Molecular Plant

Research Article

Molecular Mechanism of the Specificity of Protein

Import into Chloroplasts and Mitochondria in
Plant Cells
Dong Wook Lee1, Sumin Lee2, Junho Lee2, Seungjin Woo1, Md. Abdur Razzak1,
Alessandro Vitale3 and Inhwan Hwang1,2,*
1
Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 37673, Korea
2
Department of Life Sciences, Pohang University of Science and Technology, Pohang 37673, Korea
3
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale Delle Ricerche, Milano, Italy
*Correspondence: Inhwan Hwang (ihhwang@postech.ac.kr)
https://doi.org/10.1016/j.molp.2019.03.003

ABSTRACT
Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides
and presequences function as signal sequences for specific import into chloroplasts and mitochondria,
respectively. However, how these highly similar signal sequences confer the protein import specificity
remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct
steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal
regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and prese-
quences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions
of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the
absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers
specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast
import receptor, also contributes to determining chloroplast import specificity. We propose that common
ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by
the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the
N-terminal region.
Key words: transit peptide, presequence, protein import into chloroplasts and mitochondria, N-terminal specificity
domain, C-terminal common translocation domain, import specificity determination
Lee D.W., Lee S., Lee J., Woo S., Razzak Md.A., Vitale A., and Hwang I. (2019). Molecular Mechanism of the
Specificity of Protein Import into Chloroplasts and Mitochondria in Plant Cells. Mol. Plant. 12, 951–966.

INTRODUCTION
Eukaryotic cells contain multiple organelles that perform specific
functions. To function properly these organelles need specific
proteins, the vast majority of which are encoded by the nuclear
genome. Thus, organellar proteins must be imported from the
cytosol during or after translation. The most crucial question is
how these proteins are specifically delivered to the organelle
where they are needed. It has long been established that
organellar proteins contain signal sequences containing the infor-
mation needed for specific intracellular delivery. These signal se-
quences must be recognized by the molecular machinery of the
correct target organelle with sufficient specificity to avoid recog-
nition by the molecular machinery of nontarget organelles
(Okreglak and Walter, 2014; Ye et al., 2015). Thus, these
targeting signals must possess highly distinct features.
Plant cells contain both chloroplasts and mitochondria, which are
thought to have evolved via similar evolutionary processes,
namely, by the conversion of endosymbiotic bacteria into organ-
elles. The import signal sequences of chloroplasts and mitochon-
dria are transit peptides and presequences, respectively. They
impart specificity to the protein import process, which commonly
entails multiple steps. Transit peptides also contain information for
preprotein degradation in the cytosol via ubiquitin-dependent 26S
proteasome-mediated proteolysis when preproteins are not
efficiently imported into chloroplasts (Lee et al., 2009b, 2016).
Systematic analyses revealed that transit peptides and
presequences contain several short sequence motifs required
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 951
Molecular Plant
for various steps during protein import into chloroplasts and
mitochondria, respectively (Lee et al., 2006, 2008, 2012, 2015,
2018; Li and Teng, 2013; Holbrook et al., 2016; Razzak et al.,
2017; Lee and Hwang, 2018). However, the primary structures
of transit peptides and presequences are highly diverse and
there is no consensus sequence. Consistent with this, the
sequence motifs identified from individual transit peptides
and presequences are quite divergent at the level of primary
structure. In fact, the degree of sequence diversity among transit
peptides or among presequences is as high as that between
transit peptides and presequences. Despite this variability in
primary structure, both transit peptides and presequences have
highly similar amino acid compositions, including enrichment in
basic, hydrophobic, and hydroxylated residues (Bhushan et al.,
2006). Thus, transit peptides and presequences are highly
similar in many aspects. Moreover, certain transit peptides
deliver proteins into mitochondria in vitro or in vivo in cells
lacking plastids (Hurt et al., 1986a, 1986b; Cleary et al., 2002),
raising the possibility that specificity for chloroplasts or
mitochondria is a requirement only in organisms with both
organelles, such as plants. Thus, questions remain as to how
such diversity can support accurate protein import into
chloroplasts or mitochondria and how the transit peptide- and
presequence-mediated import specificity is determined. In silico
analyses can accurately discriminate between transit peptides
and presequences when charge properties and hydrophobicity
are used as discrimination factors (Ge et al., 2014). Thus, despite
the high degree of similarity, these two signal sequences should
contain specificity-determining motifs.
In this study, we systematically investigated the mechanism
determining the import specificity of transit peptides and prese-
quences. We focused on how these signals specifically deliver
proteins to their target organelles in vivo and when during the
import process this specificity is determined. We provide evi-
dence that both transit peptides and presequences contain the
sequence motifs necessary for nondiscriminatory protein import
into chloroplasts and mitochondria, and that the N-terminal re-
gion confers the import specificity. We identified a domain con-
taining multiple arginine residues and a relatively hydrophobic
motif in the N-terminal regions of presequences as the mitochon-
drial specificity factor. The presence of this domain and the
absence of multiple arginine residues in the N-terminal region
confer mitochondrial and chloroplast specificity, respectively.
Moreover, surface-localized import receptors such as chloro-
plast AtToc159 also contribute to determining specificity.
RESULTS
N-Terminal Domains of Transit Peptides and
Presequences Confer Protein Import Specificity for
Chloroplasts and Mitochondria, Respectively
To elucidate the mechanisms by which presequences and transit
peptides mediate protein import specifically into mitochondria and
chloroplasts, respectively, despite having similar amino acid com-
positions, we asked two questions. First, do these similarities have
any functional implication for protein import? Second, how do
these similar signal sequences support specific protein import
into the target organelle? To address these questions, we em-
ployed mutants in which portions of the domains from transit pep-
952 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria
tides and presequences were swapped. Previously we showed
that although RbcS (Rubisco small subunit) and Cab (chlorophyll
a/b-binding protein) transit peptides have different sequence
motifs, many mutant transit peptides in which portions of these do-
mains were swapped properly imported proteins into chloroplasts
(Lee et al., 2015). FA[N77] and RbcS[N79] are N-terminal domains
containing 77 residues of the F1-ATPase g subunit (FA) and 79
residues of RbcS, and are a representative presequence and
transit peptide, respectively (Lee et al., 2006, 2012). We
generated a series of mutants by swapping portions of these
two domains and tested whether the hybrid domains properly
imported proteins into either chloroplasts or mitochondria.
Previously, we showed that moderate hydrophobicity in the
N-terminal regions of transit peptides is critical for protein import
into chloroplasts (Lee et al., 2006, 2008). Because FA[N77]
contains four arginine residues in the N-terminal 12 amino
acids, which is very rare in transit peptides (Bhushan et al.,
2006; Garg and Gould, 2016), we first substituted the N-terminal
12 amino acids of FA[N77] with those of RbcS[N79]. A series of
constructs with hybrid domains fused with GFP were generated
(Figure 1A), and the specific import of the resulting fusion
proteins into chloroplasts or mitochondria was tested in
transiently transfected Arabidopsis protoplasts. All serial RbcS
[xx]/FA[xx] constructs produced fluorescent GFP signals at
chloroplasts (Figure 1B). These constructs yielded processed
forms of the GFP fusion protein, as observed when protein
extracts from protoplasts were analyzed by western blotting with
an anti-GFP antibody (Figure 1C). Upon subcellular fractionation,
the processed forms of the proteins from all these hybrid
constructs copurified with isolated chloroplasts but not with
mitochondria (Figure 1D), confirming that all RbcS[xx]/FA[xx]
domains supported chloroplast import.
Of the reciprocal serial FA[xx]/RbcS[xx] domains, FA[20] and
FA[30], but not FA[12], supported mitochondrial import
(Figure 2A and 2B). FA[1–12]/RbcS[13–79] fusion proteins
were largely unprocessed. By contrast, FA[1–20]/RbcS[21–79]
fusion proteins were present as processed forms (Figure 2C).
Moreover, processed forms of both FA[1–20]/RbcS[21–79] and
FA[1–30]/RbcS[31–79] fusion proteins were detected in the
mitochondrial fraction but not in the chloroplast fraction
(Figure 2D). These results indicate that FA[1–20]/RbcS[21–79]
and FA[1–30]/RbcS[31–79], but not FA[1–12]/RbcS[13–79],
supported mitochondrial import. Although FA[N12] was not
sufficient to change the import specificity of RbcS[N79] from
chloroplasts to mitochondria, it was crucial for protein import
into mitochondria, as FA[DN12] failed to deliver proteins into
mitochondria (Figure 2B).
The primary structures of transit peptides show no close
homology and can be classified into at least seven different sub-
groups on the basis of critical sequence motifs (Lee et al., 2008).
Thus, we examined whether small N-terminal fragments (12-
amino-acid residues) of other transit peptides alter the import
specificity of FA[13–77] from mitochondria to chloroplasts. We
generated BCCP[1–12]/FA[13–77] and E1a[1–12]/FA[13–77]
using the transit peptides of BCCP (biotin carboxyl carrier
protein) and E1a (E1a subunit of pyruvate dehydrogenase)
(Supplemental Figure 1A) (Lee et al., 2008, 2009a). The
localization of fusion proteins produced by these two
constructs in protoplasts was examined. Both BCCP[1–12]/FA
Protein Import into Chloroplasts and Mitochondria Molecular Plant
A

