CORRECTIONS
Journal of Integrative Plant Biology 2007, 49 (8): 1100–1111
1. In line 20 of the right column of page 1102, the estimated number of total proteins in
chloroplasts without most outer envelope membrane proteins that do not carry predictable
transit peptides in Arabidopsis thaliana is 2200 (2100 + 87), instead of 3000. This
number, of course, does not account for those in the chloroplast interior which are
synthesized without canonical transit peptides.
2. In Table 1 (page 1103), four homologous GTPases, Toc159, Toc132, Toc120, and
Toc90, are mistakenly predicted to be anchored to the membrane via C-terminal α-helical
regions. Although the C-terminal portions appear to anchor these proteins to the lipid
bilayers, there is no clear predictable hydrophobic domain in these sequences (Bédard
and Jarvis 2005). Thus, their structures should be denoted as “X”.
3. atToc64-I is included in Table 1, but the reference #9 (Qbadou et al. 2006) does not
discuss its localization in the outer envelope membrane at all. Chew et al. (2004 FEBS
Lett 557:109-114) showed that a fusion protein with GFP in its C-terminus was found in
nucleus, but not in plastids. Thus, the reference #9 should be deleted from the atToc64-I
line, and it should be noted that its outer envelope localization has not been confirmed yet.
Journal of Integrative Plant Biology 2007, 49 (8): 1100–1111
·Invited Review·
The Chloroplast Outer Envelope Membrane: The Edge of
Light and Excitement
∗
Kentaro Inoue
(Department of Plant Sciences, College of Agricultural & Environmental Sciences, University of California, One Shields Avenue, Davis,
CA 95616, USA)
Abstract
The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including
nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm
and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of
various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts
as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived
from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support
the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent
progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation
of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer
envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle
biogenesis, plant growth and development, and also membrane biology in general.
Key words: chloroplast envelope; lipid biosynthesis; outer membrane; protein import; putative solute channel.
Inoue K (2007). The chloroplast outer envelope membrane: The edge of light and excitement. J. Integr. Plant Biol. 49(8), 1100–1111.
Available online at www.blackwell-synergy.com/links/toc/jipb, www.jipb.net
The chloroplast is an organelle that is specific and essential
to photosynthetic eukaryotic cells. According to the endosymbiotic theory, the chloroplast shares the common ancestor with
extant cyanobacteria (for a recent review, see Larkum et al.
2007). Currently in higher plants, chloroplasts are developed
from pre-existing plastids and are never synthesized de novo.
The chloroplast is surrounded by an envelope consisting of
the outer and inner membranes which are separated only by
5–10 nm (Keegstra et al. 1984) (Figure 1). These two mem-
Received 6 Mar. 2007 Accepted 24 Apr. 2007
Supported by the Department of Pomology of the University of California
at Davis (2002–), the Agricultural Experiment Station of the University of
California (2002–), National Research Initiative of the USDA-CSREE (2003–
2006), the UC Discovery Grant (2003–2007), and the California Citrus
Research Board (2007–)
∗
Author for correspondence.
Tel: +1 530 752 7931;
Fax: +1 530 752 9659;
E-mail: <[email protected]>.
C 2007 Institute of Botany, the Chinese Academy of Sciences
doi: 10.1111/j.1672-9072.2007.00543.x
branes function as a physical barrier to separate the interior of
the organelle from the cytoplasm. In addition, the chloroplast
envelope plays a key role in communication between the
cytoplasm and the organelle. Because most proteins found
in the chloroplast are encoded in the nuclear genome and
synthesized on cytosolic ribosomes, the chloroplast depends
its biogenesis on posttranslational protein import, which requires
recognition and translocation of precursor proteins at the envelope. The contact sites of the two membranes (Figure 1) have
been shown to accumulate such a translocation intermediate
(Schnell and Blobel 1993). In addition to the precursor proteins,
the chloroplast needs to import and export numerous solutes
including ions, nucleotides, amino acids, and other metabolic
intermediates and products in order to maintain its own activities
and also to support the viability of the cell. Furthermore, a
number of metabolic reactions such as the biosynthesis of
membrane lipids take place in the chloroplast envelope.
The inner membrane of the chloroplast envelope has been
recognized as the permeability barrier. It consists of a number
of specific translocators and enzymes whose importance and
properties have been appreciated and examined very well in the
last several decades (Heber and Heldt 1981; Joyard et al. 1998;
Weber et al. 2005). By contrast, the chloroplast outer envelope
The Chloroplast Outer Envelope Membrane Proteome
1101
Schleiff 2004; Bédard and Jarvis 2005; Kessler and Schnell
2006; Smith 2006), roles of envelope lipids in protein import
(Bruce 1998), lipid metabolism and trafficking (Dörmann and
Benning 2002; Awai et al. 2006; Jouhet et al. 2007), and
solute transporters in the plastid envelope (Weber et al. 2005).
