(Received for publication, January 27, 1997, and in revised form, April 14, 1997)
From the The enzyme farnesyl-diphosphate synthase (FPS; EC
2.5.1.1./EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. FPS is
considered to play a key role in isoprenoid biosynthesis. We have
reported previously that Arabidopsis thaliana contains two differentially expressed genes, FPS1 and FPS2,
encoding two highly similar FPS isoforms, FPS1 and FPS2, (Cunillera,
N., Arró, M., Delourme, D., Karst, F., Boronat, A., and Ferrer,
A. (1996) J. Biol. Chem. 271, 7774-7780). In this
paper we report the characterization of a novel Arabidopsis
FPS mRNA (FPS1L mRNA) derived from the FPS1 gene. A
cDNA corresponding to the FPS1L mRNA was cloned using a reverse
transcription-polymerase chain reaction strategy. Northern blot
analysis showed that the two FPS1-derived mRNAs are
differentially expressed. The FPS1L mRNA accumulates preferentially
in inflorescences, whereas the previously reported FPS1 mRNA (FPS1S
mRNA) is predominantly expressed in roots and inflorescences. FPS1L
mRNA contains an in-frame AUG start codon located 123 nucleotides
upstream of the AUG codon used in the translation of the FPS1S isoform.
Translation of the FPS1L mRNA from the upstream AUG codon generates
a novel FPS1 isoform (FPS1L) with an NH2-terminal extension
of 41 amino acid residues, which has all the characteristics of a
mitochondrial transit peptide. The functionality of the FPS1L
NH2-terminal extension as a mitochondrial transit peptide
was demonstrated by its ability to direct a passenger protein to yeast
mitochondria in vivo and by in vitro import
experiments using purified plant mitochondria. The
Arabidopsis FPS1L isoform is the first FPS reported to
contain a mitochondrial transit peptide.
The enzyme farnesyl-diphosphate synthase
(FPS1; EC 2.5.1.1/EC 2.5.1.10) catalyzes
the sequential 1 Genomic and cDNA sequences encoding FPS have been isolated and
characterized from a variety of organisms, ranging from bacteria to
higher eukaryotes (7, 8). Recently, the enzyme has been cloned from
several plant species such as Arabidopsis thaliana (9, 10),
Lupinus albus (11), Zea mays (12),
Artemisia annua (13), Hevea brasiliensis (14),
and Parthenium argentatum (15). Although the complexity of
the FPS gene families has been studied in a limited number of plants,
it seems that plant FPS is encoded by multigene families like other key
enzymes of the isoprenoid biosynthetic pathway, such as
3-hydroxy-3-methylglutaryl-CoA reductase (16-18), and
geranylgeranyl-diphosphate synthase (19). We have recently shown that
Arabidopsis contains a small FPS gene family consisting of
at least two genes, FPS1 and FPS2, which have a
very similar structure and share a high level of sequence similarity
(10). At least two FPS gene copies have been detected in the maize
genome (12), and cDNA sequences encoding two highly similar FPS
isoforms have also been reported in L. albus (11) and
P. argentatum (15). The pattern of expression of individual genes encoding FPS has been reported only in Arabidopsis,
where it has been shown that the two currently characterized FPS genes (FPS1 and FPS2) are expressed differentially at
both quantitative and qualitative levels (10). At present, the
biological significance of the occurrence of highly similar FPS
isoforms in plants is still unclear, although the differential pattern
of expression of the two FPS Arabidopsis genes suggests that
each FPS isoform might have a specialized function in directing the
flux of pathway intermediates into specific classes of isoprenoid end
products.
FPS has long been considered to be a cytoplasmic enzyme. However, it
has recently been demonstrated that in mammals FPS is mainly localized
within the peroxisomes (20), although significant levels of FPS
activity have also been detected in rat liver mitochondria (21). In
plants, the only cell compartment where plant FPS has been detected is
the cytosol (6, 22, 23), although it is widely accepted that the
enzymes utilizing isopentenyl diphosphate are distributed in three
subcellular compartments, namely cytosol, mitochondria, and plastids.
All of the FPS reported to date show high amino acid sequence
similarity, and all contain several conserved domains, including the
two aspartate-rich domains involved in substrate binding and enzyme
catalysis (24, 25). However, none of the FPS characterized so far from
a number of eukaryotic organisms (including fungi, plants, and animals)
contains NH2-terminal sequences that could represent
transit peptides for targeting into plastids or mitochondria. In this
paper we report that the expression of the Arabidopsis FPS1
gene generates a novel mRNA that encodes a mitochondrial FPS
isoform.
Restriction endonucleases and
DNA-modifying enzymes were purchased from Boehringer Mannheim and
Promega. [ A. thaliana plants
(ecotype Columbia) were grown as described previously (10).
Escherichia coli strain XL1-Blue (F The cDNA encoding
FPS1L was cloned by means of a reverse transcription-polymerase chain
reaction (PCR) strategy similar to that used previously for the cloning
of the Arabidopsis FPS2 cDNA (10). The cDNA was
amplified from a single-stranded cDNA pool, obtained by reverse
transcription of 5 µg of poly(A)+ RNA from
Arabidopsis inflorescences, using an upstream primer specific for the leader region of the FPS1L mRNA
(5
Appropriate restriction fragments were
subcloned into plasmid pBluescript. Both strands of DNA were sequenced
by the dideoxynucleotide chain termination method (26) using an
automated fluorescence-based system (Applied Biosystems).
