1 Departamento de Genética, Escola Superior de Agricultura "Luiz de
Queiroz", Universidade de São Paulo, Caixa Postal 83, 13400-970,
Piracicaba, SP, Brazil
2 Departamento de Microbiologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900,
São Paulo, SP, Brazil
3 Departamento de Botânica, Instituto de Biociências, Universidade
de São Paulo, Caixa Postal 11461, 05422-970, São Paulo, SP,
Brazil
* Author for correspondence (e-mail: mdcsilva{at}esalq.usp.br)
Accepted 15 October 2002
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Summary |
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Key words: Dual targeting, THI1, Mitochondria, Chloroplasts, Import, Translational regulation
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Introduction |
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The occurrence of mitochondria and chloroplasts in plant cells requires a
higher organellar protein import specificity than in non-plant sources
(Glaser et al., 1998). Thus,
protein targeting to both endosymbiotic organelles follows distinct pathways
and involves different targeting sequences and import machineries; this
underlines the complexity of the relationship between these cell compartments
(Duchêne et al., 2001
;
Gabriel et al., 2001
). Although
some mistargeting has been reported so far
(Franzén et al., 1990
;
Hurt et al., 1986
;
Silva-Filho, 1999
;
Whelan et al., 1990
), this is
because of heterologous expression systems or unusual targeting sequences. In
homologous systems mistargeting seems not to occur in vivo
(Silva-Filho et al., 1997
);
therefore, dual targeting to mitochondria and chloroplasts is apparently not
related to mistargeting but to cell requirement
(Soll and Tien, 1998
). An
interesting report has shed light on the protein import specificity of both
organelles. It has been suggested that a mitochondrial preprotein receptor,
called Tom22, is required for cohabitation of mitochondria and plastids in the
same cell, in such a way as to prevent mistargeting of chloroplast proteins
(Macasev et al., 2000
).
The manner in which the proteins targeted to both mitochondria and
chloroplasts are recognized by both import machineries is still poorly
understood. It has been proposed that most of these proteins carry ambiguous
targeting signals (Hedtke et al.,
2000; Small et al.,
1998
). However, recent reports showed that targeting of spinach
protoporphyrinogen oxidase II (Watanabe et
al., 2001
) and of a phage-type RNA polymerase
(Kobayashi et al., 2001
) to
both mitochondria and chloroplasts is mediated by the alternative use of two
in-frame initiation codons.
The single copy thi1 gene encodes the bivalent protein THI1
protein, which is targeted both to chloroplasts and mitochondria
(Chabregas et al., 2001). This
protein plays a role in the biosynthesis of thiamine (vitamin B1) and may be
involved in protection against organellar DNA damage
(Machado et al., 1996
;
Machado et al., 1997
). The
mature THI1 protein is synthesized with a typical N-terminal chloroplastic
transit peptide, which is in agreement with the plastid location for the
pathway (Belanger et al.,
1995
). We have previously shown the presence of a sequence
adjacent to the chloroplast-targeting signal that is able to fold into an
amphiphilic
-helix. This structure was shown to be involved in THI1
mitochondrial import as well as in targeting of a reporter protein into the
organelle (Chabregas et al.,
2001
). Furthermore, THI1 is synthesized from a single nuclear
transcript, which suggests that a post-transcriptional mechanism is
responsible for the final localization of the protein in
Arabidopsis.
In the present study, we examine in greater detail the mechanism responsible for dual targeting of THI1 to mitochondria and chloroplasts. Interestingly, two translational products appeared from thi1 mRNA by the use of two inframe AUG codons. Translation initiation efficiency was significantly higher around the first AUG, which in fact presents the optimum context. Surprisingly, translation initiation still occurred at the second AUG codon notwithstanding a poor context and a strong stem-and-loop structure. Alteration of the translation initiation context around the two AUG codons by site-directed mutagenesis significantly affected the translation initiation, especially for the first AUG. In addition, we have prepared a set of truncated constructs by the fusion of THI1 transit signals to the green fluorescent protein. The results show that the distribution of THI1 in A. thaliana is determined by a differential usage of the translational initiation codons. Therefore, the evidence presented indicates that the less favorable context for translation initiation at the second AUG is used to direct translocation of THI1 to mitochondria.
