Báo cáo khóa học: Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide doc

Fidelity of targeting to chloroplasts is not affected by removal
of the phosphorylation site from the transit peptide
Kerry-Ann Nakrieko, Ruth M. Mould and Alison G. Smith
Department of Plant Sciences, University of Cambridge, UK
Phosphorylation of the transit peptide of several chloroplast-
targeted proteins enables the binding of 14-3-3 proteins.
The complex that forms, together with Hsp70, has been
demonstrated to be an intermediate in the chloroplast pro-
tein import pathway in vitro [May, T. & Soll, J. (2000) Plant
Cell 12, 53–63]. In this paper we report that mutagenesis
(in order to remove the phosphorylation site) of the transit
peptide of the small subunit of ribulose bisphosphate carb-
oxylase/oxygenase did not affect its ability to target green
fluorescent protein to chloroplasts in vivo. We also found
no mistargeting to other organelles such as mitochondria.
Similar alterations to the transit peptides of histidyl- or
cysteinyl-tRNA synthetase, which are dual-targeted to
chloroplasts and mitochondria, had no effect on their ability
to target green fluorescent protein in vivo. Thus, phos-
phorylation of the transit peptide is not responsible for the
specificity of chloroplast import.
Keywords: amino acyl-tRNA synthetase; confocal micros-
copy; dual targeting; GFP; Rubisco.
Most chloroplast and mitochondrial proteins are nuclear-
encoded and are synthesized in the cytosol. Correct
targeting of these proteins to the organelles is thus essential
for cellular function and for the biogenesis of the individual
organelles. In most cases, this is achieved by the presence of
an N-terminal extension, called a transit peptide or pre-
sequence [1–3]. Analysis of the primary amino acid sequence
has revealed that there is little conservation either in

composition or in length, although some general features
have been identified [4,5]. Chloroplast transit peptides have
few acidic residues and are rich in hydroxylated residues;
plant mitochondrial presequences share these characteristics
but also frequently form amphipathic a-helices, with
positive charges clustered on one side [3–5]. This character-
istic has been shown to be important for targeting to
mitochondria in rice [6].
The transit peptide is necessary and sufficient for fidelity
of targeting to the chloroplast or mitochondrion, as shown
most elegantly by the fact that they are able to target
passenger proteins to the appropriate organelle. The
receptor machinery on the outer membranes of chloroplasts
and mitochondria is able to discriminate between bona fide
precursors and those of the other organelle. For example,
precursors for the light-harvesting chlorophyll a/b-binding
protein and the 33 kDa photosystem II protein are not
imported into plant mitochondria [7,8]. Likewise, the transit
peptide of the b-subunit of the F
1
-ATPase (preF
1
b) will
target proteins to plant mitochondria in vitro [7,8] and
in vivo [9] but not to chloroplasts. On the other hand, there
are some dual-targeted proteins, in particular a number
of the amino acyl-tRNA synthetases, where the transit
peptides direct import into both mitochondria and chloro-
plasts with equal efficiency both in vitro and in vivo [10–12].
Despite the importance of these transit peptides in

determining the specificity of import, the mechanism of
this specificity remains uncertain, although a number of
studies have addressed this question. In one investigation,
several chloroplast precursors, including the small subunit
of ribulose bisphosphate carboxylase/oxygenase (Rubisco)
from tobacco and the 23 kDa and 33 kDa oxygen-evolving
polypeptides from pea, were incubated with pea cytosol in
the presence of [
32
P]ATP. It was found that they were
phosphorylated on a specific serine or threonine residue
within the transit peptide [13]. The consensus phosphory-
lation sequence (Fig. 1A) resembled the motif for binding of
14-3-3 proteins, and 14-3-3 proteins were shown to bind to
the phosphoserine/phosphothreonine site en route to the
chloroplast envelope [14], although before import into the
chloroplast dephosphorylation occured [13]. Subsequently,
it was demonstrated that phosphorylated precursors form a
complex with 14-3-3 proteins and a heat shock protein,
Hsp70 isoform. This complex was found to increase the rate
of translocation into the chloroplast by three to four-fold
compared to the free precursor [14], implying that this may
act as a Ôguidance complexÕ during the translocation process.
In contrast, the mitochondrial precursor preF
1
b and the
precursor for peroxisomal malate dehydrogenase were not
phosphorylated [13], nor did they associate with 14-3-3
Correspondence to A. G. Smith, Department of Plant Sciences,
University of Cambridge, Downing Street, Cambridge, CB2 3EA,

