Tài liệu Báo cáo khoa học: The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane ppt

The most C-terminal tri-glycine segment within the
polyglycine stretch of the pea Toc75 transit peptide plays
a critical role for targeting the protein to the chloroplast
outer envelope membrane
Amy J. Baldwin and Kentaro Inoue
Department of Plant Sciences, College of Agricultural & Environmental Sciences, University of California, CA, USA
Most proteins found in plastids are encoded in the
nuclear genome, translated on cytosolic ribosomes with
cleavable N-terminal transit peptides, and imported
into the organelles post-translationally. The translocon
at the outer envelope membrane of chloroplasts 75
(Toc75) is postulated to function as a general protein
translocation channel [1–4], and was also shown to be
involved in targeting of a signal-anchored outer envel-
ope membrane protein [5]. Toc75 appears to be enco-
ded by a single functional gene in Arabidopsis thaliana
[6] and its disruption by a T-DNA insertion caused an
embryo-lethal phenotype [7], indicating the essential
role of Toc75 in the viability of plants.
Unlike other proteins destined for the outer mem-
branes of chloroplasts or mitochondria, which do not
require cleavable targeting sequences [8–10], Toc75 is
synthesized with an N-terminal transit peptide that
consists of two domains (Fig. 1) [11]. The first part
behaves as a typical stromal targeting sequence [12],
and is removed by a stromal processing peptidase
Keywords
chloroplast protein translocation channel;
polyglycine; protein targeting; transit
peptide; tripeptide segment
Correspondence

K. Inoue, Department of Plant Sciences,
College of Agricultural & Environmental
Sciences, University of California, One
Shields Avenue, Davis, CA 95616, USA
Fax: +1 530 752 9659
Tel: +1 530 752 7931
E-mail:
(Received 18 January 2006, accepted
13 February 2006)
doi:10.1111/j.1742-4658.2006.05175.x
The protein translocation channel at the outer envelope membrane of
chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal
transit peptide. Within the transit peptide of the pea Toc75, a major por-
tion of the 10 amino acid long stretch that contains nine glycine residues
was shown to be necessary for directing the protein to the chloroplast outer
membrane in vitro [Inoue K & Keegstra K (2003) Plant J 34, 661–669]. In
order to get insights into the mechanism by which the polyglycine stretch
mediates correct targeting, we divided it into three tri-glycine segments and
examined the importance of each domain in targeting specificity in vitro.
Replacement of the most C-terminal segment with alanine residues resulted
in mistargeting the protein to the stroma, while exchange of either of the
other two tri-glycine regions had no effect on correct targeting. Further-
more, simultaneous replacement of the N-terminal and middle tri-glycine
segments with alanine repeats did not cause mistargeting of the protein as
much as those of the N- and C-terminal, or the middle and C-terminal seg-
ments. These results indicate that the most C-terminal tri-glycine segment
is important for correct targeting. Exchanging this portion with a repeat of
leucine or glutamic acid also caused missorting of Toc75 to the stroma. By
contrast, its replacement with repeats of asparagine, aspartic acid, serine,
and proline did not largely affect correct targeting. These data suggest that

relatively compact and nonhydrophobic side chains in this particular region
play a crucial role in correct sorting of Toc75.
Abbreviations
mtHsp70, mitochondrial heat shock protein 70; Plsp1, plastidic type I signal peptidase 1; psToc75, Toc75 from Pisum sativum; SPP, stromal
processing peptidase; Toc, translocon at the outer envelope membrane of chloroplasts.
FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS 1547
(SPP) [13]. The second domain of the pea Toc75 tran-
sit peptide consists of 96 amino acids [11]. It is neces-
sary to target the protein to the outer envelope
membrane [13], and is cleaved off by a plastidic
type I signal peptidase (Plsp1) [14,15]. There are two
conserved regions in the second part of the Toc75
transit peptide; one is a stretch of 20–27 amino acids
that is located near the N-terminus and is rich in
hydrophobic side chains. Another conserved region is
located about 10 residues apart from the hydrophobic
region towards the C-terminus of the transit peptide.
It contains 17–22 glycine residues over a stretch of
24–29 amino acids [16]. The polyglycine stretch of the
pea Toc75 transit peptide at residues 91–110 (Fig. 1)
can further be divided by four consecutive serine resi-
dues into two regions that contain nine and six glycine
residues, respectively [11,16]. By deletion and substitu-
tion mutagenesis followed by in vitro import assay, the
first part of the glycine-rich stretch, but not the
conserved hydrophobic domain or the second polygly-
cine stretch, was found to be necessary for correctly
targeting the pea Toc75 protein to the outer envelope
membrane [16].
Two scenarios have been postulated for the potential

