Chloroplast biogenesis: diversity and regulation of the protein
Felix Kessler1and Danny Schnell2
The biogenesis of chloroplasts is dependent on the coordinate
expression of genes encoded in both nuclear and plastid
genomes. The chloroplast protein import machinery plays key
roles in organelle biogenesis by mediating the import and
assembly of thousands of nuclear-encoded proteins into the
organelle. It is now apparent that multiple levels of control exist
to integrate the activities of the protein import apparatus into
the network of chloroplast-nuclear communication that is
essential to maintain organelle homeostasis. The import
apparatus has diversified into small, functionally specialized
gene families to coordinate the import of distinct classes of
differentially expressed proteins. Protein targeting to
chloroplasts also has evolved regulatory mechanisms that
respond to cellular developmental and physiological changes,
including redox sensing, phosphorylation, and dual targeting.
Recent studies also have revealed new components that could
represent additional levels of control on the import process.
1Laboratoire de Physiologie Ve ´ge ´tale, Universite ´ de Neucha ˆtel, Rue Emile-Argand 11, 2007 Neucha ˆtel, Switzerland
2Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst,
MA 01003, USA
Corresponding author: Schnell, Danny (email@example.com)
Chloroplasts evolved from a photosynthetic bacterial
symbiont  to participate in numerous essential meta-
bolic and cellular processes in plants and algae, including
photosynthesis, amino acid, and lipid metabolism, cell
signaling, and host defense. As single-celled algae
evolved into multicellular organisms, chloroplasts conco-
and cell types, giving rise to a diverse group of inter-
related organelles called plastids [2,3]. In Arabidopsis,
plastids rely on the import of ?3000 different nucleus-
encoded proteins from the cytoplasm . Three decades
of research have unraveled complex communication
networks that couple and integrate the physiological
and biogenetic status of the chloroplast with nuclear gene
protein import apparatus itself as a key regulatory entity
in mediating plastid–nuclear interactions. As such, the
protein import machinery plays a role in a network of
processes that (1) control the overall levels of nucleus-
encoded plastid proteins synthesized, (2) maintain the
stoichiometry of multi-protein complexes that contain
both plastid and nucleus-encoded subunits, (3) respond
to organelle status or dysfunction (e.g. photosynthetic
activity or organelle stress) and, (4) coordinate changes
in protein profiles during developmental transitions from
one plastid type to another. Several recent comprehen-
sive reviews cover molecular mechanisms of protein
import [7–9], and here we will focus on its control and
integration with gene expression.
Overview of protein trafficking to chloroplast/
The major pathway for protein import, in terms of sheer
volume, is mediated by the TOC and TIC translocons
(translocons at the outer and inner membrane of chlor-
oplasts, respectively) [4,7]. The TOC-TIC pathway also
represents the first step in the targeting of the majority of
inner envelope membrane. TOC-TIC substrates are dis-
tinguished by the presence of an N-terminal targeting
sequence, designated the transit peptide . Although
more than a dozen proteins are implicated in TOC-TIC
function, experimental evidence, and the analysis of
available algal and plant genomes identify a core subset
of these components that are required for import in vivo
[11,12,13??,14,15] and are conserved across the phyloge-
netic spectrum of the plant kingdom [16,17?].
channel (Toc75) that forms a stable complex with two
membrane-bound GTPases, Toc34 and Toc159 [18,19].
The TOC GTPases represent the primary receptors for
transit peptides at the chloroplast surface, and evidence
supports roles for their intrinsic GTPase activities in con-
trolling access of preproteins to the channel . Once
transferred from the receptors to Toc75, the preprotein
translocates across the outer membrane in an ATP-de-
pendent reaction that appears to involve an Hsp70-type
molecular chaperone in the intermembrane space [21?].
The core TIC translocon physically associates with the
TOC complex and preprotein translocation proceeds
Published in Current Opinion in Cell Biology 21, issue 4, 494-500, 2009
which should be used for any reference to this work
across the outer and inner envelope membranes via their
linked channels. Although the precise nature of the TIC
channel remains a matter of discussion, three conserved
multi-spanning membrane proteins are implicated in
inner membrane translocation (Tic110, Tic20, and
Tic21) [22–24]. Tic110 also contains a binding site for
the stromal chaperone, Hsp93, or ClpC , and the
chaperone is proposed to bind to preproteins and provide
the driving force for translocation through the TIC chan-
nel . This appears to account for the additional ATP
requirement for inner membrane translocation. The
coordination of Tic110-Hsp93 activities appears to be
mediated by Tic40, an inner membrane protein, via its
intrinsic TPR and Hip/Hop domains .