Excellent comprehensive reviews on the chloroplast envelope
biology were also published previously (Douce and Joyard
1990; Joyard et al. 1991, 1998; Block et al. 2007).
Unique Features of the Chloroplast Outer
Envelope Membrane
Figure 1. The chloroplast envelope.
A chloroplast of an Arabidopsis mesophyll cell examined under an
electron microscope. The part of the chloroplast envelope is enlarged
and the outer and inner membranes are indicated. The contact site of
the two membranes is circled. Bar, 1 µm. SG and M indicate a starch
grain and a mitochondrion, respectively.
membrane has often been considered a passive compartment.
It was shown to be permeable to compounds of low molecular
weight (Heldt and Sauer 1971; Flügge 2000; Soll et al. 2000). In
addition, the chloroplast outer membrane has been described
as a remnant of the vacuolar membrane, which was used by
the host cell to engulf the symbiont (Whatley and Whatley
1981). These viewpoints may have made the outer envelope a
rather non-attractive material for scientific research. However,
especially in the last decade, a number of proteins have been
identified in the chloroplast outer envelope and their intriguing
properties revealed by biochemical, biophysical, and genetic
studies. In addition, the similarity between the outer membranes
of the chloroplast envelope and those of extant cyanobacteria
in lipid and protein compositions strongly supports the common
evolutionary origin of the two membranes (Cavalier-Smith 1982;
Keegstra et al. 1984; Joyard et al. 1991; see below).
This review aims to stimulate interest in the chloroplast outer
envelope. To do so, overviews of its general features, as well as
a list of proteins identified or predicted to be in this membrane
are provided. For readers who wish to learn more, there are
outstanding reviews on chloroplast protein import (Soll and
Traditionally, leafy tissues such as pea seedlings grown in
the greenhouse or spinach purchased at local supermarkets
have been used as a source of chloroplasts. Early studies
developed procedures for fractionation of the outer envelope
membrane from these chloroplasts by density-gradient centrifugations (Cline et al. 1981; Block et al. 1983a) and revealed its
unique features in lipid and protein compositions.
First, the weight to weight ratio of lipids to proteins in the
chloroplast outer envelope is 2.5–3.0, which is comparable
to that of tonoplast (2.3) but is much higher than that of the
chloroplast inner envelope (0.8–1.0) (Joyard et al. 1991; Moreau
et al. 1998). Thus, lipid composition may be critical for the
function of the chloroplast outer membrane. Interestingly, the
outer membranes of Gram-negative bacteria consist of about
half their mass as proteins (Koebnik et al. 2000), which is similar
to the inner but not to the outer membrane of the chloroplast
envelope.
Second, the chloroplast envelope and thylakoids as well as
cyanobacterial membranes are unique biological membranes
in that they are rich in galactoglycerolipids (Wada and Murata
1989; Joyard et al. 1991; Moreau et al. 1998). The chloroplast inner envelope and thylakoids are almost identical in
their glycerolipid composition, consisting of monogalactosyl
diacylglyrecol (MGDG) which tends to form non-bilayer structures, digalactosyl diacylglycerol (DGDG), and phosphatidyl
glycerol (PG) in the molar percentage of 55–57, 27–30, and
7–9, respectively. By contrast, the outer envelope contains
a significant amount of phosphatidyl choline (PC) (32%) in
addition to MGDG (17%), DGDG (29%), and PG (10%). Interestingly, the two polar lipids, PC and PG, are distributed
asymmetrically in the outer envelope. PC is mainly located in
the cytoplasmic site (outer leaflet), whereas PG is exclusively
present in the inner leaflet (Dorne et al. 1985). Recently, PC
in the outer leaflet of the chloroplast envelope was shown
to be converted to diacylgalactolipids by cytosolic phospholipase D and phosphatidic acid phosphatase (Andersson et al.
2004). Thus, the asymmetrical distribution of PC in the chloroplast outer envelope may be relevant to the lipid metabolism.
In addition to these lipids, isoprenoid-derived compounds
(i.e. carotenoids (Douce et al. 1973; Markwell et al. 1992) and
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Journal of Integrative Plant Biology
Vol. 49
No. 8
2007
prenylquinones such as plastoquinone-9 and tocopherol (Soll
et al. 1985)) have been identified in the chloroplast outer envelope. These compounds play important roles in light-harvesting
and photoprotection in the internal chloroplast membranes (i.e.
thylakoids). The main carotenoid in the envelope membranes
violaxanthin is known to be depleted following light stress
(Jeffery et al. 1974). Violaxanthin de-epoxidase, which converts
violaxanthin to zeaxanthin was found in thylakoids, but not in
the envelope, and the exchange of violaxanthin between these
two membranes was proposed (Siefermann-Harms et al. 1978).