C D

Figure 1. N-Terminal Region of Transit Peptides Determines the Import Specificity of Chloroplast Proteins.
(A) Sequences of RbcS[N79], FA[N77], and their mutants.
(B) Localization of reporter proteins. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll auto-
fluorescence, respectively. Scale bars, 20 mm.
(C) Western blot analysis. Protein extracts from protoplasts transformed with the indicated constructs were analyzed by western blotting with an anti-GFP
antibody. Pre, precursor form; Pro, processed form.
(D) Isolation of chloroplasts and mitochondria from transformed protoplasts. Chloroplasts and mitochondria were analyzed by western blotting. LHCB4
and IDH were used as controls for chloroplast and mitochondrial proteins, respectively. Total, total fraction; CH, chloroplast fraction; MT, mitochondrial
fraction; Pre, precursor form; Pro, processed form.

Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 953
Molecular Plant Protein Import into Chloroplasts and Mitochondria
A

C D

Figure 2. N-Terminal Region of Presequences Determines the Import Specificity of Mitochondrial Proteins.
(A) Sequences of RbcS[N79], FA[N77], and their mutants.
(B) Localization of reporter proteins. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll auto-
fluorescence, respectively. Scale bars, 20 mm.
(legend continued on next page)

954 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria
[13–77] and E1a[1–12]/FA[13–77] supported chloroplast import,
confirming that the N-terminal 12-amino-acid residues of transit
peptides generally confer chloroplast specificity to FA[13–77]
(Supplemental Figure 1B). Next, we examined whether FA[1–20]
confers mitochondrial specificity to other transit peptides. We
replaced the N[1–20] of Cab, DnaJ-J8, PORA, TOCC, and Glu2,
which are representative of five of the seven subgroups we pre-
viously identified, with FA[1–20] (Lee et al., 2008). In addition,
we generated a synthetic transit peptide, SynTP[LLSSS], by
incorporating three sequence motifs from the transit peptides
of RbcS and Cab into the N-terminal 80 residues of the
vacuolar protein CPY (carboxypeptidase Y) (Lee et al., 2015).
The resulting six hybrid constructs, namely, FA[1–20]/Cab
[21–80], FA[1–20]/DnaJ-J8[21–80], FA[1–20]/PORA[21–80], FA
[1–20]/TOCC[21–80], FA[1–20]/Glu2[21–80], and FA[1–20]/SynTP
[LLSSS][21–80], were introduced into protoplasts for analysis
of targeting. All six hybrid N-terminal domains supported
mitochondrial import of GFP, similar to FA[1–20]/RbcS[21–79]
(Supplemental Figure 1C–1F).
Since the primary structures of the presequences are also quite
diverse, we tested whether other presequences also confer
mitochondrial specificity to transit peptides. We generated
LPD2[1–20]/RbcS[21–79] by using the presequence of lipoamide
dehydrogenase 2 (LPD2), another Arabidopsis mitochondrial
protein (Lutziger and Oliver, 2001), and the resulting construct
was introduced into protoplasts. LPD2[1–20]/RbcS[21–79] also
supported protein import into mitochondria (Supplemental
Figure 2A and 2B). These results indicate that transit peptides
and presequences are functionally interchangeable for protein
import, and that the import specificity for chloroplasts and
mitochondria is determined by the sequence motif(s) located in
N-terminal specificity domains such as [N12] of transit peptides
and [N20] of presequences.
Multiple-Arginine and Hydrophobic Sequence Motifs in
the N-Terminal Regions of Presequences Function as
Specificity Factors
To identify the mitochondrial and chloroplast import specificity
factors in presequences and transit peptides, respectively, we
again compared the RbcS[N12] and FA[N20] sequences at the
amino acid level. The presence of multiple arginine (R) residues
in FA[N20] but not in RbcS[N12] was the most prominent differ-
ence (Figure 1A) (Bhushan et al., 2006; Ge et al., 2014; Lee
et al., 2006, 2012). To test whether these R residues play a role
in determining protein import specificity, we replaced the four R
residues in the R7REGRR sequence of FA[N77] with alanine (A)
residues (FA[N77][4R/4A]) or deleted (FA[N77][D4R]) (Figure 3A).
These FA[N77]-based mutants imported proteins into chloro-
plasts instead of mitochondria (Figure 3A–3D). To confirm the
function of the R residues in the presequence, we also
introduced R-to-A mutations in the presequence of LPD2 to
produce LPD2[N80][2R/2A] and examined its localization. LPD2
[N80][2R/2A] also supported protein import into chloroplasts
(C) Western blot analysis. Protein extracts from protoplasts transformed with the indicated constructs were analyzed by western blotting with an anti-GFP
antibody. Pre, precursor form; Pro, processed form.
(D) Isolation of chloroplasts and mitochondria from transformed protoplasts. Chloroplasts and mitochondria were analyzed by western blotting. LHCB4
and IDH were used as controls for chloroplast and mitochondrial proteins, respectively. T, total fraction; CH, chloroplast fraction; MT, mitochondrial
fraction; Pre, precursor form; Pro, processed form.
Molecular Plant
but not mitochondria (Supplemental Figure 2C and 2D),
indicating that the motif containing multiple R residues (i.e., the
multiple-arginine motif) in presequences is essential for speci-
fying mitochondrial import. Moreover, these results strongly sug-
gest that presequences support protein import into chloroplasts
and that specific mitochondrial protein import is achieved via
multiple-arginine motif-mediated inhibition of chloroplast import.
These results are consistent with the fact that the N-terminal re-
