Báo cáo khoa học: Protein transport in organelles: The composition, function and regulation of the Tic complex in chloroplast protein import pptx

MINIREVIEW
Protein transport in organelles: The composition,
function and regulation of the Tic complex in
chloroplast protein import
J. Philipp Benz
1,2
,Ju
¨
rgen Soll
1,2
and Bettina Bo
¨
lter
1,2
1 Plant Biochemistry, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Munich, Germany
2 Munich Center for Integrated Protein Science CiPS
M
, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Munich, Germany
Introduction
To fulfil their functions correctly, plastids permanently
communicate with the surrounding cell. This requires a
substantial traffic of substances such as nutrients,
metabolites and proteins into and out of the organelle,

which have to be funnelled across the two envelope
membranes surrounding all plastid types. Among these
transport processes, the translocation of proteins is of
particular significance. Due to the loss of more than
90% of their genetic information to the host nucleus
during evolution, plastids have become almost com-
pletely dependent on the surrounding cell. Of the
approximately 3000 proteins present in chloroplasts,
typically only 50–250 (dependent on the species) are
still encoded for on the plastome [1]. The majority of
Keywords
chloroplast; import motor; preprotein
channel; redox regulation; Tic complex;
translocon
Correspondence
J. Soll, Plant Biochemistry, Ludwig-
Maximilians-Universita
¨
tMu
¨
nchen,
Großhaderner Strasse 2-4, D-82152 Munich,
Germany
Fax: +49 89 2180 74752
Tel: +49 89 2180 74750
E-mail:
Website:
(Received 31 July 2008, accepted 11
December 2008)
doi:10.1111/j.1742-4658.2009.06874.x

It is widely accepted that chloroplasts derived from an endosymbiotic event
in which an early eukaryotic cell engulfed an ancient cyanobacterial pro-
karyote. During subsequent evolution, this new organelle lost its autonomy
by transferring most of its genetic information to the host cell nucleus and
therefore became dependent on protein import from the cytoplasm. The
so-called ‘general import pathway’ makes use of two multisubunit protein
translocases located in the two envelope membranes: the Toc and Tic com-
plexes (translocon at the outer/inner envelope membrane of chloroplasts).
The main function of both complexes, which are thought to work in para-
llel, is to provide a protein-selective channel through the envelope mem-
brane and to exert the necessary driving force for the translocation. To
achieve high efficiency of protein import, additional regulatory subunits
have been developed that sense, and quickly react to, signals giving infor-
mation about the status and demand of the organelle. These include
calcium-mediated signals, most likely through a potential plastidic calmod-
ulin, as well as redox sensing (e.g. via the stromal NADP
+
/NADPH pool).
In this minireview, we briefly summarize the present knowledge of how the
Tic complex adapted to the tasks outlined above, focusing more on the
recent advances in the field, which have brought substantial progress
concerning the motor function as well as the regulatory potential of this
protein translocation system.
Abbreviations
CaM, calmodulin; ClpC, caseinolytic protease C; Cpn, chaperonin; FNR, ferredoxin-NADP
+
-oxidoreductase; Hip, Hsp70-interacting protein;
Hop, Hsp70/Hsp90-organizing protein; Hsp, heat shock protein; IEM, inner envelope membrane; OEM, outer envelope membrane; SDR,
short-chain dehydrogenase; SPP, stromal processing peptidase; Tic, translocon at the inner envelope membrane of chloroplasts; Toc,
translocon at the outer envelope membrane of chloroplasts; TPR, tetratricopeptide repeat; Trx, thioredoxin.