A recent study has demonstrated that MGDG and DGDG have
different effects on violaxanthin de-epoxidase activity as well as
violaxanthin solubility, which implies that the intra-chloroplastic
exchange of violaxanthin may be due to the difference between
the envelope and thylakoids in their galactolipid composition
(Yamamoto 2006). Furthermore, an interesting possibility was
suggested recently that carotenoids in the outer envelope may
be converted by a carotenoid cleavage dioxygenase in the cytoplasm to apocarotenoids, which may act as signaling molecules
that are important for plant development and survival (Bouvier
et al. 2005a). Functions of other isoprenoid compounds in the
chloroplast outer envelope remain largely unexplored.
Early studies have also revealed two interesting features
of the chloroplast outer envelope proteins. The first is their
structure: in addition to the proteins anchored to or embedded
in the lipid bilayer by one or more α-helical transmembrane
domains, the outer envelope consists of proteins that are
predicted to be integrated into the membrane with β-strands
that can form a stable pore-like structure called β-barrel. βBarrel membrane proteins have been found almost exclusively
in the outer membranes of Gram-negative bacteria and the
endosymbiotic organelles (i.e. chloroplasts and mitochondria)
(Wimley 2003). The second unique feature of the chloroplast
outer membrane proteins is their targeting mechanism. With a
few exceptions, nuclear-encoded proteins found in the interior
of chloroplasts are synthesized with an N-terminal extension
called a transit peptide, which is necessary and sufficient for
targeting the protein to the organelle (Chua and Schmidt 1979;
Bruce 2001). The transit peptide-dependent protein targeting
is called the general pathway and its mechanism has been
studied extensively. In addition, several prediction programs for
the transit peptide, such as ChloroP (Emanuelsson et al. 1999),
iPSORT (Bannai et al. 2002), PCLR (Schein et al. 2001), and
Predotar (http://www.inra.fr/predotar/), have been developed.
By contrast to the proteins targeted to the interior of chloroplasts,
those found in the outer envelope usually do not carry the transit
peptide (Schleiff and Klösgen 2001). Interestingly, available
data suggest that some outer envelope proteins may use part of
the general pathway for their targeting (for review, see Hofmann
and Theg 2005).
In summary, the presence of galactolipids and β-barrel proteins strongly supports the common ancestral origin of outer
membranes of chloroplasts and extant cyanobacteria. Phos-
phatidylcholine and α-helical transmembrane proteins in the
chloroplast outer envelope, which are not detected in the modern cyanobacteria, may have been added during the evolution
of the organelle.
The Chloroplast Outer Envelope Membrane
Proteome: How Many Proteins are
Required to Make the Outer Envelope?
Recent completion of genome sequencing (Arabidopsis
Genome Initiative 2000), development of genetic tools (e.g.
Alonso et al. 2003), and establishment of efficient methods for
isolation of intact chloroplasts (e.g. Fitzpatrick and Keegstra
2001) have made Arabidopsis an attractive system alternative
to pea or spinach to study biological problems of chloroplasts.
Recently, Richly and Leister (2004) evaluated several prediction
programs extensively and proposed that about 2 100 proteins
are synthesized with transit peptides on the Arabidopsis cytoplasmic ribosomes. There are 87 proteins encoded in the
Arabidopsis chloroplast genome, none of which appears to be
localized to the outer envelope (Sato et al. 1999). These studies
estimate about 3 000 proteins in chloroplasts, which, however,
do not consider most of proteins in the outer envelope that do
not contain predictable transit peptides. How many proteins,
then, are there in the outer membrane of Arabidopsis chloroplasts? Addressing this question has to depend on individual
studies. Examining the recently-developed proteome of the
“mixed” envelope from Arabidopsis chloroplasts (Ferro et al.
2003; Froehlich et al. 2003; Friso et al. 2004) is also useful.
Table 1 lists outer membrane proteins of the Arabidopsis
chloroplast envelope whose location has been confirmed by
various methods or predicted based on their sequence similarity
to known proteins. Major proteins identified and characterized
well so far are those involved in protein import, solute exchange,
and lipid biosynthesis. Addition of other proteins of known and
unknown functions makes a total of 34 proteins in the list
(Table 1).
Protein Import Components and their
Homologs
Extensive studies by various laboratories in the last decade or
so using chemical cross-linking, co-immunoprecipitation, antibody inhibition, and translocation intermediates resulted in the
identification of several components involved in protein import
at the envelope membranes of pea chloroplasts. These proteins
are designated as Toc and Tic (Translocon at the outer- and the
inner-envelope-membranes of chloroplasts) proteins (Schnell
et al. 1997). According to the original nomenclature, (1) the
designation Toc is followed by a number indicating the molecular
The Chloroplast Outer Envelope Membrane Proteome
mass of the protein in kilodalton, (2) a two-letter prefix (in lower
case) indicates the species of origin of the component, and (3)
this is restricted to proteins whose specific functions to envelope
translocation are supported by experimental evidence (Schnell
et al. 1997). For example, psToc75 is a 75-kDa component of
the Toc complex from pea (Pisum sativum) whose function has
been established by various studies (see below). Later, when
sequences of the Arabidopsis genome became available, an
additional designation system which includes the chromosomal
locations of the genes was introduced (Jackson-Constan and
Keegstra 2001). According to this, atToc75-III is a protein homologous to psToc75 and is encoded in the chromosome III of the
Arabidopsis genome. This system is, of course, restricted to proteins from organisms whose genomic information is available,
such as Arabidopsis and rice. Currently, designations depend
on individual components. This review follows the commonly-
1103
used nomenclature and defines components from pea without
the two-letter prefix (e.g. Toc75 indicates psToc75).