mechanism by which the polyglycine stretch mediates
targeting of Toc75 to the chloroplast outer envelope
[16]. In the first scenario, this region interacts with one
or more proteins either at the intermembrane space or
inner membrane of the chloroplast envelope, which
keeps Toc75 from traversing the inner membrane. In
the second scenario, the glycine-rich region prevents
the Toc75 precursor from associating with one or more
proteins that facilitate the translocation of the prepro-
tein across the inner envelope membrane. A glycine
repeat, when attached to a preprotein, was shown to
prevent the protein from binding to a mitochondrial
heat shock protein 70 (mtHsp70) [17], which exists in
the matrix and assists translocation of preproteins
across the mitochondrial membranes [18]. Thus, in the
second scenario, a mtHsp70-like protein may exist in
the intermembrane space of the chloroplast envelope
and play a key role. Nevertheless, a detailed mechan-
ism by which the polyglycine stretch mediates envelope
targeting of Toc75 remains unknown.
In this report, we extended the analysis of the poly-
glycine stretch of the Toc75 transit peptide in order to
better understand the targeting mechanism of the
chloroplast outer envelope membrane protein. We divi-
ded this region into three tri-glycine segments and
examined significance of each portion by in vitro
import assay. Interestingly, only the most C-terminal
tri-glycine was found to be important for correctly tar-
geting Toc75 to the outer envelope membrane.
Results

The most C-terminal tri-glycine segment is
important for correct targeting of Toc75 to the
chloroplast outer envelope membrane in vitro
Previously, we examined the importance of certain
regions within the pea Toc75 transit peptide for correct
targeting by import assay using chloroplasts isolated
from pea seedlings [16]. After import of radiolabeled
precursor proteins, chloroplasts were treated with tryp-
sin, a protease that can penetrate the outer but not the
inner envelope membrane [19,20]. The chloroplasts
containing imported proteins were further divided by
centrifugation into supernatant and pellet fractions,
and distribution of the imported proteins was ana-
lyzed. In the present study, we employed this assay
system to investigate the importance of certain residues
within the polyglycine stretch for targeting specificity.
First, we aimed to test whether or not the entire
polyglycine stretch within the residues 91–100 of the
pea Toc75 transit peptide is required for correct target-
ing of the protein. We divided this region into three
tri-glycine segments at 91–93, 95–97, and 98–100,
replaced each of them with a stretch of three alanine
residues (Table 1, AGG, GAG, and GGA, respect-
ively), and subjected these mutated proteins to in vitro
import assay. Generally, two forms of Toc75, the
intermediate and mature forms, were recovered after
the import reaction (Figs 1–3). This is similar to previ-
ous results [11–13,15,16], which may be due to the rela-
tively low activity of the Plsp1 homolog that is
responsible for full maturation of Toc75 in the pea

Fig. 1. The biogenesis of pea Toc75. The precursor, intermediate,
and mature forms of Toc75 are indicated as prToc75, iToc75, and
mToc75 with the numbers of the N-terminal amino acid residues,
respectively. The stromal targeting sequence and the polyglycine
stretch are indicated as black and gray boxes, respectively. The
amino acid sequence of the core polyglycine stretch at residues
91–110 is shown on top and the region where mutations were
introduced is underlined. SPP, stromal processing peptidase; Plsp1,
plastidic type I signal peptidase 1. The scale bar in the bottom is
equivalent to 100 amino acid residues.
Transit peptide of Toc75 A. J. Baldwin and K. Inoue
1548 FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS
chloroplasts used for this assay [15]. Furthermore, the
intermediate and mature forms of Toc75 in a given
supernatant or pellet fraction showed a similar pattern
of protease sensitivity (e.g., compare lanes 4 and 6, or
27 and 29 of Fig. 2A) as was shown before [11,13,16].
Thus, we did not discriminate these two forms during
analyses in the present study.
Proteins derived from AGG and GAG precursors
were exclusively recovered in the membrane and were
susceptible to trypsin (Fig. 2A, lanes 15–18, 21–24;
Fig. 2B,C), indicating that they were targeted to the
chloroplast outer envelope membrane in a way statisti-
cally indistinguishable from the wildtype precursor
(Fig. 2A, lanes 3–6; P > 0.05; Student’s t-test). By
contrast, substitution of an alanine repeat for the most
C-terminal tri-glycine segment (mutant GGA) resulted
in proteins being targeted almost equally to the soluble
and membrane fractions (Fig. 2A, lanes 27, 28;