1166 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS
proteins therefore have to be imported post-transla-
tionally from the cytoplasm, which is most generally
performed via two translocation machineries present in
the outer (OEM) and inner envelope membrane
(IEM), called Toc (translocon at the outer envelope of
chloroplasts) and Tic (translocon at the inner envelope
of chloroplasts), respectively [2–5]. In this pathway,
nuclear encoded preproteins are translated with an
N-terminal extension called transit peptide, which
allows targeting of the precursor to the organelle,
specific recognition by the receptor proteins on the
surface, and subsequent translocation through both
membranes. After successful import, the transit peptide
is cleaved off by the stromal processing peptidase
(SPP), resulting in the mature form of the protein. The
entire process is superficially reminiscent of that medi-
ated by the protein translocases at the outer and inner
mitochondrial membranes [6], but plastids have devel-
oped their own ways to solve the main three tasks of
protein translocation: (a) the formation of a prepro-
tein-specific pore in the membrane (the channel); (b)
exerting the necessary driving force (the motor); and
(c) installing components that allow regulation of the
translocation efficiency depending on developmental or
environmental conditions (the regulon).
Based on biochemical and genetic evidence, eight
proteins have been implicated with respect to prepro-
tein import at the IEM of chloroplasts: Tic110, Tic62,
Tic55, Tic40, Tic32, Tic22, Tic21 and Tic20 (Figs 1

and 2). For each component, either a direct contact
with imported precursor has been demonstrated or,
otherwise, a close interaction with one of the estab-
lished Tic core proteins (usually Tic110). Last but not
least, the chaperone heat shock protein (Hsp) 93/
caseinolytic protease C (ClpC) has been demonstrated
to be a central constituent of the Tic motor complex
(see below).
The present minireview provides a short description
of recent advances in the understanding of the chan-
nel-, motor- and regulatory components of the Tic
complex. For reference, some of the available know-
ledge, including the proposed function of all Tic
components, is summarized in Table 1.
The Tic channel
Tic110 is undoubtedly the central protein of the tran-
slocon. It is not only the largest, most abundant and
best studied of all Tic proteins, but also probably the
only component involved in translocation steps hap-
pening on both sides of the IEM. This includes the
assembly of Toc–Tic ‘supercomplexes’ [7–9], preprotein
recognition [10], translocation, and folding steps of
successfully imported precursor proteins in the stroma
[11,12]. However, the exact topology of Tic110 within
the IEM is still not completely solved. There is mutual
consent about two transmembrane-helices at the
extreme N-terminus, which anchor the protein in the
membrane. The position and function of the long
C-terminal tail on the other hand remains a matter of
controversy [10,11,13–15]. According to one hypothe-

sis, the hydrophilic Tic110-Ct faces the stroma, where
it functions as a scaffold for the organization of the
stromal processes occurring during import [10,13,14].
These include the recruitment of chaperones to the
import apparatus (see below), as well as providing a
transit peptide-docking site, which is localized next to
the exit site of the translocon [10]. Another study dem-
onstrated that the function of Tic110 could extend well
beyond this role. Full-length protein as well as Tic110-
Ct was shown to insert into liposomes and form a
cation-selective ion channel, which was sensitive to
chloroplast transit peptides [11]. Interestingly, using
structural prediction software, at least two amphi-
pathic a-helices with acidic faces could be located
around the proposed transit peptide binding site [10].
These structures have been implicated with channel
function (e.g. in ligand-gated and voltage-gated K
+
channels) [16], and thus could provide an explanation
for the observed channel activity of Tic110, as well as
for the binding of transit peptides in this region
(Fig. 1).
Another putative channel protein is Tic20. Structural
predictions place Tic20 within the large group of small
hydrophobic proteins with four transmembrane-
domains (e.g. including the channel proteins Tim17
and Tim23) (Fig. 1). Distant sequence similarity also
exists between Tic20 and two prokaryotic branched-
chain amino acid transporters [17]. No data have been
published demonstrating channel activity but, because

Tic20 has prokaryotic ancestors, this suggests that it
could have been one of the very early constituents
of an evolving protein import translocon [18]. By
contrast, only eukaryotic homologues have been found
for Tic110.
However, Tic20 and Tic110 also display some simi-
lar features. For example, tissue analysis in Arabi-
dopsis thaliana indicated that both proteins can be
detected throughout the plant and that expression does
not appear to be restricted to photosynthetic tissue,
even though absolute expression levels appear to be
much lower for Tic20 than for Tic110 [13,19]. When
expression was silenced by antisense or completely
abolished using a T-DNA knockout, both mutants
exhibit severe phenotypes in A. thaliana [13,19,20].
Tic110 was shown to be essential for chloroplast
J. P. Benz et al. Translocation across the outer chloroplast membrane
FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1167
biogenesis and embryo development. In addition, it
displays a rare semi-dominant phenotype because
plants with a heterozygous knockout are already
clearly affected [13]. Antisense plants of the pea ortho-
log and main Arabidopsis isoform of Tic20, AtTic20-I,
similarly exhibit pronounced chloroplast defects, and
attic20-I knockouts were albino even in the youngest
parts of the seedling [19,20]. The presence of at least
one other Tic20 isoform (AtTic20-IV) may prevent
attic20-I plants from lethality. Two more isoforms
have been detected in Arabidopsis, which, however, do
not possess a predicted transit peptide (Table 1) [18].