Among Toc components identified so far, three proteins are
considered the core constituents in pea chloroplasts based
on their ability to reconstitute precursor protein translocation
in artificial liposomes (Schleiff et al. 2003b). They are two
homologous Guanosine triphosphatases (GTPases), Toc159
and Toc34, which are anchored to the membrane with α-helices
and expose their large N-terminal portions to the cytoplasmic
surface, and a β-barrel membrane protein Toc75. The two
GTPases are postulated to be involved in recognition and also
in the early stage of translocation of the precursor proteins.
Mechanistic details by which Toc159 and Toc34 mediate these
processes have been a matter of debate (for reviews, see
Kessler and Schnell 2004, 2006; Li et al. 2007). Toc75 is one of
the most abundant proteins in the chloroplast outer envelope
Table 1. Proteins identified or predicted to be in the outer membrane of the Arabidopsis chloroplast envelope
AGI number
Namea
Sizeb
Structurec
Proteomed
Function
Referencee
Protein import components and their homologs
At3g46740
atToc75-III
89 (75)b
B
Yes
Preprotein conducting channel
1,2
At5g19620
atToc75-V /AtOEP80
80
B
Yes
Unknown
3,4
At4g09080
atToc75-IV
44
B
Unknown
1,5
At4g02510
atToc159
161
C
Yes
Preprotein recognition/translocation
At2g16640
atToc132
132
C
At3g16620
atToc120
120
C
1,6
At5g20300
atToc90
88
C
1,7
1,6
1,6
At1g02280
atToc33
33
C
Yes
At5g05000
atToc34
35
C
Yes
Preprotein recognition/translocation
1,8
1,8
At1g08980
atToc64-I
45
P?
Unknown
1,9
At3g17960
atToc64-III
64
N or P
Preprotein recognition/translocation
1,9
At1g80920
Toc12ha
18
X
Preprotein translocation
This review
Solute channels
At2g28900
OEP16-1
16
P
At4g16160
OEP16-2
19
P
At3g62880
OEP16-4
14
P
At1g20816
OEP21-1
20
B
At1g76405
OEP21-2
20
B
Yes
Yes
Cation-selective channel
10,11
Amino acid transport?
10,11
Import of PORA precursor?
10,11
ATP-regulated anion selective channel
12
12
At1g45170
OEP24-1
24
B
Cation-selective channel
13
At5g42960
OEP24-2
24
B
Yes
General pore?
This review
At2g43950
OEP37
32
B
Yes
Cation-selective channel
14
At3g01280
VDACa
29
B
Yes
General pore?
This review
At5g15090
VDACa
25
B
Yes
Yes
This review
Lipid metabolism
At1g77590
LACS9
76
X
At3g11670
DGD1
92
P
At4g00550
DGD2
54
X
Galactolipid biosynthesis
17
At5g20410
MGD2
53
X
Galactolipid biosynthesis
18
At2g11810
MGD3
53
X
At4g15440
HPLha
43
X
Yes
Fatty acid activation
15
Galactolipid biosynthesis
16
Galactolipid biosynthesis
18
Oxilipin metabolism
This review
1104
Journal of Integrative Plant Biology
Vol. 49
No. 8
2007
Table 1. Continued
Namea
Sizeb
Structurec
At5g53280
PDV1
31
At2g16070
PDV2
34
At2g20890
THF1
34 (27)b
P?
At3g52420
OEP7
6.8
N
Unknown
21
At5g23190
CYP86B1
64
P
Monooxygenase/unknown
22
At3g52230
OMP24ha
16
C?
Unknown
This review
AGI number
Proteomed
Function
Referencee
C?
Division
19
C?
