From the Graduate School of Biological Sciences, Nara
Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara
630-0101 and the ** Plant Function Laboratory, The Institute of Physical
and Chemical Research, Hirosawa 2-1, Wako,
Saitama 351-0198, Japan
Received for publication, February 6, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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Protoporphyrinogen oxidase (Protox) is the
final enzyme in the common pathway of chlorophyll and heme
biosynthesis. Two Protox isoenzymes have been described in tobacco, a
plastidic and a mitochondrial form. We isolated and sequenced spinach
Protox cDNA, which encodes a homolog of tobacco mitochondrial
Protox (Protox II). Alignment of the deduced amino acid sequence
between Protox II and other tobacco mitochondrial Protox homologs
revealed a 26-amino acid N-terminal extension unique to the spinach
enzyme. Immunoblot analysis of spinach leaf extract detected two
proteins with apparent molecular masses of 57 and 55 kDa in
chloroplasts and mitochondria, respectively. In vitro
translation experiments indicated that two translation products (59 and
55 kDa) are produced from Protox II mRNA, using two in-frame
initiation codons. Transport experiments using green fluorescent
protein-fused Protox II suggested that the larger and smaller
translation products (Protox IIL and IIS) target exclusively to
chloroplasts and mitochondria, respectively.
Protoporphyrinogen oxidase
(Protox,1 EC 1.3.3.4) is the
final enzyme in the common pathway of chlorophyll and heme
biosynthesis, catalyzing the six-electron oxidation from
protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX) (1).
Protox homologs have been purified to apparent homogeneity from mouse
liver mitochondria (2), yeast mitochondria (3), bovine liver
mitochondria (4, 5), and spinach chloroplasts (6). In all instances,
Protox requires a flavin cofactor for enzymatic activity and a
detergent to be extracted from the membrane. Protox genes or cDNAs
have been cloned from Escherichia coli (7), Bacillus
subtilis (8), human (9), cow (5), mouse (10), and yeast (11). The calculated molecular masses of these Protox gene products range from 50 to 60 kDa, except for the 21-kDa E. coli. isoform. The N
terminus is most widely conserved, containing a consensus sequence GXGXXG, which forms part of a Since Protox is the final common enzyme in the chlorophyll and heme
biosynthetic pathways in plants, Protox must play a role in
distributing Proto IX to both pathways. Accurate examination of the
transport and subcellular localization of plant Protox is important for
an understanding of tetrapyrrole precursor flux and regulation of
tetrapyrrole synthesis. We have recently reported the characterization
and subcellular localization of plastidal Protox (Protox I) in spinach
(14). Protox I cDNA (gene name is SO-POX1) encodes a
563-amino acid, 60-kDa protein with a putative chloroplast transit
peptide. N-terminal sequence analysis of mature Protox I purified from
chloroplasts by immunoprecipitation revealed that the Protox I
precursor is cleaved at Ser-49. The predicted transit peptide (Met-1 to
Cys-48), when analyzed as a GFP-fusion construct, was found to be
sufficient for targeting and transport of the fused protein into
chloroplasts. Furthermore, immunocytochemical analysis using
electron microscopy has shown that spinach Protox I preferentially
associates with the stromal side of the thylakoid membrane, while a
small fraction of Protox I is located on the stromal side of the inner
envelope membrane (14).
Knowledge of plant mitochondrial Protox is poor in comparison with that
of plastidal Protox. In animal and yeast cells, Protox activity was
mainly detected in the inner mitochondrial membrane and solubilized
from the membrane by several detergents (3, 4, 15), suggesting that the
Protox was intimately associated with this structure. In plant cells,
Protox activity and tobacco PPX-II protein itself have also been
observed in mitochondria (13). However, there have been no
investigations into the precise suborganellar location of Protox in
plant mitochondria. Furthermore, little is known about the transport
mechanism of mitochondrial Protox. In general, proteins transported
into the mitochondria have an N-terminal targeting peptide, which is
processed after transport is complete (16). However, tobacco PPX-II and
other known homologs of PPX-II do not possess the typical mitochondrial targeting sequence at their N termini (Ref. 17; accession nos. AB025102
and AJ225107). Actually, in vitro transport experiments have
shown that tobacco PPX-II is transported to mitochondria without any
size reduction (13). The mechanism by which this occurs is not yet understood.
Protox is the target enzyme of phthalimide-type herbicides such as
N-[4-chloro-2-fluoro-5-propagyloxy]-phenyl-3,4,5,6-tetrahydrophthalimide (S23142) and diphenylether-type herbicides such as
5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoic acid (18). In
plant cells, the inhibition of Protox by the herbicides ultimately
results in massive accumulation of Protogen, which then leaks out of
the plastid (19). Cytosolic protogen is rapidly oxidized to Proto IX by
nonspecific, herbicide-resistant peroxidases that are bound to the
plasma membrane. Highly reactive singlet oxygen generated by light
activation of Proto IX provokes membrane lipid peroxidation, leading to
cell death (18). We have previously obtained S23142-resistant tobacco
cultured cells (YZI-1S) by stepwise selection with the herbicide (20).
Several lines of data provide strong evidence that the herbicide
resistance of YZI-1S cells is due to the overproduction of
mitochondrial Protox, even though the primary target of the herbicide
is chloroplast Protox. In the resistant cells, excess Protogen
generated by inhibition of chloroplast Protox is rapidly utilized for
heme synthesis in mitochondria by the abnormally high level of
mitochondrial Protox, thus preventing the accumulation of Proto IX
(21). This suggests that mitochondrial Protox is also important in
preventing the accumulation of the photosensitizer, Proto IX, and in
maintaining the flow of tetrapyrrole precursors for heme and
chlorophyll biosynthesis. Therefore, detailed knowledge about the
subcellular localization and transport mechanism of plant mitochondrial
Protox is important for clarification of not only the control mechanism
of tetrapyrrole biosynthesis but also the mode of action of the
herbicide resistance.