Furthermore, chloroplasts from attic20-I antisense
plants, as well as from heterozygous attic110, were
Fig. 1. Schematic overview showing the predicted functional domains and topology of all Tic components. Transmembrane domains are
depicted as columns. Regions involved in membrane binding are coloured in red, motifs involved in protein–protein interaction are blue and
the dehydrogenase domains of Tic32 and Tic62 are shown in green. Tic110 contains two transmembrane domains at the proximal N-termi-
nus. The topology of the long C-terminus is still not completely solved. In this model, we tentatively tried to combine several views by add-
ing some transmembrane columns having amphipathic character (indicated by red–white colour marked with a ‘?’). Tic20 and Tic21/PIC1
both belong to the big group of four-transmembrane domain proteins. Topology-predictions indicate an N
in
/C
in
orientation. Tic62 belongs to
the extended family of SDRs and can be divided in two distinct modules. The N-terminus contains the dehydrogenase domain (green) and
might mediate membrane binding via a hydrophobic patch on the surface of the protein, whereas the C-terminus features a series of Pro/
Ser-rich repeats (blue) that allow specific binding of FNR. Tic22 is a soluble protein located in the intermembrane space (IMS) with no func-
tional domains known so far. Tic55 is a Rieske [2Fe-2S]-centre containing oxidoreductase with an additional mononuclear iron binding site
(both in brown) and two transmembrane helices at the C-terminus. The conserved cysteine pair (CXXC) possibly involved in regulation by
thioredoxins is indicated. The SDR Tic32 contains an NADPH binding site and the active site motifs characteristic for SDRs (green). A CaM
binding site was located in the extreme C-terminus (blue). Tic40 consists of an N-terminal transmembrane domain and a soluble C-terminus
protruding into the stroma. Conserved regions of the C-terminus are the TPR domain, consisting of seven predicted a-helices (blue), and the
Sti1-like Hip/Hop domain at the extreme C-terminus (yellow), involved in activation of Hsp93.
Translocation across the outer chloroplast membrane J. P. Benz et al.
1168 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS
demonstrated to be defective in preprotein import
across the IEM [13,20].
Based on these similarities, the hypothesis was pro-
posed that Tic20 and Tic110 could both dynamically
associate to co-operate in channel formation [10]. The
only real biochemical indication for this suggestion
shows that a minor fraction of Tic110 (approximately

5%) could be coeluted with Tic20 (and Tic22) in a
Toc–Tic supercomplex [21]. However, no coelution
was detected in the absence of the Toc complex,
making a direct or permanent interaction unlikely.
In summary, both Tic20 and Tic110 are clearly
important for plant viability and preprotein transloca-
tion, but only for Tic110 do the electrophysiological
and biochemical data indicate direct channel activity
as well as involvement in the import motor complex
(see below). Similar data for Tic20 are still missing,
but it can be speculated that either various translocons
exist, or that Tic20 exhibits a different kind of protein
translocation activity, which is possibly analogous to
the inner membrane of mitochondria, where the
Tim23/Tim17 and Tim22 channels exist in parallel,
each responsible for translocation of a different subset
of precursors [6].
Recently, another protein with four predicted trans-
membrane-domains, similar to Tic20, was identified as
a third putative translocon component and named
CIA5/Tic21 (Fig. 1) [19]. The phenotype of attic21
plants resembled that of attic20-I, but the affiliation
with the Tic complex was questioned by a second
Fig. 2. Schematic illustration of the Toc and Tic chloroplast import machineries with focus on the components involved in preprotein translo-
cation at the IEM. Individual Tic components are labelled with their respective names and some key functional domains are additionally indi-
cated (Tic40 and Tic62); Toc components are not labelled. The predicted transmembrane domains of Tic40 and Tic55 are shown as small
columns protruding into the IEM. Components of the channel/motor complex are depicted in yellow (Tic110, Tic40 and Hsp93), redox-regula-
tory subunits in blue (Tic62 with associated FNR, Tic55 and Tic32), the proposed alternative import channel Tic20 and the intermembrane
space (IMS) component Tic22 in red and the second involved chaperone Cpn60 in green. A cytoplasmically translated preprotein with an
N-terminal transit peptide is shown during its translocation through the Toc and Tic complexes. Tic22 may be involved in the stabilization of