Division
19
Sugar signaling; thylakoid formation
20
Others
a
Yes
Yes
Toc12h, fatty acid hydroperoxide lyase (HPLh), and OMP24h are proteins identified in this study, which are apparently homologous to Toc12 from
pea, HPL from tomato, and OMP24 from spinach, respectively. The presence of VDAC-like proteins (At3g01280 and At5g15090) in the chloroplast
outer membrane may be highly speculative.
b
Predicted size in kDa. Two proteins, atToc75-III and THF1, have been shown to contain N-terminal cleavable targeting sequences, and sizes of their
mature forms are indicated in the parentheses.
c
Proteins predicted to be embedded in the lipid bilayer via a single (N, signal-anchor means locating its N-terminus in the intermembrane space;
C, tail-anchor means locating its N-terminus in the cytoplasm), or multiple (P, polytopic) α-helices, β-strands (B) are indicated. Those without any
obvious transmembrane domains are indicated as X.
d
Proteins identified in the chloroplast envelope proteome (Friso et al. 2004) are indicated as “Yes”.
e
References that describe these Arabidopsis proteins in the chloroplast outer envelope. 1. Jackson-Constan and Keegstra 2001; 2. Inoue and
Keegstra 2003; 3. Eckart et al. 2003; 4. Inoue and Potter 2004; 5. Baldwin et al. 2005; 6. Bauer et al. 2000; 7. Hiltbrunner et al. 2004; 8. Jarvis et al.
1998; 9. Qbadou et al. 2006; 10. Reinbothe et al. 2004; 11. Philippar et al. 2007; 12. Hemmler et al. 2006; 13. Schleiff et al. 2003; 14. Goetze et al.
2006; 15. Schnurr et al. 2002. 16. Froehlich et al. 2001a; 17. Kelly et al. 2003; 18. Awai et al. 2001; 19. Miyagishima et al. 2006; 20. Wang et al. 2004;
21. Lee et al. 2001; 22. Watson et al. 2001; or this review.
DGD, digalactosyl diacyl; LAC9, long chain acyl-CoA synthetase 9; MDG, monogalactosyl diacyl; OEP, outer envelope protein; OMP, outer membrane
protein; PDV, plastid division; PORA, protocholophyllide oxidoreductase A; THF1, thylakoid formation 1; Toc and Tic, translocon at the outer- and the
inner-envelope-membranes of chloroplasts; VDAC, voltage-dependent anion channel.
(Cline et al. 1981). The presence of its apparent homologs
in modern cyanobacteria suggests the endosymbiotic origin of
Toc75 (Bölter et al. 1998; Reuman et al. 1999). In addition to
chemical cross-linking assays (Perry and Keegstra 1994; Ma
et al. 1996), reconstitution experiments using artificial liposomes
(Hinnah et al. 1997, 2002) led to the widely-accepted idea that
Toc75 forms a protein translocation pore. Interestingly, unlike
other outer membrane proteins, Toc75 requires a cleavable
transit peptide that consists of two parts for its correct targeting
(Tranel et al. 1995; Tranel and Keegstra 1996; Inoue et al.
2001; Inoue and Keegstra 2003; Baldwin and Inoue 2006),
and depends its complete maturation on a membrane-bound
type I signal peptidase in addition to a soluble stromal processing peptidase (Inoue et al. 2005). Besides the three core
components, two other proteins have been identified as Toc
components. Toc12 contains a J-domain, which can interact
with Hsp70 chaperones, and is suggested to form a multi-protein
complex in the intermembrane space to assist precursor protein
translocation (Becker et al. 2004). Toc64 has been proposed to
play two roles: it may function as a receptor to Hsp90-associated
preproteins in the cytoplasmic face of the outer envelope, and
also may be part of the precursor translocation complex in the
intermembrane space (Sohrt and Soll 2000; Qbadou et al. 2006,
2007).
Proteins from various plastid types and a number of plant
species can be targeted properly to chloroplasts isolated from
pea seedlings, and at least some of the import components
appear to be conserved (Dávila-Aponte et al. 2003). Thus, one
might assume that a non-specific general pathway mediates
protein import into plastids. This assumption was, however,
questioned when genes encoding multiple isoforms for some
of Toc components (i.e. four isoforms for Toc159 and two for
Toc34) were identified in the Arabidopsis genome (for review,
see Jackson-Constan and Keegstra 2001) (Table 1). Analyses
of gene expression patterns as well as reverse-genetic and
biochemical studies have led to a model that each isoform
has a preference for precursor substrates: namely, two Toc159
homologs, atToc159 and atToc90, and a Toc34 homolog,
atToc33, may be involved in the import of photosynthetic proteins, whereas another two Toc159 homologs, atToc132 and
atToc120, and the second Toc34 homolog, atToc34, may be
responsible for the import of proteins with non-photosynthetic
functions (Jarvis et al. 1998; Bauer et al. 2000; Kubis et al.