In the present study, we report the molecular cloning, subcellular
localization, and transport mechanism of spinach Protox II, which has
high sequence identity to the tobacco mitochondrial Protox.
Surprisingly, this protein was located not only in mitochondria but
also in chloroplasts. Two translation products of different sizes
(Protox IIL and Protox IIS) were produced from Protox II mRNA using
two in-frame initiation codons and then were transported into
chloroplasts and mitochondria, respectively. It has been thought that
the known plant proteins that are dually targeted to chloroplasts and
mitochondria have an ambiguous N-terminal targeting sequence, which
interacts nonspecifically with import machinery (or chaperon) for both
mitochondria and chloroplasts (22). The manner of the dual targeting in
Protox II is clearly different from the above system. We have shown
here for the first time the existence of a novel mechanism of
intracellular targeting for compartmentalizing protein isoforms in
chloroplasts and mitochondria.
Chemicals and Plant Material--
Spinach plants (Spinacia
oleracea L. cv. tonic, Watanabe-saishujyo Ltd.) were grown in a
greenhouse at 25 °C for 8 weeks or in a growth chamber on a 12-h
light/12-h dark cycle at 20 °C for 4 weeks at a light intensity of
140 µmol m cDNA Cloning and Sequence Analysis--
Total RNA was
isolated from 4-week-old spinach leaves using RNeasy plant kits
(Qiagen, Germany). First-strand cDNA was synthesized from total RNA
using a Ready-To-Go T-primed first-strand kit (Amersham Pharmacia
Biotech). For polymerase chain reaction (PCR) isolation of
mitochondrial Protox cDNA, one set of oligonucleotide primers was
synthesized on the basis of the nucleotide sequences of tobacco PPX-II cDNA (13, 21). The primer sequences were as
follows: 5'-GAAGG(A/G)GCAAA(C/T)AC(C/T)ATGACT-3' (SMT-1F) and
5'-ATCAGGAAG(A/T)(A/G)T(C/T)TGCATTCC-3' (SMT-1R). Taq DNA
polymerase and reaction buffer (Expand High Fidelity PCR system) were
purchased from Roche Molecular Biochemicals (Mannheim, Germany) and
used for all PCR experiments (94 °C for 5 min, 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C). PCR
reactions were terminated with a 10-min incubation at 72 °C and
stored at 4 °C. PCR fragments (0.56 kb) were cloned using a TA
cloning kit (Invitrogen, Groningen, Netherlands), and five clones from
each PCR reaction were sequenced with a DNA sequencer (model 377; PE
Applied Biosystems). PCR and cloning procedures were independently
repeated twice to confirm DNA sequences.
The 5' end of Protox cDNA was amplified by rapid amplification of
cDNA ends (RACE) using a 5'/3'-RACE kit (Roche Molecular Biochemicals). First-strand cDNA synthesized from mRNA
was dA-tailed with terminal deoxytransferase, and second-strand
cDNA was synthesized using a poly(T) cassette primer
(5'-ACTCGAATTCACGCGGCCGCAT15-3'). An initial 5'-RACE
PCR was performed using specific primer I (5'-GATCTCCACCACTTGTACCCG-3') and cassette primer I (5'-ACTCGAATTCACGCGGCCGCA-3') under the recommended PCR conditions. After the first reaction, the PCR product was diluted 2000-fold and subjected to secondary PCR
amplification. The second amplification was performed using
specific primer II (5'-TCCACCTTTTCCTTCTTGG-3') and cassette primer I. For 3'-RACE, cDNA was synthesized from mRNA with a poly(T)
cassette primer (5'-TGGAAGAATTCGCGGCGGCAGT16-3'). 3'-RACE
PCR was performed using specific primer III
(5'-GATCTCCACCACTTGTACCCG-3') and cassette primer II (5'-
TGGAAGAATTCGCGGCGGCAGT-3'). The amplified fragments from 3'-RACE (1.1 kb) and for 5'-RACE (0.65 kb) were cloned with a TA cloning kit
(Invitrogen), and five independent clones from each PCR reaction were
sequenced as described above. PCR and cloning procedures were
independently repeated twice, and DNA inserts were sequenced on
both strands to ensure that no mutations had been introduced during PCR amplification.
Southern Blot Analysis--
Genomic DNA was isolated from young
spinach leaves with a Nucleon plant-DNA extraction kit (Amersham
Pharmacia Biotech) according to standard protocol. The DNA (10 µg)
was digested with BglII, DraI, or
EcoRI and then electrophoresed on a 0.7% (w/v) agarose gel.
Fractionated DNA was transferred onto a Hybond N+ membrane
(Amersham Pharmacia Biotech). Mitochondrial Protox cDNA (1.6 kb)
was labeled with an AlkPhos Direct labeling kit (Amersham Pharmacia
Biotech) according the manufacturer's instructions. Hybridization and
wash were done at 65 °C, and the signals were detected by a CDP-Star
chemiluminescent detection reagent system (Amersham Pharmacia Biotech).
Antibody Production and Purification--
The internal region of
spinach Protox II, encompassing the region from Ser-75 to Ser-415 (see
Fig. 1), was cloned in-frame into the pET-28(a)+ vector (Novagen,
Madison, WI). The resulting His tag fusion protein was overproduced in
the BL21(DE3) strain of E. coli (Invitrogen) and purified
using a HisTrap kit according to the manufacturer's protocol (Amersham
Pharmacia Biotech). The purified recombinant protein (10 mg) was used
to immunize rabbits. Antiserum was further purified by affinity
chromatography with a HiTrap
N-hydroxysuccinide-activated Sepharose column
(Amersham Pharmacia Biotech) conjugated to the recombinant Protox II.