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Materials and Methods |
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In order to modify the translation initiation context around the two
in-frame AUG codons, three constructions were obtained by PCR-directed
mutagenesis. In the first construct, named THI64-1c, the AAAATGGCT
context in the vicinity of the first ATG (italics) was mutated to
CAAATGCCT (mutations underlined). The upstream
primer carrying a KpnI site was
5'-CCCGGTACCCCAAATGCCTGC-3' and the downstream primer which
carries a HindIII site was
5'-CCCAAGCTTCCAGGACTAACAGATTG-3'. To obtain the second construct,
named THI64-2c, in which the context around the second in-frame ATG (italics)
GAGATGACG was modified to ACCATGGCG
(mutations underlined), two PCR fragments were brought together. The first 212
bp fragment obtained (thia) has already been described. The second fragment
(called thib), corresponding to 155 bp, was obtained from the primer
5'-CCCCTCGAGGTCACGTACCATGGCGAG-3', carrying a XhoI site,
and the primer 5'-CCCAAGCTTCCAGGACTAACAGATTG-3', which carries a
HindIII site. These fragments were cut with XhoI and ligated
with T4 DNA ligase. Finally, in the third construct, called THI64-12c, both
initiation codons were altered (same modifications as described before). In
this case, a fragment similar to thia (named thia) obtained with the
upstream primer 5'-CCCGGTACCCCAAATGCCTGC-3' and downstream primer
5'-CCCCTCGAGACGATCGATTCCTTG-3' and thib were joined.
All constructs were engineered to contain KpnI and
HindIII sites at the 5' and 3' ends, respectively, in
order to clone them in-frame into the respective sites of the plasmid SK+ GUS
(Silva-Fillho et al., 1996). The resulting plasmids were as follows: SK+
THI64-GUS, SK+ THI641-GUS, SK+ THI64
2-GUS, SK+ THI64-1c-GUS, SK+
THI64-2c-GUS and SK+ THI64-12c-GUS.
In-vitro-coupled transcription and translation were performed using the T7 TNT Quick-Coupled Transcription/Translation System (PROMEGA), according to the manufacturer's instructions, in the presence of [35S] methionine (Amersham Pharmacia). One tenth of the translated proteins was run on 10% SDS-PAGE and dried before exposure. Signals were quantified using the GST-700 Imaging Densitometer and the software Molecular Analyst (BioRad).
Transport experiments using GFP fusion proteins
The sequence corresponding to the N-terminus of THI1 (nucleotides 1-315),
which includes the entire length of the chloroplastic transit peptide followed
by 50 amino acids of the mature THI1 (including the putative mitochondrial
presequence), was amplified by PCR using the A. thaliana cDNA as a
template. A similar approach to the thi1 gus constructions (see
below) was used to generate the constructs named THI50 (two ATGs in-frame),
THI501 (first ATG was mutated to ATC) and THI50
2 (second
in-frame ATG was mutated to ATC), similar to THI64, THI64
1 and
THI64
2, respectively. Furthermore, an additional construct starting
from the second in-frame ATG, named THImet2, was also generated by PCR. The
plasmid SK+ THI64
2-GUS was used as a template to obtain the
THI50
2 fragment. For all PCR reactions the downstream primer was
5'-CCCACTAGTAGGGTTCTTACTGATCTC-3'. The THI50 and THI50
2
upstream primers were 5'-CCCAGATCTCAAAATGGCTGC-3'. To obtain the
THI50
1 and THImet2 fragments the upstream primers were, respectively,
5'-CCCAGATCTCAAAATCGCTGC-3' and
5'-CCCAGATCTGAGATGACGAGAAGGTAC-3'. All DNA fragments were
engineered to contain BglII and SpeI restriction sites at
the 5' and 3' end, respectively. These fragments were cloned into
the GFP expression vector pCAMBIA1302
(Roberts et al., 1997
),
previously digested by BglII and SpeI. The resulting vectors
were designated to as pCambia-THI50-GFP, pCambia-THI50
1-GFP,
pCambia-THI50
2-GFP and pCambia-THImet2-GFP.
