Plant Physiol. (1998) 118: 935-943
Biosynthesis of Lipoic Acid in Arabidopsis:
Cloning and Characterization of the cDNA for Lipoic Acid
Synthase1
Rie Yasuno and
Hajime Wada*
Department of Biology, Faculty of Science, Kyushu University,
Ropponmatsu, Fukuoka 810-8560, Japan
 |
ABSTRACT |
Lipoic acid is a coenzyme that is
essential for the activity of enzyme complexes such as those of
pyruvate dehydrogenase and glycine decarboxylase. We report here the
isolation and characterization of LIP1 cDNA for lipoic
acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag
homologous to the lipoic acid synthase of Escherichia
coli. This cDNA was shown to code for
Arabidopsis lipoic acid synthase by its ability to complement a
lipA mutant of E. coli defective in
lipoic acid synthase. DNA-sequence analysis of the LIP1
cDNA revealed an open reading frame predicting a protein of 374 amino
acids. Comparisons of the deduced amino acid sequence with those of
E. coli and yeast lipoic acid synthase homologs showed a
high degree of sequence similarity and the presence of a leader
sequence presumably required for import into the mitochondria.
Southern-hybridization analysis suggested that LIP1 is a
single-copy gene in Arabidopsis. Western analysis with an antibody
against lipoic acid synthase demonstrated that this enzyme is located
in the mitochondrial compartment in Arabidopsis cells as a 43-kD
polypeptide.
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INTRODUCTION |
Lipoic acid (6,8-thioctic acid) is a sulfur-containing coenzyme
that is required for the activity of enzyme complexes involved in the
oxidative decarboxylation of 
-ketoacids (Reed and Hackert, 1990
;
Perham, 1991; Mattevi et al., 1992) and in the Gly-cleavage system
(Fujiwara et al., 1990; Kim and Oliver, 1990; Macherel et al., 1990).
There are five lipoyl proteins: the dihydrolipoamide acyltransferase
subunits of pyruvate, -ketoglutarate, branched-chain -ketoacid
dehydrogenase complexes, protein X of the pyruvate dehydrogenase
complex, and the H-protein of the Gly-cleavage system. Lipoic acid is
covalently bound to these proteins via an amide linkage to the
-amino group of specific Lys residues (Reed and Hackert, 1966). The
lipoyl-Lys arm functions as a carrier of reaction intermediates, and
reducing equivalents interact with the active sites of the components
of the complexes (Yeaman, 1989; Douce et al., 1994).
Despite the importance of the lipoyl-prosthetic group in the
functioning of several enzyme complexes, the biosynthesis of lipoic
acid is not well understood in any organism. Molecular genetic studies
(Vanden Boom et al., 1991; Reed and Cronan, 1993; Morris et al., 1994,
1995) of Escherichia coli have identified three genes,
lipA, lipB, and lplA that are involved
in the biosynthesis and transfer of lipoic acid. lipA
encodes a lipoic acid synthase that is required for an insertion of the
first sulfur atom into the octanoate backbone (Reed and Cronan, 1993).
lplA and lipB encode lipoate ligases that are
involved in the transfer of lipoic acid to cognate proteins (Morris et
al., 1994, 1995). LplA and LipB proteins primarily function in the
utilization of lipoic acid exogenously added to the growth medium and
endogenously synthesized lipoic acid in E. coli cells,
respectively (Morris et al., 1994, 1995). LplA protein is involved not
only in biosynthesis of lipoyl-AMP with lipoic acid and ATP but also in
the transfer of the lipoyl group from lipoyl-AMP to cognate proteins
(Morris et al., 1995). Although LipB protein also transfers the lipoyl
group to cognate protein, this transfer involves the lipoyl group in
lipoyl-ACP (Jordan and Cronan, 1997).
Biosynthesis of lipoic acid in E. coli has also been studied
using labeling experiments. Parry (1983) demonstrated that octanoic acid is a direct precursor of lipoic acid and 8-thiooctanoic and 6-thiooctanoic acids are possible intermediates in lipoic acid biosynthesis. This finding is consistent with the fact that the E. coli lipA mutant is complemented by the addition of both
thiooctanoic acids (Reed and Cronan, 1993) and suggests the presence of
a second enzyme involved in insertion of a second sulfur atom into
thiooctanoic acids.
Although the biosynthesis of lipoic acid in E. coli has been
studied, much less is known about biosynthesis of lipoic acid in
eukaryotes. Sulo and Martin (1993) isolated the LIP5 gene
from the yeast Saccharomyces cerevisiae by functional
complementation of a mutant defective in biosynthesis of lipoic acid.
DNA-sequence analysis of LIP5 revealed that it encodes a
protein homologous to lipoic acid synthase of E. coli. A
gene for lipoyltransferase, which catalyzes the transfer of the lipoic
acid to cognate proteins, has recently been cloned from another yeast,
Kluyveromyces lactis (Chen, 1997).
