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471
Protein import into chloroplasts
Kenneth Keegstra* and John E Froehlich
Three proteins from the chloroplastic outer envelope
membrane and four proteins from the inner envelope
membrane have been identified as components of the
chloroplastic protein import apparatus. Multiple molecular
chaperones and a stromal processing peptidase are also
important components of the import machinery. The
interactions of these proteins with each other and with the
precursors destined for transport into chloroplasts are
gradually being described using both biochemical and genetic
strategies. Homologs of some transport components have
been identified in cyanobacteria suggesting that at least some
of import machinery was inherited from the cyanobacterial
ancestors that gave rise to chloroplasts.
Addresses
MSU-Department of Energy Plant Research Laboratory, Michigan State
University, East Lansing, MI 48824-1312, USA
*e-mail: keegstra@pilot.msu.edu
Current Opinion in Plant Biology 1999, 2:471–476
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
Tic
translocon at the inner membrane of chloroplasts
Toc
translocon at the outer membrane of chloroplasts
Introduction
Because chloroplasts and other types of plastids contain a
genome with a limited coding capacity, the large majority
of proteins contained within these organelles must be
imported from the cytoplasm. The chloroplastic protein
import system has received considerable attention since
the first reports of its existence some 20 years ago [1,2].
This brief review focuses on recent progress in the identification and characterization of components of the
envelope-based transport apparatus. We briefly consider
interactions between these components and precursor proteins that occur during protein import, and we address the
evolutionary origins of the import apparatus.
Figure 1 presents a schematic representation of the protein
import apparatus and a working model of the interactions
that occur during the transport of precursor proteins into
chloroplasts. This schematic model is based on work from
several research groups over the past few years; other
reviews should be consulted for details and for a consideration of earlier work [3–6]. Components in the outer
envelope membrane are known as Toc (translocon at the
outer membrane of chloroplasts) proteins, whereas components in the inner membrane are called Tic (translocon at
the inner membrane of chloroplasts) proteins; the number
refers to their molecular mass [7]. Molecular chaperones are
also involved during protein import and putative participants have been identified in several locations including
the cytoplasm, the intermembrane space between the two
envelope membranes, and the stromal space.
Energy, provided by the hydrolysis of nucleoside triphosphates, is needed for at least three, and possibly more,
steps during the import process. Hydrolysis of GTP is a
unique requirement for chloroplastic protein import
[8••,9•]; GTP is not needed for mitochondrial protein
import [10]. The chloroplastic import apparatus contains
two components that have GTP-binding sites, Toc159 and
Toc34, and it is assumed that they account for the GTP
requirement. ATP is needed in at least two locations: the
early stages of import require ATP hydrolysis in the intermembrane space (Figure 1c) [11], and ATP hydrolysis is
required in the stroma to accomplish protein translocation
(Figure 1d) [12]. In each case, it is postulated, but not
proven, that this ATP is used by molecular chaperones.
Outer membrane components and the early
stages of protein import
The Toc complex fulfills three essential functions during
protein import: first, it specifically recognizes the transit
peptide of precursor proteins; second, after precursor protein binding, the complex initiates membrane
translocation; and third, it participates in the formation of
contact sites between inner and outer chloroplastic membranes. Evidence from several laboratories indicates that
the Toc complex contains three membrane proteins:
Toc159, Toc34, and Toc75 [13–15]. In addition to the Toc
proteins, lipids have also been thought to play a crucial role
in the early stages of precursor binding to chloroplasts. For
instance, transit peptides have been shown to interact with
the chloroplastic-specific lipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol [16,17]. From these
studies it was proposed that the early stages of precursor
targeting to the chloroplasts involved a specific interaction
of the transit peptide with the lipids of the outer envelope
membrane (Figure 1a). Recently, this hypothesis gained
further support when Chen and Li [18•] used a genetic
approach to dissect the early stages of precursor binding to
chloroplasts. They showed that chloroplasts isolated from
an Arabidopsis mutant deficient in digalactosyldiacylglycerol are defective in their ability to import precursors
destined for the interior of chloroplasts. The defect in
these chloroplasts appears to occur at the energy-independent binding stage of import.
After an initial interaction of precursor with the lipids of
the outer envelope membrane [16,17], the precursor interacts with both Toc159 and Toc34 during the
energy-independent stage of binding [19,20] (Figure 1b).
Both of these proteins contain GTP-binding domains
[21–23]. Toc159 was identified originally as an 86 kDa protein [21,22], designated Toc86; however, recent studies
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Figure 1
Precursor protein
(a)
(b)
(c)
(d)
Hsp
70
159
75 34
159
34
22
34
159
Hsp
22 70
22
20
55
20
110
Hsp93
OM
20
110
IM
110
Hsp93
Hsp93
Hsp
70
SPP
Hsp
60
Hsp93
Current Opinion in Plant Biology
have concluded that Toc86 is a proteolytic fragment of the
native 159 kDa protein [8••,24]. It has been proposed that
Toc159 contains a transit peptide binding site for precursor
proteins. There are two pieces of evidence for this role of
Toc159: antibodies against Toc159 block precursor binding
to isolated chloroplasts [21], and two different precursor
proteins can be cross-linked effectively to Toc159 [14,19].
The role of Toc34 during the energy-independent stage of
precursor protein binding to the Toc complex has been difficult to determine. Recent experiments show that Toc34
can be cross-linked to precursor protein during the early
stages of binding [19]. But the presence of either GTP or
ATP prevents the formation of cross-linking to Toc34. The
authors conclude that Toc34 is not a part of the transit peptide receptor but rather plays a regulatory role in precursor
binding [19].
The third component of the Toc complex that interacts with
precursor protein during the early stages of binding is Toc75
[25]. The transit peptide of precursor proteins can be crosslinked to Toc75 [14,20], thus confirming a close interaction.