the Toc/Tic/preprotein supercomplex. In this model, Tic110 forms the channel protein and also acts in the recruitment of Hsp93 in concert
with the co-chaperone Tic40. The TPR domain of Tic40 is considered to mediate the interaction with Tic110, whereas the Sti1-like Hip/Hop
domain was shown to enhance the ATPase activity of the chaperone Hsp93. The motor activity of this AAA+ ATPase probably accounts for
most of the ATP requirement of the import reaction, exerting the pulling force on the incoming precursor. The SPP is thought to act very
early after the preprotein emerges from the Tic channel, and Cpn60 (a GroEL-homologue) is probably involved in folding of the processed
precursor. The association of the redox-sensing regulatory subunits Tic62 (with the FNR bound to the C-terminus) and Tic32 appears to be
quite dynamic (double arrows). It is not known whether this is also true for the Rieske protein Tic55, but a similar behaviour is assumed in
this model.
J. P. Benz et al. Translocation across the outer chloroplast membrane
FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1169
study demonstrating that the same gene locus does not
encode a protein conducting channel, but instead an
iron permease (PIC1) [22].
Tic motor function
Early chloroplast import studies demonstrated that
cytoplasmically synthesized preproteins are imported
into the organelle in an ATP-dependent process [23].
By contrast to mitochondria, the energy is not used to
generate a membrane potential for driving the import
reaction, but exerts its effect on a stromal ATPase with
a different function [24]. Chaperones subsequently
have been the main candidates for this ATPase activity
and, indeed, members of the Hsp60 [chaperonin
(Cpn)60] and Hsp100 (Hsp93) families have been
found to interact with the Tic translocon [7,8,12]. To
date, no involvement of Hsp70s or Hsp90s with pre-
protein import has been reported, although both have
homologues present in the chloroplast stroma. This is
somewhat surprising, given that the analogous motor
of mitochondria relies solely on the activity of an

Hsp70 [6,25,26].
Cpn60 (60 kDa), a homologue to bacterial GroEL,
was the first chaperone demonstrated to specifically
co-immunoprecipitate with Tic110 in an ATP-depen-
dent manner [12]. However, analysis of the interaction
between Tic110, Cpn60 and imported preprotein
revealed that only the interaction with the mature form
is ATP-dependent and thus mediated by Cpn60. This
suggests that Tic110 serves in the recruitment of the
chaperonin, which then acts in the folding of the pro-
cessed protein.
All subsequent studies indicated that it is actually
the ternary complex of Tic110, Tic40 and Hsp93/ClpC
that comprises the import motor at the IEM of chlo-
roplasts (Fig. 2). All three proteins function at approx-
imately the same (late) stage of the import process
[27]. Genetic characterization of double mutants in
Arabidopsis revealed non-additive interactions (epista-
sis) amongst the respective knockout mutations,
providing additional support for this functional
co-operation [28].
The involvement of the AAA+ family ATPase
Hsp93/ClpC in preprotein translocation is interesting
because it also acts in intracellular degradation and
substrate turnover, which it performs in association
with its proteolytic counterpart ClpP [29,30]. Neverthe-
less, Hsp93/ClpC was also shown to display intrinsic
chaperone activity [31] and thus appears to be capable
of performing several tasks in the chloroplast, which
are probably dependent on the suborganellar compart-

