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,

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.

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-

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].

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

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-

/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)

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

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

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

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

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)

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