gions of presequences contain more R residues than those of
transit peptides (Bhushan et al., 2006; Lee et al., 2012; Ge
et al., 2014; Garg and Gould, 2016). We examined whether the
similarly positively charged lysine (K) residues can replace Rs in
multiple-arginine motifs. However, the R-to-K substitution
mutant of FA[N77] failed to deliver proteins into mitochondria.
An R-to-E (glutamic acid) substitution mutant also failed to sup-
port mitochondrial import (Figure 3E–3G), confirming the
importance of Rs in the multiple-arginine motif for mitochondrial
import. In addition, both mutant constructs yielded mainly cyto-
solic GFP signals, indicating that substitution of Rs with other
charged residues is also deleterious for chloroplast import.
Next, we examined whether the presence of the multiple-arginine
motif is sufficient to transform a transit peptide into a prese-
quence. We replaced the SS and MV dipeptides in the S7SATMV
sequence of RbcS[N79] with four Rs or with four A residues (as a
negative control), which are at positions equivalent to those of the
four Rs in the FA presequence (Figure 4A). RbcS[N79][4R]
localized to the cytosol, whereas RbcS[N79][4A] localized to
chloroplasts (Figure 4B), indicating that the multiple-arginine
motif prevents RbcS[N79]-mediated chloroplast protein import
but is not sufficient to mediate mitochondrial import. To confirm
these results biochemically, we analyzed protein extracts from
protoplasts transformed with these constructs by western blot-
ting with an anti-GFP antibody. RbcS[N79][4R] fusion proteins
were mainly present as precursors, with only a minor proportion
in the processed form. However, RbcS[N79][4A] fusion proteins
were mainly in the processed form, with only a minor proportion
detected as precursors (Figure 4C), confirming that RbcS[N79]
[4R] and RbcS[N79][4A] localize to the cytosol and chloroplast,
respectively.
The results in Figure 4 suggested that FA[N20] contains an
additional motif(s) that confers mitochondrial import. We
noticed that the sequence L13LPSIAAR largely consists of
hydrophobic residues and conforms to the consensus Tom20-
binding motif 4XX44 (Figure 4A) (Abe et al., 2000; Lee et al.,
2012; Ye et al., 2015). Although an in vitro import study
revealed the role of Arabidopsis Tom20 in the mitochondrial
import of some preproteins, the mitochondrial import of FA
[N77] was barely affected in protoplasts of tom20-2/tom20-3/
tom20-4 mutant plants (Lister et al., 2007; Lee et al., 2012).
Thus, the functional relationship between the LLPSIAAR motif
of FA[N77] and Tom20 is still not clear. Because of this, in this
study we refer to this motif as the ‘‘hydrophobic sequence
motif.’’ We examined whether this hydrophobic sequence motif
Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 955
Molecular Plant Protein Import into Chloroplasts and Mitochondria
A

B C

F
G

Figure 3. Multiple-Arginine Motif in Presequences Functions as a Chloroplast Evasion Signal.

(A and E) Sequences of FA[N77] and its substitution mutants.
(B and F) Localization of reporter proteins. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll
autofluorescence, respectively. Scale bars, 20 mm.
(C and G) Western blot analysis of reporter proteins. Protoplast extracts were analyzed by western blotting with an anti-GFP antibody. Pre, precursor
form; Pro, processed form.
(D) Isolation of chloroplasts and mitochondria from transformed protoplasts. Chloroplasts and mitochondria were analyzed by western blotting. LHCB4
and IDH were used as controls for chloroplast and mitochondrial proteins, respectively. CH, chloroplast fraction; MT, mitochondrial fraction; Pro,
processed form.

is involved in determining mitochondrial specificity. The sequence
LLPSIAAR was inserted into RbcS[N79][4R] and RbcS[N79] to
produce RbcS[N79][4R][LLPSIAAR] and RbcS[N79][LLPSIAAR],
respectively, and the resulting domains fused with GFP were
examined for their localization in protoplasts. RbcS[N79][4R]
[LLPSIAAR] and RbcS[N79][LLPSIAAR] supported protein import
into mitochondria and chloroplasts, respectively (Figure 4B),
indicating that the hydrophobic sequence motif confers
mitochondrial import specificity to a transit peptide when it is
combined with the multiple-arginine motif, but not when it is
introduced alone (Figure 4B–4D). To test whether these findings
956 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
apply to other transit peptides, we inserted the multiple-arginine
motif alone or with the hydrophobic sequence motif into PORA
[N80] to obtain PORA[N80][4R] and PORA[N80][4R][LLPSIAAR],
respectively (Supplemental Figure 3A). Fluorescence analysis
showed that reporter proteins fused with PORA[N80][4R]
[LLPSIAAR] and PORA[N80][4R] localized to mitochondria and
the cytosol, respectively, indicating that the multiple-arginine
motif alone inhibits PORA[N80]-mediated chloroplast import,
while the multiple-arginine motif and hydrophobic sequence
motif together confer mitochondrial specificity to PORA[N80]
(Supplemental Figure 3B). However, western blot analysis
Protein Import into Chloroplasts and Mitochondria Molecular Plant

Figure 4. Multiple-Arginine Motif and Hydrophobic Sequence Motif in the FA Presequence Are Sufficient for Mitochondrial Specificity
of Protein Import.
(A) Sequences of RbcS[N79] and its mutants.
(B) Localization of reporter proteins. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll auto-
fluorescence, respectively. Scale bars, 20 mm.
(legend continued on next page)

Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 957
Molecular Plant
showed that the majority of PORA[N80][4R][LLPSIAAR] fusion
proteins were present as precursor forms that copurified with
mitochondria (Supplemental Figure 3B and 3C), strongly
suggesting that PORA[N80][4R][LLPSIAAR] is associated with
mitochondria but that its translocation is compromised. To test
this, we fractionated the protein extracts from protoplasts
transformed with PORA[N80][4R] and PORA[N80][4R][LLPSIAAR]
into soluble and pellet fractions by ultracentrifugation and
analyzed these fractions by western blotting. Unlike the PORA
[N80][4R] fusion protein, the precursor of the PORA[N80][4R]
[LLPSIAAR] fusion protein was mainly detected in the pellet
fraction, supporting the idea that PORA[N80][4R][LLPSIAAR]
localized to, but was not fully translocated into mitochondria
(Supplemental Figure 3D).
Next, we focused on the role of serine (S) residues in determining
the specificity for chloroplasts and mitochondria because
previously, it was suggested that S residues are a critical factor
for chloroplast import specificity (Garg and Gould, 2016;
Ge et al., 2014). We replaced four S and one threonine (T)
residue in RbcS[1–12]/FA[13–77], and the resulting construct
was introduced into protoplasts. However, these mutations did
not affect protein import into chloroplasts (Supplemental
Figure 4A–4D), indicating that these hydroxylated S and
T residues in the sequence context of RbcS[1–12]/FA
[13–77] are not important for chloroplast import. Moreover, this
result is consistent with that of our previous study showing that
hydroxylated residues such as S in the N-terminal region of the
RbcS transit peptide are not critical for efficient import into
chloroplasts (Lee et al., 2006). On the other hand, multiple S
residues in the internal region of the DnaJ-J8 transit peptide
were essential for chloroplast import (Lee et al., 2008). FA[1–20]/
DnaJ-J8[21–80], which still contains this motif consisting of
multiple S residues, was exclusively imported into mitochondria
(Supplemental Figure 1C and 1D), suggesting that, although
S residues are abundantly present in the N-terminal specificity
domain of transit peptides, they are not involved in specifying
protein import into chloroplasts. Altogether, these results
suggest the following scenario for protein import into
mitochondria in plants: (1) the multiple-arginine motif inhibits
import into chloroplasts, thereby causing proteins to be localized
to the cytosol; (2) the hydrophobic sequence motif together with
the multiple-arginine motif leads to a commitment to mitochon-
drial import; and (3) sequence motifs in the C-terminal domains
of transit peptides or presequences support the translocation of
proteins across the mitochondrial envelope membranes.
Sequence Motifs Involved in Translocation across
Envelope Membranes of Chloroplasts and Mitochondria
Are Functionally Interchangeable
Next, we investigated how the transit peptide supports mitochon-
drial import when it contains the multiple-arginine motif and the
hydrophobic sequence motif. Transit peptides contain many
(C) Western blot analysis of reporter proteins. Protoplast extracts were analyzed by western blotting with an anti-GFP antibody. The import efficiency was
defined as the amount of the processed form relative to the total amount of expressed protein. Data represent the mean ± SD (n = 3). Pre, precursor form;
Pro, processed form.
(D) Isolation of chloroplasts and mitochondria from transformed protoplasts. Chloroplasts and mitochondria were analyzed by western blotting. LHCB4
and IDH were used as controls for chloroplast and mitochondrial proteins, respectively. T, total fraction; CH, chloroplast fraction; MT, mitochondrial
fraction; Pre, precursor form; Pro, processed form.
958 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria
small sequence motifs that are crucial for protein import into chlo-
roplasts (Lee et al., 2006, 2008, 2009a, 2015; Li and Teng, 2013;
Holbrook et al., 2016). We examined whether sequence motifs in
transit peptides that are critical for chloroplast import also play a
role in presequence/transit peptide hybrid sequence-mediated
mitochondrial import. In RbcS[N79], the sequence motifs FP-
RK, CMQVW, and KKFET are crucial for chloroplast import (Lee
et al., 2006). We deleted these motifs from FA[1–20]/RbcS
[21–79], which supported mitochondrial import (Figures 5A and
2B). The resulting mutants were introduced into protoplasts to
examine their localization. The FA[1–20]/RbcS[21–79][DFP-RK]
GFP fusion protein was almost exclusively localized in the
cytosol and was not observed in mitochondria (Figure 5B). In
addition, the majority of FA[1–20]/RbcS[21–79][DCMQVW] and
FA[1–20]/RbcS[21–79][DKKFET] fusion proteins also showed
cytosolic localization with mitochondrial localization observed in
a small proportion of transformed protoplasts (Figure 5B and
5C). Altogether, these results suggest that critical sequence
motifs in transit peptides are actually critical for the nonspecific
translocation of proteins across the envelope membranes of
both chloroplasts and mitochondria.
Proteins with Hybrid Transit Peptides Are Imported into
Chloroplasts via the General Import Pathway
To investigate whether the import machineries used for transit
peptide-mediated preprotein import into chloroplasts are also
capable of importing preproteins in which the C-terminal domain
of the signal is from a mitochondrial presequence, we character-
ized the chloroplast import pathways used by the hybrid
sequence RbcS[1–12]/FA[13–77]. Members of the AtToc159
family (AtToc159/AtToc132/AtToc120) are major chloroplast
import receptors (Bauer et al., 2000; Ivanova et al., 2004; Kubis
et al., 2004; Bischof et al., 2011). In ppi2 plants, which lack
AtToc159 (Bauer et al., 2000), the chloroplast import efficiency
of RbcS[N79] is reduced by about 30% (Bauer et al., 2000; Lee
et al., 2009a, 2015) and precursors are subjected to ubiquitin-
dependent 26S proteasome-mediated degradation (Lee et al.,
2009b, 2016). We examined whether RbcS[1–12]/FA[13–77] is
imported into chloroplasts via the AtToc159 pathway. ppi2
protoplasts transformed with the construct encoding the RbcS
[1–12]/FA[13–77] GFP fusion protein alone or together with T7-
AtToc159GM were treated with or without MG132, a proteasome
inhibitor, and chloroplast import was analyzed by western blot
analysis with an anti-GFP antibody. The levels of unimported
precursors were higher, although the amount of imported/pro-
cessed forms (quantified using a blot with a short exposure) was
slightly lower in the absence of exogenous T7-AtToc159GM
(Figure 6A). Moreover, MG132 treatment further increased the
precursor levels in ppi2 protoplasts, as observed previously
(Lee et al., 2009b). Nevertheless, in ppi2 protoplasts, the
majority of RbcS[1–12]/FA[13–77] GFP fusion protein was still
detected as the processed form, as observed with the wild-type
RbcS transit peptide, which was likely due to the compensatory
Protein Import into Chloroplasts and Mitochondria Molecular Plant

Figure 5. Sequence Motifs in the C-Terminal Region of Transit Peptide Are Functionally Interchangeable for Protein Import into
Mitochondria.
(A) Sequences of RbcS[N79], FA[N77], and their hybrid and deletion mutants.
(legend continued on next page)