Fujiwara et al. (1994, 1996) purified two isoforms of lipoyltransferase
from bovine liver mitochondria using the apoH protein of the
Gly-decarboxylase complex as an acceptor of lipoate. Both isoforms of
lipoyltransferase catalyze the transfer of the lipoyl group from
lipoyl-AMP to apoH protein but have no activity to activate lipoate to
lipoyl-AMP, in contrast to the E. coli lipoyltransferase encoded by the lplA gene (Morris et al., 1995). Furthermore,
these researchers cloned a cDNA for the bovine lipoyltransferase and found that both isoforms are derived from the same translated product
but are processed differently (Fujiwara et al., 1997). We recently
studied fatty acid synthesis in mitochondria isolated from pea and
found that a major part of the de novo-synthesized fatty acids may be
used for the biosynthesis of lipoic acid (Wada et al., 1997). Together
these results have begun to define the biosynthesis of lipoic acid and
its intracellular organization in eukaryotes.
We describe the isolation of an Arabidopsis cDNA, LIP1, that
encodes a lipoic acid synthase and show that this cDNA complements the
E. coli lipA mutant defective in lipoic acid synthase. To our knowledge, this is the first cDNA for lipoic acid synthase that has
been cloned and characterized in a higher organism. We also report the
intracellular localization of the enzyme in Arabidopsis cells and
demonstrate that lipoic acid synthase is located in the mitochondrial
compartment.
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MATERIALS AND METHODS |
Plant Material
Arabidopsis ecotype Columbia was grown under continuous
light (40 µmol photons m
2
s1) at 25°C in trays containing vermiculite.
Leaves, roots, and flowers were harvested from 4-week-old plants and
used for RNA, DNA, protein, and lipoic acid extractions.
Cell Fractionation of Arabidopsis
Twenty grams of leaves from 4-week-old Arabidopsis plants was used
for cell fractionation. Plants were placed in the dark for 12 h
prior to harvesting the leaves. All of the procedures described below
were carried out at 4°C. The leaves were homogenized with a grinder
in 40 mL of medium containing 50 mM Tes-NaOH, pH 7.2, 0.3 M mannitol, 1 mM EDTA, 1 mM
MgCl2, 0.2% (w/v) BSA, 0.5% (w/v) PVP-40, 4 mM Cys, and 10 mM 2-mercaptoethanol. The brei was filtered through eight layers of gauze and centrifuged at 3,300g for 5 min, and then the supernatant was centrifuged
at 10,500g for 20 min. The supernatant obtained after the
10,500g centrifugation was centrifuged at
100,000g for 2 h, and the supernatant and pellet
obtained were used for western analysis as the cytosol and microsome
fractions, respectively.
The pellet obtained after the 10,500g centrifugation was
used for fractionation of chloroplasts and mitochondria as
described by Day et al. (1985) with the following modifications.
The pellet was suspended in 2 mL of medium A containing 20 mM Tes-NaOH, pH 7.2, 2 mM
KH2PO4, 1 mM
EDTA, 2 mM MgCl2, 0.1% BSA, and 14 mM 2-mercaptoethanol, applied onto a stepwise gradient
composed of 2 mL of 21%, 4 mL of 26%, and 2 mL of 47% (w/v) Percoll
solutions, and centrifuged at 65,000g for 45 min in a
swinging-bucket rotor (model RPS-40T, Hitachi, Tokyo, Japan). All of
the Percoll solutions used in the gradient centrifugation were made up
in 10 mM Tes-NaOH, pH 7.2, 0.25 M Suc, and
0.1% BSA.
After the centrifugation chloroplasts and mitochondria were separated
as a green band at the interface between the 21% and 26% Percoll
layers and as a white band at the interface between the 26% and 47%
Percoll layers, respectively. Both chloroplast and mitochondria
fractions were separately recovered from the gradient and diluted six
times with medium B, which contained the same components as medium A
except that it had 2 mM DTT instead of 2-mercaptoethanol
and 2 mM KH2PO4, and then
centrifuged at 12,500g for 20 min. The obtained pellets were
suspended in medium B and used in western analysis as the chloroplast
and mitochondria fractions.
cDNA Cloning and Analysis
An Arabidopsis EST clone, 193K14, which contains an open reading
frame encoding a polypeptide homologous to lipoic acid synthase of
Escherichia coli, was obtained from the Arabidopsis
Biological Resource Center (The Ohio State University, Columbus). The
5
-terminal region of Arabidopsis LIP1 cDNA was amplified by
the 5-RACE method (5-Full RACE Core Set, Takara, Shiga, Japan). A
phosphorylated primer, 5-GTGTGTAATCTACGG-3, was used for the
synthesis of cDNA using total RNAs isolated from Arabidopsis leaves.
The following two primer sets were also used for first and second PCR
in the 5 RACE, respectively: a set of 5-CAGCCGCGTGTACATGTATCCCCA-3 and 5-CGGAATTCGAAATGGGATTCAGAT-3 and another set of
5-GACATTTCGCTTCCTCGCAGACGG-3 and 5-CGAAGCTTGATAGAAGCCGATCGA-3.