Fig. 2B). Most of the proteins recovered in the former
fraction and about half of those in the latter fraction
were resistant to trypsin (Fig. 2A, lanes 27–30;
Fig. 2C). These data indicate that the Toc75 transit
peptide with GGA mutation targeted the protein
to multiple locations: the stroma, the internal mem-
branes where trypsin cannot reach (i.e., either the inner
envelope membrane or thylakoids), and the outer
envelope membrane where trypsin can digest proteins.
This targeting pattern was statistically indistinguish-
able from that of another Toc75 mutant in which most
of the glycine residues were replaced with alanine
(Table 1, polyAla; Fig. 2A, lanes 7–12; P > 0.05;
Student’s t-test). These data suggest that the most
C-terminal tri-glycine segment within the polyglycine
stretch is necessary for correct targeting of Toc75.
Next, we wished to test whether the most C-ter-
minal tri-glycine segment is sufficient for correct tar-
geting of Toc75. To this end, we kept this segment
intact and replaced the other six glycine residues in
the polyglycine stretch with alanine residues (Table 1,
AAG). We also prepared two additional mutated pro-
teins as controls in which alanine residues were sub-
stituted for all but either the first or the second
tri-glycine segments (Table 1, GAA and AGA, respect-
ively). When tested by in vitro import assay, proteins
derived from GAA and AGA precursors were mistar-
geted both to the stroma and to the membrane frac-
tions almost evenly (Fig. 2A, lanes 33, 34, 39, 40;
Fig. 2B). Proteins imported into the membrane

derived from AGA precursor appeared to be slightly
more susceptible to trypsin than those from GAA
precursor: susceptibility of proteins in the pellet from
AGA mutant was 67%, whereas that of proteins
derived from GAA was about 50% (Fig. 2C). Over-
all, however, we were not able to detect significant
differences between the three mutants, GGA, GAA,
and AGA, in their targeting patterns (distributions to
the supernatant and pellets shown in Fig. 2B and sen-
sitivity to trypsin presented in Fig. 2C; P > 0.05;
Student’s t-test). AAG transit peptide also mistargeted
Toc75 to the stroma. However, about 10 times more
proteins were found in the membrane than those in
the supernatant (Fig. 2A, lanes 45 and 46; Fig. 2B),
and this pattern is distinct from that of the other
two mutated Toc75 precursors, GAA and AGA
(P<0.05; Student’s t -test).
We considered the possibility that the difference
between AAG and the other two mutants might be
due to kinetics of the import; i.e., AAG precursor
might be imported into the stroma more slowly than
other mutants. In order to test this possibility, we
monitored the distribution of imported proteins
derived from wildtype, AGA, and AAG precursors
into the supernatant and pellet fractions between 3
and 30 min of the reaction. If the above possibility
were correct, we should see changes in distribution of
proteins, especially those from AGA, during the time
course. As shown in Fig. 3, the ratios of proteins
recovered in the supernatant to those in the membrane

fraction appeared to be consistent among different
reaction times within a single precursor (e.g., for
AGA, compare lanes 19 and 20, 23 and 24, 27 and 28,
and 31 and 32, respectively). Furthermore, trypsin-sen-
sitivity of imported proteins was also consistent over
the time course (e.g., for AGA, compare lanes 19–34).
These data may indicate that (a) AGA precursor was
imported into the stroma so efficiently that we could
not detect translocation intermediates trapped in the
Table 1. Part of the pea Toc75 transit peptide and its derivatives
used in this study.
Name Sequence (residues 91–100)
WT GGGAGGGGGG
polyAla *SA*AAAAA*
AGG AAA*******
GAG ****AAA***
GGA *******AAA
GAA ****AAAAAA
AGA AAA****AAA
AAG AAA*AAA***
GGD *******DDD
GGE *******EEE
GGL *******LLL
GGN *******NNN
GGS *******SSS
GGP *******PPP
A. J. Baldwin and K. Inoue Transit peptide of Toc75
FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS 1549
outer membrane; (b) AAG precursor was sorted to
multiple pathways in a way distinct from that of AGA