ment (stromal versus membrane-tethered) and the
respective interaction partners.
Subsequent to the initial demonstration of a specific,
ATP-dependent association of Hsp93 with Tic110 and
incoming precursor [7,8], considerable progress has
been made, especially concerning the role of Tic40 and
Hsp93 in the motor complex [27,28,32–34] and the
possible order of events [35].
Tic40 is an integral membrane protein containing
a single transmembrane span within its extreme
Table 1. Components implicated with the Tic complex, their Arabidopsis isoforms, and the proposed function.
Tic component
Isoforms in
A. thaliana (AGI) Proposed function in the Tic complex Selected references
Tic110 AtTic110 (At1g06950) Channel protein; chaperone recruitment in motor complex [10–15,28,33,57]
Tic62 AtTic62 (At3g18890) Redox regulation: sensing of NADP
+
/NADPH ratio [47,51,53]
Tic55 AtTic55 (At2g24820) Redox regulation; possibly regulated by thioredoxins [48,49,58]
Tic40 AtTic40 (At5g16620) Co-chaperone in motor complex; Hsp93 activator; timing
device
[27,28,32,34,35,59,60]
Tic32 AtTic32-IVa (At4g23430)
AtTic32-IVb (At4g23420)
AtTic32-IVc (At4g11410)
Redox regulation: sensing of NADP
+
/NADPH ratio; site of
Ca
2+

/CaM regulation
[46,61]
Tic22 AtTic22-IV (At4g33350)
AtTic22-III (At3g23710)
Intermembrane space complex (with Toc12, imsHsp70
and Toc64)
[21,62–64]
Tic21/PIC1 AtTic21/AtPIC1
(At2g15290)
Channel protein; Fe-permease [19,22]
Tic20 AtTic20-I (At1g04940)
AtTic20-IV (At4g03320)
AtTic20-V (At5g55710)
AtTic20-II (At2g47840)
Channel protein [19–21,65]
Hsp93 (ClpC) AtHsp93-V (At5g50920)
AtHsp93-III (At3g48870)
ATPase in motor complex [7,8,28,32,33,35]
Translocation across the outer chloroplast membrane J. P. Benz et al.
1170 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS
N-terminus, anchoring it in the IEM, whereas the
C-terminus of the protein projects into the stroma
(Fig. 1) [34]. Two motifs can be located in the C-termi-
nal half of the stromal domain: (a) the last approxi-
mately 60 amino acids are weakly similar to a
conserved motif of the mammalian co-chaperones
Hsp70-interacting protein (Hip) and Hsp70/Hsp90-
organizing protein (Hop) and (b) the region immedi-
ately preceding this domain is predicted to form a
structure similar to a tetratricopeptide repeat (TRP)

motif. Hip and Hop play regulatory roles in Hsp70
and Hsp90 cycles [36–39] and, interestingly, the yeast
Hop homologue Sti1p was also shown to associate
with Hsp104, which is a member of the Hsp100 family
[40]. TPR domains are degenerate 34-amino acid
repeats forming anti-parallel a-helices known to be
involved in an array of protein–protein interactions
(generally with non-TPR proteins) [41]. Both domains
are very characteristic for co-chaperones.
Using various Tic40-deletion constructs in an
attempt to complement the pale green and slow
growing tic40 knockout phenotype, it could be
shown that the C-terminal Hip/Hop (Sti1-like)
domain, as well as the N-terminal transmembrane-
helix and a central region including the putative
TPR motifs, is essential for correct protein activity.
Only the full-length cDNA clone was able to reverse
the phenotype to wild-type growth [32]. A more
detailed characterization of the single domains pro-
vided valuable insight into the possible functional
role of Tic40: the Sti1-like region of Tic40 was
shown to be functionally equivalent to the Sti1
domain of human Hip, corroborating the role of
Tic40 as a bona fide co-chaperone [32]. Additionally,
in in vitro assays using overexpressed Hsp93 and var-
ious Tic40 deletion constructs, the same domain was
found to stimulate the ATPase activity of the chap-
erone [35]. Because this stimulating effect was only
visible with the Hip/Hop-domain alone and not with
the entire stromal domain including the TPR motifs,