These constructions were introduced into tobacco mesophyll protoplasts,
prepared as follows: Green leaves of Nicotiana tabacum (var. SRI),
grown in vitro (250 mg), were cut into small strips using a new razor
blade and incubated in 5 ml of an enzyme solution [0.2% Macerozyme R-10
(Yakult Honsha Co., Ltd., Tokyo, Japan), 1.0% Cellulase Onozuka R-10 (Merck),
0.5% Driselase (Sigma) in CPW8 (Frearson et
al., 1973
)] at room temperature for 16 hours under gentle
agitation (
30-40 rpm). After incubation, the protoplast suspension was
filtered through a 64 µm mesh, the protoplasts being collected by
centrifugation at 46 g for 5 minutes. The pelleted protoplasts
were resuspended in 5 to 10 ml of CPW8 solution and centrifuged. After
centrifugation, the protoplasts were resuspended in MKCl (500 mM mannitol, 5
mM KCl and 200 µM MOPS) at a density of 2x106
protoplasts/ml. Protoplasts were electroporated with 10-50 µg of the
respective plasmid DNAs and incubated in the dark for 24-48 hours before
analysis. Transient expression of the GFP constructs in eletroporated
protoplasts was analyzed using an epifluorescence microscope Axioplan 2
(Zeiss). For GFP fluorescence, excitation was at 450-490 nm and emission at
520 nm. Chloroplast autofluorescence was detected between 664 and 696 nm with
an excitation at 488 nm.
mRNA structure analysis
The secondary structure prediction for thi1 mRNA was performed by
informatic tools directly available at the
http://mfold2.wustl.edu/mfold/rna/for1-2.3.cgi.
and
http://www.ibc.wustl.edu/
zuker/rna/sites.
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Results |
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|
The context surrounding the first AUG plays a major role in the
regulation of translation initiation
An analysis of the translation efficiency of thi1 mRNA revealed
that translation occurs preferentially in the first AUG codon
(Fig. 1B, lane 1;
Fig. 1C, lane 1). On the other
hand, the second in-frame AUG codon is inserted into a suboptimal context
(GAGAUGAC). In order to figure out the context in the
vicinity of both AUG start codons in thi1 translation initiation, we
performed site-directed mutagenesis creating more or less favorable contexts
(Fig. 1A). A poor context
around the first AUG start codon (CAAAUGCCU)
(Fig. 1A) drastically reduced
its translation efficiency (Fig.
1B, lane 4 and Fig.
1C, lane 4). In addition, translation initiation at the second
in-frame AUG was increased. Interestingly, when an optimum context
(ACCAUGGCG) was introduced into the vicinity of the second
in-frame AUG (Fig. 1A), the
efficiency of translation was not significantly affected
(Fig. 1B, lane 5 and
Fig. 1C, lane 5). When the
translation initiation context was altered simultaneously (the suboptimal
context CAAAUGCCU was introduced around the first
start codon, and the more favorable context ACCAUGGCG
was placed around the second in-frame start codon)
(Fig. 1A), initiation at the
upstream AUG was significantly reduced, whereas translation at the second site
was increased by 50% (Fig. 1B,
lane 6; Fig. 1C, lane 6),
ensuring that in the absence of first AUG furtherance, second AUG improvement
can be accomplished.