To obtain the desired RNase
protection probe a DNA fragment extending from nucleotide positions
Total RNA from different tissues of
Arabidopsis was isolated as described (28). Thirty µg of
RNA from each sample was fractionated by electrophoresis in 1% (w/v)
agarose gels containing 2.2 M formaldehyde and blotted to
Hybond-N nylon membranes (Amersham). The 32P-labeled
(random primed) probes used were a PCR amplification fragment of 289 bp
extending from nucleotides A
SacII-SalI fragment, containing the FPS1L
cDNA, was excised from plasmid pcNC3 and cloned into the
corresponding sites of plasmid pBluescript to create plasmid pcBNC3. By
using site-directed mutagenesis (30), the ATG start codon of the FPS1S
isoform was converted to an ATC codon (encoding Ile). The resulting
plasmid was designated pcBNC3Mut. The two cDNA sequences, FPS1L and
FPS1LMut, were cut out as SacI-SalI fragments
from plasmids pcBNC3 and pcBNC3Mut, respectively, and cloned into the
corresponding sites of plasmid pSP65 (Promega). The resulting plasmids,
pcSPNC3 and pcSPNC3Mut, were used as templates for in vitro
transcription/translation using [35S]Met and the
TNTTM Coupled Wheat Germ Extract System (Promega). The
35S-labeled proteins were separated by SDS-polyacrylamide
gel electrophoresis (12% acrylamide) and detected by fluorography.
To construct plasmid
pFPS1Ltp-Y Mitochondria were
isolated from potato tubers (Solanum tuberosum var. Bintje)
as described (34). For in vitro import studies the purified
mitochondria were washed and resuspended in a buffer containing 400 mM mannitol, 10 mM potassium phosphate (pH
7.2), and 0.1% (w/v) bovine serum albumin. The FPS1L protein was
synthesized by in vitro transcription/translation of plasmid
pcSPNC3 using [35S]Met and the TNTTM Coupled
Reticulocyte Lysate System. The import reaction contained 35 µl of
purified mitochondria (10 mg of protein/ml), 160 µl of import buffer
(250 mM mannitol, 20 mM HEPES (pH 7.5), 80 mM KCl, 1 mM K2HPO4, 1 mM ATP, 1 mM malate, 2 mM NADH, and
1 mM dithiothreitol), and 15 µl of the reticulocyte
lysate translation mixture. After incubation for 30 min at 20 °C,
70-µl aliquots of the import reaction were treated with proteinase K
(20 µg/ml), either in the presence or absence of 0.5% (v/v) Triton
X-100. These aliquots and the remainder of the import reaction were
then incubated for 20 min at 20 °C. After the addition of 1 mM phenylmethylsulfonyl fluoride, samples were incubated
for further 15 min. For inhibition of mitochondrial import, purified
mitochondria were incubated for 5 min in the presence of 1 µM valinomycin prior to the import reaction. After the
different treatments, mitochondria were repurified by centrifugation through a 25% (w/v) sucrose cushion. The resulting pellets were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide), and the radiolabeled products were detected by fluorography.
We have recently reported that the
expression of the Arabidopsis FPS1 gene generates an
mRNA of approximately 1.3 kilobases which encodes the isoform FPS1
(10). A cDNA encoding this isoform had previously been cloned by
functional complementation of a mutant yeast strain defective in FPS
activity (9). A detailed analysis of the nucleotide sequence of the
5 The occurrence of an mRNA containing the upstream ATG was first
detected by using a reverse transcription-PCR strategy (data not
shown). To confirm the existence of this novel FPS1 mRNA we isolated the corresponding cDNA by reverse transcription-PCR using poly(A)+ RNA from Arabidopsis inflorescences
(for details, see "Experimental Procedures"). A cDNA fragment
of approximately 1.4 kilobases was cloned and sequenced. The cDNA
insert was found to have a nucleotide sequence of 1,396 bp, excluding a
polyadenylate tail of 30 bases, which contained the complete sequence
of the FPS1 cDNA previously characterized as well as 99 and 89 additional nucleotides at the 5 To define the 5 The
expression pattern of the Arabidopsis FPS1S and FPS1L
mRNAs was analyzed by Northern blot analysis using total RNA
isolated from roots, stems, leaves and inflorescences (Fig.
2). A PCR fragment of 289 bp extending from nucleotides
To analyze whether the upstream in-frame AUG codon in
the FPS1L mRNA is used as translational start codon, the FPS1L
transcript was synthesized in vitro from plasmid pcSPNC3
(Fig. 3A) and translated in a wheat germ
cell-free system. The products obtained were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by fluorography. Two proteins of 44 and 40 kDa resulted from the translation of the
FPS1L mRNA (Fig. 3B, lane 1). The estimated
size of the two proteins is in good agreement with the molecular mass
predicted for proteins initiated at the first (44,254 Da) and the
second (39,689 Da) AUG codons. When a similar experiment was performed using transcript FPS1LMut (plasmid pcSPNC3Mut, Fig. 3A), in
which the second AUG codon was converted to an AUC codon (encoding Ile) by site-directed mutagenesis, only the 44-kDa protein was synthesized (Fig. 3B, lane 2). Taken together, these results
show that in the wheat germ lysate system, the most 5
Analysis of the amino acid composition and the
predicted secondary structure of the NH2-terminal extension
of the Arabidopsis FPS1L isoform (Fig. 4)
revealed that it has all of the features characteristic of a
mitochondrial transit peptide (35, 36). There is a complete absence of
acidic residues and an enrichment in basic (arginine, lysine, and
histidine), hydroxylated (serine), and hydrophobic (leucine) residues.
