Molecular chaperone involvement in chloroplast protein import
Chloroplasts are organelles of endosymbiotic origin that perform essential functions in plants. They contain about 3000 different proteins, the vast majority of which are nucleus-encoded, synthesized in precursor form in the cytosol, and transported into the chloroplasts post-translationally. These preproteins are generally imported via envelope complexes termed TOC and TIC (Translocon at the Outer/Inner envelope membrane of Chloroplasts). They must navigate different cellular and organellar compartments (e.g., the cytosol, the outer and inner envelope membranes, the intermembrane space, and the stroma) before arriving at their final destination. It is generally considered that preproteins are imported in a largely unfolded state, and the whole process is energy-dependent. Several chaperones and cochaperones have been found to mediate different stages of chloroplast import, in similar fashion to chaperone involvement in mitochondrial import. Cytosolic factors such as Hsp90, Hsp70 and 14-3-3 may assist preproteins to reach the TOC complex at the chloroplast surface, preventing their aggregation or degradation. Chaperone involvement in the intermembrane space has also been proposed, but remains uncertain. Preprotein translocation is completed at the trans side of the inner membrane by ATP-driven motor complexes. A stromal Hsp100-type chaperone, Hsp93, cooperates with Tic110 and Tic40 in one such motor complex, while stromal Hsp70 is proposed to act in a second, parallel complex. Upon arrival in the stroma, chaperones (e.g., Hsp70, Cpn60, cpSRP43) also contribute to the folding, assembly or onward intraorganellar guidance of the proteins. In this review, we focus on chaperone involvement during preprotein translocation at the chloroplast envelope. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.
Molecular chaperone involvement in chloroplast protein import
Úrsula Flores-Pérez, Paul Jarvis
Department of Biology, University of Leicester, Leicester LE1 7RH, UK
Received 18 January 2012
Received in revised form 16 March 2012
Accepted 31 March 2012
Available online 12 April 2012
Chloroplasts are organelles of endosymbiotic origin that perform essential functions in plants. They contain about
3000 different proteins, the vast majority of which are nucleus-encoded, synthesized in precursor form in the
cytosol, and transported into the chloroplasts post-translationally. These preproteins are generally imported via
envelope complexes termed TOC and TIC (Translocon at the Outer/Inner envelope membrane of Chloroplasts).
They must navigate different cellular and organellar compartments (e.g., the cytosol, the outer and inner envelope
membranes, the intermembrane space, and the stroma) before arriving at their ﬁnal destination. It is generally
considered that preproteins are imported in a largely unfolded state, and the whole process is energy-
dependent. Several chaperones and cochaperones have been found to mediate different stages of chloroplast im-
port, in similar fashion to chaperone involvement in mitochondrial import. Cytosolic factors such as Hsp90, Hsp70
and 14-3-3 may assist preproteins to reach the TOC complex at the chloroplast surface, preventing their aggrega-
tion or degradation. Chaperone involvement in the intermembrane space has also been proposed, but remains un-
certain. Pr eprotein translocation is completed at thetranssideoftheinnermembranebyATP-drivenmotor
complexes. A stromal Hsp100-type chaperone, Hsp93, cooperates with Tic110 and Tic40 in one such motor com-
plex, while stromal Hsp70 is proposed to act in a second, parallel complex. Upon arrival in the stroma, chaperones
(e.g., Hsp70, Cpn60, cpSRP43) also contribute to the folding, assembly or onward intraorganellar guidance of the
proteins. In this review, we focus on chaperone involvement during preprotein translocation at the chloroplast
envelope. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and
© 2012 Elsevier B.V. All rights reserved.
Plastids are a diverse group of organelles found ubiquitously in plant
cells [1,2]. Chloroplasts, the most prominent members of the plastid
family, contain the green pigment chlorophyll and are responsible
for the reactions of photosynthesis, as well as sundry important
biosynthetic functions. Plastids entered the eukaryotic lineage through
endosymbiosis, and have evolved from an ancient photosynthetic pro-
karyote similar to extant cyanobacteria [3,4]. While plastids retain a
functional endogenous genetic system, the plastid genome is greatly re-
duced and typically encodes just ~100 different proteins [5,6].Most
(>90%) of the ~3000 different proteins that are needed to develop a
fully-functional chloroplast are encoded in the nucleus and synthesized
on free cytosolic ribosomes [7,8].
Typically, nucleus-encoded chloroplast proteins are synthesized in
precursor form, each one having an amino-terminal targeting signal
called a transit peptide. These precursors, or preproteins, are trans-
ported into the organelle post-translationally, in an energy-consuming
process termed chloroplast protein import. Import is mediated by
hetero-oligomeric protein complexes in the outer and inner envelope
membranes that surround each plastid; these complexes are termed,
respectively, TOC and TIC (Translocon at the Outer/Inner envelope
membrane of Chloroplasts) [9–12]. Once a preprotein arrives in the
chloroplast interior (the stroma), the transit peptide is proteolytically
removed by the stromal processing peptidase (SPP), allowing the pro-
tein to assume its functional conformation or engage one of several in-
ternal sorting pathways [12–14].