2003, 2004; Ivanova et al. 2004; Smith et al. 2004; Hiltbrunner
et al. 2004). The Arabidopsis genome contains four genes that
encode apparent homologs to Toc75 in the chromosomes I, III,
IV, and V, respectively (Jackson-Constan and Keegstra 2001;
Eckart et al. 2002). Among them, atTOC75-I is a pseudo-gene
The Chloroplast Outer Envelope Membrane Proteome
(Baldwin et al. 2005) and atTOC75-III appears to encode the
sole functional homolog to Toc75. The encoded protein shows
the highest identity (73%) to the pea ortholog in its predicted
primary sequence and is the only homolog that has the unique
bipartite transit peptide (Inoue and Keegstra 2003; Baldwin
et al. 2005). Functions of the other two Toc75 homologs remain
elusive. atToc75-IV appears to be a 44-kDa protein that is a
truncated form of Toc75 without the N-terminal half (Baldwin
et al. 2005). By contrast, atToc75-V is encoded as an 80-kDa
protein which shows only 22% sequence identity to Toc75. A
pea ortholog to atToc75-V is a 66-kDa protein and was not
found in the Toc complex (Eckart et al. 2002). Based on the
size of the pea ortholog and also the prediction by ChloroP,
atToc75-V was originally assumed to be synthesized with a
cleavable transit peptide of 11 kDa and processed to a mature
form of 69 kDa (Eckart et al. 2002). This assumption, however,
contradicted immunoblotting and in vitro import data, which
showed that the size of atToc75-V is 80 kDa (Inoue and Potter
2004). Inoue and Potter (2004) further proposed to rename
this protein as AtOEP80 (Arabidopsis thaliana outer envelope
protein 80), instead of atToc80 or atToc75-V, because of a
lack of experimental evidence to support its function. Detailed
phylogenetic analyses have indicated that Toc75 and OEP80
diverged early in the evolution of chloroplasts from their common
ancestor with existing cyanobacteria (Eckart et al. 2002; Inoue
and Potter 2004).
Reverse-genetic studies using Arabidopsis have demonstrated the importance of the Toc components in the viability of
plants. Disruption of the atTOC75-III gene by a T-DNA insertion
resulted in an embryo-lethal phenotype (Baldwin et al. 2005;
Hust and Gutensohn 2006). Simultaneous knockout of the two
genes for Toc159 homologs, atTOC132 and atTOC159, or that
of the genes for Toc34 homologs, atTOC33 and atTOC34, also
caused embryo-lethality, but the mutant embryos were arrested
at the developmental stage later than that of the attoc75-III
mutant (Constan et al. 2004; Kubis et al. 2004; Hust and Gutensohn 2006). Interestingly, the knockout of genes for apparent
Toc64 orthologs in the moss Physcomitrella patens (Rosenbaum Hofmann and Theg 2005) and Arabidopsis (Qbadou
et al. 2006) did not cause any visible phenotypic defects of
the organisms. Nonetheless, chloroplasts from the Arabidopsis
toc64 mutant showed a reduction in Hsp90-dependent protein
import (Qbadou et al. 2006). Whether this is the case in the
moss remains to be examined.
Putative Solute Channel Proteins
In addition to the nuclear-encoded precursor proteins, various
solutes including ions, amino acids, nucleotides, lipids, carbohydrates, and other metabolite intermediates and products pass
through the outer envelope. In mitochondria, a 30-kDa β-barrelforming protein called voltage-dependent anion channel (VDAC)
1105
or porin mediates transport of a wide variety of metabolites in the
outer membrane (for review, see Benz 1994). Flügge and Benz
(1984) showed that the chloroplast outer envelope contained
a porin-type pore which was permeable to proteins of up to
10 kD. Fisher et al. (1994b) identified in pea root plastids, but not
in chloroplasts, a VDAC-like protein that was predicted to contain 16 anti-parallel β-strands. Recently, however, Clausen et al.
(2004) demonstrated by immunoblotting and protein targeting
assays that VDAC-like proteins were undetectable not only in
chloroplasts but also in root plastids. Interestingly, two VDAChomologs encoded by genes At3g01280 and At5g15090, respectively, have been identified in the Arabidopsis chloroplast
envelope proteome (Table 1). Both of them, however, have
also been found in the mitochondrial proteome (Brugiere et al.
2004). Thus, the identification of the VDAC homologs in the
chloroplast outer envelope could be due to the contamination of
the mitochondrial fraction.
If a VDAC-like protein is not present, then, which proteins
mediate the exchange of various solutes in the chloroplast
outer envelope? This question has been extensively addressed
by Jürgen Soll and Richard Wagner’s groups who have been
reconstituting several proteins into artificial liposomes and characterizing their properties using electrophysiological measurements. They have identified four proteins of 16, 21, 24, and
37 kDa which were named OEP16 (outer envelope protein
16), OEP21, OEP24, and OEP37, respectively, from the outer
membrane of the pea chloroplast envelope. Among them,
OEP16 is folded into four-α-helical transmembrane domains
(Linke et al. 2004) and has been suggested to be responsible
for the transport of amino acids (Pohlmeyer et al. 1997). In Arabidopsis, four proteins apparently homologous to OEP16 were
identified (Reinbothe et al. 2004) (Table 1). Among them, three
proteins, AtOEP16.1, 16.2, and 16.4, were localized to chloroplasts, whereas AtOEP16.3 was found only in mitochondria
(Philippar et al. 2007). Interestingly, OEP16 was also shown
to be involved in a substrate-dependent import of protocholophyllide oxidoreductase A (PORA) by biochemical and genetic
studies (Reinbothe et al. 2004; Pollmann et al. 2007). This model
has, however, been challenged by another genetic study although they used the same mutant plants (Philippar et al. 2007).