precursor, or (c) the effect of AAG mutation on cor-
rect targeting was significantly less than that of AGA
mutation. Taken together with the previous results, we
conclude that the most C-terminal tri-glycine segment
is more important than the preceding two tri-glycine
segments, but not sufficient for correct targeting of
Toc75 completely to the chloroplast outer envelope
membrane.
A specific single glycine residue in the
tri-glycine stretch does not determine the
targeting specificity
Next, we tested the importance of individual glycine
residues at positions 98–100 in wildtype precursor for
correct targeting. The replacement of Gly98 with alan-
ine has already been shown not to affect proper target-
ing [16]. Similarly, individual substitutions of glycine
residues at 99 and 100 to alanine residues in wildtype
AB
C
Fig. 2. Effects of alanine substitutions for the three tri-glycine segments in the Toc75 transit peptide on targeting specificity. (A) After the
import of radiolabeled translation products (tl), chloroplasts were analyzed directly (imp), or subsequently treated without (–) or with (+) tryp-
sin, lysed hypotonically, fractionated into the supernatant (S) and pellet (P) fractions by centrifugation, and analyzed by SDS ⁄ PAGE followed
by fluorography. Precursor (pr), intermediate (i) and mature (m) forms of Toc75 are indicated. (B) Distribution of imported proteins in the
supernatant and pellet fractions. Values indicate total amount of the intermediate and mature proteins recovered in each fraction quantified
using
IMAGEJ version 1.34 (National Institutes of Health, USA, and shown as a percentage of the total amount of pre-
cursor subjected to the import reaction. The mean values and standard deviations represented by error bars were calculated based on at
least three independent experiments. (C) Resistance of imported proteins to trypsin. Values indicate the ratio of trypsin-resistant proteins
(both the intermediate and mature proteins) to total proteins recovered in the supernatant or pellet fractions. The mean values and error bars
were calculated based on at least three independent experiments.

Transit peptide of Toc75 A. J. Baldwin and K. Inoue
1550 FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS
precursor did not affect targeting specificity (data not
shown). In order to test if any two glycine residues are
sufficient for correct targeting of Toc75, we substituted
alanine for each glycine residue at positions 98–100 in
AAG transit peptide. Like the polyAla mutant, pro-
teins derived from all three mutants were mistargeted
mainly to the stroma, in a way distinct from the pat-
tern observed with AAG precursor (data not shown).
These data suggest that the targeting specificity does
not depend on any single residue, but requires all three
glycines at positions 98–100.
The tri-glycine stretch can be replaced with
repeats of several other nonglycine residues
In order to gain further details about the importance
of the tri-glycine segment at residues 98–100 of the
Toc75 transit peptide, we replaced this portion with
repeats of various amino acids, and examined the
effects on protein targeting. Particularly, we aimed to
test the following three hypotheses: (a) A protein with
properties similar to a known molecular chaperon,
namely mtHsp70, plays a key role in recognition of the
Toc75 transit peptide. (b) Non-glycine residues that
frequently occur within and near the glycine-rich
regions of the Toc75 transit peptides can substitute for
glycine residues. (c) Another helix breaker, proline
[21], can substitute for glycine.
The first hypothesis is based on the report that a
polyglycine stretch keeps a preprotein from binding

mtHsp70 in the mitochondrial matrix [17]. Another
amino acid repeat that was shown to disrupt the interac-
tion of a preprotein with mtHsp70 was that of glutamic
acid [17]. Hydrophobic residues, such as leucine and
alanine, were found to have a high affinity to a bacterial
mtHsp70 homolog, DnaK [22]. We sought to test the
first hypothesis based on these observations. We
replaced the tri-glycine stretch with a repeat of glutamic
acid or that of leucine (Table 1, GGE and GGL,
respectively), and examined the effects of these substitu-
tions on targeting. Proteins derived from GGL precur-
sor were targeted both to the stroma and to the
membrane (Fig. 4A, lanes 21–24; Fig. 4B,C) in a way
similar to GGA mutant (Fig. 4A, lanes 9–12;
Fig. 4B,C). Import of GGE mutant was less efficient
than that of other proteins, as evidenced by the accumu-
lation of the precursor form of Toc75 that was sensitive
to thermolysin after the import (Fig. 4A, lanes 14 and
16; data not shown). The intermediate form derived
from GGE mutant recovered both in the supernatant
and pellet fractions was resistant to trypsin (Fig. 4A,
lanes 15–18; Fig. 4B,C), indicating its localization to the
stroma, and thylakoid or inner membranes. These data
indicate that a repeat of glutamic acid cannot replace
the tri-glycine segment in the envelope targeting
sequence. Thus, mtHsp70-like protein may not be
involved in the polyglycine-mediated envelope targeting.
In order to test the second hypothesis, we analyzed
the 24–29 amino acid long regions within and nearby
the conserved polyglycine stretches of Toc75 transit