Production and purification of polyclonal antibodies raised against
recombinant spinach Protox I has been described previously (14).
Immunoblot Analysis--
For immunoblot analysis, intact
chloroplasts and mitochondria were isolated from 4-week-old spinach
leaves by centrifugation on a Percoll linear gradient according to the
method of Gualberto et al. (23). Fractionated chloroplasts
and mitochondria were judged to be intact by methods described
previously (14). Isolation of the envelope and thylakoid membranes from
intact chloroplasts was performed as described previously (14). These
samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE)
(10%, v/v). Separated proteins were electrophoretically transferred to
a nitrocellulose membrane (Hybond N-ECL, Amersham Pharmacia Biotech)
with a Bio-Rad semidry blotter. Nonspecific binding was blocked with
3% bovine serum albumin (BSA) in 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% (w/v) Tween 20 for 1 h at room
temperature. Immunoreactive polypeptides were detected using an
alkaline phosphatase-conjugated goat antibody raised against rabbit
IgG(H+L) (Jackson Immunoresearch Laboratories) and visualized by
reaction with nitro blue tetrazolium chloride and bromochloroindolyl
phosphate. Protein concentration was determined with a protein assay
kit (Bio-Rad) using BSA as the standard.
For quantitative analysis of the amount of Protox II and Protox I
within mitochondria or chloroplasts, immunodetections were performed
with an enhanced immunochemiluminescence kit (ECL-plus, Amersham
Pharmacia Biotech) and chemiluminescence was detected with a
photographic film (Hyperfilm; Amersham Pharmacia Biotech). As the
internal standard for chemiluminescence intensity, recombinant Protox
II and Protox I were used. The signal was quantified by scanning
densitometry using an NIH Image 1.61 software (National Institutes of
Health), from exposed film with the exposure time adjusted to
non-saturating conditions
Functional Complementation of hemG-deficient Mutant of E. coli--
Two cDNA clones encoding Protox II (Met-1 to Ile-562 and
Met-27 to Ile-562) were amplified by PCR using the following two sets
of primers: (5'-TATGGTAATACTACCGGTTTC-3' and
5'-ACAAGATGACCGAGGAGACTATATAA-3'); and (5'-TATGGGCAACGTTTCTGAGCGAAAT-3'
and ACAAGATGACCGAGGAGACTATATAA-3'), respectively. Amplified fragments
were cloned into a pCR 2.1 plasmid vector (Invitrogen) in-frame with
the lacZ gene. The resulting plasmids, pM1M2-POX2 and
pM2-POX2, were introduced into E. coli BT3
( In Vitro Transcription and Translation--
For construction of
native Protox II, cDNA encoding native spinach Protox II (Met-1 to
Ile-562) was PCR-amplified using one set of primer pair:
5'-ACTGATCGAATGGTAATACTACCG-3' and 5'-TTATATAGTCTCCTCGGTCATCTTGT-3'. In
order to introduce a ATG to ATC point mutation in the first initiator
Met codon, the following set of mutagenic primers was used for the PCR
amplification: 5'-ACTGATCGAATCGTAATACTACCG-3' and
5'-TTATATAGTCTCCTCGGTCATCTTGT-3'. These two constructs were then cloned
into the pGEM-T easy vector downstream of T7 RNA polymerase promoter
(Promega). The resulting plasmids were designated pM1M2-POXII and
pM2-POXII, respectively. Furthermore, the construct M1-POXII, in which
only the second ATG (encoding Met-27) of Protox II is converted to an
ATC codon (encoding Ile), was amplified by PCR-based site-directed
mutagenesis of the pM1M2-POXII plasmid using the following primer pair:
5'-CCAGTTATCGGCAACGTTTCT-3' and
5'-ACGTTGCCGATAACTGGGTTG-3'. The cDNA fragment was also
cloned into the pGEM-T easy vector, with the resulting plasmid being
designated pM1-POXII. These plasmids were digested with
SacII and ApaI to remove the extra sequences between the T7 promoter and Protox II coding regions. The termini of
DNA fragments were converted to blunt ends with a DNA blunting kit
(TaKaRa Shuzo, Kyoto, Japan) and then were self-ligated with T4 DNA
ligase (Life Technologies, Inc.). The plasmids were linearized with
MluI. The linearized plasmids (pM1M2-POX2, pM2-POX2, and pM1-POX2) were used as the templates for in vitro
transcription/translation using the T7 TNT quick-coupled wheat germ
extract systems (Promega) according to the manufacturer's
instructions. The translated proteins were separated by SDS-PAGE (10%
acrylamide gel) and detected by immunoblotting with a antibody against
spinach Protox II.
Transport Experiment Using GFP Fusion Proteins--
For
construction of a GFP expression vector, the GFP coding sequence was
PCR-amplified from the pGFP2 vector as described previously (14).
cDNA fragments containing Protox II N-terminal regions were
obtained from full-length cDNA of spinach Protox II by PCR using
two sets of specific primers as follows. For M1T97, SPMTS1 primer
(5'-GTCTAGAATGGTAATACTACCGGTTTCCC-3') and SPMTS2 primer
(5'-CAGTACTAGTGGTATTTGCCCCTTCATCCCAA-3') were used; for M2T97, SPMTS2
primer (5'-GTCTAGAATGGGCAACTGAGCGA-3') and SPMTA2 primer were used. For
construction of M1T97, the second ATG (encoding Met-27) was converted
to ATC (encoding Ile-27) by PCR-based site-directed mutagenesis as
described above. Amplified fragments were digested with NcoI
and SpeI and cloned in-frame into the XbaI and
SpeI sites of the pCR 2.1 vector containing the
GFP2 gene. The plasmid was further digested with
XbaI and SacI, and the resulting GFP fusion
constructs were cloned into the cleavage sites of the expression vector
pBI221 (Novagen). As a control for mitochondrial GFP transport, a
fusion construct encoding CoxIV-GFP (24) was used (provided by Dr.