Dual targeting of thi1 to chloroplasts and mitochondria is determined
by an alternative translation initiation mechanism
The occurrence of two translational products raises the possibility that
the two isoforms are translated from thi1 mRNA, using two different
in-frame start codons. To explore this hypothesis, we analyzed the in vivo
transport of both translational products using green fluorescence protein
(GFP) gene constructs (Fig. 2). cDNA encoding the thi1 N-terminal region (Met-1 to Pro-119) was fused
to the 5' end of the gfp gene (named pCambia-THI50-GFP)
(Fig. 2a). To confirm that
these two proteins were translated from two distinct in-frame AUG start
codons, two other constructions were prepared, pCambia-THI501-GFP
(Fig. 2b), in which Met-1 was
converted to Ile-1, and pCambia-THI50
2-GFP
(Fig. 2c), in which Met-70 was
mutated to Ile-70. In addition, a construct starting from the second in-frame
AUG (Met-70) was also obtained (pCambia-THImet2-GFP)
(Fig. 2d). All the constructs
were fused in-frame to the 5' end of the gfp gene and placed
under the control of the transcriptional 35S promoter from the cauliflower
mosaic virus. These plasmids were introduced into tobacco leaf protoplasts by
electroporation, and transient expression was analyzed by fluorescence
microscopy. After 24 hours of culture, the THI50-GFP chimeric protein could be
observed in both mitochondria and chloroplasts
(Fig. 3A). Chloroplasts could
be easily distinguished by their red autofluorescence, owing to their
chlorophyll. Similar results were obtained with the SYCO-GFP construct
(Fig. 3B), which has been shown
to be dual targeted to both organelles
(Peeters et al., 2000
). With
the THI50
1-GFP construct (first AUG converted to AUC) the fluorescence
was restricted to mitochondria (Fig.
3C). The fluorescence pattern of ß-GFP, which is known to be
selectively delivered into mitochondria
(Duby et al., 2001
), was very
similar to THI50
1-GFP (Fig.
3D). Additionally, in the tobacco protoplasts transfected with
THImet2-GFP (second in-frame AUG was fused to GFP), green fluorescence was
only seen in mitochondria (Fig.
3E). By contrast, with the THI50
2-GFP construct (in which
the second in-frame AUG was converted to AUC), fluorescence was restricted to
chloroplasts (Fig. 3F). These
results are similar to the typical plastid targeting construct RecA-GFP
(Fig. 3G)
(Köhler et al., 1997
).
With the 35S-GFP construct (not fused to any of the thi1 targeting
sequences), GFP fluorescence was found in the cytosol (data not shown).
|
|
Structural analysis of the 5' portion of the thi1 mRNA
To search for mechanisms that could explain the possible translation
initiation at the second AUG, thi1 mRNA was dissected. Two
interesting features were observed that might be linked to translation
initiation. First, A. thaliana wild-type thi1 mRNA is
predicted to form a relatively stable stem-loop structure between the two
in-frame AUG codons, particularly in the region extending from the last base
of codon 41 through the first base of the second in-frame AUG start codon
(Fig. 4A). The stability of
such a stable stem-loop structure at this position is interesting and may
interfere in the translation efficiency. Second, we examined the sequences
within the region flanked by the two in-frame AUGs start codons of
thi1, which could function as a putative internal ribosome entry site
(IRES). In previous studies, it has been shown that eukaryotic mRNAs contain
short complementary matches to 18S rRNA, suggesting that ribosome recruitment
at some cellular IRES might occur by base pairing between mRNA and 18S rRNA
(Zhou et al., 2001;
Fernandez et al., 2002
). In
fact, sequence comparison between thi1 mRNA and 18S rRNA identified
two complementary sequence matches (Fig.
4B). These matches contain stretches of 13 (nucleotides 62-74) and
12 (91-102 nt) nucleotides with 85% and 92% complementarity, respectively.
|
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Discussion |
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The finding that thi1 contains a single mRNA excludes the
possibility of transcript heterogeneity, which could result in the production
of proteins with variable N-termini. In vitro transcription/translation
experiments showed that thi1 produces two translational products with
the expected size for translation initiation for the two in-frame AUG start
codons. Interestingly, analysis of the vicinity of both start codons indicates
that the sequence context around the first AUG
(AAAAUGGC) is more favorable for translation
initiation than the second (GAGAUGAC), as it
corresponds to the reported dicot consensus
(A/GAAAUGGC)
(Joshi et al., 1997;
Lukaszewicz et al., 2000
). A
purine, preferably A, at position -3 (three nucleotides before the AUG codon,
which is numbered +1 to +3), and G at position +4, are the major determinants
for translation initiation efficiency in higher plants
(Kozak, 1991
;
Kozak, 1997
). We reasoned that
the observed low translation levels at the second AUG may, somehow, be related
to a reduced demand for the shorter translational product inside the cell.