Regulation at the TOC GTPase receptors
As with other membrane translocation systems, prepro-
tein recognition and the initiation of membrane translo-
cation at the TOC complex are the committed steps in
the import process. Consistent with their roles as gate-
keepers, the TOC GTPase receptors represent key func-
tional and regulatory points in the pathway. All
preproteins initially bind at the TOC receptors before
translocation through the TOC channel. Interestingly,
protein import in isolated chloroplasts can be driven by
the internal ATP generated in the light via photosyn-
thetic electron transport [21?]. A role for GTP in the
import process is revealed only with the addition of non-
hydrolyzable GTP analogs, which inhibit preprotein
binding and translocation [21?]. This suggests an import-
ant role for TOC GTPase activities in regulating mem-
brane transport. For the purposes of our discussion, we
willconsider theTOC GTPasesasafunctionalregulatory
unit and not concentrate on parsing the detailed
Toc34 and Toc159 both appear to be required in vivo,
recent studies suggest that their GTPase activities might
be partially overlapping [28,29??].
The foremost regulatory function of the TOC receptors
appears to be as gatekeepers to the translocon channel
(Figure 1) [28,29??]. Both receptors bind transit peptides
and are in contact with preproteins at the initial stage of
preprotein binding to the translocon. Transit peptide
binding stimulates the GTPase activity of both receptors
, and therefore has been proposed to control the GTP
switch that opens the gate to the translocon and allows
membrane translocation to proceed. The TOC receptors
also interact directly with one another via their GTPase
domains [31,32??], leading to the hypothesis that GTP-
The role of the TOC GTPase receptor families as gatekeepers to the TOC translocon. Members of the Toc159 (atToc159 and atToc132/120) and Toc34
(atToc33 and atToc34) preprotein receptor families in Arabidopsis mediate the recognition of nucleus-encoded preproteins by binding to preprotein
transit peptides at the outer envelope membrane (OM) . Preprotein binding is hypothesized to stimulate receptor GTPase activity , thereby
controlling the molecular switch (changes in receptor dimerization) that provides preprotein access to the translocon channel (atToc75). The Toc159
and Toc34 family members assemble in combination to form distinct translocons with the Toc75 channel (atToc75). The different TOC receptor
isoforms mediate the recognition of distinct classes of nucleus-encoded preproteins to maintain the proper levels of functional classes of proteins that
are required for the biogenesis and homeostasis of the organelle [13??,36].
regulated TOC receptor interactions represent a gate
across the translocon [33,34]. Although other modulators
might exist (i.e. GAP or GEF proteins) and the stoichi-
ometry of GTP hydrolytic cycles and preprotein translo-
cation is unknown, the role of the transit peptide in
controlling the GTPases is mechanistically satisfying
and provides a link between recognition of the transit
peptide and opening the translocon gate [30,35].
A second dimension to the TOC GTPase switch function
has been revealed by the biochemical and molecular
Toc159 and Toc34 receptors in Arabidopsis. These stu-
dies demonstrate that the receptors not only specifically
recognize transit peptides but also discriminate between
different types of transit peptides [36,37] (Figure 1). The
size of the Toc34 family varies from one to two in higher
plant species . Individual null mutants of the two
Toc34 genes in Arabidopsis (atToc33 and atToc34) exhi-
bit distinct phenotypes . However, this is probably
due to differential expression of the two genes, and their
functions appear to be largely redundant . In higher
plants, the Toc159gene family consists of at least three or
four genes . In contrast to the Toc34-like genes, null
mutants of individual atToc159 family members exhibit
more pronounced differential effects on plastid bio-
genesis [13??,36]. At least three of the Arabidopsis recep-
tors, atToc159, atToc132, and atToc120, bind distinct
classes of preproteins. Furthermore, atToc159 mutants
preferentially disrupt chloroplast biogenesis, whereas
atToc120/132 mutants exhibit more general effects on
plastid function. This leads to a novel hypothesis that
includes not only the role of the Toc receptors in regulat-
ing translocon access but also their role in defining dis-
tinct import pathways. The Toc159 and Toc34 family
members are envisioned to assemble in various combi-
nations to generate translocons with distinct selectivities
that would thereby control the type of cargo that gains
access to the organelle [7,8]. This additional selectivity
would be key to maintaining organelle homeostasis
during development. In particular, it allows flux of pre-
proteins within a specific class (e.g. photosynthetic
proteins) to be controlled independent of other classes
(e.g. housekeeping proteins), thereby avoiding compe-
tition at import sites (Figure 1). As such, the TOC
receptors provide an additional level of control that
balances protein import with the changes in transcrip-
tional profiles that accompany physiological and devel-
opmental changes during plastid biogenesis. It will be
important to more fully explore the mechanism of
profiles of the receptors to more clearly define the roles of
the distinct pathways in organelle development.