it was hypothesized that the protein exists in a
closed conformation, in which the TPR domain
shields the Hip/Hop-domain from the chaperone.
Surprisingly, the TPR motifs themselves appear to
mediate the interaction with Tic110 and not with the
chaperone partner (Hsp93), which is in contrast to
the function of these motifs in Hop and Hip
[40,42,43]. Interestingly, binding of Tic40 to Tic110 is
favoured when the transit peptide-binding site of
Tic110 is occupied by incoming preprotein, but inter-
action with Tic40 appears to decrease the affinity of
Tic110 for the transit peptide, which is subsequently
released and therefore accessible for processing by
the SPP and interaction with Hsp93 [35]. Conforma-
tional changes occurring upon binding of Tic40 to
Tic110 presumably also open the Hip/Hop-domain of
Tic40, allowing it to stimulate the motor activity of
Hsp93.
Obviously, the import motor is still functional in the
absence of Tic40 because tic40 knockout plants are
viable, even though the plants are very pale [27]. In
addition, dominant-negative phenotypes could be
observed in some Tic40 complementation lines, indi-
cating that the overexpressed deletion-constructs inter-
fered with some residual motor activity [32]. Thus,
Tic40 clearly enhances the operational efficiency of the
complex and was proposed to function as a timing
device, co-ordinating the sequential steps of transloca-
tion (Fig. 2) [32,35].
The function of the ATPase Hsp93 in protein import

was further analyzed using the characterization of
Arabidopsis knockout mutants [28,33]. In Arabidopsis,
two homologues of Hsp93 exist (Hsp93-III and Hsp93-
V), sharing high (approximately 91%) sequence iden-
tity. Hsp93-V is thought to be the main isoform with a
several-fold higher expression rate than Hsp93-III.
Nevertheless, some degree of redundancy appears to
exist among both proteins because the mildly chlorotic
hsp93-V knockout phenotype can be complemented by
overexpression of the other isoform. However, analysis
of double knockouts of both Hsp93 homologues did
not result in the identification of double homozygotes,
establishing that Hsp93 function is essential for viabil-
ity, just as is the case for Tic110 [28,33]. This observa-
tion indicates that Hsp93 and Tic110 are of similar
importance for the organelle.
Another finding concerns the significance of the
Hsp93-related motor activity on the overall import of
preproteins through both envelope membranes. It is
known that Tic110 and Hsp93 are constituents of
Toc–Tic supercomplexes that are associated with pre-
cursor protein [7,8]. Therefore, it could be possible that
the ATPase activity of Hsp93 exerts a pulling effect
also at the level of the OEM, similar to the situation
in mitochondria. When performing import experiments
with tightly folded as well as unfolded preprotein in
viable hsp93-III/-V double-mutant (knockdown)
chloroplasts, the use of an unfolded preprotein did
not alleviate the decreased import efficiency in
hsp93-III/-V (and tic40) plants. This implies that the

rate-limiting step for protein import in the mutant
chloroplasts is not precursor unfolding [33,44] and
could be interpreted as an indication for separate
unfolding forces (and thus motor activities) in the
outer and inner membranes of the chloroplast envelope
during preprotein import.
J. P. Benz et al. Translocation across the outer chloroplast membrane
FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1171
Possible ways of regulation
As outlined above, a great amount of protein traffic
has to take place at the envelope membranes of chlo-
roplasts, which has to be tightly regulated to ensure
that the supply correlates with the demand of the orga-
nelle at any given time. Logically, translocation across
the envelope is surely a bottleneck in the path of trans-
ported proteins from the cytosol to their final destina-
tion in the chloroplast. The Tic and Toc translocons
are therefore perfectly situated to impose a regulatory
control over incoming preproteins. Additionally,
because the demand of the chloroplast is ‘sensed’
inside the organelle, the IEM is closest to the origin of
the signal, and thus regulation at the Tic complex
could be one of the fastest ways to react efficiently.
To our current knowledge, at least two types of sig-
nals convene at the Tic complex: (a) the stromal
NADP
+
/NADPH ratio sensed via Tic62 and Tic32,
giving information about the metabolic state of the
chloroplast and (b) a calcium signal, which is mediated