Taken together, the cross-linking data support the hypothesis that Toc75 and Toc159 form a binding site that
specifically binds transit peptides. Considerable evidence
indicates that Toc75 also constitutes the major component of
the protein-conducting channel in the outer envelope membrane. The most compelling evidence comes from Hinnah et
al. [26], who showed that recombinant Toc75 has voltagesensitive ion conductance properties when reconstituted into
Stroma
A schematic working model for protein import
into chloroplasts. The numbers for the
membrane components and the molecular
chaperones (Hsps) represent their molecular
mass. Further details about these components
are given in the text. The location for the
utilization of GTP is highly speculative, but the
locations for the hydrolysis of ATP are based
upon experimental evidence that is further
described in the text. Stage (a) represents a
hypothetical step where the transit peptide
interacts only with the lipids of the envelope
membrane. Stage (b) depicts a reversible step
where the transit peptide also interacts with
proteins of the transport apparatus, but prior to
the hydrolysis of nucleoside triphosphates.
Stage (c) depicts an early translocation
intermediate, where precursor is irreversibly
associated the transport apparatus, but still
sensitive to exogenously added protease.
Stage (d) depicts the final steps of protein
transport, processing and assembly. The transit
peptide located at the amino-terminus of the
precursor protein is removed by the stromal
processing peptidase (SPP). The differences in
components present in the complexes shown
in stages (b), (c), and (d) is a deliberate attempt
to reflect the dynamic nature of the
composition of the translocation apparatus.
liposomes. The reconstituted Toc75 ion channel was calculated to have a small pore with a diameter of 8–9 Å. Such a
small opening would require that the precursor be completely unfolded during translocation across the outer membrane.
They also demonstrated that this conductance was selectively regulated by the transit peptide of precursor protein,
supporting the role of Toc75 as a transit-peptide-regulated
channel [26]. However, Toc75 may not be the only component comprising the protein-conducting channel of the outer
membrane. Ma et al. [14] have shown that during translocation, precursor proteins can be cross-linked to regions of
Toc159 other than the cytoplasmic domain. Hence, the
membrane domain of Toc159 may participate in the translocation process, possibly as part of the translocation channel.
Although only three integral membrane proteins have been
identified as components of the Toc complex, some of
them are encoded by multiple genes that show differential
expression during plastid development. For example,
Arabidopsis contains two Toc34-like genes that are differentially expressed [27••]. The protein products of these
genes, AtToc33 and AtToc34, have 61% amino acid
sequence identity to each other, and 59% and 64% amino
acid sequence identity with pea Toc34, respectively.
AtToc33 is preferentially expressed during the early stages
of seedling development, whereas AtToc34 is expressed at
constitutively low levels during all stages of leaf development. Mutant plants deficient in AtToc33 are delayed in
plastid development and show a persistent chlorophyll
deficiency; however, they are only partially defective in
Protein import into chloroplasts Keegstra and Froehlich
473
protein import. Either AtToc34 or AtToc33 can complement the mutant, demonstrating the functional redundancy
of the two proteins. This work is important on several
accounts. It is the first application of reverse genetics to the
study of protein import into chloroplasts; furthermore, it
demonstrates that some components of the transport apparatus are encoded by multiple genes and that these genes
are differentially expressed. More work is needed to understand the functional significance of these multiple genes.
using different strategies [29,30], the two disagreed on its
topology within the membrane. Jackson et al. [31] have
reinvestigated this question and concluded that the large
hydrophilic domain of Tic110 faces the stromal side of the
inner membrane. On the basis of this topology, Tic110 is
postulated to interact with stromal molecular chaperones
during protein import, possibly in a manner similar to the
way in which Tim44 interacts with Hsp70 in the mitochondrial matrix [10].
Although a role for GTP in protein import had been reported [11], the discovery that both Toc159 and Toc34 contain
GTP-binding domains [21–23] required a re-evaluation of
the role for GTP during import [8••,9•]. In the first stage,
the formation of early translocation intermediates is mediated by Toc components that bind and hydrolyze nucleoside
triphosphates. Initial interaction of the transit peptide
occurs at Toc159, with the possibility of some involvement
of Toc75. Further insertion of precursor protein into the
import apparatus requires GTP hydrolysis. GTP hydrolysis
at Toc159 and/or Toc34 may induce a conformational change
in the overall Toc complex that induces the precursor to
insert itself across the outer membrane through the proteinconducting channel. Therefore, two roles have been
proposed for the function of GTP during import. In the first,
GTP acts as a regulator for early translocation intermediate
formation. In this role, Toc159 and/or Toc34 may regulate
gating of the protein-conducting channel, thus ensuring the
specificity of precursor binding. Through a cycle of GTP
binding and hydrolysis, Toc159 and/or Toc34 may act as a
switch to ensure that precursors are committed to further
translocation. In the second role, GTP binding may initiate
the formation of contact sites, thus forming functional
translocation complexes. As the import apparatus is reconstituted, using purified components, future investigation
will begin to focus on the separate roles of Toc159 and
Toc34 during the early stages of binding.
Tic55, an iron-sulfur-containing protein of the inner envelope membrane, was identified as a translocation
component based primarily on its comigration with other
translocation components during blue-native polyacrylamide gel electrophoresis [32]. However, others have not
found this protein in transport complexes isolated by different techniques [19,33••], so more work is needed to
confirm that this protein is part of the import apparatus. If
Tic55 is confirmed to be a translocation component, it will
be interesting to determine what role, if any, the redox
center has during import.