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Molecular Plant Protein Import into Chloroplasts and Mitochondria
Figure 6. RbcS[1–12]/FA[13–77] Uses the
General Import Pathway Involving AtToc159
and Hsp93-V.
(A) AtToc159-dependent import of RbcS[1–12]/FA
[13–77] into chloroplasts. The construct encoding
RbcS[1–12]/FA[13–77] fused with GFP was co-
transformed with the empty vector (R6) or T7-
AtToc159GM into ppi2 protoplasts. Eight hours
later, the protoplasts were treated with dimethyl
sulfoxide or MG132 and further incubated for 6 h.
Protoplast extracts were analyzed by western
blotting with anti-GFP and anti-T7 antibodies. For
quantification of the amount of Pro forms, a blot
with a shorter exposure time was used. This
experiment was repeated more than three times with similar results. Pre, precursor form; Pro, processed form. The large subunit (RbcL) of the Rubisco
complex stained with Coomassie brilliant blue (CBB) was used as a loading control.
(B) Hsp93-dependent import of RbcS[1–12]/FA[13–77] into chloroplasts. A construct encoding RbcS[1–12]/FA[13–77] fused with GFP was transformed
into hsp93-V protoplasts. For quantification of the amount of Pro forms, a blot with a shorter exposure time was used. This experiment was repeated more
than three times with similar results. Proteins were analyzed as in (A). Pre, precursor form; Pro, processed form. The large subunit (RbcL) of the Rubisco
complex stained with CBB was used as a loading control.

role of AtToc132/AtToc120 in the absence of AtToc159 (Inoue
et al., 2010; Bischof et al., 2011). Coexpression of T7-
AtToc159GM rescued the defective import of RbcS[1–12]/FA
[13–77] (Figure 6A). Altogether, these results indicate that RbcS
[1–12]/FA[13–77] is imported into chloroplasts via the general
pathway involving AtToc159/AtToc132/AtToc120. Furthermore,
these results collectively suggest that the C-terminal domain of
the mitochondrial presequence can support protein import into
chloroplasts via the general Toc complexes. We also examined
the involvement of stromal Hsp93, which is associated with the
Tic complex and is a well-known general chaperone involved in
a later step during chloroplast import (Lee et al., 2015, 2016,
2018; Flores-Perez et al., 2016; Huang et al., 2016). The amount
of precursor, but not processed forms (quantified using a blot
with a short exposure), was higher in protoplasts from hsp93-V
knockout plants than in the wild-type protoplasts (Figure 6B).
Moreover, when hsp93-V protoplasts were treated with MG132,
the levels of RbcS[1–12]/FA[13–77] precursor were further
increased, as was observed with the wild-type RbcS transit
peptide (Lee et al., 2016). These results indicate that, similar to
the wild-type RbcS transit peptide, RbcS[1–12]/FA[13–77] is
imported into chloroplasts via a pathway involving Hsp93-V. Alto-
gether, these results suggest that, similar to the transit peptide of
RbcS, RbcS[1–12]/FA[13–77], which contains the C-terminal
domain from the FA presequence, uses the general chloroplast
protein import pathway involving members of the AtToc159 family
and Hsp93-V.
Import Receptors such as AtToc159 Specify Chloroplast
Import when the Signal Sequence Can Deliver Clients to
Both the Chloroplast and Mitochondria
Import factors at the surfaces of chloroplasts and mitochondria
play a crucial role in the import of cargo by binding to incoming pre-
cursors associated with chaperones from the cytosol (Young et al.,
2003; Smith et al., 2004; Qbadou et al., 2006; Ponce-Rojas et al.,
2017). To test whether the import receptor AtToc159 is involved
in specificity determination, we examined protein subcellular
localization in ppi2 mutant protoplasts. First, ppi2 protoplasts
were transformed with the construct encoding the RbcS[1–12]/
FA[13–77] fusion protein and its localization was examined.
Similar to wild-type RbcS[N79], RbcS[1–12]/FA[13–77] was not
mistargeted to mitochondria in ppi2 protoplasts (Supplemental
Figure 5). It is possible that the contribution of AtToc159 to
import specificity cannot be determined using sequences
specific for the chloroplast. Thus, we examined import in ppi2
protoplasts using a targeting signal with specificity for both
chloroplasts and mitochondria. Many targeting signals that
deliver clients to both the chloroplast and mitochondria have
been identified in previous studies (Chew et al., 2003; Rudhe
et al., 2004; Berglund et al., 2009; Baudisch and Klosgen, 2012;
Carrie and Small, 2013; Xu et al., 2013; Daras et al., 2014; Ye
et al., 2015). For our purposes, we generated an artificial
construct RbcS[1–12]/FA[1–77], which contains both the
chloroplast and mitochondrial specificity domains we defined in
this study (Figure 7A). We reasoned that the presence of both a
transit peptide N-terminal specificity domain and a presequence
N-terminal specificity domain in a signal sequence may confer
specificity to both organelles. RbcS[1–12]/FA[1–77] fused to
GFP supported protein import into both chloroplasts and
mitochondria in wild-type protoplasts (Figure 7B). However,
the signal intensity was stronger at chloroplasts than at
mitochondria, suggesting that RbcS[1–12]/FA[1–77] is slightly
biased toward chloroplast import over mitochondrial import.
To confirm the dual import of RbcS[1–12]/FA[1–77] at the
biochemical level, we prepared chloroplast and mitochondrial
fractions, and analyzed protein extracts from chloroplast and
mitochondrial fractions together with total protoplast extracts
by western blotting with an anti-GFP antibody (Figure 7C).
Organellar fractions were confirmed using anti-LHCB4 and

(B) Localization of reporter proteins. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll auto-
fluorescence, respectively. Each pattern was quantified by counting the number of 100 transformed protoplasts with the pattern. Scale bars, 20 mm.
(C) Western blot analysis of the reporter proteins. Protoplast extracts were analyzed by western blotting with an anti-GFP antibody. The import efficiency
was defined as the amount of the processed form relative to the total amount of the expressed protein. Data represent the mean ± SD (n = 3); Pre,
precursor form; Pro, processed form.

960 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria Molecular Plant

Figure 7. Organellar Receptors such as Toc159 Contribute to Chloroplast Targeting Specificity via a Dual-Targeting Hybrid
Sequence.
(A) Sequences of RbcS[N79], FA[N77], and their mutants.
(B) Localization of RbcS[1–12]/FA[1–77]. Green, red, and blue signals represent GFP, mitochondria stained with MitoTracker red, and chlorophyll au-
tofluorescence, respectively. Scale bars, 20 mm.
(C) Isolation of chloroplasts and mitochondria from transformed protoplasts. Chloroplasts and mitochondria were analyzed by western blotting. LHCB4
and IDH were used as controls for chloroplast and mitochondrial proteins, respectively. T, total fraction; CH, chloroplast fraction; MT, mitochondrial
fraction; Pro, processed form. An asterisk indicates the protein band present in mitochondria.
(D) Western blot analysis of reporter proteins. Protoplast extracts were analyzed by western blotting with an anti-GFP antibody. The mitochondrial import
efficiency was defined as the amount of the mitochondrial-specific processed form, indicated by an asterisk, relative to the total amount of protein
imported into chloroplasts and mitochondria. Data represent the mean ± SD (n = 3). Pre, precursor form; Pro, processed form.
(E) Hsp93, a stromal protein, is not involved in determining the targeting specificity of chloroplast proteins. RbcS[1–12]/FA[1–77] fused with GFP was
transformed into wild-type and hsp93-V protoplasts. Twelve hours later, the protoplasts were treated with dimethyl sulfoxide or MG132 and further
incubated for 6 h. Protoplast extracts were analyzed by western blotting with an anti-GFP antibody. Pre, precursor form; Pro, processed form.