UK. Fax: + 44 1223 333953, Tel.: + 44 1223 333952,
E-mail:
Abbreviations: GFP, green fluorescent protein; Rubisco, ribulose
bisphosphate carboxylase/oxygenase; TSSU.tp.wt, transit peptide of
the small subunit of Rubisco from tobacco; PSSU.tp.wt, transit
peptide of the small subunit of Rubisco from pea; CtRS.tp.wt, transit
peptide of cysteinyl-tRNA synthetase from Arabidopsis thaliana;
HtRS.tp.wt, transit peptide of histidyl-tRNA synthetase from
Arabidopsis thaliana; CoxIV.tp, transit peptide of cytochrome c
oxidase from yeast; wt, wild-type.
(Received 1 September 2003, revised 19 November 2003,
accepted 1 December 2003)
Eur. J. Biochem. 271, 509–516 (2004) Ó FEBS 2004 doi:10.1046/j.1432-1033.2003.03950.x
proteins [14]. Furthermore, phosphorylatable precursor
proteins translated in a wheat-germ system were stably
associated with 14-3-3 proteins, but those translated in a
reticulocyte system were not. This provided an attractive
possibility as a means of preventing mistargeting of
chloroplast precursors to other organelles such as mito-
chondria [13]. In this paper we describe experiments
conducted to investigate this possibility in vivo.Thetransit
peptide of the small subunit of Rubisco (SSU) precursor
from tobacco (Nicotiana tabacum) (TSSU.tp.wt), identical
to that used in the in vitro studies [13,14], was fused to the
green fluorescent protein (GFP) from Aequorea victoria [15],
and the phosphorylation site was mutated. After transfor-
mation of the construct into plant cells by particle
bombardment, the targeting of the GFP by the transit
peptide was viewed directly in living cells by confocal
microscopy. Similar experiments were performed with the

transit peptide of SSU from pea (Pisum sativum)
(PSSU.tp.wt), and those of Arabidopsis thaliana cysteinyl-
and histidyl-tRNA synthetases (CtRS.tp.wt and
HtRS.tp.wt, resepectively), which have been shown to be
dual-targeted to chloroplasts and mitochondria [11,12].
Materials and methods
Materials
Restriction enzymes, T4 DNA ligase and polymerase, and
dNTPs came from GibcoBRL, Life Technologies (Paisley,
UK) or New England BioLabs Inc. (Hitchin, UK). GFP-
containing plasmids (pOL-GFP.LT, pCoxIV-GFP, pCtRS-
GFP and pHtRS-GFP) encoding GFP, yeast CoxIV-GFP
and Arabidopsis thaliana cysteinyl-tRNA synthetase-GFP
and histidyl-tRNA synthetase-GFP, were obtained from
I. Small (INRA, Evry, France), together with a reverse
primer for GFP to enable sequencing of the border of the
fusion constructs. The QuikChange Site-Directed Mutagen-
esis Kit was from Stratagene (La Jolla, CA, USA). Tungsten
microcarriers, macrocarriers, stopping screens and rupture
disks were purchased from Bio-Rad Laboratories Ltd.
(Hemel Hempstead, UK). Components for in vitro tran-
scription, wheat germ extract and amino acids were
obtained from Promega (Madison, WI, USA).
L
-[
35
S]methi-
onine/cysteine PRO-Mix
TM
was purchased from Amersham

Pharmacia (Chalfont St.Giles, Bucks, UK). Oligonucleotide
primers were synthesized by MWB-Biotech AG (Ebersberg,
Germany) or Invitrogen Life Technologies (Paisley, UK).
Generation of fusion-protein constructs
and site-directed mutagenesis
The SSU transit peptide of tobacco ([16]; accession number
PSRBCS3A) was fused in frame with GFP (encoding
solubility-modified red-shifted GFP) [17], into the KpnI/SalI
sites of pOL-GFP.LT [12], yielding pTSSU.tp.wt-GFP
(Table 1). Similarly, the pea SSU transit peptide sequence
([18]; accession number P07689) was inserted in frame into
the KpnI/SphI sites of pUC18-GFP, yielding pPSSU.tp.wt-
GFP (Table 1). Constructs were verified by DNA sequence
analysis.
Site-directed mutagenesis of these chloroplast transit
peptides, together with those for histidyl-tRNA synthetase
(HtRS) and cysteinyl-tRNA synthetase (CtRS) was
designed and performed according to the guidelines sug-
gested in the manual of the Stratagene QuikChange
TM
Site-
Directed Mutagenesis Kit. For each transit peptide, a single
mutant was constructed, in which the phosphorylated serine
or threonine residue was altered to an alanine, and also a
double mutant where the upstream serine was also changed
to an alanine (Table 1). All mutations were verified by
DNA sequence analysis. Further details of cloning and
primers used for mutagenesis are available on request from
A. G. Smith (University of Cambridge).
Import assays into isolated chloroplasts