The amplified DNA was subcloned into a pCRII vector (Original TA
Cloning Kit, Invitrogen, San Diego, CA). The obtained plasmid was
designated pRACE-5 and its nucleotide sequence was determined. The
3-terminal region of LIP1 was also amplified by PCR with 193K14 as the template and subcloned into pBluescript II KS(+) (Stratagene). This clone, designated pBlue-3, was used to determine the sequence of both strands in the 3-terminal region of
LIP1 cDNA.
DNA-sequencing reactions were performed (BcaBest dideoxy
sequencing kit, Takara, Shiga, Japan; Thermo sequenase
sequencing kit, Amersham) and sequences obtained (ALF Red DNA
sequencer, Pharmacia Biotech, Tokyo, Japan; DNA sequencer DSQ-1000,
Shimadzu, Kyoto, Japan). Double-stranded DNAs were used as templates,
and the sequence of each strand was determined. Nucleotide and deduced amino acid sequences were analyzed by suitable software (GENETYX-MAC, Software Development Co., Tokyo, Japan).
Nucleic Acid Extraction and Analysis
Genomic DNA used for Southern analysis was extracted from
Arabidopsis leaves using a DNA-extraction kit (ISOPLANT, Nippon Gene,
Tokyo, Japan). The genomic DNA was digested with the appropriate restriction enzymes, separated by electrophoresis on a 0.8% (w/v) agarose gel, and transferred to a nylon membrane
(Hybond-N+, Amersham). The membrane was
hybridized using a DNA-labeling and -detection system (ECL kit,
Amersham).
Total RNAs used for cDNA synthesis and RT-PCR were extracted from
leaves, roots, and flowers of Arabidopsis using an RNA-extraction kit
(RNeasy plant kit, Qiagen, Chatsworth, CA). RT-PCR analysis of the
LIP1 gene was performed with an RT-PCR kit (SuperScript OneStep System, Life Technologies) using the following two primers: 5-TTGGGGATACATGTACACGC-3 and 5-CTGAATCCCATTTCCATGCC-3. Two micrograms of total RNAs from each organ was used for RT-PCR analysis. As a control, the expression of the RCO1 gene for the
cytosolic form of cyclophilin was also checked by RT-PCR with the same
RNA preparations using the following two primers:
5-ACTTCGACATGACCATCGAC-3 and 5-TTCCCATGAGAACACACACC-3.
Functional Complementation of the E. coli lipA
Mutant
Deletions of the LIP1 cDNA were created using PCR
with specific primers designed to remove varying numbers of residues
from the N terminus of the LIP1 protein. The eight-nucleotide sequence 5-CGCCATGG-3 including an NcoI site was added to the
5 end of each primer. PCR products were digested with
NcoI and ligated into the NcoI site of an
expression vector, pKK233-2 (Clontech, Palo Alto, CA), to give an
in-frame desired product. The obtained plasmids were designated
pLIP1-
0, pLIP1-18, and pLIP1-26, where the number after the
represents the number of amino acid residues deleted from the N
terminus of LIP1 protein. These plasmids were used for transformation
of the E. coli lipA mutant KER176 (Reed and Cronan,
1993). The transformants were plated on rich-broth medium (Davis et
al., 1992) supplemented with 50 µg mL1 ampicillin, 10 µg mL1 kanamycin, and 50 ng mL1 lipoic
acid.
To determine the effect of the deletions on the function of the
resulting LIP1 proteins, colonies of the transformants on the
rich-broth plate were streaked onto other plates, which contained M9
medium supplemented with 50 µg mL1 ampicillin, 10 µg
mL1 kanamycin, 1 mM MgSO4, 1%
(w/v) succinate, 5 mM acetate, 5 × 105
% (w/v) vitamin B1, 0.4 mM IPTG, and either 0 or 50 ng mL1 lipoic acid, and then the growth of the
transformants on the plates was checked. The transformant of the
E. coli lipA mutant with pKK233-2 was used as a negative
control in these experiments.
Overexpression of LIP1 in E. coli and Antibody
Production
The region of LIP1 cDNA encoding a putative mature
protein was amplified by PCR using the following two primers:
5-CGCCATGGGCTTCTCCTCTTCCTC-3 and 5-CGCCATGGAGTGTGTAATCTACGG-3.
The eight-nucleotide sequence 5-CGCCATGG-3 including an
NcoI site was added to the 5 end of both primers. The
amplified DNA fragment was digested with NcoI and
ligated into the NcoI site of an expression vector,
pET-30a(+) (Novagen, Madison, WI). The resultant plasmid, designated
pET-LIP1, was used for transformation of E. coli
BL21(DE3) (Studier et al., 1990). The transformant of BL21(DE3) with
pET-LIP1 was grown at 37°C in Luria-Bertani medium supplemented with
200 µg mL1 ampicillin. When
A600 of the E. coli
culture reached 0.5, IPTG was added to the medium at a final
concentration of 0.4 mM to induce expression of
LIP1, and the culture was incubated for a further
26 h at 25°C.
Overexpressed LIP1 fusion protein, which contains six His residues at
the N terminus as a tag, was purified by nickel-affinity chromatography
(His-Bind Resin and Buffer Kit, Novagen). Polyclonal antibody was
generated by injecting the purified LIP1 fusion protein into a rabbit.