Regulation via cytosolic factors
Two complexes have been proposed to assist in targeting
cytoplasmic preproteins to the TOC GTPase receptors
(Figure 2). The ‘guidance complex’ consists of a 14-3-3
The potential role of cytosolic factors in targeting preproteins to the TOC complex. A cytosolic guidance complex (14-3-3 protein and cytosolic Hsp70)
[38,39] and an Hsp90 complex (Hsp90 and Toc64) [40,41] are proposed to stimulate the delivery of preproteins to the TOC receptors. Association of
the guidance complex with preproteins is controlled by transit peptide phosphorylation. Cytosolic Hsp90 is proposed to bind preproteins and deliver
them to Toc64 at the chloroplast surface for subsequent transfer to the TOC translocon. The activities of neither complex are essential in Arabidopsis
[42–44] suggesting that they play specialized roles in the targeting of specific preproteins. Alternatively, these factors could function as parts of a
cytosolic quality control system (broken arrows) to avoid the accumulation of preproteins in the cytoplasm if the preprotein is damaged or the levels of
translation exceed the capacity of the import apparatus.
protein and a cytoplasmic Hsp70 chaperone . This
complex increased the efficiency of protein import in
vitro. Recognition of preproteins by the guidance com-
plex centers on potential serine/threonine phosphoryl-
ation sites within transit peptides, suggesting that this
interaction is controlled by a phosphorylation/depho-
sphorylation cycle . Thesecond cytoplasmic targeting
complex involves cytoplasmic Hsp90 and a putative
chloroplast outer membrane receptor, Toc64 [40,41].
Toc64 is proposed to function as an alternative preprotein
receptor for preproteins bound to Hsp90. Both targeting
complexes are proposed to subsequently deliver prepro-
teins to the TOC complex for translocation.
Interestingly, neither the guidance complex nor Hsp90
complex appear to be essential for protein import in vivo.
Many transit peptides lack the phosphorylation sites for
guidance complex binding, and mutations in the sites in
potential guidance complex substrates do not affect
protein import in vivo . Insertional mutants in the
Toc64 genes in Arabidopsis and moss (Physcomitrella
patens) exhibit no detectable physiological or protein
import defects [43,44]. These observations suggest that
their primary roles might be to assist the targeting of
specific proteins, for example, highly abundant prepro-
teins, such as the small subunit of rubisco. Although
speculative, the guidance complex and Hsp90 complex
could play roles in targeting to the specific pathways
represented by different TOC GTPases (Figure 1) or
participate in quality control systems that monitor these
pathways (Figure 2). The activity of the Hsp90 complex
in quality control systems that monitor protein levels or
protein damage (e.g. misfolding) in other cellular com-
partments . The proposed phosphorylation cycle
regulating guidance complex binding also is consistent
with a monitoring function. These systems could
represent a mechanism to avoid the accumulation of
newly synthesized preproteins in the cytoplasm under
conditions when plastid integrity is compromised or
when the levels of protein synthesis exceed the capacity
of protein import.
Regulation at the level of dual targeting
Dozens of different proteins have been shown to exhibit
dual targeting to chloroplasts and mitochondria, and there
have also been reports of dual targeting between chlor-
oplasts and the nucleus, peroxisomes and the ER .
Many dual targeted proteins are those involved in shared
processes within the different organelles, including tran-
scription, translation, protein turnover, and shared meta-
bolic or enzymatic activities. Dual targeting provides a
mechanism for single genes to encode conserved func-
tions within different organelles and is proposed to con-
stitute a mechanism of inter-organellar regulation that
coordinates these functions within the two organelles
Dual targeting has been best characterized for chloro-
plasts and mitochondria and is achieved by a number of
mechanisms. These include alternative mRNA splicing
to generate a mitochondrial presequence or a chloroplast
transit peptide, ambiguous (dual) targeting signals that
target to both organelles, or the selection of alternative
translational start sites [47,48,49?]. Although the mech-
anisms of regulating dual targeting have not been
examined in detail, the distribution of these proteins
between organelles appears to be closely monitored.
General mechanisms of regulating alternative splicing
are well known, and might apply to the generation of
alternative targeting signals by this mechanism. Much
less is known about the control of distribution of proteins
carrying dual targeting signals.However,there are reports
that the degree of dual targeting of specific proteins is cell
type specific, indicating that the distribution is tightly
regulated . It also has been noted that many dual
targeting signals contain potential phosphorylation sites,
and this modification could regulate the interaction of the
targeting signal with cytosolic factors or receptors at the
organelle surface .