in vitro
For chloroplast import experiments in vitro, the constructs
encoding the wild-type and modified transit peptides fused
to GFP were subcloned into pBluescript, such that the genes
were under the control of the T7 or T3 promoter. Methods
Table 1. Plasmids generated in this study.
Name Encoding Mutation
pTSSU.tp.wt-GFP Transit peptide of tobacco
SSU fused to GFP
None
pTSSU.tp.S34A-GFP As above Serine 34 changed to alanine
pTSSU.tp.S31A/S34A-GFP As above Serine 31 and serine 34 changed to alanine
pPSSU.tp.wt-GFP Transit peptide of pea
SSU fused to GFP
None
pPSSU.tp.T34A-GFP As above Threonine 34 changed to alanine
pPSSU.tp.S32A/T34A-GFP As above Serine 32 and threonine 34 changed to alanine
pCtRS.tp.wt-GFP Transit peptide of cysteinyl-tRNA
synthetase fused to GFP
None
pCtRS.tp.S22A-GFP As above Serine 22 changed to alanine
pCtRS.tp.S21A/S22A-GFP As above Serine 21 and serine 22 changed to alanine
pHtRS.tp.wt-GFP Transit peptide of histidyl-tRNA
synthetase fused to GFP
None
pHtRS.tp.S52A-GFP As above Serine 52 changed to alanine
pHtRS.tp.S50A/S52A-GFP As above Serine 50 and serine 52 changed to alanine
510 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004
for in vitro transcription, in vitro translation and isolation
of chloroplasts were as described [19].

Expression of GFP-fusion constructs
in vivo
For transient expression in plant tissues of the GFP-fusion
protein constructs in pUC18-GFP or pOL-GFP.LT,
tobacco (Nicotiana tabacum) and pea (Pisum sativum)plants
were grown as described [19], and onion (Allium cepa)was
obtained from a local market. The constructs were intro-
duced into the plant material by biolistic transformation,
and the location of GFP fluorescence was determined by
confocal microscopy as described [19].
Results
Generation of fusion constructs and mutagenesis
of the phosphorylation signal
The consensus phosphorylation site in chloroplast transit
peptides has been identified as (P/G)X
n
(K/R)X
n
(S/
T)X
n
(S*/T*) [13], where the asterisk indicates the site of
phosphorylation and n is a spacer of 0–3 residues (Fig. 1A).
Figure 1B indicates the phosphorylation motifs in the
transit peptides of tobacco and pea preSSU, and the dual-
targeted CtRS and HtRS from Arabidopsis. For each transit
peptide, two mutant forms were generated – one in which
the phosphorylated threonine or serine was altered to an
alanine, and a double mutant, where the upstream serine
was also altered to an alanine. This serine has been

suggested to affect the efficiency of phosphorylation [13],
and might also be able to be phosphorylated itself. The
sequences encoding the wild-type and mutant forms of the
transit peptides were fused in frame to the cDNA encoding
GFP such that the fusion proteins were under the control of
the ubiquitous CaMV 35S promoter and nopaline synthase
(nos) terminator (Fig. 1C).
Import of TSSU.tp-GFP fusion proteins into isolated
chloroplasts
in vitro
Mutation of the phosphorylated serine in the transit peptide
of tobacco SSU did not alter the efficiency of import into
isolated chloroplasts in vitro [13]. We wanted to ensure that
our constructs, in which the mature small subunit had been
replaced with GFP, behaved similarly, before we carried out
our experiments in vivo. Accordingly, the constructs enco-
ding the fusion proteins were subcloned into a vector for
transcription in vitro, and radiolabelled fusion proteins were
made by translation into a wheat germ system in the
presence of [
35
S]methionine and [
35
S]cysteine. The radio-
labelled precursors were incubated with isolated pea chlo-
roplasts, followed by reisolation of the organelles, and the
proteins were analysed by SDS/PAGE and fluorography
(Fig. 2). Figure 2A shows the results for GFP alone. The
translation product of 27 kDa, corresponding to the size
of GFP, does not associate with chloroplasts (+ Cp). In