Kinya Akashi, Nara Institute of Science and Technology, Nara, Japan).
Transient expression of GFP constructs by particle bombardment in
spinach leaf cells was carried out as described previously (14). GFP
fluorescence was observed using a fluorescence microscope (Leica) with
a Micro Mover-W (Photometrics, Tucson, AZ) fitted with a triple-band
filter (81 series Pinkel no. 1 filter set; Chroma Technology,
Brattleboro, VT). Autofluorescence in chloroplasts was observed at an
excitation wavelength of 540 nm and an emission wavelength of 600 nm,
and the GFP fluorescence was observed at an excitation wavelength of
495 nm and emission wavelength of 530 nm. Two images were acquired
separately by the IP Lab-PVCAM system with a cooled CCD camera
(Photometrics) and pseudo-colored based on the original emission
fluorescence. The composite images were printed with Pictrography 3000 (Fuji, Tokyo, Japan).
Immunogold Electron Microscopy--
For immunogold electron
microscopy of Protox II distribution, spinach leaves of 1-month-old
seedlings were fixed with 0.6% glutaraldehyde and 4% paraformaldehyde
in a 0.05 M sodium cacodylate buffer (pH 7.4) for 5 min at
4 °C under a vacuum and stored at 4 °C for 1 h. The tissues
were rinsed with a 0.05 M sodium cacodylate buffer (pH 7.4)
for 2 h at 4 °C. The fixed materials were then dehydrated in an
ethanol series at 4 °C, embedded in LR White resin (London Resin
Co., London, United Kingdom), and polymerized by UV irradiation at room
temperature. Ultrathin sections were cut with a diamond knife and
mounted on uncoated nickel grids.
Prior to incubation in the primary antibody, the sections were blocked
with 1% BSA and 1% goat serum in PBS for 30 min at room temperature.
Specific antibody against plastidal Protox was diluted in PBS
supplemented with 1% goat serum. Incubation with the primary antibody
was performed overnight at 4 °C, followed by washing with 0.05%
Tween 20 in PBS (PBST, pH 7.4). The secondary antibody, goat
anti-rabbit IgG conjugated to 15-nm gold particles (Biocell Research
Laboratories, Cardiff, United Kingdom), was diluted in PBST. Sections
were incubated in the secondary antibody for 90 min at room
temperature. The sections were then washed in PBST and in distilled
water, followed by staining with uranyl acetate for 20 min. The
sections were observed under a transmission electron microscope
(H-7100; Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV.
Isolation and Characterization of Spinach cDNA Encoding a
Tobacco Mitochondrial Protox Homolog--
To isolate cDNA encoding
the spinach homolog of tobacco mitochondrial Protox, one set of
specific oligonucleotide primers (SMT-1F and SMT-1R) was synthesized
based on the PPX-II sequence from N. tabacum
(tobacco) (13, 21). PCR amplification of cDNA from spinach produced
a 565-bp product. The nucleotide sequence of the cDNA clone was
highly homologous to tobacco PPX-II (67%). Full-length
cDNA was obtained by 5'- and 3'-RACE PCR. The 1896-bp cDNA
contained an open reading frame of 1593 bp, which encoded a 531-amino
acid protein with a calculated molecular mass of 58,319 Da (Fig.
1). The deduced amino acid sequence shows
a high degree of identity to tobacco mitochondrial Protox (70%), while
the sequence identity to Protox I (plastidal Protox of spinach) (14) is
relatively low (28%). The glycine-rich motif
GXGXXG that had been previously proposed as a
dinucleotide binding site of many flavin-containing proteins (25) was
also found in the sequence (Fig. 1). All of the sequence data suggest
that this cDNA encodes the spinach homolog of tobacco mitochondrial
Protox, resulting in its designation as Protox II (gene name is
SO-POX2).
N-terminal sequence alignment between Protox II and other tobacco
PPX-II homologs (mitochondrial Protox isoforms) revealed the existence
of 26- and 33-amino acid N-terminal extensions in the spinach and maize
proteins, respectively (Fig. 2).
Furthermore, the internal sequence following the second Met (Met-27 of
spinach Protox II and Met-34 of maize Protox-2) aligned with N-terminal sequences of other plant mitochondrial Protoxes. The extension sequences were enriched in Ser and Thr but did not contain any acidic
amino acids such as Asp or Glu. Such features are characteristic of
chloroplast transit peptides (26). In fact, ChloroP, a program that
searches for putative chloroplast targeting sequences (27), predicted
that a chloroplast transit peptide is contained within both extension
sequences (data not shown). On the other hand, a typical mitochondrial
import signal sequence was not found in either the extension or in the
subsequent region following the second Met.
It was reported that cDNA encoding tobacco mitochondrial Protox is
able to complement the hemG mutation in E. coli
(13). BT3 (
To determine the number of copies of the Protox II gene in spinach, we
performed Southern blot analysis of complete spinach genomic DNA
digested with BglII or DraI, which have no
recognition sites within Protox II gene, and EcoRI, which
has one site within the gene. Under high stringency hybridization
conditions, a single band in the BglII and DraI
digests, and two bands in the EcoRI digest were observed,
indicating that only one copy of the Protox II gene exists in the
spinach genome (Fig. 3).
