Molecular chaperone involvement in chloroplast protein import☆
Úrsula Flores-Pérez, Paul Jarvis⁎
Department of Biology, University of Leicester, Leicester LE1 7RH, UK
a b s t r a c ta r t i c l ei n f o
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).
Theymust navigatedifferent cellular and organellar compartments (e.g., the cytosol, the outerand 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-
certain. 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 com-
plex,while stromal Hsp70 isproposedto act in a second, parallelcomplex. Upon arrival inthe 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.
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
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
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-
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.
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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-
portofa6.5 kD(23 Åindiameter)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.
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 theformation 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 deficientin 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
specificity 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-shockprotein,70 kD) isone ofthe chaperones thought
to facilitate the cytosolic phase of chloroplast protein transport. Most
chloroplast transit peptides are predicted to possess at least one
Hsp70 bindingsite, while direct interactions between Hsp70s and tran-
of such Hsp70 binding for protein import remains uncertain, as it is not
essential for protein translocation in vitro [45,46]. Moreover, a recent
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,
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
Differentiatingbetween two distinct,endosymbiotically-derivedor-
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 factthat the protein import receptors in plant mitochondria
well as from those in chloroplasts [17,36]. In spite of these receptor dif-
ferences, some chloroplast preproteins can be efficiently imported into
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-
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-
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
plasts that lack Toc64 protein [59,60], indicating that this putative tar-
geting mechanism is also not essential. It is conceivable that such
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