Download Structure and function of the chloroplast signal recognition particle

Curr Genet (2004) 44: 295–304
DOI 10.1007/s00294-003-0450-z
R EV IE W A RT I C L E
Danja Schünemann
Structure and function of the chloroplast signal recognition particle
Received: 16 July 2003 / Revised: 5 September 2003 / Accepted: 7 September 2003 / Published online: 21 October 2003
Springer-Verlag 2003
Abstract The targeting of proteins, including the insertion and translocation of proteins in or across membranes, is a fundamental process within a cell, and a
variety of specialized mechanisms for protein transport
have been developed during evolution. The signal recognition particle (SRP) is found in the cytoplasm of
most, if not all, eukaryotes and prokaryotes where it
plays a central role in the co-translational insertion of
membrane proteins into the endoplasmic reticulum and
plasma membrane, respectively. SRP is a ribonucleoprotein consisting of an RNA and at least one polypeptide of 54 kDa (SRP54). Interestingly, chloroplasts
contain a specialized type of signal recognition particle.
Chloroplast SRP (cpSRP) contains a SRP54 homologue
but differs strikingly from cytosolic SRP in various aspects of structure and function. In contrast to cytosolic
SRP, it contains a novel protein subunit (cpSRP43) and
lacks RNA. CpSRP is also distinctive in its ability to
interact with its substrate, light-harvesting chlorophyll
a/b-binding protein, post-translationally. Furthermore,
it is remarkable that the 54 kDa subunit of cpSRP is
also involved in the co-translational transport of chloroplast-encoded thylakoid proteins, and is therefore able
to switch between the co- and post-translational means
of interaction with its respective substrate proteins.
Keywords Chloroplast Æ Thylakoid Æ Signal recognition
particle Æ Protein transport Æ LHCP Æ ALB3
Communicated by S. Hohmann
D. Schünemann
Lehrstuhl für Allgemeine und Molekulare Botanik,
Ruhr-Universität Bochum, 44780 Bochum, Germany
E-mail: [email protected]
Tel.: +49-234-3228561
Fax: +49-234-3214184
Introduction
A mechanism of protein transport that has been extensively studied is the co-translational protein transport to
the endoplasmic reticulum (ER) of eukaryotic cells. The
cytosolic components, as well as the membrane components like the receptors and the translocation pore,
have been characterized (reviewed in Martoglio and
Dobberstein 1996; Rapoport et al. 1996; Johnson and
van Waes 1999). The targeting process is initiated when
the signal peptide of the protein emerges from the
ribosome and is recognized by the signal recognition
particle (SRP). SRP is a cytosolic ribonucleoprotein
consisting of a 7S-RNA and six polypeptides. The
54-kDa subunit (SRP54) mediates the binding to the
signal peptide, whereas the subcomplex SRP9/14 causes
an elongation arrest. The complex of ribosome, SRP,
and the nascent protein binds to the SRP receptor (SR)
in the ER. The SRP receptor consists of an integral
membrane protein (SRb) and a peripheral membrane
protein (SRa). Upon dissociation of SRP from the signal
peptide, translation resumes and the protein is translocated co-translationally through the translocation pore
(Sec61p) into the ER, and SRP dissociates from the
receptor in a GTP requiring step.
The Escherichia coli SRP pathway is related to the
eukaryotic SRP pathway and mediates primarily the cotranslational targeting of most plasma membrane proteins (reviewed in Bernstein 2000; de Gier and Luirink
2001; Valent 2001). Like the eukaryotic SRP, the E. coli
SRP is also a ribonucleoprotein. It consists of an RNA
component (4.5S-RNA) and one protein component
[Ffh (fifty four homologue)]. Both components are
homologous to the 7S-RNA and SRP54 of the eukaryotic SRP, respectively. The E. coli protein FtsY was
identified as a SRP receptor based on sequence similarity
to the a-subunit of the eukaryotic SRP receptor (SRa).
In contrast to SRa, FtsY is localized as a peripheral
protein at the plasma membrane as well as a soluble
protein in the cytoplasm (Luirink et al. 1994). It was
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shown that the cytoplasmic FtsY binds to the SRP/
ribosome/nascent protein complex and delivers this
complex to the plasma membrane via interaction of the
N-terminal domain of FtsY with membrane components
(Zelazny et al. 1997; Valent et al. 1998; de Leeuw et al.
2000). The direct contact of FtsY with acidic phospholipids in the membrane might be the reason why no
homologous protein for the b-subunit of the eukaryotic
SRP receptor has yet been identified. SRP is released at
the plasma membrane from the translating ribosome in a
GTP-dependent reaction, and the protein uses the SecYEG translocon for insertion into the membrane
(Valent et al. 1998).