Since THI1 presents two targeting sequences in tandem, we hypothesized that
translation initiation in the first AUG would deliver the protein to
chloroplasts. In addition, the presence of a second in-frame AUG at the
beginning of a sequence able to fold into an amphiphilic -helix, and
usually found in mitochondrially imported proteins
(Neupert, 1997
), suggested
that this codon could be a site for translation initiation. The presence of
both in-frame AUG codons fused to the 5' end of GFP directed
translocation of the reporter protein to mitochondria and chloroplasts of
tobacco leaf protoplasts. Supporting evidence for this mechanism was obtained
from the observation that mutation in the first AUG to AUC abolished
importation of GFP to chloroplasts, and the fluorescent protein was found to
be only associated with mitochondria. Thus, the second AUG can also be
recognized in vivo by the plant translation machinery and the product indeed
delivered to mitochondria. In accordance with this, the construct starting
directly from the second in-frame AUG fused to GFP confirmed that this
amphiphilic
-helix secondary structure functions as a mitochondrial
presequence. Another important result was obtained by the conversion of the
second in-frame AUG to AUC: GFP was then found associated to the plastids and
the targeting to mitochondria completely lost. The fact that GFP was targeted
specifically to particular organelles is not direct proof of targeting, but
does provide strong evidence that the different isoforms of THI1 are produced
in vivo and that a translational initiation mechanism is involved in the dual
targeting of this protein.
According to the scanning model, translation is initiated at the first AUG
codon contained in a particular context
(Kozak, 1991). Why would
translation initiation occur at the second AUG of thi1 mRNA if its
context were not favored? It has been suggested that initiation sites in
eukaryotic mRNAs are reached via a scanning mechanism that predicts that
translation should start at the AUG codon nearest the 5' end of the mRNA
(Kozak, 1999
). However, a
recent survey on translation initiation in vertebrates indicates that
translation initiation from downstream AUGs is more common than generally
believed (Peri and Pandey,
2001
). It is suggested that mechanisms such as leaky scanning,
reinitiation or internal initiation of translation might have a greater role
than previously reported (for a review, see
Gray and Wickens, 1998
).
Translation at the second AUG, in which the surrounding sequence is not
suitable for efficient initiation, might indicate a leaky scanning mechanism,
where the ribosome does not always recognize the initial AUG. Alternatively,
thi1 mRNA contains short complementary matches to 18S rRNA, raising
the possibility that ribosome recruitment at some cellular IRESes might occur
by base pairing between thi1 mRNA and 18S rRNA. Placing these
observations together, translation of both in-frame start codons would involve
a cap-dependent translation mechanism around the first AUG and a
cap-independent translation process at the second start codon. Although
speculative, these assumptions can be tested experimentally. Further studies
are necessary to determine the mechanism for initiating translation of
Arabidopsis thaliana thi1 mRNA.
Although the function of THI1 in mitochondria and chloroplasts is not
completely understood, it seems that THI1 requirement is increased in
chloroplasts. In fact, thiamine biosynthesis has been associated to plastids,
where it participates as a cofactor for the two enzyme complexes involved in
the citric acid cycle, pyruvate dehydrogenase and -ketoglutarate
dehydrogenase (Belanger et al.,
1995
). The need for thiamine is thus essential for plant
metabolism, which suggests that the enzymes involved in its biosynthetic
pathway are required at high levels. On the other hand, it has been suggested
that THI1 has a second role in protecting mitochondrial DNA from damage
(Machado et al., 1996
;
Machado et al., 1997
). One
reasonable possibility is that THI1 participates in independent reactions in
two organelles at different requirements. Evaluation of the precise role of
THI1 in mitochondria and chloroplasts would certainly help in understanding
the differential requirements of this protein and its effect on translation
regulation.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Belanger, F. C., Leustek, T., Chu, B. and Kriz, A. L. (1995). Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation. Plant Mol. Biol. 29,809 -821.[Medline]
Chabregas, S. M., Luche, D. D., Farias, L. P., Ribeiro, A. F., van Sluys, M.-A., Menck, C. F. M. and Silva-Filho, M. C. (2001). Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts. Plant Mol. Biol. 46,639 -650.[CrossRef][Medline]
Danpure, C. (1995). How can the products of a single gene be localized to more than one intracellular compartment? Trends Cell Biol. 5,230 -238.[CrossRef]
Duby, G., Oufattole, M. and Boutry, M. (2001).