Chloroplast import bears considerable similarity to mitochondrial
protein import, which is mediated by translocon complexes termed
TOM and TIM (Translocase of the Outer/Inner Mitochondrial mem-
brane) [15–17]. In both cases, preproteins are threaded through the
membranes in an unfolded state, amino-terminus ﬁrst. Both import sys-
tems comprise multiple preprotein receptors that project large domains
into the cytosol, both possess channel components in the outer and
inner membranes, and both are powered, to a greater or less extent,
by ATP hydrolysis (see below). However, the principal components of
the TOC/TIC and TOM/TIM systems are not closely related. The core
components of the TOC complex are Toc159, Toc34 and Toc75 (the
numbers indicate size in kD). The ﬁrst two are receptor components
that mediate transit peptide recognition via their cytosolically-
oriented GTPasedomains, while Toc75 forms a β-barrel channel for pre-
protein conductance. Electrophysiological analysis of the Toc75 channel
indicated a narrow pore ~14 Å in diameter, ﬂanked on either side by
Biochimica et Biophysica Acta 1833 (2013) 332–340
This article is part of a Special Issue entitled: Protein Import and Quality Control in
Mitochondria and Plastids.
⁎ Corresponding author. Tel.: +44 116 223 1296; fax: +44 116 252 3330.
E-mail address: firstname.lastname@example.org (P. Jarvis).
0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
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two wider vestibules . Such a pore would be wide enough only to
accept largely unfolded preprotein clients. However, the successful im-
port of a 6.5 kD (23 Å in diameter) tightly-folded, internally-crosslinked
protein domain  suggests either that the pore is somewhat larger
than the aforementioned estimate, or that the channel has a degree of
ﬂexibility. Critical components of the TIC apparatus include Tic110
and Tic40, the roles of which will be discussed later.
As already mentioned, protein import into chloroplasts is an energy-
dependent process. According to energy requirements determined in
vitro, three distinguishable stages of import have been deﬁned. Firstly,
the binding of preproteins with the TOC receptors is a reversible and
energy-independent step called energy independent binding .Sub-
sequently, initial translocation leads to the formation of an early import
intermediate. This irreversible second step requires GTP (for the recep-
tors) and a low concentration of ATP (≤ 100 μM) in the intermembrane
space [21–23]. Finally, preproteins are completely translocated into the
stroma at the expense of high concentrations of ATP (~1 mM) in the
stroma . The latter energy requirement is attributed to stromal
The presumed need for preproteins to be in a largely unfolded
state during import is dictated by physical characteristics of the im-
port machinery, as discussed earlier, and this in turn necessitates
the involvement of molecular chaperones — a diverse group of factors
that facilitate folding processes and conformational changes in other
proteins . In fact, a variety of different chaperones are required
during chloroplast protein import, and these are employed at differ-
ent stages in the process: in the cytosol following ribosomal release,
to prevent misfolding or aggregation of preproteins and to guide
them to the chloroplast surface; during the import process itself, to
maintain translocation competence of the preproteins and to drive
transport at the expense of ATP hydrolysis; and, following the com-
pletion of import, to assist with folding, assembly or onward trans-
port to internal destinations. In this review, we will touch on all of
these aspects, focusing in particular on chaperone involvement dur-
ing envelope translocation.
2. Chaperone involvement in the cytosol
Notwithstanding recent evidence that some chloroplast proteins
are translated near the border of chloroplasts in the green alga,
Chlamydomonas reinhardtii, suggesting mRNA transport as a compo-
nent of the overall targeting scheme , chloroplast protein import
is generally considered to be a post-translational process (in contrast
with signal recognition particle [SRP]-dependent translocation into
the endoplasmic reticulum, for example, which is co-translational).
Thus, cytosolic factors are required to facilitate the passage of prepro-
teins from the ribosome to the chloroplast surface, and to prevent
their aggregation or premature degradation [28–30]. The transit pep-
tide, as the ﬁrst part of the preprotein to emerge from the ribosome,
plays a critical role in the interactions with such components.
Transit peptides are to a large extent responsible for the targeting
properties of chloroplast preproteins. Indeed, they are very effective at
mediating the import of heterologous passenger proteins into chloro-
plasts [31,32]. And yet, despite the apparent speciﬁcity of the chloro-
plast import process, transit peptides are remarkably diverse in both
length and sequence [33,34]. They vary from 20 to >100 residues, are
rich in hydroxylated residues, and are deﬁcient in acidic residues giving
them a net positive charge. In this respect, transit peptides are rather
similar to the functionally-analogous presequences of mitochondrial
preproteins (raising puzzling questions about how organellar targeting
ﬁcity is achieved in plants [35,36]). While mitochondrial prese-
quences share a characteristic secondary structure (they form amphi-
pathic helices that are important for interaction with receptors of the
TOM machinery ), chloroplast transit peptides do not seem to pos-
sess this property [38,39]. Instead, it has been hypothesized that they
speciﬁcally evolved to have “perfect random coil” properties, perhaps
to aid interaction with cytosolic factors .