Localization of Spinach Protox II--
To examine the localization
of spinach Protox II, we prepared a polyclonal antibody against a
recombinant fragment of spinach Protox II (Ser-101 to Ser-443) and
purified it by affinity chromatography (see "Experimental
Procedures"). The purified anti-Protox II antibody did not
cross-react with recombinant spinach Protox I (data not shown).
Immunoblot analysis with the antibody revealed the presence of two
proteins with apparent molecular masses of 57 and 55 kDa in total
spinach leaf extract (Fig. 4A,
lane 1). When separate analyses of chloroplast
and mitochondrial fractions were conducted, however, the 55-kDa band
was found only in mitochondria, whereas the 57-kDa isoform was observed
exclusively in chloroplasts (lanes 2 and
3). To identify more precisely the subcellular localization of these proteins, membrane fractions were prepared from mitochondria and chloroplasts, with further processing of the chloroplasts into
thylakoid, envelope, and stroma fractions. Immunoblot analysis showed
the presence of the 55-kDa protein in the mitochondrial membrane
fraction and the 57-kDa protein in the chloroplast envelope fraction
(Fig. 4B). These results suggest that Protox II exists as
two isoforms with different molecular masses in spinach leaf and that
the smaller and larger isoforms are located on the mitochondrial membrane and chloroplast envelope membrane, respectively.
In Vitro Translation Products from Transcripts of Protox II
Gene--
Although immunoblot analysis revealed the presence of two
Protox II isoforms in spinach (Fig. 4), Protox II gene exists as one
copy gene in the spinach genome (Fig. 3). Moreover, spinach Protox II
has an N-terminal extension composed of 26 amino acids, which precedes
a region that is initiated by a second methionine (Met-27) and is
highly identical to the N-terminal sequences of other plant
mitochondrial Protox proteins (Fig. 2). The calculated molecular masses
of Protox II with and without the extension (58,319 and 55,615 Da)
correlate well to the observed masses of the two isoforms (57 and 55 kDa) observed in immunoblot analysis (Fig. 4). This correlation raises
the possibility that the two isoforms were translated from the same
spinach Protox II mRNA using two different in-frame AUG initiator
codons. To explore this hypothesis, we analyzed the translation
products from Protox II mRNA using an in vitro
transcription/translation-coupled system from wheat germ lysate. When
the pM1M2-POXII vector, containing the two putative in-frame initiation
codons (Fig. 5A) was used as a
template, two protein bands with apparent molecular masses of 59 and 55 kDa were detected by anti-Protox II antibody (Fig. 5B,
lane 1). To confirm that these two proteins were
translated separately from two in-frame initiation codons of one
mRNA, two other vectors were constructed, pM1-POXII, in which the
second ATG codon was mutated to ATC, and pM2-POXII, in which the first
ATG codon was mutated to ATC (Fig. 5A). When the pM2-POXII
vector was used as a template, only the 55-kDa protein band was
detected (Fig. 5B, lane 2), whereas a 59-kDa
protein was exclusively generated from pM1-POXII (lane
3). In a control experiment lacking plasmid, no protein was
detected (data not shown). These results clearly indicate that two
proteins of 59 and 55 kDa are translated from a single Protox II
mRNA using two in-frame AUGs. The larger and smaller forms of
Protox II were named Protox IIL and Protox IIS, respectively.
Transport of Protox IIL and Protox IIS into Chloroplasts and
Mitochondria--
In vivo transport of Protox IIL and
Protox IIS was studied using green fluorescence protein (GFP). cDNA
encoding the N-terminal region of Protox IIL (Met-1 to Thr-97) in which
Met-27 was converted to Ile-27 (M1T97) and cDNA encoding the
N-terminal region of Protox IIS (Met-27 to Thr-97) (M27T97) were each
fused with the 5' end of the GFP gene. Each construct was placed under
the control of the cauliflower mosaic virus 35 S promoter. These
plasmids were introduced into spinach leaves by bombardment, and
transient expression was observed by fluorescence microscopy. When the
Protox IIL construct, pM1T97-GFP, was introduced into a spinach leaf,
green fluorescence was seen in chloroplasts (Fig.
6A). In contrast, in the
spinach leaf transfected with pM27T97-GFP, the Protox IIS construct,
the green fluorescence was observed only in punctate organelles (Fig. 6B). The fluorescence pattern of yeast CoxIV-GFP, which is
known to be efficiently delivered into mitochondria (24) was very similar to that of pM27T97-GFP (Fig. 6C), indicating that
M27T97-GFP protein does indeed co-localize with mitochondria in
spinach. Transfection of control GFP, lacking an N-terminal tag, gave a diffuse pattern, with green fluorescence spread out over the
guard cell (Fig. 6D).
Spatial Distribution of Protox II in Chloroplasts and
Mitochondria--
To identify the spatial distribution of Protox II
within chloroplasts and mitochondria, we performed an immunogold
electron microscopic analysis. When ultrathin sections of spinach leaf tissue were incubated with specific anti-Protox II antibody and gold-conjugated antiserum to rabbit immunoglobulin, gold particles were
observed in chloroplasts and mitochondria. In chloroplasts, most of the
gold particles were conjugated to the stromal side of the inner
envelope membrane (Fig. 7A),
whereas gold particles were found on the inner mitochondrial membrane
(Fig. 7B). When preimmune serum was used, there was no
appreciable binding of gold particles and the omission of primary
antibody yielded negative results (data not shown). The observations
suggest that Protox IIL associates with the stromal side of the inner
envelope membrane, and that Protox IIS is found on the inner
mitochondrial membrane.