In contrast to OEP16, all of the other three putative channel proteins are predicted to form β-barrel structures. Interestingly, however, extant cyanobacteria do not seem to
contain apparent homologs to any of them (Hsu and Inoue,
unpubl. obs. 2007). OEP21 orthologs from pea and Arabidopsis have been predicted to traverse the membrane with
eight β-strands and were shown to form rectifying adenosine
triphosphate (ATP)-regulated anion selective channels in artificial liposomes (Bölter et al. 1999; Hemmler et al. 2006).
OEP24 was predicted to contain seven (Pohlmeyer et al. 1998)
or twelve (Schleiff et al. 2003a) transmembrane β-strands.
It was reconstituted to form a slightly cation-selective high
conductance channel, which allowed a flux of a wide range of
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Journal of Integrative Plant Biology
Vol. 49
No. 8
2007
solutes (Pohlmeyer et al. 1998). Quite remarkably, expression
of OEP24 cDNA rescued the respiratory deficiency of a yeast
mutant that lacked VDAC (Rohl et al. 1999). This is in contrast
to the report showing that expression of a potato mitochondrial
VDAC cDNA could not complement another porin-deficient
mutant of yeast (Heins et al. 1994). Finally, OEP37, which was
originally identified by Schleiff et al. (2003a), was reconstituted
into liposomes as a cation-selective channel (Goetze et al.
2006). Interestingly, the OEP37-channel showed an affinity to
an inner envelope protein Tic32 in vitro although knockout of the
atOEP37 gene did not cause any obvious phenotypic defects
including import of Tic32 in Arabidopsis chloroplasts (Goetze
et al. 2006). Overall, biophysical studies have helped generate
ideas regarding the physiological functions of these putative
channel proteins.
Proteins Involved in Lipid Metabolism
The chloroplast outer envelope contains various lipids,
which are represented by galactolipids, carotenoids, and
prenylquinones. A number of enzymes that are responsible
for their biosynthesis have been identified (for reviews, see
Dörmann and Benning 2002; Bouvier et al. 2005b; DellaPenna
and Pogson 2006), but most of them appear to be located
in compartments other than the outer envelope. Nonetheless,
limited numbers of lipid biosynthetic enzymes have been identified in the Arabidopsis chloroplast outer envelope including
four galactolipid galactosyltransferases, DGD1, DGD2, MGD2,
and MGD3. The DGD and MGD genes were originally identified
by genetic screening of mutant plants that were deficient in
DGDG and MGDG, respectively (Dörmann et al. 1995; Jarvis
et al. 2000). DGD1 and MGD1 provide the bulk of galactolipids
in green tissues, whereas DGD2 and MGD2/3 appear to be
responsible for the biosynthesis of galactolipids in non-green
plastids and in extraplastidic membranes on phosphate starvation (for review, see Benning and Ohta 2005). Both DGD1
and DGD2 were localized in the chloroplast outer envelope
(Froehlich et al. 2001a; Kelly et al. 2003). MGD1 was found in
the chloroplast inner envelope, whereas MGD2 and MGD3 were
suggested to be located in the outer envelope (Awai et al. 2001).
Thus, the chloroplast outer envelope clearly plays important
roles in the biosynthesis of galactoglycerolipids.
Some fatty acids synthesized in chloroplasts are postulated
to be exported to the cytoplasm where they are used as
precursors to glycerolipids at the endoplasmic reticulum. This
process requires reactivation of the fatty acids by acyl-CoA
synthethase in the chloroplast outer membrane (Andrews and
Keegstra 1983; Block et al. 1983b). Recently, long chain acylCoA synthetase 9 (LACS9) was localized to the chloroplast
envelope by in vitro import assay and its in vivo targeting as
a green fluorescent protein (GFP)-fusion, and was suggested
to be responsible for this fatty acid reactivation (Schnurr et al.
2002). However, its localization in the outer envelope has not
been proven yet (Koo et al. 2004).
Finally, two cytochrome P450s have been identified in the
chloroplast outer envelope membranes. One of them, fatty
acid hydroperoxide lyase, is involved in oxylipin biosynthesis
in tomato (Froehlich et al. 2001b), and its apparent Arabidopsis ortholog At4g15440 has been identified in the chloroplast
envelope proteome (Table 1). The physiological role of another
outer membrane cytochrome P450 CYP86B1 remains unknown
(Watson et al. 2001).