GTP hydrolysis is required for early import intermediate
formation, but it is not sufficient for complete translocation
across the outer membrane. The stability of early translocation intermediates is enhanced by ATP at low levels
[11]. The presence of Hsp70 at the inner surface of the
outer membrane may explain the need for ATP during
early translocation intermediate formation. Young et al. [9•]
have further shown that GTP has no effect on the translocation steps that occur after the early import intermediate
has been formed (Figure 1c,d). From this study, they conclude that once the early import intermediates are formed,
ATP hydrolysis, not GTP hydrolysis, mediates translocation. This ATP is most probably utilized by stromal
chaperones thought to be involved in transport across the
chloroplastic envelope membranes [13,15,28].
Inner membrane components and the
formation of contact sites
Although Tic110, the first inner membrane component to
be described, was identified independently by two groups
Kouranov and Schnell [19] identified Tic22 and Tic20, two
inner envelope membrane proteins, as translocation components using a chemical cross-linking strategy. More
recently, Kouranov et al. [33••] isolated cDNA clones
encoding Tic22 and Tic20, provided preliminary characterization of these proteins, and offered hypotheses
regarding their role in protein import. Tic22 is a peripheral membrane protein associated with the outer surface of
the inner envelope membrane. It does not contain readily
recognizable sequence motifs, such as nucleotide binding
domains, and does not possess sequence similarity to components of other transport systems; it does, however,
possess 19% sequence identity over 176 amino acid
residues with an open reading frame in the cyanobacterium Synechocystis 6803 genome [34••]. Tic20 is an integral
membrane protein that is predicted to span the inner envelope membrane via three hydrophobic helical domains.
Proteolytic digestion studies provide evidence that the
amino-terminal domain, which is very basic, extends into
the stroma, whereas the carboxy-terminal hydrophilic
domain extends into the intermembrane space [33••].
As noted earlier, Tic22 and Tic20 were first identified as
translocation components by their ability to be cross-linked
to precursors during import. Kouranov et al. [33••] also used
immunoprecipitation of detergent-solubilized envelope
membranes to demonstrate that a small proportion of Tic22
and Tic20 were present in complexes that also contained
Tic110, Toc159, Toc75 and Toc34. Interestingly, they could
not find complexes of Tic22 and Tic20 with Tic110, suggesting that the inner membrane components do not form
complexes in the absence of association with the Toc complex to form putative contact sites. Kouranov et al. [33••]
speculate that Tic20 may serve as part of the proteintranslocating channel in the inner envelope membrane,
whereas Tic22 may recognize precursor proteins as they
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Cell biology
enter the intermembrane space and direct them to the
inner membrane import machinery. In this role, Tic22 may
be the factor that connects the two envelope membranes,
causing the formation of contact sites. If this is the case, one
important unsolved question is what factors regulate the
ability of Tic22 to mediate the apparently dynamic interaction between the components from the inner and outer
envelope membranes.
Molecular chaperones and the translocation
process
By analogy with mitochondrial protein import where a
matrix Hsp70 supports transport across the inner membrane [10], a stromal Hsp70 [35] was an obvious candidate
for this role in chloroplasts; however, the stromal Hsp70
could not be found in translocation complexes [13,15].
Rather, translocation complexes contain Hsp93, a stromal
protein in the Hsp100 family of molecular chaperones [36].
This observation has been confirmed by the recent studies
of Kouranov et al. [33••]. Moreover, both groups find evidence of an association between Hsp93 and Tic110,
suggesting that these two proteins may have roles similar
to that suggested for Hsp70 and Tim44 in mitochondria:
providing the driving force for pulling proteins into the
matrix [10]. Kessler and Blobel [29] also found Hsp60 associated with translocation complexes via an association with
Tic110, a result that was confirmed by Kouranov et al.
[33••]. Defining the role of each of the chaperones in protein translocation will require additional studies. However,
it is tempting to speculate that Hsp93, and possibly Hsp70,
provide the driving force for translocation across the inner
envelope membrane, whereas Hsp60 is involved in folding
the protein following its translocation in an unfolded state.
Evolutionary origins of the protein
import apparatus
It is widely accepted that chloroplasts evolved from
cyanobacteria following an endosymbiotic event. As bacterial genes were transferred into the nucleus, a system was
needed for delivering the proteins back into the evolving
organelle. The origins of this protein transport system poses
interesting questions regarding the early evolution of
chloroplasts. Bölter et al. [41••] and Reumann et al. [42••]
provided evidence that at least one modern translocation
component, that is Toc75, is related to a cyanobacterial
outer membrane protein. Reumann et al. [42••] argued that
this cyanobacterial protein is related to a family of outer
membrane proteins of unknown function whose members
are widespread in the Gram-negative bacteria. This family,
in turn, is related to another group of proteins involved in
secretion from Gram-negative bacteria. Moreover, Bölter
et al. [41••] demonstrated that the cyanobacterial protein is
capable of channel activity. Thus, it seems likely that
Toc75, the putative outer membrane channel of the import
system, evolved from an outer membrane channel involved
in secretion from the free-living cyanobacterium. More
recently, Reumann and Keegstra [34••] presented evidence
that both Tic22 and Tic20 have cyanobacterial homologs,
whereas Toc159, Toc34/33 and Tic110 do not. They conclude that the protein import apparatus has a dual origin,
with some components deriving from cyanobacteria, possibly from an ancient protein secretion mechanism, and other
components having been added to the system from other
sources during the course of evolution.
Conclusions and prospects
An Hsp70 protein present in the intermembrane space has
also been identified as a component of translocation complexes (Figure 1c) [28]. Although this is an ideal candidate
for providing the driving force for transport across the outer
envelope membrane, a cDNA encoding this protein has
not yet been identified. Thus, more work is needed to
determine the role, if any, of this protein in the import
process. Another Hsp70 associated with the surface of
chloroplasts (Figure 1b) has been identified [37,38],
although its role in protein import is also unclear.