The antiserum was prepared from the rabbit and the anti-Arabidopsis
LIP1 fusion protein IgG was purified using a kit (ImmunoPure, Pierce).
SDS-PAGE and Western Analysis
SDS-PAGE was performed according to the method of Laemmli (1970)
using a 12% (w/v) polyacrylamide gel. Protein concentration was
determined by the method of Bradford (1976) using BSA as the standard.
Western analysis was carried out according to the method of
Post-Beittenmiller et al. (1991).
Biological Assay of Lipoic Acid
For extraction of lipoic acid, leaves, roots, and flowers of
Arabidopsis were collected from 4-week-old plants, frozen in liquid
nitrogen, and ground with a mortar and pestle. The obtained powders
were suspended in 6 N
H2SO4 and autoclaved for
2 h to release lipoic acids from proteins. After autoclaving, the
suspension was adjusted to pH 7.0 with 4 N NaOH, made up to
a known volume, and filtered to remove any insoluble materials. The
obtained solution was used for biological assay by the turbidimetric
method (Herbert and Guest, 1970) using the E. coli lipA
mutant (Reed and Cronan, 1993) as the test organism.
| |
RESULTS |
Identification of the cDNA Coding for Arabidopsis Lipoic Acid
Synthase
A BLAST search (Pearson and Lipman, 1988) of the database of the
National Center for Biotechnology Information using the amino acid
sequence of E. coli lipoic acid synthase (LipA) identified an Arabidopsis EST clone containing an open reading frame encoding a
polypeptide homologous to lipoic acid synthase of E. coli.
This clone, 193K14, was obtained from the Arabidopsis Biological
Resource Center, and the complete sequence of the 1224-bp cDNA in the
EST clone was determined. The cDNA contains an open reading frame that
codes for a protein with an amino acid sequence similar to those of
lipoic acid synthases of E. coli (Reed and Cronan, 1993) and
yeast (Sulo and Martin, 1993).
Since this open reading frame began at the 5 terminus of the cDNA and
did not begin with a Met residue, it appeared that the cDNA was not a
full-length copy of the corresponding mRNA. Therefore, the 5-terminal
region of the corresponding mRNA was amplified by the 5-RACE method
using cDNAs prepared with total RNAs from leaves. The PCR products
obtained by the 5-RACE method were subcloned into the vector pCRII.
Five cloned PCR products were isolated and sequenced. The sequences of
all the clones overlapped with each other and with the 5-terminal
region of 193K14. The sequence of the longest clone, pRACE-5, extended
the sequence of 193K14 by 105 nucleotides to a total length of 1330 nucleotides.
The 3 region of LIP1 cDNA was also amplified by PCR and
subcloned into pBluescript II KS(+) to determine the nucleotide
sequence of both strands of this region. The obtained clone was
designated pBlue-3. The sequence of pBlue-3 perfectly overlapped with
the 3-terminus region of LIP1 cDNA in 193K14. The
nucleotide sequence of full-length LIP1 cDNA was obtained by
a combination of sequences of 193K14, pRACE-5, and pBlue-3. This
entire nucleotide sequence of the LIP1 cDNA was deposited in
the DDBJ, EMBL, and GenBank nucleotide sequence databases with the
accession no. AB007987. This LIP1 cDNA encodes a polypeptide
of 374 amino acids, which corresponds to a molecular mass of 41,344 D.
The amino acid sequence of LIP1 protein is compared in Figure
1 with those of lipoic acid synthases of
E. coli and yeast. The amino acid sequence identity between
the Arabidopsis LIP1 protein and the lipoic acid synthases of E. coli and Saccharomyces cerevisiae are 44% and 67%,
respectively. Several stretches of conserved residues that may be
important for the function of this enzyme were found. Two conserved C
motifs, C-E-E-A-X-C-P-N-X-X-E-C and C-T-R-X-C-X-F-C, were found at
positions from 103 to 114 and 135 to 142 in the Arabidopsis LIP1
protein, respectively. The second C motif is also conserved in biotin
synthases of Arabidopsis (Patton et al., 1996; Weaver et al., 1996) and
microorganisms (Otsuka et al., 1988; Zhang et al., 1994; Bower et al.,
1996). Since lipoic acid synthase and biotin synthase catalyze a
similar reaction that inserts a sulfur atom into a hydrocarbon chain, the conserved C residues may play an important role in this process. Comparison of the N-terminal region of the sequences clearly indicates that Arabidopsis LIP1 protein contains a 36-residue extension relative
to the lipoic acid synthase of E. coli. The amino acid sequence of this region has some characteristics in common with transit
peptides for targeting to mitochondria (von Heijne et al., 1989; von
Heijne, 1992): an overall positive charge due to the relatively high
proportion of R residues, the lack of negatively charged residues, and
the relatively high proportion of hydroxylated residues. In
addition, the sequence R17-C-F-S is similar to a
motif (R-X-
-X-S), which may represent the potential
cleavage site for a mitochondrial transit peptide (von Heijne, 1992).