It contains the sequence RIQS (amino acid residues 36-39 in Fig.
4A), which fits the most commonly reported mitochondrial
targeting peptide cleavage motif RX/XS (where
X represents any amino acid) (36), preceded by two arginine residues located at positions
To study the possible role of the FPS1L NH2-terminal
extension as a mitochondrial targeting sequence, we analyzed the
ability of this predicted transit peptide to direct the CoxIV subunit of cytochrome c oxidase into yeast mitochondria in
vivo. This experimental approach was based on previous
observations demonstrating the interchangeability of mitochondrial
targeting sequences between plants and yeast (31, 38-41). For this
purpose, an in-frame fusion was made between the fragment of the
Arabidopsis FPS1L cDNA coding for the 41 NH2-terminal residues of the FPS1L isoform (FPS1Ltp) and
the S. cerevisiae COXIV gene lacking the region coding for its own mitochondrial transit peptide (
To
confirm further the functional role of the FPS1L
NH2-terminal extension as a mitochondrial targeting
sequence we analyzed the import of the Arabidopsis FPS1L
isoform into plant mitochondria (Fig. 6). The FPS1L
mRNA was in vitro translated using a rabbit reticulocyte
lysate, and the resulting products were analyzed by SDS-polyacrylamide
gel electrophoresis and fluorography. The translation mixture contained
similar amounts of radiolabeled FPS1L (44 kDa) and FPS1S (40 kDa)
isoforms (Fig. 6, lane 2), which were completely digested
after incubation with proteinase K (Fig. 6, lane 1). When
the translation mixture was incubated with purified potato mitochondria
followed by treatment with proteinase K, a protected polypeptide of
approximately 40 kDa was detected (Fig. 6, lane 4). The
protection was abolished when mitochondria were solubilized with Triton
X-100 prior to proteinase K treatment (Fig. 6, lane 5). When
the import experiment was performed in the presence of valinomycin,
which collapses the membrane potential required for protein import into
mitochondria, the protected 40-kDa polypeptide was not detected (Fig.
6, lane 6). No import into mitochondria was observed when
similar experiments were performed using as a precursor the 40-kDa
FPS1S isoform generated from the FPS1S mRNA (data not shown). Taken
together, these results demonstrate that the 44-kDa
Arabidopsis FPS1L isoform is targeted into plant mitochondria and processed to a 40-kDa mature FPS1 isoform by cleavage
of a transit peptide of approximately 4 kDa.
The great complexity of the plant isoprenoid biosynthetic pathway
has led to the suggestion that its regulation requires the coordinated
activity of several key enzymes (6, 42, 43). FPS, a prenyltransferase
that catalyzes the synthesis of farnesyl diphosphate from isopentenyl
diphosphate and dimethylallyl diphosphate, is considered to play a
central role in the overall control of the plant isoprenoid pathway.
This assumption has been primarily made on the basis that farnesyl
diphosphate is the starting point of different branches of the plant
isoprenoid pathway leading to the synthesis of essential end products
(6, 42, 43). The proposed regulatory role of plant FPS is further
supported by the observation that in mammals FPS is a highly regulated
enzyme that plays a relevant role in the control of sterol biosynthesis (2-5). We have recently shown that Arabidopsis contains a
small FPS gene family consisting of at least two genes, FPS1
and FPS2. These genes are differentially expressed and
encode two highly similar FPS isoforms (10). At least two FPS isoforms
have also been reported to occur in other plant species (11, 15). Thus, the occurrence of FPS isoforms is a general feature of higher plants,
although the specific role of each individual FPS isoform in the
isoprenoid biosynthetic pathway is currently unknown.
In this paper we report that the expression of the Arabidopsis
FPS1 gene generates a previously undetected mRNA that encodes a novel FPS1 isoform (FPS1L) with an NH2-terminal extension
of 41 amino acids with respect to the FPS1 isoform (FPS1S) previously characterized (9, 10). The occurrence of the FPS1L mRNA was demonstrated by the cloning of its corresponding cDNA. RNA blot analysis using a FPS1L mRNA-specific probe and an
FPS1-derived probe that recognizes both FPS1S and FPS1L
mRNAs simultaneously showed that FPS1S and FPS1L mRNAs are
present in all tissues analyzed, although FPS1L mRNA accumulates
preferentially in inflorescences, and FPS1S mRNA is mainly
expressed in roots and inflorescences. The fact that the
FPS1 gene generates two mRNAs that are also differentially expressed suggests that the two transcripts are under
the control of alternative promoters. The results obtained in the RNase
protection analysis show that FPS1S and FPS1L mRNAs have
heterogeneous 5 At the protein level, the novel Arabidopsis FPS1L isoform is
identical in sequence to FPS1S but is extended at its NH2
terminus by an additional sequence of 41 amino acids. This
NH2-terminal extension has no counterpart among the FPS
characterized so far from a number of eukaryotic organisms (7-15).