contrast, the 33 kDa precursor of pTSSU.tp.wt-GFP is
imported into the chloroplast and processed to the size of
GFP alone (Fig. 2B). The two mutant forms of the tobacco
SSU transit peptide (Fig. 2C,D) behave like the wild-type,
andineachcase, 2% of added precursor is imported
(estimated by densitometry). We performed time-course
experiments to investigate the rate of import, where we
observed no difference between the constructs encoding the
single and double mutants and the wild-type transit peptides
(data not shown). Using the same approach, we tested the
wild-type and mutant forms of the pea SSU transit peptide.
Again, identical import reactions in vitro were observed
for all three constructs. From these experiments we can
conclude that the presence of the GFP as passenger protein,
rather than the mature SSU protein, did not affect the
ability of the SSU transit peptide to target proteins to
chloroplasts in vitro. Furthermore, mutations in the transit
peptide had no significant effect on this ability.
Fig. 1. Identification of a phosphorylation motif in the transit peptides of tobacco SSU, pea SSU and dual-targeted Arabidopsis CtRS and HtRS.
(A) The consensus phosphorylation motif described by Waegeman and Soll [13]. (B) The transit peptides of tobacco and pea SSU and Arabidopsis
CtRS and HtRS, with the phosphorylation motif underlined and the predicted site of phosphorylation marked with an asterisk. (C) Schematic of
the GFP-fusion constructs with the individual transit peptides (TP), such that the genes were under the control of the CaMV 35S promoter and
nopaline synthase (nos)terminator.
Ó FEBS 2004 Chloroplast targeting fidelity (Eur. J. Biochem. 271) 511
Targeting of GFP
in vivo
by wild-type and mutated
transit peptides of tobacco SSU
The ability of the SSU transit peptides to target GFP in vivo
was investigated by transient expression in plant tissues. The

constructs in pUC18-GFP or pOL-GFP.LT were intro-
duced into four-week old tobacco leaves by biolistic
transformation, and after 16–24 h in the dark at 25 °C,
cells exhibiting GFP fluorescence were identified by epiflu-
orescence. These were examined further by confocal micro-
scopy (Fig. 3). For each construct, the GFP fluorescence,
chlorophyll autofluorescence, and the overlay of the two,
are shown in a single guard cell; the other cell of the pair
was not transformed. As expected, when GFP alone is
expressed, it is found throughout the cytosol and in the
nucleoplasm, but is excluded from the chloroplasts
(Fig. 3A). In contrast, GFP expressed as a fusion protein
with the wild-type tobacco SSU transit peptide is found
exclusively in chloroplasts, as evidenced by the exact
superposition of GFP and chlorophyll fluorescence in the
overlay (Fig. 3B). The single and double mutants of this
transit peptide, TSSU.tp.S34A-GFP and TSSU.tp.S31A/
S34A-GFP give essentially identical patterns (Fig. 3C,D);
all the GFP fluorescence was localized in chloroplasts, and
none was seen in either the cytosol or other organelles, such
as mitochondria. A punctate pattern of GFP fluorescence in
much smaller organelles, as demonstrated in Fig. 3E, is seen
where the cell is expressing mitochondrially targeted
CoxIV.tp-GFP [12]. In order to ensure that this pattern
was representative of the targeting properties of the transit
peptides in other cells, the targeting properties of
TSSU.tp.wt, TSSU.tp.S31A and TSSU.tp.S31A/S34A were
observed in onion epidermal cells, which are nonphoto-
synthetic (Fig. 4). Although there is no chlorophyll fluor-
escence, the plastids can be identified by the virtue of

