstrating that both enzymes are localized in
peroxisomes of S. cerevisiae (Fig. 12). These data suggest
that peroxisomes of S. cerevisiae harbor the entire enzyme
equipment needed for the utilization of unsaturated fatty acids,
including an NADP-dependent isocitrate dehydrogenase, a
putative component of an NADPH-regenerating system.
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DISCUSSION |
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Here we report on the molecular identification and functional
characterization of a peroxisomal NADP-dependent isocitrate dehydrogenase of S. cerevisiae. In line with a role in the
peroxisomal metabolism of unsaturated fatty acids, the Idp3p has been
demonstrated to be exclusively peroxisomal, and the protein was shown
to be essential for the growth of S. cerevisiae on
unsaturated fatty acids but dispensable for growth on saturated fatty
acids. The supposed function of the protein in peroxisomal fatty acid
metabolism is the regeneration of NADPH that is needed by the
NADPH-dependent 2,4-dienoyl-CoA reductase for the reductive
elimination of double bonds of unsaturated fatty acids. This reductase
and the 2,
3-enoyl-CoA isomerase, another auxiliary enzyme needed
for the degradation of unsaturated fatty acids, have been localized to
yeast peroxisomes (Fig. 12). The presence of these enzyme activities in
peroxisomes has far reaching implications for our understanding of the
peroxisomal metabolism and transport of metabolites across the
peroxisomal membrane. The data presented are consistent with the
assumption that peroxisomes of S. cerevisiae maintain the
entire enzyme equipment needed for the degradation of unsaturated fatty
acids, including an NADP-dependent isocitrate
dehydrogenase, a putative constituent of a peroxisomal
NADPH-regenerating redox system.
Idp3p was isolated from peroxisomes of oleic acid-induced yeast cells (Fig. 2), and peptide sequence data of the protein were instrumental in cloning the corresponding gene from a genomic yeast library (Fig. 3). Idp3p is exclusively localized in peroxisomes, and consistent with its function in peroxisomal fatty acid metabolism, Idp3p was highly induced upon growth on oleic acid (Figs. 5-7). Protease protection data suggested that the protein resides in the peroxisomal lumen (Fig. 10), which is further supported by the observation that peroxisomal targeting of Idp3p depends on the presence of a C-terminal type 1 peroxisomal targeting signal (data not shown), known to target proteins from the cytosol across the peroxisomal membrane barrier into the peroxisomal matrix (41, 45, 46). That the peroxisomal Idp3p indeed is an NADP-dependent isocitrate dehydrogenase was confirmed by the characterization of the enzymatic properties of purified, recombinant Idp3p (Fig. 8). Interestingly, an NADP-dependent isocitrate dehydrogenase has also been detected in peroxisomes of the n-alkane-utilizing yeast C. tropicalis (44).
Beside the peroxisomal Idp3p, three yeast isoenzymes of isocitrate
dehydrogenase have been described that catalyze the oxidative decarboxylation of isocitrate to -ketoglutarate. The NAD-specific mitochondrial isoenzyme is an octamer of two nonidentical subunits designated Idh1p and Idh2p (47, 48) and is believed to catalyze a key
regulation step in the tricarbonic acid cycle. Less clear are the
functions of the two NADP-specific isoenzymes located in mitochondria
and the cytosol (17, 18). The glutamate auxotrophy upon deletion of
both Idp1p and Idh1p suggest that both enzymes contribute to the
anaplerotic supply of
-ketoglutarate for glutamate formation (40).
Furthermore, as isocitrate and
-ketoglutarate can traverse the
mitochondrial membrane via specific transporters (49), it has been
suggested that the proteins may participate in an intercompartmental
exchange of reducing equivalents (18). This raises the question of
whether Idp3p might play a comparable role in the peroxisomal
metabolism. In the cytosol, NADPH is generated by, for instance, the
pentose phosphate pathway. However, because of the impermeability of
the peroxisomal membrane for pyrimidine nucleotides (15), the cytosolic
NADPH pool cannot directly account for the peroxisomal need for NADPH.
This emphasizes the necessity for an NADPH-regenerating system in the
peroxisomal lumen. Because the formation of
-ketoglutarate for the
production of glutamate is primarily catalyzed by the yeast
mitochondrial NAD-dependent and NADP-dependent
isocitrate dehydrogenases (40), the most likely biological function of
Idp3p is the regeneration of NADPH. The involvement of Idp3p in the
intraperoxisomal regeneration of NADPH, which is necessary for the
degradation of unsaturated fatty acids, is also more in agreement with
the peroxisomal localization and with the oleic acid inducibility of
the protein.
The requirement of the peroxisomal degradation of fatty acids with
even-numbered double bonds for NADPH is well established (8, 12).