If this sequence motif is the actual cleavage site, the mature form of
LIP1 protein is a polypeptide of 356 amino acid residues with a
molecular mass of 39,197 D.
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| Figure 1.
Comparison of the amino acid sequences of lipoic
acid synthases. The deduced amino acid sequence of lipoic acid synthase
encoded by Arabidopsis (Arabi.) LIP1 cDNA is compared
with that of lipoic acid synthases of S. cerevisiae
(Yeast) (Sulo and Martin, 1993) and E. coli (Reed and
Cronan, 1993). The amino acid residues conserved in all sequences are
indicated by asterisks. Hyphens represent gaps to maximize the
alignment of the sequences. The conserved Cys motifs are underlined.
The putative cleavage site for the mitochondrial transit peptide is
indicated by an arrowhead.
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Complementation of the E. coli lipA Mutant
To confirm that LIP1 protein is a lipoic acid synthase of
Arabidopsis, the LIP1 cDNA was expressed in the
E. coli lipA mutant, which is defective in lipoic acid
synthase (Reed and Cronan, 1993). As shown in Figure
2, when the E. coli lipA
mutant was transformed with pKK233-2 (control vector) and plated on
lipoic-acid-free medium, no growth of the transformant was observed,
although the transformant could grow on medium containing lipoic acid.
By contrast, the transformant with the plasmid (pLIP1-0) carrying
the full-length LIP1 cDNA could grow on lipoic-acid-free
medium. These findings clearly demonstrate that Arabidopsis
LIP1 cDNA functionally complements the E. coli
lipA mutant and that the LIP1 cDNA encodes a
lipoic acid synthase.
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| Figure 2.
Complementation of the lipA mutant
of E. coli by expression of Arabidopsis
LIP1 cDNA. The E. coli lipA mutant
(KER176), which is defective in lipoic acid synthase (Reed and Cronan,
1993), was transformed with the plasmids pKK233-2 (control, plates A
and B) and pLIP1-0 (plates C and D). The colonies of the each
transformant were streaked onto plates containing 50 µg
mL1 ampicillin, 50 µg mL1 kanamycin, and
either 50 ng mL1 lipoic acid (plates A and C) or no
lipoic acid (plates B and D), and the plates were incubated at 37°C
for 3 d.
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Deletions of N-terminal residues in the LIP1 protein also resulted in
functional complementation of the E. coli lipA mutant (data
not shown). However, the deletion of 18 residues, which gives a
putative, mature LIP1 protein, enhanced the growth of the mutant, and
the deletion of 26 residues slightly inhibited the growth of the
transformant compared with the full-length clone. These results suggest
that the deleted amino acid residues are not required for catalytic
activity of LIP1 protein.
Expression and Organization of the LIP1 Gene for
Arabidopsis Lipoic Acid Synthase
Figure 3 shows genomic Southern
analysis of the LIP1 gene of Arabidopsis. The 1.1-kb
EcoRI fragment containing Arabidopsis LIP1 cDNA was obtained by digestion of 193K14 with
EcoRI and was used as a probe. When the genomic DNA was
digested with EcoRI, BamHI, or
SalI, a single hybridizing band was detected in all cases, indicating that in Arabidopsis lipoic acid synthase is encoded
by a single gene.
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| Figure 3.
Genomic Southern analysis of the Arabidopsis
LIP1 gene. Genomic DNA was extracted from Arabidopsis
leaves and digested with EcoRI (lane 1),
BamHI (lane 2), and SalI (lane 3). Three
micrograms of DNA was applied to each lane. The 1.1-kb
EcoRI fragment containing LIP1 cDNA was
prepared from the 193K14 clone and used as a probe. The positions of
the DNA size markers (in kb) are indicated on the left.
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To investigate the organ-specific expression of the LIP1
gene, northern analysis was carried out using total RNAs prepared from
leaves, roots, and flowers. However, no clear signal was detected in
the analysis (data not shown), suggesting that the level of expression
of LIP1 gene was low. Therefore, RT-PCR analysis was used to
estimate the level of expression of LIP1 gene in each organ.
Figure 4 shows the results of RT-PCR. In
all tested organs a 628-bp DNA fragment corresponding to
LIP1 cDNA was detected at the same level (lanes 2-4). When
the genomic DNA was used as the template instead of RNA (lane 1), a
700-bp DNA fragment was amplified. The difference between the 628- and
700-bp DNA fragments was most likely caused by the presence of an
intron in this region of the LIP1 gene. The 700-bp DNA
fragment detected with genomic DNA was not detected with total RNAs
prepared from any of the tested organs, suggesting that the total RNAs
used for RT-PCR were not contaminated with genomic DNA. As a control
the level of expression of the RCO1 gene for the cytosolic
form of cyclophilin, which is expressed at the same level in leaves,
roots, and flowers (Lippuner et al., 1994), was also checked by RT-PCR
of the same RNA samples (lanes 5-7). In all tested organs, 628 bp,
which corresponds to RCO1 cDNA, was detected at same level.