Considerable progress has been made in recent years in
identifying the components of the chloroplastic protein
import system. Although it is difficult to know how many
components are required for the import apparatus, it is reasonable to argue that the majority of the transport proteins
have been identified and that relatively few additional
components will be found. However, at least in
Arabidopsis, several components are encoded by a small
family of related genes ([27••]; D Jackson, K Keegstra,
unpublished observations). Thus, it will be important to
determine the functional and developmental significance
of these various family members.
Either during import or immediately afterwards, the transit peptide is removed by a stromal processing protease
(Figure 1d) [39]. The same metalloprotease removes the
transit peptide from many different precursors, including
acting on its own precursor in trans [39]. The importance
of this enzyme for the import process, and consequently
for the overall operation of the plant, is demonstrated by
the recent generation of plants containing an antisense
version of the protease gene and having reduced levels of
the protein. These plants have a severe phenotype,
including chlorotic leaves, reduced growth, a reduced
number of plastids per cell and lower amounts of internal
membranes [40•].
In the immediate future, the major challenge will shift
away from identification of new components to determining the functions of those components shown in
Figure 1 as well as newly discovered components. This
is likely to involve two efforts that have just begun. The
first is in vitro reconstitution of the protein import
process or at least of particular partial reactions. A start
has been made by Hinnah et al. [26], who have reconstituted purified Toc75 into lipid bilayers to show that it
has channel activity. However, it is unlikely that Toc75 is
the only constituent of the channel because the reconstituted activity does not behave like the Toc complexes in
outer envelope membranes. The challenge will be to
Protein import into chloroplasts Keegstra and Froehlich
define various partial reactions and reconstitute them
using purified components.
475
the formation of early-import intermediates, but GTP does not play a role during the translocation of precursors from the intermediate state.
10. Pfanner N, Craig EA, Hönlinger A: Mitochondrial preprotein
translocase. Annu Rev Cell Dev Biol 1997, 13:25-51.
A second important strategy will be the use of genetics or
reverse genetics to suppress or eliminate a particular component, following on the important work of Jarvis et al.
[27••], who identified Arabidopsis plants lacking Toc33. With
the new availability of several strategies for isolating plants
with disruptions in a particular gene of interest, approaches
similar to those employed by Jarvis et al. [27••] for Toc33 will
certainly be applied to other transport components. An alternative is to use the antisense method, as illustrated by the
work of Wan et al. [40•] with plants containing reduced levels of the stromal processing protease. The availability of
reverse genetic approaches and the power of in vitro reconstitution assays should lead to models for the mechanism of
protein import showing much more detail than the rather
crude scheme shown in Figure 1.
Acknowledgements
The authors thank Danny Schnell for making available unpublished
information from his laboratory. Work from the authors’ laboratory was
supported by grants from the Cell Biology Program at the National Science
Foundation and from the Energy Biosciences Program at the Department of
Energy.
11. Olsen LJ, Keegstra K: The binding of precursor proteins to
chloroplasts requires nucleoside triphosphates in the
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•
Young ME, Keegstra K, Froehlich JE: GTP promotes formation of
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••
Jarvis P, Chen LJ, Li HM, Pete CA, Fankhauser C, Chory J: An
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A T-DNA tagged mutant that showed delayed greening led to the identification of Toc33, a translocation component closely related to Toc34. Although
the two genes are functionally interchangeable they have different patterns
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development. Chloroplasts from Toc33 mutant plants have a reduced capac-
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Cell biology
ity to import precursors and at least one precursor, NADPH:protochlorophyllide oxidoreductase (POR), accumulates in vivo in mutant plants.
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33. Kouranov A, Chen XJ, Fuks B, Schnell DJ: Tic20 and Tic22 are new
•• components of the protein import apparatus at the chloroplast
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Expanding upon the label-transfer cross-linking approach of Kouranov and
Schnell [19] and Perry and Keegstra [20], the authors investigated the interaction of a precursor protein with the chloroplastic protein import apparatus.
The authors identified two new components of the import apparatus, Tic22
and Tic20, that are involved in protein translocation across the inner membrane. cDNA clones were isolated for two new import components from the
inner envelope membrane. Tic20 is an integral membrane protein and may
form part of the protein translocating channel in the inner envelope membrane. Tic22 is a peripheral membrane protein associated with the outer surface of the inner envelope membrane and may be involved in the formation
of contact sites.
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•• import machinery from chloroplastic envelope membranes.
Trends Plant Sci 1999, 8:302-307.
Sequence analysis programs were used to identify putative homologs of genes
encoding protein import components in a cyanobacterial genome. Because
not all components have homologs in cyanobacteria, the authors argue that the
import apparatus has a dual origin with some components being inherited from
cyanobacteria and other components arising from other origins, probably during the evolutionary pathway leading to modern chloroplasts.
35. Marshall JS, DeRocher AE, Keegstra K, Vierling E: Identification of
heat shock protein Hsp70 homologues in chloroplasts. Proc Natl
Acad Sci USA 1990, 87:374-378.
36. Schirmer EC, Glover JR, Singer MA, Lindquist S: Hsp100/Cpl
proteins: A common mechanism explains diverse functions.
Trends Biochem Sci 1997, 21:289-296.
37.
Ko K, Bornemisza O, Kourtz L, Ko ZW, Plaxton WC, Cashmore AR:
Isolation and characterization of a cDNA clone encoding a
cognate 70-kDa heat shock protein of the chloroplast envelope.
J Biol Chem 1992, 267:2986-2993.
38. Kourtz L, Ko K: The early stage of chloroplast protein import
involves Com70. J Biol Chem 1997, 272:2808-2813.