Quantitation of Two Protox II Isoforms and Protox I in Chloroplasts
and Mitochondria--
The above results provide strong evidence that
Protox II proteins are located not only in the mitochondria but also in
chloroplasts. Spinach plastidal Protox, Protox I, associates primarily
with the stromal side of the thylakoid membrane, with a small portion localizing to the stromal side of the inner envelope membrane (14).
These results raise a new question as to how the two isoforms of Protox
II and Protox I are distributed and how the function of each Protox is
coordinated in spinach cells. To address the question, we performed
quantitative antibody analysis of Protox I and Protox II. The level of
Protox IIL in chloroplasts (1.2 ± 0.03 pmol/mg of chloroplast
protein) was about 10 times lower than that of Protox I (14.7 ± 2.5 pmol/mg of chloroplast protein) (Table
I). The amounts of Protox IIL and Protox
I in the chloroplast envelope membrane are very similar (44.3 ± 7.8 and 48 ± 10.8 pmol/mg of envelope protein, respectively),
whereas the amount of Protox I in thylakoid membrane was estimated at
28.4 ± 5.9 pmol/mg of thylakoid protein (Table I). Since almost
half of all spinach chloroplast proteins exist in the thylakoid
membrane and only 2% of total chloroplast proteins are present in the
envelope membrane (28), the total amount of thylakoid-associated Protox
I was estimated at about 14 pmol/mg of chloroplast protein, and the relative amount of Protox IIL and Protox I in the envelope membrane was
estimated to be about 10 times less, at 1.2 pmol/mg of chloroplast protein. On the other hand, about 12.2 ± 0.1 pmol of Protox
IIS/mg of mitochondria protein was estimated to exist in mitochondria. Since the ratio of chloroplast to mitochondrial proteins in one cell is
not known, a precise ratio of chloroplast Protox to mitochondrial Protox in one cell could not be obtained. However, it is interesting that the amount of Protox IIS per mitochondria protein is similar to
the amount of the two chloroplast Protoxes per chloroplast protein
(Table I).
Alternative Translation Initiation at Two In-frame
AUGs--
Alternative transcription initiation and alternative
translation initiation are well known mechanisms for dual protein
production from a single gene (29). Some genes can produce more than
one mRNA by use of alternative transcription initiation sites.
Frequently, the production of multiple mRNAs has no functional
consequence. However, if transcript heterogeneity leads to polypeptide
heterogeneity, then it can result in the production of proteins with
markedly different structures and properties (22, 29). The region
upstream of the transcription start site often contains
homopurine/homopyrimidine sequences, which are recognized by a protein
factor and are able to initiate gene transcription (30, 31). Such a
homopurine/homopyrimidine sequence was not found between the first and
second ATGs of the Protox II genome DNA (data not shown). Moreover,
only the longer of the cDNA variants, containing the first and
second ATGs, was obtained in the process of Protox II cDNA cloning
(Fig. 1), suggesting that the dual production of Protox IIL and IIS
would not be a result of alternative transcription initiation.
Another mechanism by which variability can be introduced into the N
terminus of a polypeptide encoded by a single gene is alternative
translation initiation from a single transcript using two potential
in-frame translation initiation sites (29). The eukaryotic mechanism of
translation initiation is distinctive in that the 40 S ribosomal
subunit normally binds at the 5' end of the mRNA. The small
ribosomal subunit then migrates through the 5'-untranslated region
until it encounters the first AUG codon. Flanking sequences modulate
the efficiency with which the first AUG codon is recognized as a stop
signal during the scanning phase of initiation. Initiation sites
usually conform to all or part of a GCCRCCaugG sequence (32). The most
highly conserved position within this consensus sequence is the purine
at positions
Among other homologs of tobacco mitochondrial Protox, maize Protox-2
has a 33-amino acid N-terminal extension that is similar to that of
Protox II (Fig. 2). Analysis of the upstream sequences of the first and
the second AUG codons showed that, as in spinach Protox, the second AUG
also possesses a stronger initiator sequence than the first AUG,
suggesting that the two isoforms may also be synthesized from a single
maize Protox-2 mRNA. It is not known why only two Protoxes (spinach
and maize) have N-terminal extensions. Several PPX-II homologs may
actually have unknown N-terminal extensions, which would require more
careful sequence analysis to identify.
Intracellular Targeting of Two Protox II Isoforms--
GFP fused
to the N-terminal region of spinach Protox IIL (pM1T97-GFP) was
transported into chloroplasts, whereas GFP fused to the homologous
region from Protox IIS (pM27T97) was targeted to mitochondria (Fig. 6).
This implies that the extension may serve as a chloroplast transit
sequence. The N-terminal transit peptide is usually cleaved off the
precursor protein as it is transported across the two membranes of
chloroplast (33). There was a small difference in the molecular sizes
estimated by SDS-PAGE between the in vitro translation
product (59 kDa) and mature protein identified in a chloroplast
fraction (57 kDa). This size difference was also confirmed by
co-electrophoresis of both proteins in the same lane (data not shown),
suggesting that Protox IIL is processed after transport into the chloroplast.
A great majority of mitochondrial proteins is also encoded in the
nuclear genome, synthesized in the cytosol, and then transported to the
mitochondria. Most mitochondrial proteins are synthesized as larger
precursors containing N-terminal cleavable extension peptides called
presequences (34). However, MitoP, a commonly used computer
program predicting mitochondrial presequences (35), predicted that
spinach Protox IIS has no typical cleavable mitochondrial presequence.
This prediction is in agreement with our experimental data that the
molecular size of mature Protox IIS in leaves is identical with that of
an in vitro synthesized product (Figs. 4 and 5B).