by a still elusive chloroplast calmodulin (CaM), associ-
ated with Tic32 (Fig. 3).
Redox regulation
Redox regulation is long known to play a prominent
role in the chloroplast metabolism, and also at least
two preproteins (the nonphotosynthetic ferredoxin
FdIII and the ferredoxin-NADP
+
-oxidoreductase iso-
form II of maize) were demonstrated to be differen-
tially imported in the light compared to the dark [45].
Diurnal changes in the thylakoids or, more generally,
the stromal redox system (e.g. the NADP
+
/NADPH
pool) thus appear to have an impact on the import
characteristics of the organelle. It is therefore not sur-
prising to find proteins with redox-active domains as
Tic constituents. Up to now, the ‘regulon’ of the Tic
complex comprises three proteins: Tic62, Tic32 and
Tic55. The former two proteins belong to the
(extended) family of short-chain dehydrogenases/reduc-
tases (SDRs) and have already been demonstrated to
possess dehydrogenase activity in vitro [46,47]. Less is
known about the redox properties of Tic55. Sequence
analysis revealed the presence of a Rieske-type [2Fe-
2S] cluster and a mononuclear iron-binding site [48].
Database research classifies Tic55 as a member of the
chlorophyll a oxygenase/pheophorbide a oxygenase-
like oxygenases, which act for example in chlorophyll

biogenesis or oxygen-dependent degradation pathways.
Rieske proteins generally play important roles in elec-
tron transfer (e.g. in the cytochromes present in the
respiratory chain of mitochondria or in the thylakoids
of chloroplasts). Whether Tic55 acts as an oxygenase
in vitro or in vivo has not been studied to date, but the
close proximity of the Rieske protein Tic55 and the
two bona fide dehydrogenases Tic32 and Tic62 at
the Tic complex holds the intriguing possibility of a
Fig. 3. Schematic model of the proposed regulatory signals sensed by the Tic complex and their effect on the involved subunits. Three sig-
nals are thought to convene at the Tic complex: (1) information about the chloroplast metabolic redox state, represented by the stromal
NADP
+
/NADPH ratio and sensed by the two dehydrogenases Tic62 and Tic32; (2) a calcium signal, mediated by a still unknown plastidic
CaM or CaM-like protein binding to Tic32; and (3) a second redox-related signal, in which a stromal thioredoxin interacts with a conserved
cysteine pair (CXXC) of the Rieske protein Tic55. The redox state of the NADP
+
/NADPH pool was demonstrated to have a drastic effect on
the association of Tic62 and Tic32 with the Tic complex. Both components dissociate from the complex at high NADPH concentrations.
Tic62 was shown to reversibly shuttle between the stroma the IEM dependent on the NADP
+
/NADPH ratio. For Tic32, a similar relocaliza-
tion as for Tic62 is assumed in this model.
Translocation across the outer chloroplast membrane J. P. Benz et al.
1172 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS
small electron transfer chain being present at the Tic
translocon [47]. In addition, a very recent study identi-
fied Tic55 as a target of stromal thioredoxins (Trx) in
barley chloroplasts [49]. Trxs are small ubiquitous pro-
teins with redox-active disulfide bridges that regulate

enzyme activities (e.g. in the Calvin cycle or the oxida-
tive pentose phosphate cycle) by dithiol oxidoreduction
of their target proteins [50]. Necessary for this reaction
is a conserved pair of cysteines, which can be detected
in Tic55 (CXXC motif; Fig. 1). However, no further
conclusion about how the oxidoreduction affects Tic55
function in the Tic complex could be drawn from this
analysis.
Investigation of the Tic complex under changing
redox conditions revealed a high degree of dynamics.
For example, addition of NADPH leads to dissocia-
tion of the two dehydrogenases Tic32 and Tic62 from
the complex, indicating that the metabolic state of the
organelle appears to have a profound influence on Tic
composition [46]. Further studies with Tic62 corrobo-
rated this finding and revealed that this protein shut-
tles between the chloroplast membrane compartment
and the stroma dependent on the stromal NADP
+
/
NADPH ratio [47] (Fig. 3). Oxidizing conditions lead
to fast membrane binding and integration into the Tic
complex. Reducing conditions on the other hand lead
to solubilization into the stroma and increased interac-
tion with its other known interaction partner ferre-
doxin-NADP
+
-oxidoreductase (FNR). Interestingly,
this membrane binding was found to be reversible, and
is assumed to be mediated by a hydrophobic patch on