39. Richter S, Lamppa GK: A chloroplast processing enzyme functions
as the general stromal processing peptidase. Proc Natl Acad Sci
USA 1998, 95:7463-7468.
40. Wan JX, Bringloe D, Lamppa GK: Disruption of chloroplast
•
biogenesis and plant development upon down-regulation of a
chloroplast processing enzyme involved in the import pathway.
Plant J 1998, 15:459-468.
Plants containing the gene for the chloroplast processing enzyme in an antisense orientation had reduced levels of this protein and reduced levels of
the processing enzyme activity. These plants had severe phenotypes including chlorotic leaves, stunted growth, reduced numbers of chloroplasts and
aberrant chloroplast morphology. Chloroplasts isolated from these plants
had severely reduced ability to import precursors, thereby implicating the
processing enzyme directly in the import process.
41. Bölter B, Soll J, Schulz A, Hinnah S, Wagner R: Origin of a
•• chloroplast protein importer. Proc Natl Acad Sci USA 1998,
95:15831-15836.
A homolog of Toc75 was identified in a cyanobacterial genome. Experimental
evidence demonstrated the cyanobacterial protein, SynToc75, was present in
the outer envelope membrane. Reconstitution of SynToc75 into liposomes
produced a voltage-gated channel. These data plus the data from [42••] provide the first evidence that one of the chloroplastic protein import components was derived from the cyanobacterial ancestor of chloroplasts.
42. Reumann S, Davila-Aponte J, Keegstra K: The evolutionary origin of
•• the protein-translocating channel of chloroplastic envelope
membranes: identification of a cyanobacterial homolog.
Proc Natl Acad Sci USA 1999, 96:784-789.
The authors identified the same cyanobacterial homolog of Toc75 reported
in [41••]. Attempts to disrupt the chromosomal copy of SynToc75 were
unsuccessful, unless the cell contained a plasmid copy of the gene, leading
to the unexpected conclusion that SynToc75 is an essential protein. More
detailed sequence analysis demonstrated that SynToc75 is related to a family of genes found in all Gram-negative bacteria examined to date. Combined
with results from [41••], these data provide the first evidence that at least
portions of the chloroplastic protein import apparatus was derived from a
cyanobacterial ancestor.
Protein import into chloroplasts
Kenneth Keegstra* and John E Froehlich
Three proteins from the chloroplastic outer envelope
membrane and four proteins from the inner envelope
membrane have been identified as components of the
chloroplastic protein import apparatus. Multiple molecular
chaperones and a stromal processing peptidase are also
important components of the import machinery. The
interactions of these proteins with each other and with the
precursors destined for transport into chloroplasts are
gradually being described using both biochemical and genetic
strategies. Homologs of some transport components have
been identified in cyanobacteria suggesting that at least some
of import machinery was inherited from the cyanobacterial
ancestors that gave rise to chloroplasts.
Addresses
MSU-Department of Energy Plant Research Laboratory, Michigan State
University, East Lansing, MI 48824-1312, USA
*e-mail: keegstra@pilot.msu.edu
Current Opinion in Plant Biology 1999, 2:471–476
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
Tic
translocon at the inner membrane of chloroplasts
Toc
translocon at the outer membrane of chloroplasts
Introduction
Because chloroplasts and other types of plastids contain a
genome with a limited coding capacity, the large majority
of proteins contained within these organelles must be
imported from the cytoplasm. The chloroplastic protein
import system has received considerable attention since
the first reports of its existence some 20 years ago [1,2].
This brief review focuses on recent progress in the identification and characterization of components of the
envelope-based transport apparatus. We briefly consider
interactions between these components and precursor proteins that occur during protein import, and we address the
evolutionary origins of the import apparatus.
Figure 1 presents a schematic representation of the protein
import apparatus and a working model of the interactions
that occur during the transport of precursor proteins into
chloroplasts. This schematic model is based on work from
several research groups over the past few years; other
reviews should be consulted for details and for a consideration of earlier work [3–6]. Components in the outer
envelope membrane are known as Toc (translocon at the
outer membrane of chloroplasts) proteins, whereas components in the inner membrane are called Tic (translocon at
the inner membrane of chloroplasts) proteins; the number
refers to their molecular mass [7]. Molecular chaperones are
also involved during protein import and putative participants have been identified in several locations including
the cytoplasm, the intermembrane space between the two
envelope membranes, and the stromal space.
Energy, provided by the hydrolysis of nucleoside triphosphates, is needed for at least three, and possibly more,
steps during the import process. Hydrolysis of GTP is a
unique requirement for chloroplastic protein import
[8••,9•]; GTP is not needed for mitochondrial protein
import [10]. The chloroplastic import apparatus contains
two components that have GTP-binding sites, Toc159 and
Toc34, and it is assumed that they account for the GTP
requirement. ATP is needed in at least two locations: the
early stages of import require ATP hydrolysis in the intermembrane space (Figure 1c) [11], and ATP hydrolysis is
required in the stroma to accomplish protein translocation
(Figure 1d) [12]. In each case, it is postulated, but not
proven, that this ATP is used by molecular chaperones.
Outer membrane components and the early
stages of protein import
The Toc complex fulfills three essential functions during
protein import: first, it specifically recognizes the transit
peptide of precursor proteins; second, after precursor protein binding, the complex initiates membrane
translocation; and third, it participates in the formation of
contact sites between inner and outer chloroplastic membranes. Evidence from several laboratories indicates that
the Toc complex contains three membrane proteins:
Toc159, Toc34, and Toc75 [13–15]. In addition to the Toc
proteins, lipids have also been thought to play a crucial role
in the early stages of precursor binding to chloroplasts. For
instance, transit peptides have been shown to interact with
the chloroplastic-specific lipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol [16,17]. From these
studies it was proposed that the early stages of precursor
targeting to the chloroplasts involved a specific interaction
of the transit peptide with the lipids of the outer envelope
membrane (Figure 1a). Recently, this hypothesis gained
further support when Chen and Li [18•] used a genetic
approach to dissect the early stages of precursor binding to
chloroplasts. They showed that chloroplasts isolated from
an Arabidopsis mutant deficient in digalactosyldiacylglycerol are defective in their ability to import precursors
destined for the interior of chloroplasts. The defect in
these chloroplasts appears to occur at the energy-independent binding stage of import.