anti-IDH (isocitrate dehydrogenase) antibodies that are specific to
chloroplasts and mitochondria, respectively. Protoplast extracts
contained four processed forms recognized by the anti-GFP
antibody: the highest molecular weight band and the two lowest
molecular weight bands were specific to the chloroplast fraction,
and the band with the second highest molecular weight (indicated
by an asterisk in Figure 7C) was specific to the mitochondria
fraction, confirming that RbcS[1–12]/FA[1–77] supports dual
import into chloroplasts and mitochondria. The size of the
mitochondrial-specific band was the same as that of the mature
form of the FA[N77] GFP fusion protein (Figure 7D), indicating
that the processing site at FA[N77] is used in the processing of
proteins delivered into mitochondria by RbcS[1–12]/FA[1–77].
The amount of the mitochondria-specific processed form was
much lower than that of the chloroplast-specific processed form
(Figure 7C and 7D), consistent with the signal intensities in the
GFP images (Figure 7B). In wild-type protoplasts, the fusion pro-
teins encoded by the reciprocal constructs FA[1–12]/RbcS[1–77]
Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 961
Molecular Plant
and FA[1–20]/RbcS[1–79] localized to the cytosol and exclusively
to mitochondria, respectively (Supplemental Figure 6A and 6B).
This confirmed our results presented above that multiple R
residues play a role in evading protein import into chloroplasts
and that both the multiple-arginine residue motif and the Tom20-
binding motif are required for specifying import into mitochondria.
Given these findings, we examined the behavior of RbcS[1–12]/FA
[1–77] in ppi2 protoplasts. Western blotting analysis of protein ex-
tracts from transformed ppi2 protoplasts showed that the levels of
the mitochondrial-specific processed form (indicated by an
asterisk in Figure 7D) were greatly increased with respect to
those in wild-type protoplasts, with a concomitant decrease in
the chloroplast-specific processed forms (Figure 7D). It should
be also noted that, in ppi2 protoplasts, the cytosolic precursor
form of RbcS[1–12]/FA[1–77] did not accumulate (Figure 7D),
whereas precursors of RbcS-nt:GFP and Cab-nt:GFP were
clearly detected (Lee et al., 2009a, 2015). These results indicate
that, in the absence of AtToc159, some RbcS[1–12]/FA[1–77]
fusion proteins, which were destined for the chloroplast, were
instead targeted to mitochondria thanks to the N-terminal
specificity domain of FA[N77].
Next, we examined whether Hsp93-V contributes to specificity
determination. Hsp93 is localized in the stroma and thus func-
tions at later steps during chloroplast import (Huang et al.,
2016; Lee et al., 2018). The construct encoding the RbcS[1–12]/
FA[1–77] fusion protein was transformed into hsp93-V proto-
plasts, and protein extracts were analyzed by western blotting
with an anti-GFP antibody. The levels of chloroplast-specific pro-
cessed forms were lower in hsp93-V protoplasts than in wild-type
protoplasts and the levels of the mitochondria-specific pro-
cessed form remained unchanged, indicating that mutation of
Hsp93-V does not lead to an increase in delivery to mitochondria
(Figure 7E). Instead, mutation of Hsp93-V caused an increase in
the precursor levels of the RbcS[1–12]/FA[1–77] fusion protein,
suggesting that in the absence of Hsp93-V a portion of the fusion
protein accumulates in the cytosol. Together these results sug-
gest that the import receptors at the surface of the chloroplast,
but not protein factors functioning at a later step during protein
import into chloroplasts, contribute to the prevention of import
into mitochondria in vivo.
Reporter Proteins Expressed Using the RbcS Promoter
Show the Same Localization Patterns as Those
Expressed Using the Strong CaMV 35S Promoter
Expression of constructs using the very strong CaMV (cauliflower
mosaic virus) 35S promoter in protoplasts could result in misloc-
alization of expressed proteins (Dixit et al., 2006; Tanz et al., 2013;
Ling et al., 2017). We therefore examined whether this affected the
localization of reporter proteins used in this study. We substituted
the CaMV 35S promoter with the promoter of Arabidopsis
RbcS for six representative constructs: RbcS[N79], FA[N77],
and four hybrid constructs (Supplemental Figure 7). The
localization patterns of reporter proteins expressed using these
six RbcS promoter-driven constructs were the same as those of
reporter proteins expressed using the corresponding CaMV 35S
promoter-driven constructs (Supplemental Figure 7A). Analysis
of protein extracts by western blotting confirmed that the
expression levels of the constructs driven by the RbcS promoter
962 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria
were much lower than those of the constructs driven by the
CaMV 35S promoter (Supplemental Figure 7B). These results
confirmed that the expression levels did not affect the
localization pattern of reporter proteins used in this study.
DISCUSSION
In this study, we provide evidence that presequences for mito-
chondrial import and transit peptides for chloroplast import are
composed of two functional domains. The N-terminal domain
contains sequence motifs for import specificity, whereas the
C-terminal domain contains sequence motifs that are cross-func-
tional, supporting protein translocation across the envelope
membranes of both chloroplasts and mitochondria. The speci-
ficity is determined by two sequence motifs, the multiple-arginine
motif and the hydrophobic sequence motif, identified in the N-ter-
minal regions of presequences. The presence of these two motifs
was sufficient to confer mitochondrial specificity to the C-termi-
nal regions of both transit peptides and presequences. By
contrast, the absence of the multiple-arginine motif in the N-ter-
minal domains of presequences was sufficient for conferring
chloroplast specificity. Consistent with this finding, transit pep-
tides generally lack a multiple-arginine motif in the N-terminal
region (Bhushan et al., 2006; Garg and Gould, 2016). Thus,
the presence or absence of a multiple-arginine motif and hydro-
phobic sequence motif functions as an on/off switch for mito-
chondria/chloroplast targeting in the signal sequences that
support protein translocation across the envelope membranes
of chloroplasts and mitochondria. Interestingly, it appears that
SPP (stromal processing peptidase) in chloroplasts and MPP
(mitochondrial processing peptidase) in mitochondria faithfully
recognize and cleave the mitochondrial presequence and chloro-
plast transit peptide, respectively. Considering the pivotal roles of
SPP in preprotein import and chloroplast biogenesis (Zhong
et al., 2003; Trosch and Jarvis, 2011), the cleavage of hybrid
signal sequences in which the cleavage site came from the
mitochondrial presequence might also have contributed to
efficient import into chloroplasts.
The results from previous studies showed that R residues are more
prevalent in the N-terminal regions of presequences than in those
of transit peptides (Bhushan et al., 2006; Ge et al., 2014) and
contribute to efficient protein import into mitochondria (Duby
et al., 2001). Here, we defined these R residues as one of two
motifs that function as the mitochondrial specificity-determining
factor. However, this new role may not be incompatible with their