These results suggest that the LIP1 gene is expressed at the
same level in all organs of Arabidopsis.
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| Figure 4.
Organ-specific expression of the
LIP1 gene. The level of LIP1 mRNA for
lipoic acid synthase was analyzed by RT-PCR with total RNAs extracted
from leaves (lane 2), roots (lane 3), and flowers (lane 4). In lane 1, genomic DNA was used as the template for RT-PCR instead of RNA. The
level of mRNA for the cytosolic form of cyclophilin was also analyzed
as a control with the same total RNAs extracted from leaves (lane 5),
roots (lane 6), and flowers (lane 7). The positions of the DNA size
markers (in kb) are indicated on the left.
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To prepare polyclonal antibody against Arabidopsis LIP1 protein, LIP1
was overexpressed in E. coli BL21(DE3) as a fusion protein containing a His tag at the N terminus. Figure
5A shows SDS-PAGE analysis of proteins
from BL21(DE3) cells that were transformed with pET-30a(+) and
pET-LIP1. In the transformant with pET-LIP1, a 46-kD protein was
detected, and the level of the protein was dramatically increased upon
addition of IPTG. The molecular mass of the protein was close to that
of the Arabidopsis LIP1 fusion protein encoded in pET-LIP1. By
contrast, in the transformant with pET-30a(+), the 46-kD protein was
not detected and the protein composition did not significantly change
upon addition of IPTG. These findings suggest that the 46-kD protein is
an Arabidopsis LIP1 fusion protein.
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| Figure 5.
Overexpression of Arabidopsis LIP1
cDNA in E. coli. A, Changes in protein composition in
E. coli BL21(DE3) cells transformed with pET-30a(+) and
pET-LIP1. Total proteins from BL21(DE3)/pET-30a(+) (lanes 1 and 3) and
BL21(DE3)/pET-LIP1 (lanes 2 and 4) were analyzed by SDS-PAGE on a 12%
(w/v) polyacrylamide gel. IPTG (0.4 mM) was added to the
growth medium to induce expression of LIP1, and then the
culture was incubated for 0 h (lanes 1 and 2) and 26 h (lanes
3 and 4) at 25°C. An arrowhead indicates the position of the LIP1
protein. The positions of protein molecular mass markers are indicated
on the left (in kD). B, Purification of LIP1 fusion protein from the
homogenates of BL21(DE3)/pET-LIP1. Total proteins before (lane 1) and
after (lane 2) purification with nickel-affinity chromatography were
analyzed by SDS-PAGE. C, Western analysis of total proteins from
BL21(DE3)/pET-30a(+) (lane 1) and BL21(DE3)/pET-LIP1 (lane 2). The
samples corresponding to lanes 3 and 4 in A were applied to SDS-PAGE,
blotted to the nitrocellulose membrane, and used for western analysis.
The blot was probed with anti-Arabidopsis LIP1 IgG.
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The overexpressed LIP1 fusion protein containing a His tag was purified
to near homogeneity by nickel-affinity chromatography from the E. coli homogenate (Fig. 5B) and the purified protein was used for
preparation of polyclonal antibody against Arabidopsis LIP1 protein.
Figure 5C shows the results of western analysis of total proteins from
BL21(DE3)/pET-30a(+) and BL21(DE3)/pET-LIP1 with the anti-Arabidopsis
LIP1 IgG. In BL21(DE3)/pET-LIP1, signals were detected at the position
of 46 and 37 kD, whereas no signal was detected in BL21(DE3)/pET30a(+).
The strong signal at 46 kD corresponds to the position of LIP1 fusion
protein. The weak signal at 37 kD may have been caused by the
degradation of the LIP1 fusion protein in E. coli cells,
because the size of the signal was lower than that of LIP1 fusion
protein and no signal was detected in the control BL21(DE3)/pET30a(+).
These findings demonstrate that the reactivity of the prepared IgG is
specific to the Arabidopsis LIP1 protein.
To investigate the organ-specific localization of Arabidopsis lipoic
acid synthase, total proteins were prepared from leaves, roots, and
flowers and used for western analysis. Figure
6A shows the results of western analysis
with the anti-Arabidopsis LIP1 IgG. Two major signals were detected at
positions 43 and 67 kD in leaves and flowers, but not in roots. The
size of the signal at 43 kD is close to the molecular mass of mature
lipoic acid synthase calculated from the deduced amino acid sequence,
and the signal was not detected when the same sample was analyzed using
a rabbit preimmune serum instead of anti-LIP1 IgG (data not shown). The
signal at 67 kD was not always detected, and the size of the signal was
much higher than the molecular mass of mature lipoic acid synthase
calculated from the deduced amino acid sequence. These results suggest
that the signal at 43 kD but not the one at 67 kD corresponds to lipoic
acid synthase and the content of lipoic acid synthase is higher in
leaves and flowers than in roots.
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| Figure 6.
Western analysis of lipoic acid synthase in
Arabidopsis. A, Organ-specific localization of lipoic acid synthase.