stromules [20], clearly visible as long protrusions (arrowed)
from the plastids in the higher magnification pictures
(Fig. 4B,D,F).
Effect of alteration of phosphorylation site
on GFP-targeting by transit peptides of pea SSU
and dual-targeted CtRS & HtRS
Our results with the tobacco SSU transit peptide constructs
were reproducible and clearly demonstrated that alteration
of the phosphorylation signal had no effect on the efficacy
or specificity of targeting in vivo. To determine if this were
true for other transit peptides, we chose three others to
investigate using the same approach. The transit peptide for
pea SSU has 64% sequence identity to the tobacco SSU,
with the phosphorylation motif at an identical position.
In addition, we chose the transit peptides of two amino
Fig. 3. Targeting of tobacco SSU-GFP fusion proteins (pTSSU.tp.wt-
GFP, pTSSU.tp.S34A-GFP and pTSSU.tp.S31A/S34A-GFP) in
tobacco guard cells in vivo. In each panel, the left column is a false-
colour image of the GFP channel, the middle column is chlorophyll
autofluorescence and the right column is the GFP and chlorophyll
channels superimposed. All images are multiprojections of six or eight
scans of the depth of a whole tobacco guard cell. (A) GFP alone. (B)
pTSSU.tp.wt-GFP. The GFP is clearly targeted to the chloroplasts as
the GFP fluorescence overlays precisely with that of the chlorophyll
autofluorescence. (C) Mutant transit peptide pTSSU.tp.S34A-GFP.
(D) The double mutant transit peptide pTSSU.tp.S31A/S34A-GFP.
(E) A mitochondrial-targeted CoxIV-GFP [12] is shown where the
typical punctate pattern of these smaller organelles is apparent. The
scale bar in all images is 10 lm.
Fig. 2. Import experiment with isolated chloroplasts and tobacco SSU-

GFP fusion proteins. (A) Incubation of the translation product of GFP
(27 kDa), and with isolated pea chloroplasts (+ Cp). (B) Incubation
of the 33 kDa precursor of pTSSU.tp.wt-GFP (Twt), and with isolated
chloroplasts. In this case, the precursor is imported into chloroplasts
and processed to the size of GFP alone. (C) and (D) Incubation of
GFP fused to the two mutant forms of the tobacco SSU transit peptide
(pTSSU.tp.S34A-GFP and pTSSU.tp.S31A/S34A), and with isolated
pea chloroplasts. Import is essentially the same as for the wild-type
transit peptide.
512 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004
acyl-tRNA synthetases, CtRS and HtRS, from A. thaliana.
These proteins have been shown to be dual-targeted both
in vivo and in vitro [10–12], so the transit peptides (65 and 73
residues long, respectively) must contain targeting informa-
tion for both mitochondria and chloroplasts. They both
contain phosphorylation motifs, but these are not in
equivalent positions: in CtRS it is in the first third of the
sequence (residues 17–22), whereas in HtRS it is towards the
end, at position 48–52 (Fig. 1B).
For each of these three transit peptides, both the
phosphoacceptor residue and the upstream serine were
mutated to alanine to generate single and double mutants
(Table 1). These constructs were introduced into pea or
tobacco guard cells by biolistic transformation and the
location of the GFP fluorescence viewed by confocal
microscopy (Fig. 5). The results with the pea SSU transit
peptides were identical to those for tobacco SSU transit
peptides. Alteration of the phosphorylation site did not
impede targeting of the passenger GFP to the chloroplasts,
nor was there any mistargeting to mitochondria.

The pattern of GFP fluorescence after targeting by either
CtRS.tp.wt or HtRS.tp.wt differed from that with SSU.tp.
As well as being in large round organelles that coincided
with the chlorophyll fluorescence, it was also seen in small
punctate organelles that correspond to mitochondria (com-
pare with Fig. 3E). Again, modification of the phosphory-
lation motif had no effect on the efficacy of chloroplast
targeting, or indeed to mitochondria. Identical results were
obtained using Arabidopsis leaf material for biolistic trans-
formation (data not shown).
Discussion
In this paper, we have used the ability to image GFP
fluorescence in living plant tissue by confocal microscopy,
in order to test the role of a phosphorylation motif in the
transit peptides of several precursor proteins. This phos-
phorylation motif has been shown to be necessary to form
a complex with 14-3-3 proteins and Hsp70, which make
the precursor more import-competent [14]. This charac-
teristic has been proposed as a possible means of ensuring
specificity for chloroplast import. However, our results
demonstrate that removal of the phosphorylation motif
from the transit peptides of tobacco and pea SSU did not
Fig. 4. The effect of mutagenesis of the phosphorylation site in the transit peptide of tobacco SSU on the targeting of GFP fusion proteins in onion
epidermal cells. The tobacco SSU.tp-GFP constructs were transiently expressed in nonphotosynthetic onion epidermis. The figures are multi-
projections of 14 or 16 scans through two adjacent cells expressing the fusion proteins, superimposed on a single bright field scan, allowing the
outline of the cells to be visualized easily. (A) and (B) pTSSU.tp.wt-GFP. (C and D) pTSSU.tp.S34A-GFP. (E and F) pTSSU.tp.S31A/S34A-GFP.
For each construct, GFP is localized in plastids, which are easily identified as such by the presence of stromules [20], indicated by the arrows in the
higher magnification images. The scale bar is 50 lm in the images on the left, and 10 lm in the higher magnification images from regions in (A), (C)
and (E), shown on the right.
Ó FEBS 2004 Chloroplast targeting fidelity (Eur. J. Biochem. 271) 513