role in import efficiency. The R residues cannot be replaced by Ks,
raising the possibility of specific interactions with molecular ma-
chinery. The specific requirement of Rs in the multiple-arginine
motif is intriguing and not fully understood. Similarly, TAT (twin-
arginine translocation) signal sequences in bacteria and chloro-
plasts also have an invariable R requirement (Alami et al., 2002;
Cline, 2015). The insertion of multiple-arginine motifs into transit
peptides prevented transit peptide-mediated protein import into
chloroplasts and caused precursors to accumulate in the cytosol,
raising the possibility that a certain unidentified factor(s) may
function early in the protein import process in the cytosol before
or during protein binding to the surfaces of organelles.
The other motif that played a crucial role in mitochondrial speci-
ficity was the hydrophobic sequence motif, which conforms to
Protein Import into Chloroplasts and Mitochondria
the consensus Tom20-binding motif and was also identified in
presequences of animal and yeast mitochondrial proteins
(Fukasawa et al., 2015). In the Tom20-binding motif, the hydro-
phobic residues are involved in binding to Tom20 (Abe et al.,
2000; Yamano et al., 2008). However, regardless of sequence
conservation, it is not clear whether the presequences of plant
mitochondrial proteins also contain genuine Tom20-binding mo-
tifs, because plant Tom20 differs from animal and yeast Tom20 in
several aspects (Macasev et al., 2000) and, moreover, the import
of FA[N77] into mitochondria is hardly affected in tom20-2/
tom20-3/tom20-4 protoplasts (Lee et al., 2012). Thus, given its
importance in determining the specificity of mitochondrial
import, it is possible that the hydrophobic sequence motif in FA
[N77] plays another critical role during protein import into
mitochondria in addition to binding to Tom20.
The determination of chloroplast specificity seems simpler than
that of mitochondria specificity, because the absence of the
multiple-arginine motif in presequences was sufficient for chloro-
plast import. In fact, the N-terminal regions of transit peptides
should have moderate hydrophobicity for efficient chloroplast
import (Lee et al., 2006, 2008) and play a role early during
protein import into chloroplasts. The N-terminal region was also
suggested to be involved in recognition by stromal Hsp70 and
efficient translocation (Chotewutmontri and Bruce, 2015). Thus,
the role of the transit peptide N-terminal region in determining
chloroplast specificity is consistent with its role in the early
steps of protein import into chloroplasts (Lee et al., 2006, 2008).
However, hydroxylated amino acids such as S that are regarded
as the sequence determinant for chloroplast import (Ge et al.,
2014; Garg and Gould, 2016) did not determine import into
chloroplasts (Lee et al., 2006). Moreover, the absence of S
residues in the transit peptide/presequence hybrid sequences
did not inhibit chloroplast import (Supplemental Figure 4).
One important finding here is that the C-terminal domains of transit
peptides and presequences are functionally interchangeable,
supporting translocation into both organelles. The sequence mo-
tifs of transit peptides critical for protein import into chloroplasts
were also crucial for the import of presequence/transit peptide
hybrid sequences into mitochondria. This is consistent with
previous findings that transit peptides deliver proteins into
mitochondria in vitro and in cells without plastids in vivo (Hurt
et al., 1986a, 1986b; Cleary et al., 2002), experimental
conditions under which competition between the two organelles
is not present and therefore specificity might not be critical.
This raises many new questions, such as how sequence motifs
in transit peptides are recognized by the import machinery of
mitochondria and vice versa. We showed that presequences
without the multiple-arginine motif deliver proteins into chloro-
plasts via the general import pathway involving members of the
AtToc159 family and Hsp93. One possible explanation is that the
molecular machineries for protein translocation across the enve-
lope membranes of both chloroplasts and mitochondria are highly
flexible and recognize the motifs in transit peptides and prese-
quences. Consistent with this idea, the primary structures of
transit peptide motifs are highly variable (Lee et al., 2008, 2015),
and this is also true for presequences (Lee et al., 2012).
A more fundamental question is how transit peptides and prese-
quences evolved to have this relationship. We propose two
Molecular Plant
possible explanations. One is that, because both organelles
evolved from Gram-negative bacteria (Gupta, 2000), the original
signal sequences responsible for crossing bacterial envelope
membranes evolved by maintaining sequence motifs with
similar characteristics. The other possibility is that the signal
sequence of the first organelle that evolved was recruited for
the second organelle and, subsequently, the N-terminal
domains evolved to acquire the multiple-arginine motif and the
hydrophobic sequence motif, or lost them, leading to specific
import into mitochondria or chloroplasts. One good example of
how adding a sequence motif confers targeting specificity is
the targeting of proteins to the lysosome or vacuole through en-
domembrane compartments; the secretion of proteins from the
trans-Golgi network is the default pathway, and the addition of
a new sorting motif to secretory proteins diverts them to the lyso-
some/vacuole (Wang et al., 2014).
With this in mind, which molecular machinery recognizes the
specificity-determining motifs? Cytosolic proteins such as
Hsp70, Hsp90, and 14-3-3 contribute to the import of proteins
into chloroplasts (May and Soll, 2000; Qbadou et al., 2006). In
addition, mitochondrial import stimulating factor and nascent
polypeptide-associated complex, as well as Hsp70 and Hsp90,
contribute to the import of proteins from the cytosol into mito-
chondria (Young et al., 2003; Lee et al., 2013). However, it is
not known whether these factors are involved in determining
the specificity for chloroplasts and mitochondria. To address
this question, we used an artificial signal sequence with dual
specificity. In fact there are many examples of single species
of proteins that are imported into both chloroplasts and
mitochondria (Rudhe et al., 2004; Berglund et al., 2009;
Baudisch and Klosgen, 2012; Carrie and Small, 2013; Xu et al.,
2013; Daras et al., 2014; Ye et al., 2015; Garg and Gould,
2016). These proteins may have no or weaker import specificity
for chloroplasts or mitochondria. We showed here that when
RbcS[1–12]/FA[1–77], the artificial signal sequence with dual
specificity, was used as the signal sequence for import in ppi2
protoplasts, the amount of proteins imported into mitochondria
was greatly increased with a concomitant decrease in the
amount of proteins imported into chloroplasts. However, in
hsp93-V protoplasts, despite the fact that the amount of proteins
imported into chloroplasts decreased, the amount of reporter
proteins imported into mitochondria did not increase, but instead
reporter proteins accumulated as precursor forms. Thus, the two
chloroplast import factors have a differential effect on protein