the protein surface, located in the N-terminal half of
the protein, including the dehydrogenase domain. Spe-
cific binding of the FNR is mediated by a unique series
of proline/serine-rich repeat motifs located in the
C-terminus. For the integration into the Tic complex
finally, a central region of the protein was shown to be
sufficient, which contains parts of both, the N-termi-
nus and C-terminus (Fig. 1). These results demonstrate
that Tic62 is able to react very sensitively to redox
changes in the chloroplast stroma and that it adjusts
its localization accordingly. These features would allow
it to fulfil its proposed role as a redox-sensor protein
in the chloroplast [47,51]. How exactly changes in the
redox state of the chloroplast affect the translocation
is not yet known, but it has been suggested that the
dynamic Tic composition could influence the import
characteristics of a certain subset of preproteins, which
might also act in redox-dependent pathways [47].
The reason for the strong association of Tic62 with
the FNR still remains one of many open questions.
Because flavin-containing proteins have already been
described to be present in redox chains in chloroplast
envelope membranes [52], one possibility is the recruit-
ment of FNR from the stroma or even thylakoids to
the Tic complex in order to become part of the hypo-
thetical electron transfer chain mentioned above. How-
ever, the involvement of the FNR appears to be an
evolutionary young mode of regulation. This notion
derives from an extensive database analysis of the
Tic62 protein looking for homologues in other

sequenced organisms [53]. It was found that the N-ter-
minal half of the protein, comprising the dehydro-
genase domain, is highly conserved in all
oxyphototrophs, and homologues can be found even
in green sulfur bacteria. The C-terminus, containing
the FNR binding repeats, on the other hand, is present
only in higher plants. This C-terminal extension there-
fore appears to have been added only recently in evo-
lution, which could make Tic62 one of the youngest
Tic constituents.
Ca
2+
/CaM regulation
Calcium is a common secondary messenger that regu-
lates many biochemical processes (e.g. relaying envi-
ronmental signals to various cellular response
pathways). This is generally achieved through binding
to calcium sensing proteins such as CaM, which subse-
quently change their affinities to downstream target
proteins, leading to further responses [54,55]. Even
though regulation by calcium/CaM is considered to be
a eukaryotic trait, import analyses into chloroplasts
could demonstrate that organellar processes have been
integrated into the calcium signalling network of the
cell [56]. Calcium ionophores as well as the CaM-
inhibitor ophiobolin A affected the translocation of
preproteins containing a cleavable N-terminal transit
peptide. This indicates that: (a) the general Toc/Tic
pathway is involved in calcium regulation and (b) a
CaM or CaM-like protein is the most likely mediator

of this regulation. In an attempt to isolate CaM-bind-
ing proteins, Tic32 was identified as the only IEM
protein specifically interacting with CaM in a calcium-
dependent manner, corroborating the idea that the Tic
complex is the site of calcium regulation (Fig. 3). Fur-
ther binding assays employing several Tic32-deletion
constructs allowed the localization of the CaM-binding
site to the 26 most C-proximal amino acids (Fig. 1).
This region was predicted to form a basic amphipathic
helical structure characteristic for CaM-binding
domains, and contains at least one conserved potential
CaM-binding motif [46]. Additionally, the binding of
CaM at the C-terminus and the binding of NADPH at
the extreme N-terminus appear to be mutually exclu-
sive, suggesting that two different signalling pathways
J. P. Benz et al. Translocation across the outer chloroplast membrane
FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1173
convene at Tic32 and are integrated at the Tic
complex.
Conclusions
Increasing evidence is accumulating to suggest that we
experience not only the one Tic complex, but also that
the composition and activity of the Tic machinery can
be adapted (regulated). Distinct regulatory circuits
might sense distinct organellar requirements via: (a) a
Ca
2+
/CaM; (b) a metabolic NADP
+
/NADPH; or (c)

an environmental Trx mediated signal. These signals,
either alone or in combination, could influence the
import of preproteins. A prominent and difficult task
for future studies will therefore be to determine how
organelle metabolism and physiology influences protein
import by the Tic complex and by the Toc–Tic trans-
locon as a whole.
Acknowledgements
We would like to thank our colleagues from the labo-
ratory for helpful discussions, and especially Anna
Stengel for critical reading of the manuscript. Finan-
cial support was provided by the Deutsche Fors-
chungsgemeinschaft Grant SFB594 and the Elite
Network of Bavaria (to J. P. Benz).
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