Hsp70 (Heat-shock protein, 70 kD) is one of the chaperones thought
to facilitate the cytosolic phase of chloroplast protein transport. Most
chloroplast transit peptides are predicted to possess at least one
Hsp70 binding site, while direct interactions between Hsp70s and tran-
sit peptides have beendemonstrated [41–44]. However, the importance
of such Hsp70 binding for protein import remains uncertain, as it is not
essential for protein translocation in vitro [45,46]. Moreover, a recent
study showed that cytosolic Hsp70 associates withaccumulated precur-
sors that are targeted for degradation via the ubiquitin proteasome sys-
tem , indicating that Hsp70 binding does not necessarily serve to
escort preproteins to the chloroplast surface. Nevertheless, Hsp70
does appear to play a role in protein import in cooperation with other
cytosolic factors, such as 14-3-3 (see below; Fig. 1). It is conceivable
that different isoforms of Hsp70 are responsible for these different
The 14-3-3 protein family includes regulatory molecules and
chaperones that speciﬁcally bind to phosphorylated proteins in order
to mediate various signal transduction processes, as well as protein
translocation . Many chloroplast transit peptides contain a 14-3-3-
binding phosphopeptide motif [28,48]. It was reported that 14-3-3
can form a “guidance complex” together with Hsp70 and preproteins,
and that this signiﬁcantly increases in vitro import efﬁciency for certain,
phosphorylatable preproteins . The 14-3-3-containing guidance
complex was also hypothesized to play a role in determining the
speciﬁcity of targeting to chloroplasts versus mitochondria in plants,
as 14-3-3 cannot bind plant mitochondrial preproteins .However,
mutation of the putative 14-3-3-binding site in transit peptides did
not affect import efﬁciency or ﬁdelity in vivo [49,50], indicating that
the 14-3-3 guidance complex system is dispensable. It is possible that
this mechanism is important only under certain conditions; it was re-
cently reported that the loss of a kinase thought to be responsible for
transit peptide phosphorylation results in an inefﬁcient de-etiolation
Differentiating between two distinct, endosymbiotically-derived or-
ganelles (i.e., chloroplasts and mitochondria) is a unique problem faced
by protein transport systems in plant cells. Related to this issue, per-
haps, is the fact that the protein import receptors in plant mitochondria
are signiﬁcantly different from those in yeast or animal mitochondria, as
well as from those in chloroplasts [17,36]. In spite of these receptor dif-
ferences, some chloroplast preproteins can be efﬁciently imported into
plant mitochondria in vitro, butnot in vivo . This implies that special
mechanisms are employed to achieve import speciﬁcity in vivo, and
that components of such mechanisms are absent or inactive in vitro.
Aside from the 14-3-3 guidance hypothesis discussed above, one strat-
egy that might contribute to targeting speciﬁcity is mRNA transport to-
wards the targetdestination, such that preproteins are produced only at
the periphery of the correct organelle [27,53,54]. However, the general
signiﬁcance of mRNA targeting in plants remains to be seen.
In mitochondrial protein import in animal cells, Hsp90 is an addi-
tional chaperone involved in cytosolic guidance, directing some prepro-
teins to the Tom70 receptor . Similarly, Hsp90 has also been
implicated in the delivery of certain preproteins to chloroplasts as part
of a second guidance complex, which was recently reported to also in-
volve the cochaperone Hop (Hsp70/Hsp90-organizing protein) and
the immunophilin FKBP73 [56–58]. There are two important differ-
ences between this guidance complex and the one discussed earlier:
ﬁrstly, Hsp90 binds to preproteins that are not necessarily phosphory-
lated; secondly, unlike the 14-3-3 complex which carries preproteins
directly to the Toc34 receptor, Hsp90 employs Toc64 (see below) as
an initial docking site before preproteins are passed on to Toc34 
(Fig. 1). However, preproteins proposed to follow the Hsp90–Toc64
pathway were found to be imported with normal efﬁciency into chloro-
plasts that lack Toc64 protein [59,60], indicating that this putative tar-
geting mechanism is also not essential. It is conceivable that such
333Ú. Flores-Pérez, P. Jarvis / Biochimica et Biophysica Acta 1833 (2013) 332–340
guidance systems are only important under speciﬁc conditions that
have so far eluded analysis [49,59,60], or that a certain amount of re-
dundancy between these (and perhaps other) guidance systems exists
such that loss of one does not have an appreciable effect.
The Toc64 protein was identiﬁed by its co-puriﬁcation with the
TOC complex after chemical cross-linking . It is described as a pe-
ripheral component that dynamically associates with the complex, in
contrast with the stably-associated core components . The pro-
tein is proposed to have three transmembrane spans, thereby pre-
senting a carboxy-terminal TPR (TetratricoPeptide Repeat) domain
to the cytosol and a central domain (with amidase homology) to
the intermembrane space . Biochemical studies indicated that
the two domains might enable bipartite functionality, the TPR domain
acting as a receptor for the Hsp90 guidance complex [56,57], and the
central domain aiding transport through the intermembrane space
(see Section 3) . However, as already mentioned, in vivo studies
using knockout mutants did not support the importance of Toc64
for protein import [59,60]. Nonetheless, because its supposed mito-
chondrial counterpart, Tom70 (a TPR-domain receptor of well-
established function), is also non-essential , further work is needed
before a ﬁnal conclusion can be reached on Toc64's role. Interestingly,
one of the three Toc64 homologues in Arabidopsis (atToc64-V/
mtOM64) is localized in the mitochondrial outer membrane, perhaps
replacing the Tom70 receptor which is absent in plants [64,65];but
like its chloroplast relative, this protein too is dispensable .
More recently, an additional protein was identiﬁed that shares fea-
tures with Toc64: the OEP61 (Outer Envelope Protein, 61 kD) protein
possesses an amino-terminal TPR domain with similarity to that of
Toc64, and it is localized in the chloroplast outer envelope membrane
by a single carboxy-terminal span [66,67]. This protein was shown to
interact with chloroplast preproteins and Hsp70, but not Hsp90, sug-
gesting that it might be an additional docking site at the chloroplast
Another cytosolic factor involved in chloroplast protein targeting
is AKR2 (AnKyrin Repeat-containing protein 2), which was identiﬁed
as having an important role in the insertion of outer envelope mem-
brane proteins [68,69]; such outer membrane proteins do not have
transit peptides, and follow a pathway different from the canonical
TOC/TIC route discussed above 
(Fig. 1). The AKR2 protein is
proposed to act as a chaperone, preventing aggregation of these client
proteins and guiding them to the envelope membrane, where Toc75
is thought to assist their insertion . It was recently shown that
this guidance function of AKR2 is assisted by Hsp17.8, which is a
member of the small heat shock protein (sHsp) family . Interest-
ingly, AKR2 is also reported to have a role in the insertion of peroxi-
somal membrane proteins , suggesting that it may be important
for the targeting of a broad class of membrane proteins .