peptides from six plant species. As shown in Table 2,
three residues, asparagine, aspartic acid, and serine,
occur relatively frequently in these regions. Together
they account for 17% of residues within this stretch.
We replaced the critical tri-glycine within the pea
Toc75 transit peptide with repeats of these three
Fig. 3. Time course of the distribution of imported proteins. Radiolabeled Toc75 precursors were incubated with intact chloroplasts at room
temperature for the time indicated, then chloroplasts were reisolated and analyzed as described in the legend to Fig. 2A.
A. J. Baldwin and K. Inoue Transit peptide of Toc75
FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS 1551
AB
C
Fig. 4. Effects of replacements of the most C-terminal tri-glycine segment within the polyglycine stretch of the Toc75 transit peptide with
repeats of various amino acids on targeting specificity. (A) Radiolabeled Toc75 precursors were incubated with isolated chloroplasts and ana-
lyzed as described in the legend to Fig. 2A. (B) Distribution of imported proteins in the supernatant and pellet fractions quantified as de-
scribed in the legend to Fig. 2B. The mean values and standard deviations represented by error bars were calculated based on at least three
independent experiments. (C) Resistance of imported proteins to trypsin quantified as described in the legend to Fig. 2C. The mean values
and error bars were calculated based on at least three independent experiments.
Table 2. Amino acid compositions in the polyglycine stretch of Toc75 transit peptides. Sequences are those reported previously [16].
Plant species Amino acid # Gly Asn Asp Ser Phe Trp Ala Thr His Tyr
Pea 91–118 17 1 1 5 1 1 1 0 0 0
Arabidopsis 101–126 19 2 3 0 2 0 0 0 0 0
Soybean 84–112 21 2 2 0 1 1 0 1 1 0
Lotus 64–91 22 1 1 0 1 1 0 2 0 0
Potato 101–124 19 1 1 2 0 0 0 0 0 1
Tomato 101–124 19 1 1 3 0 0 0 0 0 0
Total 117 8 9 10 5 3 1 3 1 1
% 73.6 5.0 5.7 6.3 3.1 1.9 0.6 1.9 0.6 0.6
Transit peptide of Toc75 A. J. Baldwin and K. Inoue
1552 FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS

residues. All three mutants, GGN, GGD, and GGS
showed targeting patterns similar to AAG mutant:
proteins were targeted mainly to the membrane frac-
tion and were susceptible to trypsin, indicating that
they were in the outer envelope (Fig. 4A, lanes 25–42;
Fig. 4B,C). Small portions (1–2%) of proteins were
also recovered in the soluble fraction (Fig. 4B). Inter-
estingly, they showed different susceptibility to trypsin
(Fig. 4C): those derived from GGD and GGS were
resistant, indicating their location in the stroma,
whereas GGN-derived proteins were degraded, imply-
ing their location in the intermembrane space. Another
interesting observation was that overall import effi-
ciency of GGN mutant was 18%, which was signifi-
cantly higher than those of other precursors (3–12%;
Fig. 4B).
Finally, we prepared a construct in which the glycine
repeat was replaced with a repeat of proline (Table 1,
GGP). The distribution of proteins derived from GGP
precursor to the supernatant and the membrane frac-
tions was similar to those of GGN, GGD, GGS, and
AAG (Figs 2B and 4B). Interestingly, similar to the
case of GGN mutant, a small but significant amount
of proteins from GGP found in the supernatant were
susceptible to trypsin (Fig. 4B,C), indicating that they
did not traverse the inner envelope membrane.
Taken together, tri-proline and tri-asparagine can
substitute for tri-glycine in envelope targeting to an
extent somewhat better than repeats of aspartic acid
and serine residues.