Therefore, spinach Protox IIS would be put into the rare class of
mitochondrial proteins lacking a cleavable presequence. The signal for
mitochondrial targeting and import in such non-cleavable mitochondrial
proteins is often still contained within the N-terminal region (36). In
a transport experiment using a GFP fusion protein, it was revealed that
a peptide consisting of residues from Met-27 to Thr-97 of Protox IIS is
sufficient for mitochondrial transport (Fig. 6B). Such
activity was not observed when only the first 23 amino acids (Met-27 to
Val-49) were used for fusion with GFP (data not shown), suggesting that
the information for mitochondrial targeting is contained over a wide
area within the N terminus of Protox IIS.
Protein targeting to plant mitochondria and chloroplasts is usually
very specific. Recently, the specificity of chloroplast and
mitochondria targeting sequences has been studied using tandem fused
proteins of both targeting sequences (37). The sequences coding for the
presequence of the mitochondrial F1-ATPase Control of Chlorophyll and Heme Biosynthesis Pathways--
Two
tetrapyrrole molecules, chlorophyll and heme, are synthesized in
chloroplasts. A crucial branch point of the tetrapyrrole synthetic
pathway in higher plants is the chelation of either Mg2+ to
make chlorophyll or Fe2+ for heme catalyzed by magnesium
chelatase or ferrochelatase, respectively (38). One model that has been
proposed for the control of this branchpoint, based on biochemical
studies, is that the two enzymes are spatially separated within the
chloroplast, magnesium chelatase being associated with the envelope
membrane (39), and ferrochelatase existing exclusively in the
thylakoids (40). However, recent studies using radiolabeled molecules
have shown that ferrochelatase protein is located on both the thylakoid and envelope membranes, suggesting that heme biosynthesis occurs on the
chloroplast envelope membrane and also that control of the branchpoint
cannot be due to spatial separation of the two chelatases (41).
Although this mechanism is far from being completely understood, our
chloroplast Protox localization data present yet another attractive
model for this branchpoint control. Spinach Protox I is preferentially
associated with the stromal side of the thylakoid membrane, with a
small portion of Protox I located on the stromal side of the inner
envelope membrane (14). This implies that Protox I and Protox IIL are
located at the same site of the chloroplast inner membrane. Protox must
play the important role of supplying Proto IX to the magnesium
chelatase and ferrochelatase in chloroplasts. It seems to be
advantageous that Protox and chelatases are located close in space for
profluent flow of tetrapyrrole synthesis. In fact, mammalian Protox
interacts directly with ferrochelatase to facilitate the supply of
Proto IX (15). The possibility exists, then, that each Protox isozyme
(Protox I or Protox IIL) directly interacts with either magnesium
chelatase or ferrochelatase and that the branchpoint is regulated by
independently controlling the activity of each Protox isoenzyme.
Evaluation of this hypothesis would depend on finding evidence of a
direct, specific interaction between each Protox and a chelation enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ADP
binding fold found in many flavoproteins (12). In plants, two cDNAs
have been cloned from tobacco (Nicotiana tabacum) by
functional complementation of the heme auxotrophic, Protox-deficient
E. coli hemG mutant (13). One cDNA encodes a 548-amino
acid protein (PPX-I) containing a putative 50-amino acid plastidal
targeting sequence, whereas the other cDNA encodes a 504-amino acid
protein (PPX-II). The deduced amino acid sequences of PPX-I and PPX-II
are only 27.3% identical. The translation product of PPX-I cDNA
translocates to chloroplasts, whereas PPX-II are targeted to
mitochondria, suggesting that tobacco Protox exists in chloroplasts and
mitochondria as isoenzymes (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1.
The reagents used were of special or analytical grade from Wako Pure
Chemicals (Osaka, Japan) and Nakarai Tesque Co. Ltd. (Kyoto, Japan).
hemG::Kmr), which is obtained by
replacement of the wild-type hemG gene with the
hemG::Kmr allele by homologous
recombination. Complementation tests were performed as described
previously (14).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of the SO-POX2
cDNA clone and the amino acid sequence deduced from the
cDNA. Two start codons and the stop codon are indicated by
bold letters and asterisk,
respectively. The underline indicates the
GXGXXG motif.
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Fig. 2.
Alignment of the N-terminal region of the
predicted amino acid sequences of mitochondrial Protox from various
eukaryotes. Sequences are from spinach (AB046993),
Arabidopsis (D83139), tobacco (Y13466), potato (AJ225108),
soybean (AB025102), yeast (P40012), and human (D38537). Identical amino
acid residues for all the sequences are indicated by
asterisks, and similar residues are indicated by
dots.
hemG::Kmr), a strain of
E. coli defective in the hemG gene, grows very poorly even in rich media (14). To confirm the enzymatic activity of
the cDNA gene products, two Protox II cDNAs of different
lengths (Met-1 to Ile-531 and Met-27 to Ile-531) were ligated into
vector pCR 2.1 in-frame with lacZ, and introduced into BT3
(
hemG::Kmr) E. coli
cells. The mutation responsible for poor growth of the mutant was
complemented by each cDNA, suggesting that both constructs have
Protox activity.
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Fig. 3.
Genomic DNA Southern blot analysis of Protox
II in spinach. 20 µg of spinach genomic DNA was digested with
restriction enzymes (B, BglII; D,
DraI; E, EcoRI) and subjected to
Southern blot analysis. Hybridization was performed using full-length
cDNA of spinach Protox II labeled nonradioactively by random
priming. The sizes (in kb) of standard DNA fragments (1-kb ladder
marker, Life Technologies, Inc.) are shown at left.
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Fig. 4.