3. Crossing the intermembrane space
To complete their translocation across the two envelope mem-
branes, preproteins must traverse the intermembrane space (IMS)
after their recognition at the TOC complex. Four components have
been proposed to assist such transfer through the IMS: Toc64,
Toc12, Tic22 and imsHsp70, with the latter putatively accounting
for the ATP requirement in the IMS [75,76] (Fig. 1). However, the lo-
calization and functions of several of these proteins are controversial.
Toc64 was proposed to participate in the formation of an IMS com-
plex through its central amidase domain, and to directly interact with
Toc12 . However, as described earlier, this protein does not seem
to play an appreciable role in protein transport in vivo [59,60].The
Toc12 component was ﬁrst described as a J-domain protein anchored
to the outer membrane by its amino-terminal end, and with its
carboxy-terminal J-domain localized in the IMS. In the original model,
the latter domain would speciﬁcally stimulate the ATPase activity of
an Hsp70 chaperone (imsHsp70) to facilitate its interaction with trans-
locating preproteins. The preproteins would then complete their trans-
port across the intermembrane space after contacting the soluble Tic22
protein, which is peripherally associated with the outer face of the TIC
Toc12 was ﬁrst identiﬁed by proteomic analysis of the outer enve-
lope of pea chloroplasts. Through immunoprecipitation experiments,
it was found that Toc12 interacts with Toc64 and core TOC components,
Tic22 and imsHsp70, but not with other TIC complex components such
as Tic110 . However, more recent evidence indicates that Toc12 is
an unlikely participant in IMS transport. It was found that Toc12 is a
truncated form of a larger protein (DnaJ-J8) in pea .Moreover,pro-
tein import analyses indicated that Toc12/DnaJ-J8 possesses a cleavable
Fig. 1. Chaperone involvement in the cytosol and intermembrane space during protein transport to chloroplasts. Nucleus-encoded proteins destined for the chloroplast are recog-
nized in the cytosol by soluble factors such as Hsp70, Hsp90, 14-3-3 and AKR2. Hsp70 may form a guidance complex together with 14-3-3 that delivers phosphorylated preproteins
to the Toc34 receptor. Hsp70 is also proposed to deliver preproteins to the OEP61 protein. An alternative Hsp90 complex is reported to deliver a different subset of preproteins (that
need not be phosphorylated) to the peripheral TOC component Toc64, before their onward passage to the core TOC machinery. The AKR2 protein guides outer membrane protein
(OMP) clients to the chloroplast surface, with assistance from Hsp17.8 and possibly other, unknown factors, whereupon OMP insertion into the membrane is facilitated by Toc75.
Transport of preproteins through the intermembrane space was proposed to involve imsHs70 and a J-protein called Toc12. However, the existence of imsHsp70 is now uncertain,
while evidence suggests that “Toc12” is simply a truncated form of the stromal protein DnaJ-J8; whether some other J-protein acts in the IMS remains to be seen. A translocating
preprotein is represented by a wavy black line, while its transit peptide is shown as a thick gray line. Functional domains of the proteins are indicated in white text. TOC, translocon
at the outer envelope membrane of chloroplasts; OM, outer envelope membrane; IMS, intermembrane space.
334 Ú. Flores-Pérez, P. Jarvis / Biochimica et Biophysica Acta 1833 (2013) 332–340
transit peptide, and that the imported mature protein is soluble and
localized in the stroma .InArabidopsis,theAT1G80920 gene was
proposed to encode the orthologue of pea Toc12 . Recent studies
designated At1g80920 as AtJ8, and showed that it too is a soluble pro-
tein of the chloroplast stroma [77,78]. Furthermore, AtJ8 T-DNA inser-
tion mutants did not show any defect in the import of various
chloroplast preproteins, suggesting that AtJ8 is unlikely to be involved
in protein import .
Another critical component of the putative IMS complex is the
imsHsp70. The existence of three pea chloroplastic Hsp70 isoforms
was originally reported: two soluble proteins located in the stroma,
and one isoform tightly associated to the outer membrane but not
exposed at the outer surface of the chloroplasts . This latter
imsHsp70 isoform is supposed to interact with translocating prepro-
teins in pea chloroplasts [75,80]. Recent studies aimed to identify the
Arabidopsis homologue of the imsHsp70, and described the subcellular
localization of the three putative chloroplastic Hsp70 proteins. Two of
them, AtHsp70-6 and AtHsp70-7 (alternatively called cpHsc70-1 and
cpHsc70-2, respectively, for chloroplast Heat shock cognate protein,
70 kD; ) were found to be localized in the soluble fraction of chloro-
plasts while the third, AtHsp70-8, was not even imported into chloro-
plasts in vitro [81,82]. Thus, the gene that encodes the imsHsp70
As described above, the involvement of Hsp70 in the translocation
steps that mediate passage through the IMS is still inconclusive. How-
ever, recent evidence does support the participation of Hsp70 in chlo-
roplast protein import in the stroma, as detailed in Section 4 below.