Total proteins prepared from leaves (lane 1), roots (lane 2), and
flowers (lane 3) were applied to SDS-PAGE, blotted to a nitrocellulose
membrane, and used for western analysis with anti-Arabidopsis LIP1 IgG.
The arrowhead indicates the position of lipoic acid synthase. The
positions of protein molecular mass markers (in kD) are indicated on
the left. Fifty-five micrograms of protein was applied to each lane. B,
Processing of lipoic acid synthase. Total proteins prepared from the
E. coli lipA mutant cells transformed with plasmids
pKK233-2 (lane 1), pLIP1-0 (lane 2), pLIP1-18 (lane 3), and
pLIP1-26 (lane 4) and Arabidopsis leaves (lane 5) were used for
western analysis with anti-Arabidopsis LIP1 IgG.
|
|
Arabidopsis lipoic acid synthase contains a leader sequence, presumably
required for import into mitochondria. To determine whether this leader
sequence is processed in Arabidopsis cells, we analyzed total proteins
prepared from Arabidopsis leaves by western analysis with
anti-Arabidopsis LIP1 IgG and compared the size of mature lipoic acid
synthase in leaves with those of LIP1 proteins expressed in the
E. coli lipA mutant cells transformed with pLIP1-0,
pLIP1-18, and pLIP1-26. As shown in Figure 6B, the size of mature
lipoic acid synthase in leaves was close to that of the LIP1 protein
expressed in the E. coli cells transformed with pLIP1-18,
which encodes a LIP1 protein in which 18 amino acid residues were
deleted at the N terminus. This result suggests that lipoic acid
synthase is N-terminally processed in Arabidopsis cells.
Intracellular Localization of Lipoic Acid Synthase in
Arabidopsis
As described above, Arabidopsis lipoic acid synthase contains a
putative mitochondria-targeting transit peptide and is N-terminally processed in Arabidopsis cells. These findings raise the possibility that lipoic acid synthase is located in mitochondria. To check this
possibility we prepared cytosol, microsome, chloroplast, and
mitochondria fractions from Arabidopsis leaves and investigated the
intracellular localization of lipoic acid synthase by western analysis
with anti-Arabidopsis LIP1 IgG. Figure 7,
top, shows the results of the western analysis. A strong band at 43 kD,
corresponding to lipoic acid synthase, was detected in the
mitochondrial fraction (lane 4), whereas a faint band at the same
position was detected in the chloroplast fraction (lane 3), which
suggests that lipoic acid synthase is mainly located in the
mitochondria.
View larger version (42K):
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[in a new window]
| Figure 7.
Intracellular localization of lipoic acid synthase
in Arabidopsis. Proteins prepared from cytosol (lanes 1), microsome
(lanes 2), chloroplast (lanes 3), and mitochondria (lanes 4) fractions
were applied to SDS-PAGE, blotted to a nitrocellulose membrane, and
used for western analysis with anti-Arabidopsis LIP1 IgG (top) and with
an antibody against pea H-protein (bottom). The positions of protein
molecular mass markers (in kD) are indicated on the left. One-hundred
micrograms of protein was applied to each lane.
|
|
To confirm the localization of lipoic acid synthase in mitochondria,
the same fractionated samples were analyzed by western analysis with an
antibody against pea H-protein, which is a subunit of the
Gly-decarboxylase complex and is present in the matrix of mitochondria
(Oliver, 1994). As shown in Figure 7, bottom, the H-protein was
detected in the mitochondrial and chloroplast fractions, although the
intensity of the H-protein band in the mitochondrial fraction was much
stronger than that of the band in the chloroplast fraction. This
suggests that the mitochondrial fraction is abundant in mitochondria,
that the chloroplast fraction is slightly contaminated with
mitochondria, and that the faint band of lipoic acid synthase detected
in the chloroplast fraction is caused by the contamination of
mitochondria into the chloroplast fraction. Together, these results
demonstrate that lipoic acid synthase is located in mitochondria in
Arabidopsis cells.
The content of lipoic acids in leaves, roots, and flowers was
determined to be 170, 90, and 290 ng g1 fresh
weight, respectively. These results indicate that the content of lipoic
acid is correlated with the content of lipoic acid synthase in each
organ but not with the level of the expression of the LIP1
gene.
| |
DISCUSSION |
In this study we cloned the LIP1 cDNA from Arabidopsis
and demonstrated that it encodes a lipoic acid synthase based on the sequence similarity to lipoic acid synthases of E. coli and
yeast and on its functional complementation of an E. coli
lipA mutant defective in lipoic acid synthase. In all eukaryotes
except plants, all of the known lipoic-acid-containing proteins and
lipoyltransferases are located in the mitochondria (Fujiwara et al.,
1990; Kim and Oliver, 1990; Macherel et al., 1990; Reed and Hackert,
1990; Perham, 1991; Mattevi et al., 1992). Therefore, it is reasonable
to assume that the biosynthesis of lipoic acid takes place in the
mitochondria. In fact, lipoic acid synthases of yeast (Sulo and Martin,
1993) and Arabidopsis (this study) contain leader sequences at the N terminus that are similar to transit peptides for targeting to mitochondria. However, it is worthwhile to mention that in plants the
pyruvate-dehydrogenase complex, which contains lipoic acid as an enzyme
cofactor, is present both in mitochondria and in plastids (Lernmark and
Gardeström, 1994), raising the possibility that lipoic acid is
also synthesized in plastids, or it is synthesized in mitochondria and
then transported to plastids. In this study we prepared a polyclonal
antibody specific to lipoic acid synthase of Arabidopsis and
investigated its intracellular localization in Arabidopsis cells. The
obtained results demonstrated that lipoic acid synthase is present in
mitochondria, suggesting that the biosynthesis of lipoic acid takes
place in mitochondria, which is consistent with the previous
observation that a major part of de novo synthesized fatty acids in
mitochondria may be used for biosynthesis of lipoic acid (Wada et al.,
1997).