prevent accurate targeting of the passenger GFP to
plastids in either leaf cells (Figs 3 and 5A) or nonphoto-
synthetic cells (Fig. 4). Similarly, this caused no alteration
in the dual-targeting to chloroplasts and mitochondria
by the transit peptides of A. thaliana CtRS and HtRS
(Fig. 5B,C). We therefore conclude that this signal is not
involved in determining the specificity of import into
chloroplasts.
Instead, the guidance complex may be important to
ensure high rates of translocation for highly expressed
chloroplast proteins, or to prevent the accumulation of
nonimport competent protein in the cytosol. Cytosolic
chaperones, mitochondrial import stimulating factor [21]
(now known to be a 14-3-3 protein [22]) and presequence
binding factor [23], have been proposed to prevent aggre-
gation of mitochondrial precursors in the cytosol [24].
Interestingly, although many chloroplast-targeted proteins
have the motif, it is not present in all plastid-targeted transit
peptides. For instance, it is not present in the transit peptides
for light-harvesting chlorophyll a/b binding proteins from
pea and Arabidopsis, although as these are very hydro-
phobic proteins they might be a special case. It is absent
from the transit peptide of ferredoxin from Silene pratensis,
a soluble stromal protein, whereas ferredoxins from other
higher plants do contain the motif [25]. A notable group of
proteins that do not contain the motif are the type-2
ferrochelatases (the terminal enzyme of haem biosynthesis),
which are targeted exclusively to chloroplasts in vitro [26,27].
In contrast, the type-1 ferrochelatases, which are imported
into both chloroplasts and mitochondria in vitro [26,28],

contain the phosphorylation motif.
In fact, import into some isolated plant mitochondria has
been shown to not be robust, as photosynthetic protein
precursors like plastocyanin are imported and processed
[19,29]. In an attempt to overcome this apparent lack of
specificity, a competitive import assay was developed [30], in
which isolated mitochondria and chloroplasts from pea are
mixed together and incubated with the precursor proteins,
and then the organelles are re-separated. The authors report
that this allowed them to distinguish genuinely dual-
targeted precursors from chloroplast- or mitochondria-
destined precursors. In this study we have taken an
alternative approach using GFP as a marker to track
targeted proteins in vivo. The fact that GFP can be used to
image the location of targeted proteins in living tissue avoids
the potential artefacts of in vitro systems, in particular their
lack of specificity. Furthermore, the stability of GFP
ensures that problems of degradation of mistargeted
proteins, which is characteristic of the in vitro system, do
not occur.
Fig. 5. Effect of mutagenesis of the phosphorylation site in transit peptides of pea SSU, and Arabidopsis CtRS and HtRS on their ability to target GFP
in vivo. All images are overlays of the GFP channel and the red chlorophyll fluorescence channel. They are multiprojections of six or eight scans of
the depth of the guard cell(s) expressing the GFP-fusion constructs. (A) The targeting of pPSSU.tp.wt-GFP (wild-type), pPSSU.tp.T34A-GFP
(single mutant) and pPSSU.tp.S32A/T34A-GFP (double mutant). (B) CtRS.tp.wt-GFP (wild-type), CtRS.tp.S21A-GFP (single mutant) and
CtRS.tp.S21A/S22A-GFP (double mutant). (C) Wild-type HtRS (HtRS.tp.wt-GFP), single mutant (HtRS.tp.S50A-GFP) and double mutant
(HtRS.tp.S50A/S52A). The scale bar in all images is 10 lm.
514 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004
As well as the role of the transit peptide itself, several
other processes may play a role in the specificity of targeting
to organelles, including interactions with organellar surface