4. Chaperone involvement in the stroma
4.1. Inner membrane components
Preprotein translocation is completed by passage through the TIC
complex of the inner envelope. The putative or actual members of the
TIC machinery that have been identiﬁed to date are Tic110, Tic62,
Tic55, Tic40, Tic32, Tic22, Tic21 and Tic20 (reviewed in [11,83]); it
should be noted that Tic21 was alternatively identiﬁed as an iron trans-
porter, PIC1 (Permease In Chloroplasts 1) [84,85]. Three components
(i.e., Tic110, Tic20 and Tic21/PIC1) have been proposed to contribute
to channel formation at the inner membrane [76,86–88]; however, ev-
idence suggests that Tic110 is not present in the same complex as Tic20
and Tic21, and so it is unlikely that the three proteins cooperate in this
function (Fig. 2). The Tic40 protein, together with thestromal chap-
erone Hsp93, may constitute a motor providing the driving force for
protein import dependent upon ATP hydrolysis (discussed in detail in
the next section) (Fig. 2
). Finally, Tic62, Tic55 and Tic32 constitute a pu-
tative redox-regulator of the TIC apparatus [11,83].
Tic110 is the second most abundant protein of the inner envelope
membrane , and it plays an essential role in plastid biogenesis
since knockout mutants abort during embryogenesis [91,92].The
amino-terminus of Tic110 contains two membrane-spanning α-
helices, while its largely hydrophilic carboxy-terminal region is oriented
towards the stroma [92–94]; alternatively, the latter domain may
contain four amphipathic transmembrane helices that enable it to
form a cation-selective channel . The conformation of Tic110 results
in one or more regions facing the IMS, that might interact with Tic22
and/or TOC components enabling the formation of TOC–TIC supercom-
plexes, or receive the transit peptides of incoming preproteins, as well
as a large region facing the stroma that may interact with molecular
chaperones such as Hsp93 and Cpn60 (Chaperonin, 60 kD) [88,95 ]
Tic40, another inner envelope membrane protein, has been proposed
to function as a cochaperone in the stromal complex that facilitates pro-
tein translocation across the inner membrane . It possesses a single
α-helical transmembrane span at its amino-terminal end, while the
rest of the protein is hydrophilic and oriented towards the stroma
[96,97]. This stromal part contains a putative TPR domain (in a central re-
gion of the sequence) followed by a C-terminal domain with sequence
similarity to the eukaryotic cochaperones Hip (Hsp70-interacting pro-
tein) and Hop/Sti1 [96,98]. While the so-called Sti1 (Hip/Hop) domain
can be functionally replaced by that of human Hip , the identity of
the central region as a true TPR domain has been drawn into question
by the alternative suggestion that this region of Tic40 contains a second
Sti1 domain .Regardlessofthisissue,thecentral(putativeTPR)re-
gion of Tic40 is involved in its binding to Tic110, which is stimulated in
the presence of a preprotein, while the C-terminal Sti1 domain engages
with Hsp93 .
Cross-linking, pull-down, yeast two-hybrid, and bimolecular ﬂuores-
cence compleme nta tion assays all demonstrated the clos e proximi ty of
Tic110 with Tic40 [97,98,100]. Additional crosslinking showed that
Tic110 and Tic40 associate with preproteins at a late stage in the import
mechanism, similar to Hsp93 .InArabidopsis, tic40 null mutants are
pale due to retarded chloroplast biogenesis, and display defects in pro-
tein import at the level of translocation across the inner envelope mem-
brane . Arabidopsis double mutants (i.e., tic40 tic110
and tic40 hsp93)
did not display additive effects, supporting the idea that these proteins
cooperate functionally in vivo .
4.2. Protein import motors
4.2.1. Hsp93 as the driving force
Until recently, stromal ATP consumption in the protein import
motor was attributed primarily to the chaperone Hsp93. Immunopre-
cipitation analysis, in the presence or absence of cross-linkers, revealed
Fig. 2. Chaperone involvement in the stroma during chloroplast protein import. Prepro-
teins arriving from the intermembrane space pass through the TIC channel, which may
be formed byTic110, Tic20 and/or Tic21; the white, verticaldotted line throughthe center
of the channel indicates that Tic110 and Tic20/21 are unlikely to cooperate in channel for-
mation. It is proposed that passage through the import machinery is facilitated by two dif-
ferent ATP-driven, stromal motor complexes. Both are associated with the TIC apparatus,
and it is suggested that they act in parallel. In the ﬁrst of these motors, Tic110 cooperates
with Tic40 and the Hsp100-type chaperone, Hsp93. The Tic40 cochaperone associates
with Tic110 via its putative TPR domain, while its Sti1 (Hip/Hop) domain engages
Hsp93 to stimulate ATP hydrolysis, such that the preprotein is pulled into the stroma. In
the second import motor, Hsp70 supplies the driving force for protein import. Whether
Tic110 and Tic40 are functional components of this motor remains to be seen. Evidence
suggeststhat Hsp70 activity isfacilitated by the nucleotide exchange factor CGE, but a pos-
sible J-domain protein has not yet been identiﬁed. Upon arrival in the stroma, the transit
peptide is cleaved by the stromalprocessing peptidase (SPP). Folding, assembly oronward
transport inside the chloroplast is facilitated by other chaperones, including Hsp70, cha-
peronins (Cpn60/10) and cpSRP43. Translocating preproteins are represented by wavy
black lines, while their transit peptides are shown as thick gray lines. Functional domains
of certain proteins are indicated in white text. TIC, translocon at the inner envelope mem-
brane of chloroplasts; IMS, intermembrane space; IM, inner envelope membrane.
335Ú. Flores-Pérez, P. Jarvis / Biochimica et Biophysica Acta 1833 (2013) 332–340
the interaction of Hsp93 with Tic110 and components of the TOC com-
plex [101,102]. The Hsp93 protein was also found in precursor-
containing complexes under limiting ATP conditions, and could be
immunoprecipitated with proteins that utilize the TOC/TIC import ap-
paratus, but not with an outer membrane protein that does not use
this import machinery . In addition, a stable association of Hsp93
with transit peptides in vitro has been described .Itwasrecently
shown that it is the amino-terminal region of Hsp93 that is important
for its association with the inner envelope membrane in vivo .