Hydrophobic residues within the predicted N-terminal amphiphilic -helix
of a plant mitochondrial targeting resequence play a major role in in
vivo import. Plant J.
6, 539-549.
Duchêne, A.-M., Peteres, N., Dietrich, A., Cosset, A.,
Small, I. D. and Wintz, H. (2001). Overlapping destinations
for two dual targeted glycil-tRNA synthetases in Arabidopsis thaliana
and Phaseolus vulgaris. J. Biol. Chem.
276,15275
-15283.
Fernandez, J., Yaman, I., Merrick, W. C, Koromilas, A., Wek, R.
C., Sood, R., Hensold, J. and Hatzoglou, M. (2002).
Regulation of internal ribosome entry site-mediated translation by eukaryotic
initiation factor-2 phosphorylation and translation of a small upstream
open reading frame. J. Biol. Chem,
277,2050
-2058.
Franzén, L. G., Rochaix, J. D. and von Heijne, G. (1990). Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. FEBS Lett. 260,165 -168.[CrossRef][Medline]
Frearson, E. M., Power, J. B. and Cocking, E. C. (1973). The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol. 33,130 -137.[CrossRef][Medline]
Gabriel, K., Buchanan, S. K. and Lithgow, T. (2001). The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes. Trends Biochem. Sci. 26,36 -40.[CrossRef][Medline]
Gebhardt, J. S., Wadsworth, G. J. and Matthews, B. F. (1998). Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase. Plant Mol. Biol. 37,99 -108.[CrossRef][Medline]
Glaser, E., Sjöling, S., Tanudji, M. and Whelan, J. (1998). Mitochondrial protein import in plants. Plant Mol. Biol. 38,311 -338.[CrossRef][Medline]
Gray, N. K. and Wickens, M. (1998). Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol. 14,399 -458.[CrossRef][Medline]
Hedtke, B., Börner, T. and Weihe, A.
(2000). One polymerase serving two genomes. EMBO
Rep. 1,435
-440.
Hurt, E. C., Soltanifar, N., Goldschmidt-Clermont, M., Rochaix, J.-D. and Schatz, G. (1986). The clevable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria. EMBO J. 5,1343 -1350.
Joshi, C. P., Zhou, H., Huang, X. and Chiang, V. L. (1997). Context sequences of translation initiation codon in plants. Plant Mol Biol. 35,993 -1001.[CrossRef][Medline]
Kobayashi, Y., Dokiya, Y. and Sugita, M. (2001). Dual targeting of phagetype RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts. Biochem. Biophys. Res. Commun. 289,1106 -1113.[CrossRef][Medline]
Köhler, R. H., Zipfel, W. R., Webb, W. W. and Hanson, M. R. (1997). The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J. 11,613 -621.[CrossRef][Medline]
Kozak, M. (1991). Structural features in
eukaryotic mRNAs that modulate the initiation of translation. J.
Biol. Chem. 266,19867
-19870.
Kozak, M. (1997). Recognition of AUG and
alterantive initiator codons is augmented by G in position +4 but is not
generally affected by the nucleotides in positions +5 and +6. EMBO
J. 16,2482
-2492.
Kozak, M. (1999). Initiation of translation in prokaryotes and eukaryotes. Gene 234,187 -208.[CrossRef][Medline]
Lukaszewicz, M., Feuermann, M., Jérouville, B., Stas, A. and Boutry, M. (2000). In vivo evaluation of the context sequence of the translation initiation codon in plants. Plant Sci. 154,89 -98.[CrossRef][Medline]
Macasev, D., Newbigin, E., Whelan, J. and Lithgow, T.