Although lipoic acid is an essential cofactor for several enzymes
involved in central metabolism and is synthesized in most organisms,
the mechanism of lipoic acid synthesis is not well understood. Octanoic
acid has been shown to be the precursor of the carbon chain (White,
1980; Parry, 1983), but neither the origin nor the mechanism whereby
the sulfur atoms are inserted into the carbon chain is known. We
recently investigated the function of mitochondrial ACP, which was
discovered in the mitochondria of several organisms (Shintani and
Ohlrogge, 1994; Schneider et al., 1997), and found that it is involved
in the biosynthesis of fatty acids in mitochondria of pea and
Neurospora (Wada et al., 1997). We also found that
octanoyl-ACP is synthesized in mitochondria as an intermediate of fatty
acid synthesis and may be used for the biosynthesis of lipoic acid
(Wada et al., 1997).
This involvement of mitochondrial ACP in lipoic acid production was
recently demonstrated in yeast by Brody et al. (1997). Jordan and
Cronan (1997) reported that fatty acid synthesis in E. coli
is directly linked to the biosynthesis of lipoic acid and that lipoate
is donated from lipoyl-ACP to the pyruvate-dehydrogenase complex by
lipoyl-ACP-protein N-lipoyltransferase. This enzyme activity
was also discovered in mitochondria of both plants and fungi. These
findings suggest that lipoic acid synthesis may proceed through
ACP-bound intermediates and that lipoic acid synthase may insert a
sulfur atom into octanoic acid that has been bound to ACP.
Biotin synthase also catalyzes a reaction similar to that catalyzed by
lipoic acid synthase, the insertion of a sulfur atom into dethiobiotin
(Eisenberg, 1987; Baldet et al., 1993). Birch et al. (1995) studied the
in vitro assay system for biotin synthase and found that this enzyme
requires several low-molecular-mass cofactors and protein components.
Although it can be expected that lipoic acid synthase requires similar
enzyme cofactors as those required for biotin synthase, an in vitro
assay system for lipoic acid synthase has not yet been established;
therefore, it has not been possible to measure the activity of lipoic
acid synthase in vitro. Biotin synthase is a [2Fe-2S] cluster protein (Sanyal et al., 1994) and the Cys residues may coordinate to the Fe atom of the Fe-S cluster. In this study we found that two conserved Cys motifs are present in lipoic acid synthases of E. coli,
yeast, and Arabidopsis, and one of the Cys motifs is also conserved in the biotin synthases of Arabidopsis and microorganisms. Although it is
not known whether lipoic acid synthase is an [Fe-S] cluster protein,
it can be assumed that the conserved Cys residues may play a role in
the enzyme activity, possibly in the coordination of the Fe atom. In
this study we overexpressed Arabidopsis lipoic acid synthase in
E. coli and purified the overexpressed protein from the
E. coli homogenate. This system may help to establish an
assay system for lipoic acid synthase to aid in the study of the
function of the conserved Cys residues and how lipoic acid synthase
inserts a sulfur atom into octanoic acid.
| |
FOOTNOTES |
1
This work was supported by a Grant-in-Aid for
Scientific Research (no. 10640636) from the Ministry of Education,
Science, Sports, and Culture (Japan) and by a grant from the Asahi
Glass Foundation to H.W.
*
Corresponding author; e-mail wadarcb{at}mbox.nc.kyushu-u.ac.jp; fax
81-92-726-4761.
Received April 30, 1998;
accepted August 12, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ACP, acyl carrier protein.
EST, expressed
sequence tag.
IPTG, isopropyl-1-thio-
-D-galactoside.
RACE, rapid amplification of cDNA ends.
RT-PCR, reverse
transcription-PCR.
| |
ACKNOWLEDGMENTS |
This work would not have been possible without the support and
helpful suggestions of John Ohlrogge and the technical assistance of
Linda Savage (Michigan State University). We acknowledge Sean Jordan
and John E. Cronan, Jr. (University of Illinois at Urbana-Champaign), for providing the E. coli lipA mutant (KER176) and the
Arabidopsis Biological Resource Center (The Ohio State University) for
providing the Arabidopsis EST clone used in this study. We also thank
Dr. David J. Oliver (Iowa State University) for providing the antibody against pea H-protein.
| |
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Abstract
Introduction