After an initial interaction of precursor with the lipids of
the outer envelope membrane [16,17], the precursor interacts with both Toc159 and Toc34 during the
energy-independent stage of binding [19,20] (Figure 1b).
Both of these proteins contain GTP-binding domains
[21–23]. Toc159 was identified originally as an 86 kDa protein [21,22], designated Toc86; however, recent studies
472
Cell biology
Figure 1
Precursor protein
(a)
(b)
(c)
(d)
Hsp
70
159
75 34
159
34
22
34
159
Hsp
22 70
22
20
55
20
110
Hsp93
OM
20
110
IM
110
Hsp93
Hsp93
Hsp
70
SPP
Hsp
60
Hsp93
Current Opinion in Plant Biology
have concluded that Toc86 is a proteolytic fragment of the
native 159 kDa protein [8••,24]. It has been proposed that
Toc159 contains a transit peptide binding site for precursor
proteins. There are two pieces of evidence for this role of
Toc159: antibodies against Toc159 block precursor binding
to isolated chloroplasts [21], and two different precursor
proteins can be cross-linked effectively to Toc159 [14,19].
The role of Toc34 during the energy-independent stage of
precursor protein binding to the Toc complex has been difficult to determine. Recent experiments show that Toc34
can be cross-linked to precursor protein during the early
stages of binding [19]. But the presence of either GTP or
ATP prevents the formation of cross-linking to Toc34. The
authors conclude that Toc34 is not a part of the transit peptide receptor but rather plays a regulatory role in precursor
binding [19].
The third component of the Toc complex that interacts with
precursor protein during the early stages of binding is Toc75
[25]. The transit peptide of precursor proteins can be crosslinked to Toc75 [14,20], thus confirming a close interaction.
Taken together, the cross-linking data support the hypothesis that Toc75 and Toc159 form a binding site that
specifically binds transit peptides. Considerable evidence
indicates that Toc75 also constitutes the major component of
the protein-conducting channel in the outer envelope membrane. The most compelling evidence comes from Hinnah et
al. [26], who showed that recombinant Toc75 has voltagesensitive ion conductance properties when reconstituted into
Stroma
A schematic working model for protein import
into chloroplasts. The numbers for the
membrane components and the molecular
chaperones (Hsps) represent their molecular
mass. Further details about these components
are given in the text. The location for the
utilization of GTP is highly speculative, but the
locations for the hydrolysis of ATP are based
upon experimental evidence that is further
described in the text. Stage (a) represents a
hypothetical step where the transit peptide
interacts only with the lipids of the envelope
membrane. Stage (b) depicts a reversible step
where the transit peptide also interacts with
proteins of the transport apparatus, but prior to
the hydrolysis of nucleoside triphosphates.
Stage (c) depicts an early translocation
intermediate, where precursor is irreversibly
associated the transport apparatus, but still
sensitive to exogenously added protease.
Stage (d) depicts the final steps of protein
transport, processing and assembly. The transit
peptide located at the amino-terminus of the
precursor protein is removed by the stromal
processing peptidase (SPP). The differences in
components present in the complexes shown
in stages (b), (c), and (d) is a deliberate attempt
to reflect the dynamic nature of the
composition of the translocation apparatus.
liposomes. The reconstituted Toc75 ion channel was calculated to have a small pore with a diameter of 8–9 Å. Such a
small opening would require that the precursor be completely unfolded during translocation across the outer membrane.
They also demonstrated that this conductance was selectively regulated by the transit peptide of precursor protein,
supporting the role of Toc75 as a transit-peptide-regulated
channel [26]. However, Toc75 may not be the only component comprising the protein-conducting channel of the outer
membrane. Ma et al. [14] have shown that during translocation, precursor proteins can be cross-linked to regions of
Toc159 other than the cytoplasmic domain. Hence, the
membrane domain of Toc159 may participate in the translocation process, possibly as part of the translocation channel.
Although only three integral membrane proteins have been
identified as components of the Toc complex, some of
them are encoded by multiple genes that show differential
expression during plastid development. For example,
Arabidopsis contains two Toc34-like genes that are differentially expressed [27••]. The protein products of these
genes, AtToc33 and AtToc34, have 61% amino acid
sequence identity to each other, and 59% and 64% amino
acid sequence identity with pea Toc34, respectively.
AtToc33 is preferentially expressed during the early stages
of seedling development, whereas AtToc34 is expressed at
constitutively low levels during all stages of leaf development. Mutant plants deficient in AtToc33 are delayed in
plastid development and show a persistent chlorophyll
deficiency; however, they are only partially defective in
Protein import into chloroplasts Keegstra and Froehlich
473
protein import. Either AtToc34 or AtToc33 can complement the mutant, demonstrating the functional redundancy
of the two proteins. This work is important on several
accounts. It is the first application of reverse genetics to the
study of protein import into chloroplasts; furthermore, it
demonstrates that some components of the transport apparatus are encoded by multiple genes and that these genes
are differentially expressed. More work is needed to understand the functional significance of these multiple genes.
using different strategies [29,30], the two disagreed on its
topology within the membrane. Jackson et al. [31] have
reinvestigated this question and concluded that the large
hydrophilic domain of Tic110 faces the stromal side of the
inner membrane. On the basis of this topology, Tic110 is
postulated to interact with stromal molecular chaperones
during protein import, possibly in a manner similar to the
way in which Tim44 interacts with Hsp70 in the mitochondrial matrix [10].