import into mitochondria; the absence of AtToc159, but not
Hsp93-V, leads to a change in the destination of reporter
proteins with dual import specificity from chloroplasts to mito-
chondria without accumulation of precursors in the cytosol.
Moreover, these results raise an intriguing possibility that, for
the dual-specificity proteins, protein import into chloroplasts
involves a commitment step, most likely involving AtToc159.
However, it is not known whether a commitment step is also
involved in mitochondrial import of dual-specificity proteins,
and whether the import of proteins with transit peptides with
chloroplast specificity or presequences with mitochondrial
specificity also involves a similar commitment step. Further
studies are necessary to identify the factor(s) for presequence-
or transit peptide-mediated import and enable a deeper under-
standing of how proteins are specifically imported into chloro-
plasts and mitochondria.
Molecular Plant 12, 951–966, July 2019 ª The Author 2019. 963
Molecular Plant
METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana (Colombia ecotype) was grown on Gamborg B5
plates (G0210.0050; Duchefa, Haarlem, the Netherlands) under 40% rela-
tive humidity, 22 C, and a 16-h light/8-h dark cycle in a growth chamber.
Protoplasts were prepared from leaf tissues of 2- to 3-week-old plants.
Plasmid DNA Construction and PCR-Based Mutagenesis
DNA fragments encoding protein import signals were PCR-amplified from
Arabidopsis genomic DNA or cDNA using gene-specific primers. For tran-
sient expression analysis in Arabidopsis protoplasts, PCR products were
digested with restriction endonucleases and subcloned into pUC-GFP
containing the CaMV 35S promoter, GFP, and Nos terminator (Jin et al.,
2001). Site-specific amino acid substitution mutants were generated by
performing PCR-mediated mutagenesis. Each pair of complementary up-
per and lower primers were custom-designed with the central regions
mutated to replace the encoded amino acid residues (Supplemental
Table 1). To isolate the RbcS promoter, we amplified the 3-kb region
upstream of the RbcS protein-coding region from Arabidopsis genomic
DNA by PCR using the primers PstI-RbcS promoter-F and SalI-RbcS
promoter-R. After verifying the sequence of the RbcS promoter, the
RbcS promoter was digested with PstI and SalI, and ligated into reporter
plasmids digested with PstI and SalI.
Transient Expression in Protoplasts
All DNA plasmids were amplified in Escherichia coli (JM109 strain) and
purified using the Qiagen plasmid purification kit (Basel) according to
the manufacturer’s protocol. Purified plasmid DNA was introduced
into protoplasts isolated from leaf tissues by polyethylene glycol PEG-
mediated transformation (Jin et al., 2001). To examine mitochondria
under a fluorescence microscope, we incubated protoplasts in W5
medium containing 100 nM MitoTracker for 10 min at room temperature
in the dark. Stained protoplasts were washed twice with fresh W5
medium and then observed under a fluorescence microscope after
incubating in the dark at 22 C for 1 h.
Isolation of Chloroplast and Mitochondrial Fractions from
Protoplasts
Transformation was performed using 1.5 3 106 cells incubated with 15 mg
of plasmid DNA in a 15-ml tube. Five identical samples were prepared for
each construct at each time. After incubation for 12 h, total protein
extracts were prepared from protoplasts in one sample. Protoplasts
in the remaining samples were combined and resuspended in 5 ml of
13 HMS buffer (50 mM HEPES, 3 mM MgSO4, and 300 mM
sorbitol [pH 8.0]) supplemented with 25 mM EDTA, 10 mM EGTA, and
13 protease inhibitor cocktail (Roche). Resuspended protoplasts were
filtered through two layers of 11-mm nylon net filter (Millipore, Billerica,
MA, USA) three times for homogenization, followed by centrifugation at
2000 g for 6 min. The pellet (P1) and supernatant (S1) fractions were
used to isolate the chloroplast and mitochondrial fractions, respectively,
as described below.
The P1 fraction was resuspended in 2.5 ml of 13 HMS buffer supple-
mented with 7 mM MgCl2, 10 mM KCl, 2 mM EDTA, and 13 protease in-
hibitor cocktail, and was loaded onto 5 ml of 40% Percoll (Sigma-Aldrich,
St. Louis, MO, USA) in HMS buffer and centrifuged at 6000 g at 4 C for 1 h
(SW41 swing rotor; Beckman Coulter, Brea, CA, USA). The upper fraction
was removed carefully with a pipette and the loose pellet was collected
and diluted with 1 ml of HMS buffer in an Eppendorf tube and centrifuged
at 7500 g at 4 C for 10 min. After centrifugation, the supernatant was
discarded and the pellet at the bottom of the tube was saved as the chlo-
roplast fraction. The S1 fraction was centrifuged at 5000 g for 7 min to
completely remove the residual debris. Thereafter, the supernatant frac-
tion was further centrifuged at 16 000 g for 15 min and the pellet was saved
as the mitochondrial fraction.
964 Molecular Plant 12, 951–966, July 2019 ª The Author 2019.
Protein Import into Chloroplasts and Mitochondria
Immunoblotting
Total, chloroplast, and mitochondrial fractions were resuspended in
300 ml of buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA,
1% Triton X-100, and 13 protease inhibitor cocktail), followed by brief
sonication. Sonicated samples were subjected to centrifugation at
3000 g for 10 min to remove debris. Supernatants were mixed with pro-
tein loading buffer and boiled for 5 min. After separation by SDS–PAGE,
proteins were analyzed by western blotting. The intensities of bands on
western blots were quantified using the Multi Gauge program (Fujifilm) of
LAS3000.
Antibodies
Western blot analysis was performed using anti-GFP (Clontech, 1:5000
dilution), anti-Lhcb4 (Agrisera, AS04 045, 1:2500 dilution), and anti-IDH
(Agrisera, AS06 203A, 1:2000 dilution) antibodies.
Microscopic Analysis
Images of protoplasts with GFP, red fluorescent protein (RFP), and
MitoTracker red were obtained using a Zeiss Axioplan fluorescence micro-
scope and a cooled CCD camera (Zeiss). The filter settings were XF116
(exciter, 474AF20; dichroic, 500DRLP; emitter, 510AF23), XF33E (exciter,
535DF35; dichroic, 570DRLP; emitter, 605DF50), and XF137 (exciter,
540AF30; dichroic, 570DRLP; emitter, 585ALP) (Omega) for GFP, RFP,
and autofluorescence of chlorophyll, respectively.
SUPPLEMENTAL INFORMATION
Supplemental Information is available at Molecular Plant Online.
FUNDING
This work was supported by the Cooperative Research Program for Agricul-
ture Science and Technology Development (project no. PJ010953012019),
Rural Development Administration, Republic of Korea, and the National
Research Foundation of Korea (NRF) grant funded by the Korea Govern-
ment, Ministry of Science and ICT (project no. 2016R1E1A1A02922014).
D.W.L. was supported by grant no. NRF-2017R1C1B1006784 from the Na-
tional Research Foundation, the Ministry of Science and ICT, Korea.
AUTHOR CONTRIBUTIONS
D.W.L., S.L., and I.H. conceived this project. D.W.L., S.L., J.L., S.W., and
M.A.R. carried out experiments in protoplasts. D.W.L., S.L., A.V., and I.H.
interpreted the data and participated in discussion. D.W.L., A.V., and I.H.
wrote the manuscript.
ACKNOWLEDGMENTS
No conflict of interest declared.
Received: February 19, 2019
Revised: February 19, 2019
Accepted: March 10, 2019
Published: March 16, 2019
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