Plant cells are characterized by the presence of specific organelles: the plastids. Chloroplasts are the site of
photosynthesis and of various essential metabolic pathways, e.g., fatty acid and amino acid biosynthesis. It is
estimated that a chloroplast contains up to 3,500
Fig. 1 Targeting pathways for nuclear encoded proteins from the
chloroplast stroma to the thylakoid lumen or thylakoid membrane.
Transport of nuclear encoded proteins from the stroma to the
thylakoid lumen is mediated via the cpTat pathway and the cpSec
pathway (cp chloroplast, Tat twin arginine translocation, Sec
secretory). Substrate proteins for these pathways (i.e., OE23 23kDa subunit of the oxygen-evolving complex, PC plastocyanin)
require an N-terminal targeting domain (TD) that directs the
protein to either pathway. After translocation, the signal peptide is
cleaved off by a thylakoidal processing peptidase. A twin arginine
motif (RR) is the main characteristic of the signal peptide for the
Tat pathway. Insertion of nuclear encoded proteins into the
thylakoid membrane is mediated via the cpSRP pathway (cpSRP
chloroplast signal recognition particle) and by a spontaneous
insertion process. Proteins that insert spontaneously into the
membrane also require a specific signal peptide and are mostly
single-span membrane proteins. As substrate proteins for the
cpSRP pathway, only members of the light-harvesting chlorophyll
a/b binding proteins (LHCPs) are known, whose targeting
information resides within the mature protein. In contrast to the
spontaneous insertion process, several soluble and membrane
proteins (yellow boxes) in addition to specific energy conditions
(white boxes) are required for the three assisted pathways. The
different targeting pathways are reviewed in, for example, Eichacker and Henry 2001; Mori and Cline 2001; Robinson et al. 2001;
Schleiff and Klösgen 2001, and this work
proteins (Abdallah et al. 2000), the vast majority of
which are encoded in the nucleus with only about 100
encoded in the plastid genome. Most of the nuclearencoded proteins must be imported post-translationally
across the translocon of the inner and outer envelope
into the organelle, and thereafter distributed to the
different subcompartments within the plastid. Four different pathways have been described so far for the posttranslational translocation of proteins from the stroma
to the thylakoid membrane or across the membrane into
the thylakoid lumen: the Sec pathway and the Tat
pathway are mainly involved in the transport of lumenal
proteins, whereas the spontaneous pathway and the
cpSRP pathway are used for the insertion of integral
membrane proteins into the thylakoid membrane (reviewed in, e.g., Eichacker and Henry 2001; Mori and
Cline 2001; Robinson et al. 2001; Schleiff and Klösgen
2001). The main characteristics of these different targeting mechanisms are summarized in Fig. 1.
The post-translational cpSRP pathway
The post-translational chloroplast SRP (cpSRP) pathway is involved in targeting of members of the nuclear
encoded light-harvesting chlorophyll a/b-binding protein (LHCP) family from the stroma to the thylakoid
membrane. LHCPs constitute approximately one-third
of the total thylakoid protein, and therefore, are the
major integral thylakoid membrane proteins (Yamamoto and Bassi 1996). The LHC super-gene family of
Arabidopsis contains more than 30 members (Jansson
1999). The major LHCP (Lhcb1) belongs to photosystem II and represents perhaps the most abundant
membrane protein in nature. LHCPs are encoded in the
nucleus and synthesized in the cytosol with an N-terminal transit sequence that codes for the post-translational transport across the envelope membranes into the
stroma of the chloroplast. In the stroma, the transit sequence is cleaved off and LHCP is bound by stromal
297
components. This complex was designated ‘‘transit
complex’’ and maintains the solubility of the hydrophobic LHCPs as they are transported through the
stroma (Payan and Cline 1991). The first stromal component of the transit complex was identified as a soluble
54 kDa GTPase (cpSRP54), which is a homologue of the
54-kDa subunit of the eukaryotic signal recognition
particle (SRP54) and the Ffh subunit of the prokaryotic
SRP (Franklin and Hoffman 1993; Li et al. 1995).
Purification of the chloroplast signal recognition particle
(cpSRP) by immunoprecipitation of stroma using anticpSRP54 antibodies (Schünemann et al. 1998) and a
genetic approach using transposon tagging (Klimyuk
et al. 1999) led to the identification of the second subunit
of cpSRP, a stromal 43-kDa protein (cpSRP43). In vitro
reconstitution experiments demonstrated that both
cpSRP subunits, cpSRP54 and cpSRP43, are necessary
and sufficient to form the transit complex with LHCP
(Schünemann et al. 1998; Yuan et al. 2002). It should be
noted that cpSRP43 shows no homologues in the databases and therefore represents a novel type of SRP
subunit with an unclear evolutionary origin.