This observation raised the question about its possible function as a
transit peptide for targeting the enzyme into subcellular organelles.
The accumulation of sequence data on organellar transit peptides has
revealed that these targeting sequences share a very low level of
sequence conservation, although they have a number of common features
in terms of amino acid composition, positional amino acid preferences,
and secondary structure (35, 36). Interestingly, the FPS1L
NH2-terminal extension nicely fits all known requirements
to be a mitochondrial transit peptide. First, there is a lack of acidic
amino acid residues and an enrichment in hydroxylated and hydrophobic
residues. Second, the sequence motif RIQS, which fits the consensus
mitochondrial targeting peptide cleavage motif RXXS (where
X represents any amino acid), is found just two residues
upstream of the NH2-terminal methionine residue of FPS1S
isoform. This motif is preceded by two arginine residues located at
positions To analyze whether the FPS1L NH2-terminal extension could
function as a mitochondrial transit peptide we first studied its ability to direct a passenger protein into yeast mitochondria in
vivo. To this end, a construct expressing a chimeric protein containing the 41 NH2-terminal amino acid residues of FPS1L
fused to the CoxIV subunit of yeast cytochrome c oxidase
lacking its own mitochondrial transit peptide (plasmid
pFPS1Ltp- Our results indicate that Arabidopsis FPS1 is a bifunctional
gene encoding cytosolic and mitochondrial FPS isoforms. Consequently, FPS1 belongs to the increasing group of eukaryotic genes
encoding isozymes that perform analogous functions at different
intracellular locations (44). These genes usually have more than one
ATG start codon in their 5 A general feature of the enzymes involved in plant isoprenoid
biosynthesis is their occurrence in multiple isoforms (10, 16-19).
Although the biological significance of this fact has not yet been
fully evaluated, it is likely that the multiplicity of isoforms
reflects the great complexity of plant isoprenoid biosynthesis, concerning not only the regulation of the overall pathway but also its
subcellular compartmentalization. In contrast to the controversy
surrounding the subcellular location of the enzymes involved in the
synthesis of isopentenyl diphosphate in plants, it is widely accepted
that the enzymes utilizing isopentenyl diphosphate are distributed in
at least three cellular compartments: the cytosol, mitochondria, and
plastids. However, the cytosol is the only cell compartment in which
plant FPS has been detected (6, 22, 23). It is worth noting that a
significant level of FPS activity (approximately 13% of the total
cellular FPS activity) has been detected in rat liver mitochondria
(21). Interestingly, none of the previously reported FPS from a number
of eukaryotic organisms (including fungi, plants, and animals) (7-15)
contains NH2-terminal sequences that could represent
transit peptides for targeting to mitochondria. The
Arabidopsis FPS1L isoform described here represents the
first reported eukaryotic FPS that contains a mitochondrial transit
peptide. This finding reinforces the view that plant mitochondria can
use isopentenyl diphosphate as a precursor for the synthesis of
farnesyl diphosphate, which in turn could be utilized for the production of mitochondrial isoprenoid compounds such as ubiquinone, heme a, tRNA species, and prenylated proteins. On the basis of these
observations, it is reasonable to speculate that FPS isoforms with
mitochondrial targeting peptides might be found not only in other plant
species but also in eukaryotic organisms other than plants. We are
currently applying different molecular and cellular approaches to
assess the possible regulatory role of FPS1L in the synthesis of
mitochondrial isoprenoid compounds in plants.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80605[GenBank]. We thank Dr. Ian D. Small for providing
plasmids pYCOX and pY
Unitat de Bioquímica,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-4 condensation of two molecules of isopentenyl
diphosphate with both dimethylallyl diphosphate and the resultant
10-carbon compound geranyl diphosphate to produce the 15-carbon
compound farnesyl diphosphate (1). Because of the central branch point
location of farnesyl diphosphate in the isoprenoid pathway, FPS is
considered to play a key role in isoprenoid biosynthesis. It has been
shown that in mammals FPS is a highly regulated enzyme involved in the
control of the sterol biosynthetic pathway (2-5). FPS is also
considered to play an important role in the control of plant isoprenoid
biosynthesis. In plants, farnesyl diphosphate is the starting point of
different branches of the isoprenoid pathway leading to the synthesis
of key end products that are required for normal growth and
development, such as phytosterols, dolichols, ubiquinone,
plastoquinone, sesquiterpenoid phytoalexins, and prenylated proteins.
Therefore, changes in FPS activity could alter the flux of isoprenoid
compounds down the different branches of the pathway in competition for
the available farnesyl diphosphate and, hence, play a central role in
the regulation of a number of essential functions in plant cells (6).
However, more experimental data are still required before obtaining a
clear picture of the regulatory significance of FPS in the overall
control of isoprenoid biosynthesis in plants.