Discussion
In this report, we aimed to get insights into the mech-
anism by which the polyglycine stretch within the
Toc75 transit peptide mediates targeting of the protein
to the chloroplast outer envelope membrane. Through
mutagenesis and protein import assay in vitro, we were
able to show that the most C-terminal tri-glycine
within the polyglycine stretch is important for correctly
targeting the protein to the outer membrane. Interest-
ingly, replacements of the tri-glycine with repeats of
asparagine, aspartic acid, serine, and proline caused a
lesser degree of mistargeting compared to those with
alanine, leucine, and glutamic acid repeats. We have
not been able to identify a possible structure conserved
and specific between repeats of the former four amino
acid residues and glycine. Nevertheless, they are relat-
ively small and not hydrophobic compared to the three
amino acids that could not replace glycine. Thus, it
may be possible that the compact and hydrophilic
properties of this region are important for envelope
targeting.
How would this region keep the protein from cros-
sing the inner envelope membrane? Neither of the two
scenarios postulated before [16] can be excluded at this
point. In the first scenario, there may be a proteina-
ceous component either in the chloroplast envelope
intermembrane space or in the inner membrane that
binds to the flexible, small, and hydrophilic pocket
that corresponds to 98–100 of the pea Toc75 transit
peptide, and holds the protein at the envelope mem-

brane. In the second scenario, this pocket could pre-
vent Toc75 from interacting with a proteinaceous
component that directs the protein to the stroma.
Potential candidates for this component include a sub-
unit of Toc complex such as Toc12 [23], a Hsp70
homolog in the intermembrane space of the chloro-
plast envelope [23–26] that may have a different fea-
ture than mtHsp70, and components of the translocon
at the inner envelope membrane of chloroplasts such
as Tic22 [27,28]. Constructs generated in this study
should be useful to address these hypotheses.
Experimental procedures
Preparation of plasmids containing cDNAs for
mutated pea Toc75
Substitutions of amino acid residues in the pea Toc75 pre-
cursor were made using a QuikChangeÒ Site-Directed Mut-
agenesis Kit (Stratagene, Cedar Creek, TX, USA). Sets of
primers with variations of a sequence corresponding to nuc-
leotide numbers 277–314 of the pea Toc75 (psToc75) cod-
ing sequence and the plasmid pET-psToc75 as a template
[12] were used to generate constructs for mutants GGA,
GGE, GGL, GGN, GGD, GGS, and GGP. cDNA
sequences for GAA and AGA mutants were prepared using
a plasmid encoding GGA as a template and primers that
anneal to nucleotide numbers 277–314 of psToc75. Plas-
mids carrying sequences encoding AGG and GAG were
prepared using sets of primers corresponding to nucleotide
numbers 256–293 and 268–299 of psToc75, respectively,
and pET-psToc75 as a template. A plasmid encoding the
AGG mutant and primers corresponding to 256–293 of

psToc75 were used to prepare a construct encoding AAG.
Identities of all the clones were confirmed by sequencing of
the entire coding sequence.
Protein import assay using isolated chloroplasts
Chloroplasts were isolated from 10- to 14-day old soil-
grown pea as described [29]. Radiolabeled precursor pro-
teins were prepared from cDNA constructs using T
N
TÒ
Coupled Reticulocyte Lysate System (Promega, Madison,
WI, USA) with [
35
S]Met and T7 RNA polymerase. Import
and trypsin treatment were performed essentially as
A. J. Baldwin and K. Inoue Transit peptide of Toc75
FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS 1553
described [16]. Briefly, the radiolabeled precursor proteins
(10 lL) were incubated with chloroplasts containing
12.5 lg chlorophyll, import buffer (50 mm Hepes⁄ KOH,
330 mm sorbitol, pH 8.0) and 3 mm Mg-ATP in a total vol-
ume of 50 lL in the light for 30 min at room temperature.
After the reaction, intact chloroplasts were re-isolated by
40% (v ⁄ v) Percoll and washed once with the import buffer.
The re-isolated chloroplasts were further resuspended into
100 lL of the import buffer with or without 1.25 lg trypsin
(Sigma, St. Louis, MO, USA), incubated for 30 min on ice,
and to which 100 lL of the import buffer containing
1.25 lg trypsin inhibitor (Sigma) was added. Chloroplasts
were re-isolated by 40% (v ⁄ v) Percoll, washed with the
import buffer, and lysed with 10 mm Hepes ⁄ KOH, pH 8.0,

and 10 mm MgCl
2
. Soluble and membrane fractions were
obtained after centrifugation at 16 000 g at 4 °C for
30 min. Proteins from the soluble fraction were precipitated
with 80% (v ⁄ v) acetone. Both fractions were resuspended
in the sample buffer and analyzed by SDS ⁄ PAGE followed
by fluorography.
Acknowledgements
We thank Dr Daniel Potter for his critical reading of
the manuscript, and also members of the Inoue labor-
atory for their helpful discussions. The project was
supported by the National Research Initiative of the
USDA Cooperative State Research, Education and
Extension Service, grant number 2003-02860 to K.I.
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