Although a role for GTP in protein import had been reported [11], the discovery that both Toc159 and Toc34 contain
GTP-binding domains [21–23] required a re-evaluation of
the role for GTP during import [8••,9•]. In the first stage,
the formation of early translocation intermediates is mediated by Toc components that bind and hydrolyze nucleoside
triphosphates. Initial interaction of the transit peptide
occurs at Toc159, with the possibility of some involvement
of Toc75. Further insertion of precursor protein into the
import apparatus requires GTP hydrolysis. GTP hydrolysis
at Toc159 and/or Toc34 may induce a conformational change
in the overall Toc complex that induces the precursor to
insert itself across the outer membrane through the proteinconducting channel. Therefore, two roles have been
proposed for the function of GTP during import. In the first,
GTP acts as a regulator for early translocation intermediate
formation. In this role, Toc159 and/or Toc34 may regulate
gating of the protein-conducting channel, thus ensuring the
specificity of precursor binding. Through a cycle of GTP
binding and hydrolysis, Toc159 and/or Toc34 may act as a
switch to ensure that precursors are committed to further
translocation. In the second role, GTP binding may initiate
the formation of contact sites, thus forming functional
translocation complexes. As the import apparatus is reconstituted, using purified components, future investigation
will begin to focus on the separate roles of Toc159 and
Toc34 during the early stages of binding.
Tic55, an iron-sulfur-containing protein of the inner envelope membrane, was identified as a translocation
component based primarily on its comigration with other
translocation components during blue-native polyacrylamide gel electrophoresis [32]. However, others have not
found this protein in transport complexes isolated by different techniques [19,33••], so more work is needed to
confirm that this protein is part of the import apparatus. If
Tic55 is confirmed to be a translocation component, it will
be interesting to determine what role, if any, the redox
center has during import.
GTP hydrolysis is required for early import intermediate
formation, but it is not sufficient for complete translocation
across the outer membrane. The stability of early translocation intermediates is enhanced by ATP at low levels
[11]. The presence of Hsp70 at the inner surface of the
outer membrane may explain the need for ATP during
early translocation intermediate formation. Young et al. [9•]
have further shown that GTP has no effect on the translocation steps that occur after the early import intermediate
has been formed (Figure 1c,d). From this study, they conclude that once the early import intermediates are formed,
ATP hydrolysis, not GTP hydrolysis, mediates translocation. This ATP is most probably utilized by stromal
chaperones thought to be involved in transport across the
chloroplastic envelope membranes [13,15,28].
Inner membrane components and the
formation of contact sites
Although Tic110, the first inner membrane component to
be described, was identified independently by two groups
Kouranov and Schnell [19] identified Tic22 and Tic20, two
inner envelope membrane proteins, as translocation components using a chemical cross-linking strategy. More
recently, Kouranov et al. [33••] isolated cDNA clones
encoding Tic22 and Tic20, provided preliminary characterization of these proteins, and offered hypotheses
regarding their role in protein import. Tic22 is a peripheral membrane protein associated with the outer surface of
the inner envelope membrane. It does not contain readily
recognizable sequence motifs, such as nucleotide binding
domains, and does not possess sequence similarity to components of other transport systems; it does, however,
possess 19% sequence identity over 176 amino acid
residues with an open reading frame in the cyanobacterium Synechocystis 6803 genome [34••]. Tic20 is an integral
membrane protein that is predicted to span the inner envelope membrane via three hydrophobic helical domains.
Proteolytic digestion studies provide evidence that the
amino-terminal domain, which is very basic, extends into
the stroma, whereas the carboxy-terminal hydrophilic
domain extends into the intermembrane space [33••].
As noted earlier, Tic22 and Tic20 were first identified as
translocation components by their ability to be cross-linked
to precursors during import. Kouranov et al. [33••] also used
immunoprecipitation of detergent-solubilized envelope
membranes to demonstrate that a small proportion of Tic22
and Tic20 were present in complexes that also contained
Tic110, Toc159, Toc75 and Toc34. Interestingly, they could
not find complexes of Tic22 and Tic20 with Tic110, suggesting that the inner membrane components do not form
complexes in the absence of association with the Toc complex to form putative contact sites. Kouranov et al. [33••]
speculate that Tic20 may serve as part of the proteintranslocating channel in the inner envelope membrane,
whereas Tic22 may recognize precursor proteins as they
474
Cell biology
enter the intermembrane space and direct them to the
inner membrane import machinery. In this role, Tic22 may
be the factor that connects the two envelope membranes,
causing the formation of contact sites. If this is the case, one
important unsolved question is what factors regulate the
ability of Tic22 to mediate the apparently dynamic interaction between the components from the inner and outer
envelope membranes.
Molecular chaperones and the translocation
process
By analogy with mitochondrial protein import where a
matrix Hsp70 supports transport across the inner membrane [10], a stromal Hsp70 [35] was an obvious candidate
for this role in chloroplasts; however, the stromal Hsp70
could not be found in translocation complexes [13,15].
Rather, translocation complexes contain Hsp93, a stromal
protein in the Hsp100 family of molecular chaperones [36].
This observation has been confirmed by the recent studies
of Kouranov et al. [33••]. Moreover, both groups find evidence of an association between Hsp93 and Tic110,
suggesting that these two proteins may have roles similar
to that suggested for Hsp70 and Tim44 in mitochondria:
providing the driving force for pulling proteins into the
matrix [10]. Kessler and Blobel [29] also found Hsp60 associated with translocation complexes via an association with
Tic110, a result that was confirmed by Kouranov et al.