Enzymes and Biochemicals
-32P]dCTP (3,000 Ci/mmol),
[
-32P]rUTP (3,000 Ci/mmol) and [35S]Met
(1,000 Ci/mmol) were obtained from Amersham. Amino acids, glucose,
ammonium sulfate, ampicillin, and adenine were from Sigma. Yeast
extract, Bacto-peptone, Bacto-tryptone, and yeast nitrogen base without
amino acids and ammonium sulfate were from Difco Laboratories. All
other chemicals were of the highest commercial grade available.
(proAB lacIqZD M15, Tn10 (tetr)) recA1
gyrA96 thi-1 hsdR17 supE44 relA1 lac) (Stratagene) was used for
cloning, maintenance, and propagation of plasmids. S. cerevisiae strain WSR (Mat
, his3-11, leu2-3, 112, ade2-1, ura3-1, trp1-1, can1-100,
COXIV::LEU2) was used for tests of
complementation of respiratory deficiency.
-GGCGTTTTCGGGAGAAGAAGG-3
, nucleotides
97 to
77 in Fig.
1A) and an adaptor oligonucleotide (5
-GACTCGAGTCGACATCGGG-3
) as a downstream primer. The resulting PCR
product (approximately 1.4 kilobases) was gel purified and ligated into
plasmid pGEM-T (Promega) prior to sequencing. The resulting plasmid was
designated pcNC3.
Fig. 1.
Analysis of the 5-region of the FPS1
gene. Panel A, nucleotide sequence of the 5
-region of
the FPS1 gene and deduced amino acid sequence of the FPS1L
NH2-terminal region. The transcription start sites of the
FPS1S (TS1 and TS2) and the FPS1L (TS3) mRNAs are denoted by
arrowheads. Nucleotides are numbered (left) by assigning position +1 to the most internal transcription start site
(TS1) of the FPS1 gene. The 5
-end of the FPS1S cDNA is
indicated by an asterisk. ATG start codons are
boxed. The deduced amino acid sequence (numbered on the
right) is shown below the nucleotide sequence. The amino
acid sequence corresponding to the FPS1L NH2-terminal extension is boxed. Panel B, autoradiography of the labeled
antisense RNA probe and the protected fragments. Lane 1,
poly(A)+ RNA from Arabidopsis inflorescences;
lane 2, control yeast tRNA; lane 3,
undigested probe. The size of the undigested probe and the
protected fragments is indicated on the left in
nucleotides (nt).
[View Larger Version of this Image (34K GIF file)]
201 to +141 in the FPS1 gene was generated by PCR. The
amplified fragment was cloned into the EcoRI and
HindIII sites of plasmid pSP65 (Promega). To rule out the
existence of PCR artifacts, the cloned fragment was sequenced. The
resulting plasmid was linearized by digestion with HindIII and used as a template for in vitro transcription using SP6
RNA polymerase (Promega) and [
-32P]rUTP. The
351-nucleotide RNA antisense probe contained 342 nucleotides corresponding to the FPS1 gene and 9 additional nucleotides
derived from plasmid pSP65. RNase protection experiments were performed as described (27), except that the RNA probe was purified on a 5%
polyacrylamide, 8 M urea gel before use. The probe was
eluted by diffusion at room temperature in 600 µl of 0.5 M ammonium acetate, 1 mM EDTA, and 0.1% SDS.
After precipitation, the antisense RNA probe (2.5 × 105 cpm) was hybridized overnight at 42 °C with 8 µg
of poly(A)+ RNA from Arabidopsis inflorescences
or yeast tRNA. Digestion was performed for 2 h with 15 units of
RNase ONE (Promega) according to the manufacturer's recommendations.
Analysis of the protected fragments was performed by electrophoresis on
a 5% polyacrylamide, 8 M urea gel. The gel was dried and
exposed to x-ray film. Variation of the quantity of RNA or the
digestion conditions did not alter the pattern of protected bands. A
known DNA sequencing reaction was included as a marker. The size of the
RNA protected fragments was initially calculated according to the
sequencing reaction and corrected as described (27).
309 to
20 in the FPS1 gene,
and a 370-bp BglII-HindIII fragment from the
3
-flanking region of the FPS1 gene (10). The conditions of
hybridization and washing of the filters were as described previously
(10). Filters were reprobed with a 32P-labeled 900-bp
BamHI-EcoRI fragment of the gene for the wheat 25 S cytoplasmic rRNA (29).
COX, a cDNA fragment encoding the 41 NH2-terminal amino acid residues of the FPS1L isoform was
amplified by PCR using a forward primer
(5
-GGGAATTCAAAAATGTCTGTGAGTTGTTGTTGTAGG-3
) extending from nucleotide positions
74 to
47 in the
Arabidopsis FPS1 gene (Fig. 1A), a reverse primer
(5
-AGCTCTAGATGAAGAGCTTTGGATACG-3
) complementary to the
nucleotide sequence +36 to +53 in the FPS1 gene (Fig.
1A), and plasmid pcSPNC3 (see above) as a template. EcoRI and XbaI sites (shown in italic) were added
to the 5
-end of the primers, respectively. The 144-bp PCR product was
digested with EcoRI and XbaI and cloned into the
corresponding sites of plasmid pY
COX (kindly provided by Ian D. Small; Institut National de la Recherche Agronomique, Versailles,
France), which contains the presequence-less yeast COXIV
gene under the control of the alcohol dehydrogenase gene promoter (31).