The involvement of this chaperone in protein import has been
supported by genetic studies. In Arabidopsis, there are two isoforms
of Hsp93, termed Hsp93-V and Hsp93-III (or ClpC1 and ClpC2, respec-
tively). The two proteins share very high levels of amino acid se-
quence identity, but the expression of Hsp93-V is much higher than
that of Hsp93-III [91,105]. Arabidopsis hsp93-V knockout plants are
pale, with underdeveloped chloroplasts containing fewer thylakoid
membranes and displaying reduced protein import efﬁciency. In con-
trast, hsp93-III knockout mutants are indistinguishable from wild
type; this can be explained by redundancy, as hsp93-III hsp93-V dou-
ble mutants are embryo lethal and overexpression of Hsp93-III can
complement hsp93-V, suggesting that the two proteins have overlap-
ping functions and are able to partially substitute for each other in the
single mutants [91,105–107].
Hsp93/ClpC is a member of the Hsp100 family of chaperones,
which itself belongs to the broader AAA+ (ATPases Associated with
various cellular A ctivities) superfamily. Hsp100 proteins contain one
or two AAA+ domains, and typically assemble into hexameric rings
with a central pore through which substrate proteins can be threaded
(reviewed in [26,108]). Hsp100 proteins mediate ATP-dependent
unfolding of proteins, in processes linked to protein degradation, pro-
tein disassembly, or protein trafﬁcking across membranes .In
chloroplasts, Hsp93 can form part of the Clp protease complex (in a
second role, additional to that in preprotein import), which recog-
nizes and unfolds substrate proteins that are destined for degradation
; interaction of Hsp93 with the proteolytic ClpP core is ATP de-
pendent . The Hsp93-V/ClpC1 isoform has been identiﬁed as a
dimeric complex of ~200 kD in the stroma of Arabidopsis chloroplasts
, but the hexameric state has proved difﬁcult to detect in vivo
[113,114]. Recently, recombinant Hsp93-III/ClpC2 in solution was
shown to be in dimeric form; upon addition of ATP, the hexamer
state was observed .
The current model for protein import assumes that, as the transit
peptide of an importing preprotein emerges from the TIC channel, it
binds to the stromal domain of Tic110. This binding causes a conforma-
tional change in Tic110 and enables recruitment of Tic40, which binds
Tic110 via its putative TPR domain [94,100]. The Tic110–Tic40 interac-
tion triggers the release of the transit peptide from Tic110 and enables
the association of the preprotein with Hsp93. Tic110 may dissociate
from Tic40 when there is no transit peptide bound. The Tic40 Sti1 do-
main then stimulates ATP hydrolysis by Hsp93, which acts to pull the
preprotein into the stroma using the released energy . Subse-
quently, Hsp93-ADP may dissociate from Tic40. In this model, Tic40
seems to arrange the last steps of envelope translocation, by regulating
the interaction of the preprotein with Tic110 and Hsp93, and by con-
trolling the activity of Hsp93 [96,100] (Fig. 2).
By analogy with other Hsp100 proteins, TIC-associated Hsp93 may
act as a hexamer, and by threading incoming preproteins through the
axial channel of the complex. Such a threading mechanism would be fa-
cilitated by oscillating loops within the central channel (reviewed in
[108,109]). Structural and functional studies on other Hsp100s led to
the proposal that such loops translocate bound sections of a client pro-
tein axially down the channel, in response to the hydrolysis of ATP, thus
applying a mechanical pulling force; this pulling action is associated
with an unfolding force, since the substrate protein is forced to enter a
narrow channel [26,108]. Such a model would make sense in relation
to chloroplast import, considering the involvement of an Hsp100
protein (Hsp93) and that preproteins presumably emerge from the
TIC channel in a largely unfolded state.
Based on genetic evidence derived using mutant yeast, an Hsp100
protein (Hsp78) was also proposed to act in mitochondrial protein
import, by substituting for the main motor chaperone (mtHsp70;
see Section 4.2.2) under certain conditions . However, later ana-
lyses supported an alternative interpretation of the data that did not
link Hsp78 to import .
4.2.2. Hsp70 as the driving force
Unlike Hsp100s, Hsp70-type chaperones do not oligomerize. They
transiently associate with exposed hydrophobic segments of client
proteins via a carboxy-terminal substrate binding domain (SBD),
thereby preventing aggregation and promoting proper folding. Bind-
ing of ATP to the Hsp70 amino-terminal nucleotide binding domain
(NBD) induces conformational changes in the adjacent SBD, opening
up the substrate binding pocket, whereas ATP hydrolysis leads to clo-
sure of the pocket and stabilizes the client interaction . Chloro-
plast stromal Hsp70s are believed to exist in a variety of plant
species, including Arabidopsis, pea, poplar, rice, sorghum and moss
. And yet, the involvement of Hsp70 chaperones in chloroplast
protein import was, until recently, only suggested to occur in the
cytosolic and IMS compartments, as discussed above. This is because
an early study could detect Hsp93, but not Hsp70, in association
with translocating preproteins ; the failure of this early work
to detect Hsp70 in import complexes was recently attributed to an in-
adequacy of the antibody originally used .