Other Proteins
In addition to the proteins described above, various proteins
of known and unknown functions have been identified in the
chloroplast outer envelope (Table 1). Among them, PDV1
(plastid division 1) and PDV2 were found to be responsible for
the recruitment of a cytoplasmic dynamin-related protein ARC5
(accumulation and replication of chloroplasts 5) during plastid
division (Miyagishima et al. 2006). A hexokinase, which may
be involved in the energization of glucose export, was found in
the outer envelope of spinach chloroplasts (Wiese et al. 1999).
Recently, however, this protein was demonstrated to be located
in mitochondria (Damari-Weissler et al. 2007).
Thylakoid formation 1 (THF1) is the only known protein which
is present both in the stroma and outer envelope of chloroplasts,
and also one of few outer envelope proteins that are synthesized
as a larger precursor with a transit peptide in the cytoplasm
(Wang et al. 2004; Huang et al. 2006). The stroma-located THF1
appears to be important for thylakoid formation (Wang et al.
2004; Keren et al. 2005), whereas the outer-envelope-located
THF1 may interact in the stromule with a plasma membrane Gprotein GPA1, which plays roles in the sugar signaling pathway
(Huang et al. 2006).
Outer envelope protein 14 (OEP14) from pea, which is actually a 7-kDa protein but migrates with an apparent molecular
mass of 14 kDa on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, is anchored to the membrane via a single αhelical domain in its N-terminus and is facing its soluble domain
to the cytoplasm. A 6.7-kDa protein orthologous to OEP14 is
one of predominant proteins in the outer membrane of the
spinach chloroplast envelope (Koike et al. 1998). Although their
functions remain unknown, OEP14 orthologs from pea, spinach,
and Arabidopsis have been used as a model to study protein
targeting and insertion to the chloroplast outer membrane
(Salomon et al. 1990; Li et al. 1991; Li and Chen 1996; Tu
and Li 2000; Schleiff et al. 2001; Lee et al. 2001; Tu et al. 2004).
Another protein of unknown function, OMP24 (outer membrane protein 24), which is distinct from OEP24, was identified
as one of the major outer membrane proteins of spinach
chloroplasts (Joyard et al. 1983). It is unique in its high content
of proline and acidic residues (Fischer et al. 1994a). Its putative
The Chloroplast Outer Envelope Membrane Proteome
homolog At3g52230 has been identified in the Arabidopsis
chloroplast envelope proteome (Table 1).
1107
Andersson MX, Kjellberg JM, Sandelius AS (2004). The involvement
of cytosolic lipases in converting phosphatidyl choline to substrate for
galactolipid synthesis in the chloroplast envelope. Biochim. Biophys.
Acta 1684, 46–53.
Conclusions and Future Perspectives
Andrews J, Keegstra K (1983). Acyl-CoA synthetase is located in the
outer membrane and acyl-CoA thioesterase in the inner membrane
Recognition and translocation of nuclear-encoded precursor
proteins, the exchange of various ions and metabolites between
the cytoplasm and the organelle, and lipid biosynthesis have
been the major focuses of the research on the chloroplast outer
envelope. Recent multidisciplinary studies have identified proteinaceous components responsible for these processes. In addition, proteins involved in division, sugar-signaling, and those
with unknown functions have been found in the chloroplast outer
envelope. Because of the lack of predictable transit peptides,
identification of chloroplast outer membrane proteins requires
vigorous biochemical experiments and also can be assisted by
proteomic studies. Table 1 lists 34 proteins (including two VDAC
homologs, whose chloroplast localization may be questionable),
which should cover the main components of the Arabidopsis
chloroplast outer envelope. This list underlines the importance
of the chloroplast outer envelope not only for translocation of a
variety of molecules but also as a critical cellular compartment
to regulate various metabolic activities and signaling processes.
The list also provides us with a number of intriguing questions.
An obvious first question concerns the physiological roles of
the proteins of unknown functions including the putative solute
channels. The second question is how these proteins are
targeted to and assembled in the chloroplast outer envelope.
It has been known that distinct but partly shared pathways
are present for the targeting of signal-anchored, tail-anchored,
polytopic, and β-barrel membrane proteins to the mitochondrial
outer membrane (Rapaport 2003, 2005; Neupert and Herrmann
2007). By contrast, our understanding of protein targeting to the
chloroplast outer membrane is limited virtually only to a β-barrel
protein with the unusual bipartite transit peptide Toc75 and a
signal-anchored protein OEP14 and its orthologs. More work
is definitely required to address the question of how unique
the protein targeting to the chloroplast outer membrane is.
Finally, evolutionary origins of the protein import machineries,
especially that of Toc75, have been discussed but not completely understood yet (Reumann et al. 2005; Schleiff and Soll
2005). Addressing these questions requires multidisciplinary approaches and should contribute to advancing our understanding
of organelle biogenesis, plant growth and development, and
membrane biology in general.
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