To optimize the synthesis of the chimeric FPS1Ltp-Y
COX protein in
yeast, four changes (underlined) were introduced in the sequence of the
forward primer to convert the nucleotide sequences surrounding the ATG
start codon of the Arabidopsis FPS1L cDNA
(GAATATGAG) into the consensus reported for functional
translational start codons in yeast (AAAAATGTC) (32).
Changes in the second triplet of the Arabidopsis FPS1L cDNA coding sequence did not alter the amino acid residue encoded (Ser). Yeast strain WSR was transformed with plasmids pYCOX, pY
COX, or pFPS1Ltp-Y
COX using the modified lithium acetate procedure described by Gietz et al. (33). Ura+
transformants were selected at 28 °C on agar plates containing minimal medium (0.16% (w/v) yeast nitrogen base without amino acids
and ammonium sulfate, 0.5% (w/v) ammonium sulfate and 1% (w/v)
glucose) supplemented with histidine (20 µg/ml), tryptophan (40 µg/ml), and adenine (40 µg/ml). After 4 days of growth, selected colonies were subcultured on N3 medium (1% (w/v) yeast extract, 1%
(w/v) Bacto-peptone, 2% (v/v) glycerol, and 50 mM
potassium phosphate (pH 6.25)) to test for complementation of
respiratory deficiency.
Identification and Cloning of a Novel FPS mRNA Derived from the
Arabidopsis FPS1 Gene
-flanking region of the FPS1 gene (Fig. 1A)
revealed the presence of an in-frame ATG codon located 123 bp upstream
of the ATG start codon used in the translation of the reported FPS1
isoform (9). The utilization of the upstream ATG codon as a translation
start site would generate a different FPS1 isoform containing an
NH2-terminal extension of 41 amino acids with respect to
the previously described FPS1 isoform.
- and 3
-ends, respectively. The open
reading frame starting at the most 5
ATG triplet encodes a protein of
384 amino acid residues, with a predicted molecular mass of 44,254 Da,
which differs from the previously reported FPS1 isoform in having an NH2-terminal extension of 41 amino acids. The cDNA
clone also contains a 5
-untranslated region of 27 bp and a
3
-untranslated region of 214 bp. The polyadenylate tail was located 89 bp downstream of the polyadenylation site of the FPS1 cDNA (9),
thus indicating that different polyadenylation sites are used in the
FPS1 gene. These two FPS1 mRNAs will be hereafter
referred to as FPS1S and FPS1L, with FPS1S mRNA encoding the
previously reported FPS1 isoform (9, 10) (FPS1S) and FPS1L mRNA
encoding the novel FPS1 isoform (FPS1L) containing the
NH2-terminal extension.
-ends of the two FPS1 mRNAs (FPS1S and FPS1L) we
performed RNase protection analysis using poly(A)+ RNA
isolated from Arabidopsis inflorescences. The results
obtained are shown in Fig. 1B. The estimated size of the
most intense protected bands indicated the occurrence of a major FPS1L
transcript (band of 269 nucleotides) with a 5
-end located 62 nucleotides upstream of the FPS1L ATG start codon (TS3, Fig.
1A) and two major FPS1S mRNAs (bands of 137 and 154 nucleotides) with 5
-ends located 53 and 70 nucleotides upstream of the
FPS1S ATG start codon (TS1 and TS2, Fig. 1A). Taken
together, these results indicated that the expression of the
Arabidopsis FPS1 gene generates two mRNAs encoding two
FPS1 isoforms (FPS1S and FPS1L) that differ only in their
NH2 terminus.
309 to
20 in the FPS1 gene was used as an FPS1L
mRNA-specific probe. A 370-bp BglII-KpnI genomic fragment corresponding to the 3
-end of the FPS1
gene (10) was used as an FPS1 gene-specific probe that
recognizes both FPS1L and FPS1S mRNAs simultaneously. Similar
amounts of RNA were present in each lane, as confirmed by hybridization
of the filters with a probe derived from the wheat 25 S rRNA gene, and
radiolabeled probes of equivalent specific activity were used. Comparison of the results indicated that FPS1S and FPS1L mRNAs are
detected in all tissues analyzed although they have a different pattern
of expression. FPS1L mRNA accumulates preferentially in inflorescences, whereas FPS1S mRNA is detected mainly in roots and
inflorescences. No significant change in the levels of FPS1L mRNA
was detected when RNA samples were prepared from light- or dark-grown
seedlings.
Fig. 2.
Northern blot analysis of Arabidopsis
FPS1-derived mRNAs. Total RNA samples from different
tissues of Arabidopsis (30 µg/lane) were electrophoresed
in 1% agarose-formaldehyde gels and transferred onto nylon membranes.
Filters were hybridized with the FPS1L mRNA-specific probe and the
FPS1 gene-specific probe described under "Experimental
Procedures." The FPS1 probe hybridizes to both FPS1S and FPS1L
mRNAs. Exposure time was 9 days. To confirm that equivalent amounts
of RNA were present in each lane, filters were reprobed with a fragment
of the gene for the wheat 25 S cytoplasmic rRNA.