As was discussed earlier, most chloroplast transit peptides have
the capacity to bind Hsp70s [42–44]. The interaction between stromal
Hsp70 and the transit peptide, however, seems not to be crucial for
protein import, since preproteins with decreased Hsp70 binding af-
ﬁnity in their transit peptides are efﬁciently imported into pea chloro-
plasts in vitro [45,46]. Nonetheless, recent studies provided genetic
and biochemical evidence for the participation of stromal Hsp70 in
the process of protein import [117,118] (Fig. 2).
Shi and Theg  identiﬁed three stromal Hsp70s in the moss
Physcomitrella patens. The Hsp70-2 isoform is essential for moss
viability, as Hsp70-2 knockout plants could not be isolated; this also
indicated that neither of the other two isoforms, Hsp70-1 and
Hsp70-3, can substitute the loss of Hsp70-2 despite their high
degree of similarity. In order to study the participation of Hsp70-2
in protein import, a temperature-sensitive hsp70-2
was generated. After heat-shock treatment, it was observed that
isolated chloroplasts containing the temperature-sensitive Hsp70-2
protein display lower import competence when compared with wild-
type chloroplasts . In addition, moss Hsp70-2 could be immuno-
precipitated together with preproteins arrested as early import inter-
mediates, as well as with other translocon components (i.e., Hsp93
and Tic40), supporting the participation of Hsp70 in the TOC/TIC import
It is well established that Hsp70s invariably require a J-domain pro-
tein and, almost always, a nucleotide exchange factor (e.g., GrpE) as
partners in orderto be completely functional . Thus, a secondstrat-
egy in the aforementioned moss study  was to generate a mutant
(lcge)withsigniﬁcantly reduced levels of stromal cochaperones termed
CGEs (Chloroplast GrpE homologues). Chloroplasts isolated from the
lcge mutant were defective in protein import, strongly suggesting that
one of the CGE chaperone partners (i.e., an Hsp70) has an essential
role in protein import  (Fig. 2).
Studies in Arabidopsis and pea also supported the involvement of
stromal Hsp70sin the protein importmechanism. Protein import assays
using chloroplasts isolated from the Arabidopsis Hsp70 knockout mu-
tants, cpshsc70-1 and cphsc70-2, showed that stromal Hsp70 is impor-
tant for the import of both photosynthetic and non-photosynthetic
precursor proteins, especially at early developmental stages .
What is more, pea cpHsc70 could be immunoprecipitated together
336 Ú. Flores-Pérez, P. Jarvis / Biochimica et Biophysica Acta 1833 (2013) 332–340
with the newly-imported preproteins and translocon components
(such as Tic110), in the presence or absence of cross-linkers, strongly
suggesting that cpHsc70 is part of the translocon .
The relationship between Hsp70 involvement and the Hsp93/Tic40
system discussed earlier was also assessed genetically in Arabidopsis
. The function of Hsp70 seems to be related to that of Hsp93 in
driving translocation, as cphsc70-1 hsp93-V double mutants showstron-
ger defects in chlorophyll content and import efﬁciency than single mu-
tants. More strikingly, the cphsc70-1 tic40 double mutation is lethal,
indicating that Hsp70 and Tic40 share an overlapping and essential
function. Perhaps Hsp70 and Hsp93/Tic40 perform the same function
in parallel during protein import. Were the role of cpHsc70 only to
facilitate the assembly or functioning of Hsp93/Tic40, then the
cphsc70-1 tic40 double mutant would be expected to display the same
phenotype of the tic40 single mutant, not lethality.
Considering all of the in vivo and in vitro data presented above, it
seems that the stromal Hsp70 system is involved in protein transloca-
tion into chloroplasts, and that this mechanism is conserved from
moss to higher plants [117,118]. It is not surprising that this Hsp70
function exists in chloroplasts, as the protein import motor in mito-
chondria is driven by a matrix Hsp70 chaperone termed mtHsp70.
Like the TIC complex, the presequence translocase in mitochondria con-
sists of several subunits, which contribute to the import channel com-
plex (e.g., Tim23 and Tim17) or act as an import motor (Tim44,
Tim16, Tim14, mtHsp70, Mge1) [15–17]. The resemblance between
the Hsp70-driven motors in chloroplasts and mitochondria is evident
from the following. After initial translocation through the Tim23/
Tim17 core complex, mitochondrial preproteins contact subunits of
the PAM (Presequence translocase-Associated import Motor) machin-
ery, which provides the driving force for import. The Tim44 component
is an essential protein peripherally associated with the inner surface of
the inner membrane, and it serves to recruit mtHsp70 from the matrix
to the import complex. Once recruited, the mtHsp70 chaperone binds to
an emerging precursor, and then hydrolyses ATP, which provides the
driving force for import, as discussed below. Additional PAM compo-
nents are Tim14 and Tim16 (also called Pam18 and Pam16, respective-
ly), which are integral inner membrane cochaperones that regulate the
ATP-hydrolysis cycle of mtHsp70: Tim14 presents a J-domain at the ma-
trix side of the inner membrane that, in cooperation with Tim16, stim-
ulates the ATPase activity of mtHsp70. The nucleotide exchange factor
Mge1 (Mitochondrial GrpE-related protein 1) is also required in this
There are competing models for the delivery of power in the mito-
chondrial import motor [120,121]. In the Brownian ratchet model,
precursor-bound mtHsp70-ADP immediately dissociates from Tim44
so that random motion (Brownian oscillations) may cause a segment
of the precursor to move forwards into the matrix. A second mtHsp70
associated with Tim44 then passively traps the newly-translocated
segment, preventing backsliding into the channel. The alternative
power-stroke model suggests that the import motor acts as a me-
chanical machine that actively pulls on the incoming precursor.