(2000). How do plant mitochondria avoid importing chloroplast
proteins? Components of the import apparatus Tom20 and Tom22 from
Arabidopsis differ from their fungal counterparts. Plant
Physiol. 123,811
-816.
Machado, C. R., Costa de Oliveira, R. L., Boiteux, S., Praekelt, U. M., Meacock, P. A. and Menck, C. F. M. (1996). Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements defects in DNA repair. Plant Mol. Biol. 31,585 -593.[Medline]
Machado, C. R., Praekelt, U. M., Costa de Oliveira, R. L., Barbosa, A. C. C., Byrne, K. L., Meacock, P. A. and Menck, C. F. M. (1997). Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance. J. Mol. Biol. 273,114 -121.[CrossRef][Medline]
Neupert, W. (1997). Protein import into mitochondria. Annu. Rev. Biochem. 66,863 -917.[CrossRef][Medline]
Peeters, N. M., Chapron, A., Giritch, A., Grandjean, O., Lancelin, D., Lhomme, T., Vivrel, A. and Small, I. (2000). Duplication and quadruplication of Arabidopsis thaliana cysteinyl- and asparaginyl-tRNA synthetase genes of organellar origin. J. Mol. Evol. 50,413 -423.[Medline]
Peeters, N. and Small, I. (2001). Dual targeting to mitochondria and chloroplasts. Biochim. Biophys. Acta. 1541,54 -63.[Medline]
Peri, S. and Pandey, A. (2001). A reassessment of the translation initiation codon in vertebrates. Trends Genet. 17,685 -687.[CrossRef][Medline]
Roberts, C. S., Rajagopal, S., Smith, L. A., Nguyen, T. A., Yang, W., Nugroho, S., Ravi, K. S., Cao, M.-L., Vijayachandra, K., Patell, V. et al. (1997). A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants by both Agrobacterium and direct DNA uptake methods. In Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, 15-19. Mallaca, Malaysia.
Sambrook, J. and Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Sass, E., Blachinsky, E., Karniely, S. and Pines, O.
(2001). Mitochondrial and cytosolic isoforms of yeast fumarase
are derivatives of a single translation product and have identical amino
termini. J. Biol. Chem.
276,46111
-46117.
Silva-Filho, M. C. (1999). Translocation of a reporter protein into mitochondria is mediated by a chloroplast transit peptide and follows a normal import route. J. Plant Physiol. 151,51 -54.
Silva-Filho, M. C., Chaumont, F., Leterme, S. and Boutry, M. (1996). Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles. Plant Mol. Biol. 30,769 -780.[Medline]
Silva-Filho, M. C., Wieërs, M.-C., Flügge, U.-I.,
Chaumont, F. and Boutry, M. (1997). Different in
vitro and in vivo targeting properties of the transit peptide of
a chloroplast envelope inner membrane protein. J. Biol.
Chem. 272,15264
-15269.
Small, I., Wintz, H., Akashi, K. and Mireau, H. (1998). Two birds with one stone: genes that encode products targeted to two or more compartments. Plant Mol. Biol. 38,265 -277.[CrossRef][Medline]
Soll, J. and Tien, R. (1998). Protein translocation into and across the chloroplastic envelope membranes. Plant Mol. Biol. 38,191 -207.[CrossRef][Medline]
Watanabe, N., Che, F.-S., Iwano, M., Yakayama, S., Yoshida, S.
and Isogai, A. (2001). Dual targeting of spinach
protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative
use of two in-frame initiation codons. J. Biol. Chem.
276,20474
-20481.
Whelan, J., Knorpp, C. and Glaser, E. (1990). Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts. Plant Mol. Biol. 14,977 -982.[Medline]
Zhou, W., Edelman, G. E. and Mauro, V. P.
(2001). Transcript leader regions of two Saccharomyces
cerevisiae mRNAs contain internal ribosome entry sites that function in
living cells. Proc. Natl. Acad. Sci USA
98,1531
-1536.