[View Larger Version of this Image (48K GIF file)]
AUG codon in the
FPS1L mRNA is used preferentially to initiate translation, although the second AUG codon is also used, giving rise to significant levels of
the FPS1S protein.
Fig. 3.
Functional in vitro analysis of
the upstream AUG codon in the FPS1L mRNA. Panel A,
schematic representation of the FPS1L and FPS1LMut cDNAs used in
the in vitro transcription/translation experiments. The
regions of the cDNAs corresponding to the 5-untranslated region
(open), the NH2-terminal extension
(hatched), and part of the coding region common to FPS1L and
FPS1S (black solid) are shown. Arrows indicate
the transcription start site of the SP6 RNA polymerase promoter.
Lines represent the NH2-terminal region of the
proteins resulting from the in vitro
transcription/translation of plasmids pcSPNC3 and pcSPNC3Mut.
Panel B, FPS1L (lane 1) and FPS1LMut (lane
2) transcripts were translated in vitro in a wheat germ
cell-free system using [35S]Met as labeled precursor.
Samples were separated in a SDS, 12% polyacrylamide gel. Bands
corresponding to FPS1S and FPS1L are indicated by arrows.
The estimated molecular masses are also indicated.
[View Larger Version of this Image (25K GIF file)]
2 and
12 relative to the predicted cleavage site RI/QS. Finally, analysis of the FPS1L
NH2-terminal extension using an improved method of protein
structure prediction (37) shows that amino acid residues 8-17 can form
a positively charged amphiphilic
-helix, in which hydrophobic
residues are clustered on one face of the helix while the basic and
polar residues are on the other face (Fig. 4B).
Fig. 4.
Analysis of the Arabidopsis FPS1L
NH2-terminal extension. Panel A, amino acid
sequence of the FPS1L NH2-terminal region. Amino acid
positions are indicated below the sequence. The potential charge is
shown above each residue. The conserved cleavage motif is
underlined, and the predicted cleavage site is indicated by an arrowhead. The NH2-terminal methionine
residue of the FPS1S isoform is indicated by an asterisk.
Secondary structure prediction for the FPS1L NH2-terminal
region is shown below the amino acid sequence. H represents
helical structure, L represents looped structures, and · represents extended conformation. Panel B, helical wheel
representation of amino acids 8-17 in the FPS1L sequence. Hydrophobic
residues are boxed.
[View Larger Version of this Image (21K GIF file)]
COXIV). The
resulting plasmid (pFPS1Ltp-Y
COX, Fig. 5A)
was used to transform the yeast strain WSR, which contains a disrupted
copy of the COXIV gene and, consequently, is unable to grow
on medium containing glycerol as energy source. The results shown in
Fig. 5B indicate that plasmid pFPS1Ltp-Y
COX efficiently
complements strain WSR. The same result was obtained when strain WSR
was transformed with plasmid pYCOX, which encodes the complete amino
acid sequence of the S. cerevisiae CoxIV subunit. In
contrast, plasmid pY
COX, which encodes a truncated form of the CoxIV
subunit lacking the mitochondrial transit peptide, does not complement
the respiratory-deficient strain WSR. These results clearly demonstrate
that the NH2-terminal extension of the
Arabidopsis FPS1L isoform is able to replace the
mitochondrial targeting sequence of the CoxIV subunit of yeast
cytochrome c oxidase, thus demonstrating that it is a
functional mitochondrial transit peptide.
Fig. 5.
Functional complementation of a
CoxIV-deficient yeast strain by plasmid pFPS1Ltp-YCOX. Panel
A, schematic representation of the plasmids used in this study.
Plasmid pYCOX contains the wild-type COXIV gene from
S. cerevisiae, including the region encoding its own
mitochondrial transit peptide, which has been deleted in plasmid
pY
COX. Plasmid pFPS1Ltp-Y
COX contains the region of the
Arabidopsis FPS1L cDNA coding for the 41 NH2-terminal amino acid residues of FPS1L ligated in front
of the partially deleted COXIV gene from yeast. Panel
B, complementation analysis of yeast strain WSR transformed with
the plasmids described in panel A. Strains WSR,
WSR[pY
COX], WSR[pYCOX], and WSR[pFPS1Ltp-Y
COX] were
streaked onto N3 medium, containing glycerol as energy source, and
incubated at 28 °C for 2 days.
[View Larger Version of this Image (37K GIF file)]
Fig. 6.
Import of Arabidopsis FPS1L into
purified potato mitochondria. Lane 2 shows the radiolabeled
FPS1L (44 kDa) and FPS1S (40 kDa) isoforms (precursor, Pre),
resulting from in vitro translation of the FPS1L mRNA.
The in vitro translated FPS1 isoforms were completely
digested after the addition of proteinase K (lane 1). For
the import reaction, the in vitro labeled polypeptides were incubated with purified potato mitochondria. An aliquot of the import
reaction was left untreated (Imp, lane 3). Two
aliquots were made from the remainder of the import reaction and were
further processed by treatment either with proteinase K (lane
4) or proteinase K and Triton X-100 (lane 5). The
in vitro labeled polypeptides were also incubated with
mitochondria previously treated with valinomycin. Half of the reaction
was left untreated (Imp, lane 7), and half of the
reaction was digested with proteinase K (lane 6). Samples
were separated in a SDS, 10% polyacrylamide gel and analyzed by
fluorography. The estimated molecular masses are indicated on the
left.