lipids, subcellular location of translation, and the receptors
at the outer organellar translocon. Lipids that are present at
the chloroplast outer envelope and not on the outer
membrane of the mitochondria, such as digalactosyl
diacylglycerol [31], represent a possible means for chloro-
plast precursor discrimination. In yeast, an mRNA-binding
protein has been shown to bind to transcripts of mito-
chondrial preproteins, directing them to ribosomes in closer
proximity to mitochondria [32]. Also in fungi, the acidity of
Tom22 at the mitochondrial surface is thought to provide a
binding site for basic mitochondrial presequences [33]. The
lack of Tom22 in higher plant mitochondria has been
proposed as a means of preventing chloroplast precursors
from entering mitochondria [33]. These few examples
illustrate clearly that a complete understanding of organelle
targeting requires a multifaceted approach in order to
integrate studies of transit peptide structure and character-
istics with those of the import machinery and other cellular
factors. The approach described in this paper provides a
quick, versatile and unambiguous means of testing the effect
of altering the components of the targeting and import
process.
Acknowledgements
We thank Dr Ian Small (INRA, Evry, France) for the GFP plasmids
and Professor Colin Robinson (University of Warwick, UK) for help
with in vitro import experiments. We are grateful to the Royal Society
for funding. K A. N. was in receipt of a studentship from the
Cambridge Commonwealth Trust.
References
1. Jarvis, P. & Soll, J. (2001) Toc, Tic, and chloroplast protein

import. Biochim. Biophys. Acta 1541, 64–79.
2. Glaser, E., Sjoling, S., Tanudji, M. & Whelan, J. (1998)
Mitochondrial protein import in plants – signals, sorting, target-
ing, processing and regulation. Plant Mol. Biol. 38, 311–338.
3. Zhang, X.P. & Glaser, E. (2002) Interaction of plant mitochon-
drial and chloroplast signal peptides with the Hsp70 molecular
chaperone. Trends Plant Sci. 7, 14–21.
4. von Heijne, G. & Niskikawa, K. (1991) Chloroplast transit pep-
tides: the perfect random coil? FEBS Lett. 278,1–3.
5. Emanuelsson, O. & von Heijne, G. (2001) Prediction of organellar
targeting signals. Biochim. Biophys. Acta 1541, 114–119.
6. Kubo, N., Arimura, S., Tsutsumi, N., Hirai, A. & Kadowaki, K.
(2003) Involvement of N-terminal region in mitochondrial tar-
geting of rice RPS10 and RPS14 proteins. Plant Sci. 164,1047–
1055.
7. Chaumont, F., O’Riordan, V. & Boutry, M. (1990) Protein
transport into mitochondria is conserved between plant and yeast
species. J. Biol. Chem. 265, 16856–16862.
8.Whelan,J.,Knorpp,C.,Harmey,M.A.&Glaser,E.(1991)
Specificity of leaf mitochondrial and chloroplast processing sys-
tems for nuclear-encoded precursor proteins. Plant Mol. Biol. 16,
283–292.
9. Boutry, M., Nagy, F., Poulsen, C., Aoyagi, K. & Chua, N.H.
(1987) Targeting of bacterial chloramphenicol acetyltransferase to
mitochondria in transgenic plants. Nature 328, 340–342.
10. Duchene, A.M., Peeters, N., Dietrich, A., Cosset, A., Small, I.D.
& Wintz, H. (2001) Overlapping destinations for two dual targeted
glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus
vulgaris. J. Biol. Chem. 276, 15275–15283.
11. Peeters,N.M.,Chapron,A.,Giritch,A.,Grandjean,O.,Lancelin,

D., Lhomme, T., Vivrel, A. & Small, I. (2000) Duplication and
quadruplication of Arabidopsis thaliana cysteinyl- and aspar-
aginyl-tRNA synthetase genes of organellar origin. J. Mol. Evol.
50, 413–423.
12. Akashi, K., Grandjean, O. & Small, I. (1998) Potential dual tar-
geting of an Arabidopsis archaebacterial-like histidyl-tRNA syn-
thetase to mitochondria and chloroplasts. FEBS Lett. 431, 39–44.
13. Waegemann, K. & Soll, J. (1996) Phosphorylation of the transit
sequence of chloroplast precursor proteins. J.Biol.Chem.271,
6545–6554.
14. May, T. & Soll, J. (2000) 14-3-3 proteins form a guidance complex
with chloroplast precursor proteins in plants. Plant Cell 12, 53–63.
15. Davis, S.J. & Vierstra, R.D. (1998) Soluble, highly fluorescent
variants of green fluorescent protein (GFP) for use in higher
plants. Plant Mol. Biol. 36, 521–528.
16. Mazur, B.J. & Chui, C.F. (1985) Sequence of a genomic DNA
clone for the small subunit of ribulose bis-phosphate carboxylase-
oxygenase from tobacco. Nucleic Acids Res. 13, 2373–2386.
17. Sheen, J., Hwang, S.B., Niwa, Y., Kobayashi, H. & Galbraith,
D.W. (1995) Green fluorescent protein as a new vital marker in
plant cells. Plant J. 8, 777–784.
18. Anderson, S. & Smith, S.M. (1986) Synthesis of the small
subunit of ribulose bisphosphate carboxylase from genes cloned
into plasmids containing the SP6 promoter. Biochem. J. 240,
709–715.
19. Cleary, S.P., Tan, F.C., Nakrieko, K.A., Thompson, S.J., Mulli-
neaux, P.M., Creissen, G.P., von Stedingk, E., Glaser, E., Smith,
A.G. & Robinson, C. (2002) Isolated plant mitochondria import
chloroplast precursor proteins in vitro withthesameefficiencyas
chloroplasts. J. Biol. Chem. 277, 5562–5569.