[33••]. Defining the role of each of the chaperones in protein translocation will require additional studies. However,
it is tempting to speculate that Hsp93, and possibly Hsp70,
provide the driving force for translocation across the inner
envelope membrane, whereas Hsp60 is involved in folding
the protein following its translocation in an unfolded state.
Evolutionary origins of the protein
import apparatus
It is widely accepted that chloroplasts evolved from
cyanobacteria following an endosymbiotic event. As bacterial genes were transferred into the nucleus, a system was
needed for delivering the proteins back into the evolving
organelle. The origins of this protein transport system poses
interesting questions regarding the early evolution of
chloroplasts. Bölter et al. [41••] and Reumann et al. [42••]
provided evidence that at least one modern translocation
component, that is Toc75, is related to a cyanobacterial
outer membrane protein. Reumann et al. [42••] argued that
this cyanobacterial protein is related to a family of outer
membrane proteins of unknown function whose members
are widespread in the Gram-negative bacteria. This family,
in turn, is related to another group of proteins involved in
secretion from Gram-negative bacteria. Moreover, Bölter
et al. [41••] demonstrated that the cyanobacterial protein is
capable of channel activity. Thus, it seems likely that
Toc75, the putative outer membrane channel of the import
system, evolved from an outer membrane channel involved
in secretion from the free-living cyanobacterium. More
recently, Reumann and Keegstra [34••] presented evidence
that both Tic22 and Tic20 have cyanobacterial homologs,
whereas Toc159, Toc34/33 and Tic110 do not. They conclude that the protein import apparatus has a dual origin,
with some components deriving from cyanobacteria, possibly from an ancient protein secretion mechanism, and other
components having been added to the system from other
sources during the course of evolution.
Conclusions and prospects
An Hsp70 protein present in the intermembrane space has
also been identified as a component of translocation complexes (Figure 1c) [28]. Although this is an ideal candidate
for providing the driving force for transport across the outer
envelope membrane, a cDNA encoding this protein has
not yet been identified. Thus, more work is needed to
determine the role, if any, of this protein in the import
process. Another Hsp70 associated with the surface of
chloroplasts (Figure 1b) has been identified [37,38],
although its role in protein import is also unclear.
Considerable progress has been made in recent years in
identifying the components of the chloroplastic protein
import system. Although it is difficult to know how many
components are required for the import apparatus, it is reasonable to argue that the majority of the transport proteins
have been identified and that relatively few additional
components will be found. However, at least in
Arabidopsis, several components are encoded by a small
family of related genes ([27••]; D Jackson, K Keegstra,
unpublished observations). Thus, it will be important to
determine the functional and developmental significance
of these various family members.
Either during import or immediately afterwards, the transit peptide is removed by a stromal processing protease
(Figure 1d) [39]. The same metalloprotease removes the
transit peptide from many different precursors, including
acting on its own precursor in trans [39]. The importance
of this enzyme for the import process, and consequently
for the overall operation of the plant, is demonstrated by
the recent generation of plants containing an antisense
version of the protease gene and having reduced levels of
the protein. These plants have a severe phenotype,
including chlorotic leaves, reduced growth, a reduced
number of plastids per cell and lower amounts of internal
membranes [40•].
In the immediate future, the major challenge will shift
away from identification of new components to determining the functions of those components shown in
Figure 1 as well as newly discovered components. This
is likely to involve two efforts that have just begun. The
first is in vitro reconstitution of the protein import
process or at least of particular partial reactions. A start
has been made by Hinnah et al. [26], who have reconstituted purified Toc75 into lipid bilayers to show that it
has channel activity. However, it is unlikely that Toc75 is
the only constituent of the channel because the reconstituted activity does not behave like the Toc complexes in
outer envelope membranes. The challenge will be to
Protein import into chloroplasts Keegstra and Froehlich
define various partial reactions and reconstitute them
using purified components.
475
the formation of early-import intermediates, but GTP does not play a role during the translocation of precursors from the intermediate state.
10. Pfanner N, Craig EA, Hönlinger A: Mitochondrial preprotein
translocase. Annu Rev Cell Dev Biol 1997, 13:25-51.
A second important strategy will be the use of genetics or
reverse genetics to suppress or eliminate a particular component, following on the important work of Jarvis et al.
[27••], who identified Arabidopsis plants lacking Toc33. With
the new availability of several strategies for isolating plants
with disruptions in a particular gene of interest, approaches
similar to those employed by Jarvis et al. [27••] for Toc33 will
certainly be applied to other transport components. An alternative is to use the antisense method, as illustrated by the
work of Wan et al. [40•] with plants containing reduced levels of the stromal processing protease. The availability of
reverse genetic approaches and the power of in vitro reconstitution assays should lead to models for the mechanism of
protein import showing much more detail than the rather
crude scheme shown in Figure 1.
Acknowledgements
The authors thank Danny Schnell for making available unpublished
information from his laboratory. Work from the authors’ laboratory was
supported by grants from the Cell Biology Program at the National Science
Foundation and from the Energy Biosciences Program at the Department of
Energy.
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chloroplasts requires nucleoside triphosphates in the
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476
Cell biology
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A homolog of Toc75 was identified in a cyanobacterial genome. Experimental
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the outer envelope membrane. Reconstitution of SynToc75 into liposomes
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in [41••]. Attempts to disrupt the chromosomal copy of SynToc75 were
unsuccessful, unless the cell contained a plasmid copy of the gene, leading
to the unexpected conclusion that SynToc75 is an essential protein. More
detailed sequence analysis demonstrated that SynToc75 is related to a family of genes found in all Gram-negative bacteria examined to date. Combined
with results from [41••], these data provide the first evidence that at least
portions of the chloroplastic protein import apparatus was derived from a
cyanobacterial ancestor.