Immunoblot analysis of Protox II in spinach
leaf. Protein samples were separated by 10% (v/v) SDS-PAGE and
transferred to a nitrocellulose membrane. Immunodetection was performed
with a polyclonal antibody raised against recombinant Protox II of
spinach. The positions of molecular mass markers (in kDa) are given at
left. A, localization of Protox II in spinach
leaf. Lane 1, total leaf extract (60 µg); lane
2, purified chloroplast fraction (20 µg); lane
3, mitochondrial fraction (10 µg). B,
localization of Protox II within chloroplasts and mitochondria of
spinach leaf. Lane 1, total purified chloroplast
(20 µg); lane 2, stroma (10 µg);
lane 3, envelope (1 µg); lane
4, thylakoids (10 µg); lane 5, mitochondrial
membrane (10 µg).
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Fig. 5.
Functional in vitro analysis
of the two potential AUG codons in the SO-POX2
mRNA. A, schematic representation of
constructs used for the in vitro transcription/translation
experiments. The pM1M2-POXII construct contains both 5' ATG codons,
whereas pM1-POXII contains an intact first codon and a mutated second
codon and pM2-POXII contains a mutated first codon and an intact second
codon. B, immunodetection of in vitro translation
products from the different constructs. The translation products of
M1M2-POXII (lane 1), M2-POXII (lane
2), and M1-POXII (lane 3) were
separated by 10% (v/v) SDS-PAGE and transferred to a nitrocellulose
membrane. Immunodetection was performed with an anti-spinach Protox II
antibody. The positions of molecular mass markers (kDa) are given at
left.
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Fig. 6.
Transport of green fluorescent protein (GFP)
fused to the N-terminal peptides of spinach Protox II.
A, GFP fused to the full-length N-terminal peptide of
spinach Protox II (M1T97-GFP); B, GFP fused to the
alternative N-terminal peptide of spinach Protox II (M27T97-GFP);
C, GFP fused to the N-terminal peptide of yeast Cox IV
(pCoxIV-GFP); D, unfused GFP (pGFP). The fluorescence of GFP
was observed at an excitation wavelength of 495 nm and emission
wavelength of 530 nm (A-D, left
panel). The autofluorescence of chloroplasts was observed at
an excitation wavelength of 540 nm and emission wavelength of 600 nm
(A-D, middle panel). The merged
images of GFP and chlorophyll fluorescence are shown in the
right panel. Bar represents 10 µm.
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Fig. 7.
Immunogold labeling of Protox II in a
spinach mesophyll cell from sponge tissue of 4-week old
seedlings. Sections were immunostained with an
anti-Protox II antibody. Bar represents 0.5 µm.
Quantitation of the contents of three Protox isoforms in subcellular
fractions
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and +4 (with a preference for G at +4 and no
preference at
3). If a strong recognition sequence around the first
AUG codon is absent, some small ribosomal subunits bypass the first AUG
codon and initiate instead at a second or, rarely, even a third AUG.
Consequently, such leaky scanning produces two or more proteins from
one mRNA (32). Mutation analysis of the two putative AUG initiator
codons indicated that initiation of translation of the two Protox II isoforms relies on both the first and second AUG codons in Protox II
mRNA (Fig. 5). Moreover, analysis of the nucleotide sequence surrounding these two in-frame AUGs reveals that the second AUG has a
stronger initiation recognition sequence
(GTTATGGGC) than the first
(CGAATGGTA) (Fig. 1). These results strongly
suggest that Protox IIL and IIS are translated from one mRNA using
the first and the second AUG codons and that the mechanism of the dual
translation is leaky ribosomal scanning. It is still not known whether
such leaky ribosomal scanning is controlled or uncontrolled. The
in vitro translation system of Protox II must be useful to investigate the control mechanism of the leaky scanning.
-subunit and the transit
peptide of the chloroplast chlorophyll a/b-binding protein
were fused in tandem and introduced into tobacco. When the
mitochondrial presequence was inserted downstream to the chloroplast sequence, import into chloroplasts was observed. A mitochondrial presequence alone was able to direct transport to mitochondria; however, mitochondrial import of fusion proteins in which the chloroplast targeting sequence was linked downstream of the
mitochondrial presequence was dramatically increased. These results
indicate the importance of the more extreme N-terminal position of the targeting sequence in determining protein import specificity. The N
terminus of the Protox IIL must exert a dominant influence, directing
transport into chloroplasts, whereas its internal targeting sequence
for mitochondrial transport may not be recognized by its cognate
chaperone or receptor. The dual compartmentation of Protox II would
thus be controlled in both translation and transport steps.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Takeshi Nakano and Dr. Yoshihiro Nakajima for valuable advice. We are grateful to Tokiko Nakanishi and Hiroko Sato for technical assistance. We also thank Dr. Kinya Akashi for the kind gift of the plasmid pCoxIV-GFP and for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by a Grant-in-aid for Encouragement of Young Scientists 09760304 from the Ministry of Education, Science, Sports and Culture of Japan.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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB046993.
§ Recipient of postdoctoral research fellowships from the Japan Society for the Promotion of Science for Young Scientists.
¶ The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-743-72-5452; Fax: 81-743-72-5459; E-mail:
fsche@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M101140200
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ABBREVIATIONS |
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The abbreviations used are: Protox, protoporphyrinogen oxidase; bp, base pair(s); kb, kilobase pair(s) or kilobase(s); BSA, bovine serum albumin; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline containing Tween 20; PCR, polymerase chain reaction; PPX-II, tobacco mitochondrial protoporphyrinogen oxidase; Proto IX, protoporphyrin IX; Protogen, protoporphyrinogen IX; Protox I, plastidal protoporphyrinogen oxidase; Protox II, PPX-II homolog of spinach; Protox IIL, the larger form of Protox II; Protox IIS, the smaller form of Protox II; RACE, rapid amplification of cDNA ends; S23142, N-[4-chloro-2-fluoro-5-propagyloxy]-phenyl-3,4,5,6-tetrahydrophthalimide.
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