20. Gray,J.C.,Sullivan,J.A.,Hibberd,J.M.&Hansen,M.R.(2001)
Stromules: mobile protrusions and interconnections between
plastids. Plant Biol. 3, 223–233.
21. Mihara, K. & Omura, T. (1996) Cytoplasmic chaperones in pre-
cursor targeting to mitochondria: the role of MSF and hsp70.
Trends Cell Biol. 6, 104–108.
22. Alam, R., Hachiyan, N., Sakaguchi, M., Kawabata, S., Iwanaga,
S., Kitajima, M., Mihara, K. & Omura, T. (1994) cDNA cloning
and characterization of mitochondrial import stimulation factor
(MSF) purified from rat liver cytosol. J.Biochem.116, 416–425.
23. Murakami,H.,Pain,D.&Blobel,G.(1988)70-kDheatshock-
related protein is one of at least two distinct cytosolic factors
stimulating import into mitochondria. J.CellBiol.107, 2051–
2057.
24. Iwahashi, J., Furuya, S., Mihara, K. & Omura, T. (1992) Char-
acterization of adrenodoxin precursor expressed in E. coli.
J. Biochem. 111, 451–455.
25. Wienk, H.L.J., Czisch, M. & de Kruijff, B. (1999) The structural
flexibility of the preferredoxin transit peptide. FEBS Lett. 453,
318–326.
26. Chow, K.S., Singh, D.P., Walker, A.R. & Smith, A.G. (1998) Two
different genes encode ferrochelatase in Arabidopsis: mapping,
expression and subcellular targeting of the precursor proteins.
Plant J. 15, 531–541.
27. Suzuki, T., Masuda, T., Singh, D.P., Tan, F.C., Tsuchiya, T.,
Shimada, H., Ohta, H., Smith, A.G. & Takamiya, K. (2002) Two
types of ferrochelatase in photosynthetic and non-photosynthetic
tissues of cucumber; their difference in phylogeny, gene expression
and localization. J. Biol. Chem. 277, 4731–4737.
28. Chow,K.S.,Singh,D.P.,Roper,J.M.&Smith,A.G.(1997)A

single precursor protein for ferrochelatase-I from Arabidopsis is
imported in vitro to both chloroplasts and mitochondria. J. Biol.
Chem. 272, 27565–27571.
Ó FEBS 2004 Chloroplast targeting fidelity (Eur. J. Biochem. 271) 515
29. Lister, R., Chew, O., Rudhe, C., Lee, M.N. & Whelan, J. (2001)
Arabidopsis thaliana ferrochelatase-I and -II are not imported into
Arabidopsis mitochondria. FEBS Lett. 506, 291–295.
30. Rudhe, C., Chew, O., Whelan, J. & Glaser, E. (2002) A novel
in vitro system for simultaneous import of precursor proteins into
mitochondria and chloroplasts. Plant J. 30, 213–220.
31. Chen, L.J. & Li, H.M. (1998) A mutant deficient in the plastid
lipid DGD is defective in protein import into chloroplasts. Plant
J. 16, 33–39.
32. Ellis, E.M. & Reid, G.A. (1993) The Saccharomyces cervisiae mts1
gene encodes a putative RNA-binding protein involved in
mitochondrial protein targeting. Gene 132, 175–183.
33. Macasev, D., Newbigin, E., Whelan, J. & 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.
516 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004