Degradation of these fatty acids in the -oxidation spiral leads to
2,4-dienoyl-CoA intermediates that are reduced to
3-trans-enoyl-CoA in a redox reaction that requires NADPH
and that is catalyzed by the peroxisomal NADPH-dependent
2,4-dienoyl-CoA reductase. The resulting 3-trans-enoyl-CoA
is subsequently isomerized to 2-trans-enoyl-CoA, which can
be reintroduced into the
-oxidation spiral (Fig. 1). The assumption
that Idp3p provides the NADPH for this chain of reactions is supported
by the observation that cells lacking the protein grow normally on
stearate (C18:0) but have lost the ability to grow on petroselinic acid
(
6-C18:1; Fig. 11).
Until recently, it was generally believed that unsaturated fatty acids
with double bonds extending from odd-numbered carbon atoms are
chain-shortened to 3-cis-enoyl-CoA esters, which after isomerization to 2-trans-enoyl-CoA are further degraded by
the -oxidation spiral (Fig. 1B) (11). According to this
pathway, NADPH would not be needed for the metabolization of these
unsaturated fatty acids (Fig. 1). However, Tserng and Jin (50) reported that in mammalian cells also the degradation of unsaturated fatty acids
with double bonds extending from odd-numbered carbon atoms requires
NADPH. This observation gained support by the exploration of a novel
pathway for the reductive removal of odd-numbered double bonds of fatty
acids (Fig. 1C) (13). According to this pathway, a
3,5,
2,4-dienoyl-CoA isomerase, together with the
NADPH-dependent 2,4-dienoyl-CoA reductase and the
3,
2-dienoyl-CoA isomerase facilitate the reduction of
odd-numbered double bonds as illustrated in Fig. 1. Recently, it has
been suggested that this novel pathway might also be responsible for
the degradation of odd-numbered double bonded fatty acids in mammalian
peroxisomes (8, 14). In this respect, it is interesting to note that
also yeast cells lacking Idp3p are less capable than the wild type of
growing on oleic acid (
9-C18:1) as the single carbon source (Fig.
11).
The peroxisomal localization of the Idp3 leads to questions about the
origin of the isocitrate and the fate of the -ketoglutarate that
is produced. The most simple explanation would be that
-ketoglutarate is exported directly in exchange for isocitrate as
has been demonstrated for mitochondria (49). In principle, isocitrate
could also form in peroxisomes from the citrate that is generated by
the fusion of acetyl-CoA with oxalacetate, catalyzed by the peroxisomal
citrate synthase (Cit2p) (15, 51). However, despite efforts, an
aconitase activity has not yet been detected in yeast peroxisomes, thus making the peroxisomal formation of isocitrate from citrate rather unlikely. The direct import of isocitrate from the cytosol into the
peroxisomal lumen would predict the existence of a peroxisomal membrane
transporter for isocitrate; however, experimental evidence for such a
transporter is still missing. In general, our knowledge on the influx
and efflux of peroxisomal metabolites and especially on the nature of
the carriers involved is still rather limited. For S. cerevisiae, only two peroxisomal metabolite carriers have been
described. The heterodimeric Pat1p/Pat2p ABC-transporter has been
suggested to participate in the peroxisomal import of acyl-CoA esters
(52), and the peroxisomal carnitin acetyl transferase is involved in
the export of the
-oxidation-derived acetyl-CoA (15, 53).
The data presented here are consistent with the idea that peroxisomes of S. cerevisiae maintain the entire enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system. The latter supports the notion of an involvement of peroxisomes in an intercompartmental exchange of reducing equivalents and predicts novel peroxisomal metabolite transporters as constituents of a redox shuttle across the peroxisomal membrane.
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ACKNOWLEDGEMENTS |
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We thank Lee McAlister-Henn for providing the yeast idp1 and idp2 mutant strains. We thank Ulrike Freimann and Uta Ricken for technical assistance and Siegrid Wüthrich for photograph illustration service. We are grateful to Wolf-Hubert Kunau, Andreas Hartig, Rainer Rhodemann, and Ursula Dorpmund for reagent supply. We are grateful to Kai Erdmann, Peter Rehling, and Jürgen Saidowsky for fruitful discussions. We thank Gabi Dodt, Michael Schwierskott, and Michael Linkert for reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Er 178/2-1.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.
Present address: Institut de Genetique et Microbiologie,
Universite Paris Sud, 91405 Orsay Cedex, France.
§ To whom correspondence should be addressed: Ruhr-Universität Bochum, Insitut für Physiologische Chemie, Universitätsstr. 150, 44780 Bochum, Germany. Tel.: 0234-700-4947; Fax: 0234-709-4279; E-mail: Ralf.Erdmann{at}rz.ruhr-uni-bochum.de.
1 The abbreviations used are: HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PTS1, peroxisomal targeting signal 1.
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REFERENCES |
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