[View Larger Version of this Image (40K GIF file)]
-ends, which correlates with the lack of consensus TATA
box located at appropriate distances from the corresponding transcription start sites.
2 and
12 from the putative cleavage site (RI/QS). It has
been proposed that arginine residues located around positions
2 and
10 from the processing site play a role in defining the precise
cleavage site of targeting peptides by mitochondrial matrix proteases
(35). Third, secondary structure analysis indicates that a sequence of
10 amino acid residues located at the NH2-terminal part of
the FPS1L NH2-terminal extension can potentially fold into
a positively charged amphiphilic
-helix. Taken together, all these
observations strongly suggested that the FPS1L NH2-terminal
extension was a mitochondrial transit peptide.
COXIV) was assayed for its ability to complement the mutant
yeast strain WSR. This strain is unable to grow in a medium containing
glycerol as energy source because of a disruption of the
COXIV gene, which encodes the CoxIV subunit of mitochondrial
cytochrome c oxidase. Plasmid pFPS1Ltp-
COXIV completely
restored the respiratory activity of strain WSR, thus indicating that
the yeast CoxIV subunit was properly targeted into mitochondria. Since
the interchangeability of mitochondrial transit peptides between plants
and yeast is well documented (31, 38-41), the observation that the
NH2-terminal extension of the Arabidopsis FPS1L
isoform acts as a mitochondrial transit peptide in yeast indicates that
Arabidopsis FPS1L is the precursor of a mitochondrial FPS
isoform. This fact was confirmed further by in vitro import
studies of the Arabidopsis FPS1L into plant mitochondria.
In vitro translated FPS1L protein was imported into purified
potato mitochondria and processed to a mature mitochondrial FPS1
isoform of 40 kDa by cleavage of a transit peptide of approximately 4 kDa. From this result and assuming that FPS1L is processed by mitochondrial matrix proteases at the predicted processing site (see
above), the mature mitochondrial FPS1 isoform would contain four
additional amino acid residues at its NH2 terminus (QSSS) with respect to the FPS1S isoform.
-region and, depending on the translation
start codon used to initiate translation of the corresponding
mRNAs, have the potential to encode enzymes differing only at their
NH2 terminus. These isozymes can be targeted to different
intracellular compartments. Genes of this type have recently been
reported in plants. For example, the Arabidopsis alanyl-tRNA
synthetase gene encodes both mitochondrial and cytosolic forms of the
enzyme (31), and the Arabidopsis glutathione reductase gene
encodes both mitochondrial and chloroplastic isoforms and possibly also
a cytosolic isoform (45). In the case of the Arabidopsis
FPS1 gene, the strategy used to generate the cytosolic and the
mitochondrial FPS1 isoforms is the use of alternative transcription
start sites, resulting in the synthesis of mRNAs that differ in the
presence or absence in the 5
-region of the sequence encoding the
mitochondrial transit peptide. However, the in vitro
translation results showed that FPS1L mRNA can give rise not only
to the FPS1L isoform but also to significant levels of the FPS1S
isoform. It is thus possible that the choice of cytosolic or
mitochondrial location of FPS might involve not only the use of
alternative transcription start sites but also a mechanism of AUG
selection during the translation initiation process. It is remarkable
that a similar situation has been reported to occur in the
Arabidopsis HMG1 gene, which encodes another key regulatory
enzyme of the isoprenoid pathway (46). This gene contains two
alternative promoters that direct the expression of different mRNAs
encoding two 3-hydroxy-3-methylglutaryl-CoA reductase isoforms (HMGR1L
and HMGR1S), which differ only in the presence or absence of an
NH2-terminal extension of 50 amino acids. In this case both
3-hydroxy-3-methylglutaryl-CoA reductase isoforms are targeted
primarily to the endoplasmic reticulum membrane, although it has been
proposed that the different NH2-terminal sequences might
direct each 3-hydroxy-3-methylglutaryl-CoA reductase isoform to
specific subdomains of the endoplasmic reticulum (46, 47). These
observations give further support to the view that the regulation of
plant isoprenoid biosynthesis is under the control of complex
regulatory mechanisms operating at both transcriptional and
post-transcriptional level.
*
This work was supported in part by Grants PB93-0753 from the
Dirección General de Investigación Científica y
Técnica and GRQ94-1034 and 95SGR-00457 from the Comissión
Interdepartamental de Recerca i Innovación Tecnològica de
la Generalitat de Catalunya.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.
§
Recipient of a predoctoral fellowship from the Dirección
General de Recerca de la Generalitat de Catalunya.
To whom correspondence should be addressed. Tel.:
34-3-4024522; Fax: 34-3-4021896; E-mail:
aferrer{at}farmacia.far.ub.es.
1
The abbreviations used are: FPS,
farnesyl-diphosphate synthase; PCR, polymerase chain reaction; bp, base
pair(s).
COX and yeast strain WSR and Dr. Wolfgang
Schuster for help and advice on potato mitochondria
isolation and import experiments. We also thank Robin Rycroft for
editorial help.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.