According to this model, ATP hydrolysis induces a conformational
change in mtHsp70 that generates inward movement of the bound
precursor. It is possible that elements of both models are correct, or
that the two mechanisms cooperate as follows: for loosely-folded or
unfolded preproteins, the ratchet mechanism could be sufﬁcient,
whereas for preproteins with stably-folded domains an additional
pulling force might be required [120,121].
Stromal Hsp70 might provide the driving force in chloroplast pro-
tein import in a similar fashion to mtHsp70 in the mitochondrial im-
port motor. However, the extent to which the chloroplast Hsp70
system requires similar cochaperones needs to be established: while
the involvement of the Mge1-related nucleotide exchange factor
CGE1 has been shown in moss , information on J-protein in-
volvement remains elusive . Moreover, it remains to be deter-
mined whether a recruiting protein, similar to Tim44 for mtHsp70,
is involved. Despite the evident similarities between the chloroplast
and mitochondrial motors, a unique feature of the chloroplast system
is the fact that there are two chaperone systems working in parallel
(Fig. 2). The biological meaning of the presence of two motors has
yet to be established, but it may relate to the fact that the two chap-
erones types (i.e., Hsp100 and Hsp70) have very different modes of
action, as was discussed earlier. Perhaps the two chaperone systems
provide necessary versatility to the organelle and its import machin-
ery . It is well known that both Hsp93 and Hsp70 have other
functions in chloroplasts. Hsp93/ClpC acts as a regulatory chaperone
in the Clp protease. Several genetic and biochemical studies have
shown that the Clp protease is essential for chloroplast development
and plant viability . Hsp70 also displays other essential roles in
chloroplast development . Whether the Hsp93 and Hsp70 mo-
tors have different preprotein binding preferences, or are required
in different plastids or developmental conditions, remains to be seen.
4.3. Chaperone involvement following the completion of import
After preproteins delivered into the stroma have been processed by
SPP, they may require assistance to fold properly into their functional
conformation, or to reach their ﬁnal intraorganellar destination. The
stromal molecular chaperones Hsp70, Cpn60 and Cpn10 are all believed
to mediate the folding or onward guidanceof newly-imported polypep-
tide chains [29,124] (Fig. 2). In addition, it has been reported that Hsp93
facilitates the stromal passage of imported proteins destined to the
inner envelope membrane via the post-import (or conservative sorting)
Chloroplast Cpn60 is a homologue of the bacterial Hsp60-type chap-
erone GroEL [26,108]. Such chaperones assemble into two stacked
heptameric rings, and cooperate with a cochaperone termed Cpn10
(an Hsp10/GroES homologue), which may form heptameric caps at
either end of the Cpn60 tetradecamer. Client proteins enter a central
cavity in the Cpn60 complex, providing a protected environment in
which folding can take place, which is controlled by the nucleotide-
dependent cycling of Cpn60 subunits between binding-active and
folding-active states [26,108]. The role of the Cpn60/Cpn10 chaperonin
system in the folding of Rubisco subunits has been well documented
. Cpn20 is a chloroplast-speciﬁc cochaperone comprising two,
tandemly-arranged Cpn10-like units; while in vitro assays have
shown that it is a functional homologue of Cpn10, its speciﬁcrolein
vivo remains uncertain .
Immunoprecipitation experiments revealed that Cpn60 is in close
proximity with Tic110 . This interaction was disrupted in the
presence of ATP, while import experiments revealed a transient asso-
ciation of mature, newly-imported proteins with the Cpn60–Tic110
complex, suggesting that Tic110 can recruit Cpn60 in an ATP-
dependent manner for the folding of proteins upon their arrival in
the stroma. Biochemical data also support the interaction of newly-
imported proteins with stromal Hsp70, either before or after the in-
teraction with Cpn60 [127,128]. It has been suggested that stromal
Hsp70 and Cpn60 act sequentially to assist the maturation of
imported proteins, particularly those destined for the thylakoid mem-
branes. Another factor that facilitates the passage of new proteins
through the stroma to the thylakoids is cpSRP (chloroplast Signal
article), the clients of which are light-harvesting
chlorophyll-binding proteins. In fact, one of the cpSRP components,
a protein termed cpSRP43, was recently reported to have unique
chaperone-like properties [129,130] (Fig. 2).
Molecular chaperones participate in a host of different and essential
processes in cells. Their ﬂexibility allows them to act at various, distinct
stages throughout the processofchloroplast protein import, where they
perform a diversity of roles that include guidance, maintenance of
337Ú. Flores-Pérez, P. Jarvis / Biochimica et Biophysica Acta 1833 (2013) 332–340
structural competence for transport, and provision of a driving force.
Cytosolic chaperones may be the ﬁrst contact for chloroplast prepro-
teins following their emergence from the ribosome, and these are pro-
posed to assist docking at different receptors at the chloroplast
surface. Although the involvement of Hsp70 in the intermembrane
space remains inconclusive, the participation of other molecular chap-
erones at the stromal side of the envelope, in so-called motor com-
plexes, is strongly supported by different lines of evidence. While
Hsp93 was for many years considered to be the primary chaperone of
stromal motor complexes, recent genetic and biochemical studies
strongly suggest that stromal Hsp70 also has a signiﬁcant role to play.
Challenges for the future include the elaboration of cytosolic events
leading to docking, identiﬁcation of the putative intermembrane space
ATPase, elucidation of the mechanisms that enable two different
stromal motor complexes to operate, and the deﬁnition of the speciﬁc
biological conditions during which the different motors operate.
We are grateful to the Biotechnology and Biological Sciences
Research Council (BBSRC; grant no. BB/F020325/1) for ﬁnancial sup-
port. We thank Qihua Ling for helpful comments on the manuscript.
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