Regulation in response to organelle metabolic
Redox regulation plays a key role in the biogenesis and
metabolic regulation of chloroplasts . More recently,
the role of redox regulation in plastid function has been
Potential redox control of protein import. Three membrane associated
redox proteins (Tic55, Tic32, and Tic62) have been shown to associate
with the Tic translocon [54,55]. The association of Tic62 and Tic32 with
the inner envelope membrane appears to be controlled by their redox
state [56?]. All three proteins contain redox cofactors, leading to the
hypothesis that they modulate protein import in response to the redox
state (i.e. photosynthetic activity) of the chloroplast. Tic32 also contains
a potential calmodulin binding site . Redox control would couple the
import of photosynthetic preproteins with the photosynthetic activity of
extended to the protein import apparatus (Figure 3). The
import of two chloroplast redox proteins, ferredoxin FdIII
and ferredoxin-NADP-oxidoreductase II, has been shown
to be regulated in response to light [52,53]. This suggests
that the import apparatus responds directly to the physio-
logical state of the organelle, in particular, the level of
photosynthesis as indicated by redox state. Three inner
envelope proteins carrying redox cofactors, Tic32, Tic62,
and Tic55, are associated with the TIC translocon [54,55],
and evidence suggests that the association of Tic62 and
Tic32 with the inner envelope is regulated by the NADP/
no direct evidence that Tic32, Tic62, and Tic55 modulate
TIC activityorareinvolvedintheregulation offerredoxin
FdIII or ferredoxin oxidoreductase II import, their
dynamic association with the translocon provides a poten-
tial mechanism whereby protein import is modulated in
is known to regulate aspects of mitochondrial protein
import, suggesting a similar system for regulating traffick-
ing in energy generating organelles .
Conclusions and perspective
Import through the TOC-TIC translocons is a common
step in the expression, targeting, and assembly of
thousands of distinct nucleus-encoded plastid proteins.
A comparison of plastid to nucleus signaling pathways
with the numerous potential control points in the import
process begins to identify sites of regulatory integration
between the processes (Figure 4). The overall levels and
functional classes of proteins that accumulate within the
organelle appear to be regulated not only at the level of
nuclear transcription but also by a diverse set of import
translocons that control the flux of these protein classes.
This balance may beaccompanied bymonitoring systems
in the cytoplasm (e.g. soluble factors) that ensure that
excess or damaged preproteins are degraded and not
targeted to the TOC-TIC system. The control of pre-
protein targeting at both of these levels would comp-
lementthe signaling and control systemsthat regulatethe
translation of plastid proteins and influence nuclear gene
expression , thereby ensuring the proper stoichi-
ometry of proteins containing subunits encoded by
both genomes. Redox also provides a mechanism of
integrating chloroplast metabolic activity with gene
expression and import. Numerous photosynthetic pro-
cesses are regulated directly in response to the bioener-
getic status of the organelle as reflected in the redox
potential of the stroma. Redox signals also feedback on
nuclear transcription to control the expression of a variety
Potential sites for integration of protein import into the known pathways of chloroplast nuclear signaling. (1) Preprotein import is controlled by the
intrinsic GTPase activity of the TOC receptors. Distinct isoforms of TOC receptors provide a mechanism to modulate the levels and types of
preproteins that accumulate within the organelle in response to physiological and developmental changes in gene expression . (2) Cytosolic factors
[38–41] could monitor the balance between the levels of preprotein translation and the capacity of the import apparatus to avoid the accumulation of
preproteins in the cytosol. (3) The proper stoichiometry of proteins containing subunits encoded by both plastid and nuclear genomes would be
maintained by the rate of preprotein import and signals from the chloroplast to the nucleus that report on the levels of plastid-encoded subunits . (4)
The import apparatus can be directly modulated by the redox state of the chloroplast [52,53]. Redox signals also control the levels of expression of
specific nuclear genes [5,6]. This combined regulatory mechanism could couple the levels of preprotein expression directly to the activity of the import
of plastid proteins [5,6]. The potential control of import
via redox sensitive TIC components provides a potential
mechanism to integrate metabolic status, gene expres-
sion, and protein import. The variety of known and
potential regulatory sites of protein targeting to plastids
wasunimaginablewhentheimport apparatus wasinitially
envisioned as a housekeeping function for organelle
biogenesis and maintenance. Research in the field will
undoubtedly expand from basic mechanistic studies to
include investigations of the extent of regulation and its
role in plastid biogenesis and development.
This work was supported by National Institutes of Health grant GM61893
to DJS, Swiss National Science Foundation grant 3100A0-109667 (FK), and
funding from the National Centre of Competence in Research (NCCR)
Plant Survival (FK).
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