From these results, it became evident that chloroplasts contain a specialized SRP that contains a homologue of SRP54, but differs strikingly from the cytosolic
SRPs as it (1) lacks an RNA, (2) contains a novel 43kDa subunit, and (3) interacts with its substrate proteins
post-translationally (Table 1).
The transit complex is thought to be an intermediate in
the targeting of LHCP to the thylakoid membrane, because it was shown that the transit complex contains a
productive form of LHCP capable of integrating into the
membrane upon addition of stroma and GTP (Payan and
Cline 1991; Hoffman and Franklin 1994). Besides
cpSRP43 and cpSRP54, the third stromal protein
required for LHCP integration was identified as cpFtsY,
a chloroplast homologue of the bacterial SRP receptor
(FtsY). Initially, this finding was based on the observation that anti-cpFtsY antibodies inhibited LHCP
integration into isolated thylakoid membranes (Kogata
et al. 1999). Later it was demonstrated that the soluble
phase required for LHCP integration could be reconstituted by cpSRP (cpSRP54 and cpSRP43), cpFtsY, and
GTP (Tu et al. 1999; Yuan et al. 2002). CpFtsY is
localized mainly in the stroma, but a significant fraction is
associated with the thylakoid membrane (Tu et al. 1999).
Currently, nothing is known about the precise role of
cpFtsY in LHCP integration. By analogy, with the bacterial system it can be speculated that stromal cpFtsY
binds to subcomponents of the transit complex and
delivers it to the translocon of the thylakoid membrane.
However, no experimental data exist to support this
assumption and it will be an interesting question for the
future to analyze the mode of interaction between transit
complex, cpFtsY, and the translocon.
In order to understand the integration mechanism of
LHCP into the thylakoid membrane in detail, it will also
be important to elucidate the role of the nucleotides
involved in this process. So far, it is known that GTP
hydrolysis is absolutely required for LHCP integration
and that ATP stimulates this process if it is present in
combination with GTP (Hoffman and Franklin 1994;
Yuan et al. 2002). As GTP and ATP do not seem to have
a significant influence on transit complex formation,
they are apparently involved in the later stages of LHCP
integration. By analogy, with the bacterial SRP pathway, it is feasible that GTP is required to regulate the
possible interaction of the GTPases cpSRP54 and
cpFtsY during delivery of LHCP to the translocon. The
role of ATP in LHCP insertion is completely unresolved
as no ATP-binding protein involved in this process has
yet been identified.
Structure of cpSRP and transit complex
Database analysis of the cpSRP43 sequence identified
two types of motifs, the ankyrin repeat domain and the
Table 1 Subunit composition of cytosolic and chloroplast SRPs. The individual components of cytosolic and chloroplast SRPs and their
main functional characteristics are listed
Mammaliana
SRP54
7S RNA
–
SRP9/14
SRP19
SRP68/72
Binding to substrate
Elongation
Elongation Assembly
proteins, SRP-RNA and arrest; catalytic
arrest
of SRP, promotes
SRP receptor; GTPase
influence on SRP
association
and SR interaction
of SRP54
a
Bacterial
Ffh
4.5S RNA
–
–
–
–
Binding to substrate
Catalytic
proteins, SRP-RNA and influence on Ffh
SRP receptor (FtsY);
and FtsY interaction
GTPase
cpSRP54
–
cpSRP43
–
–
–
Chloroplastb
post-translational Binding to
Binding
LHCPs, cpSRP43
to L18 domain of
and cpFtsY; GTPase
LHCP and cpSRP54
cpSRP54
?
–
?
?
?
Chloroplastb
co-translational Binding to TM1
of D1, GTPase
a
Reviewed in Keenan et al. 2001
Reviewed in Eichacker and Henry 2001 and this work
b
298
chromodomain, which are known to mediate proteinprotein interactions. The ankyrin repeat, an approximately 33-amino acid long motif, is found in a very large
number of proteins, where it generates the dimerization
interface for a variety of different protein substrates
(reviewed in Bork 1993; Sedgwick and Smerdon 1999).
The chromodomain is a 30–70 amino acid motif found
in various proteins involved in the regulation of chromatin structure by protein-protein interactions (Koonin
et al. 1995; Eissenberg and Elgin 2000). Four ankyrin
repeats were found in the N-terminal region of
cpSRP43, and two closely spaced chromodomains were
identified in the C-terminal region (Klimyuk et al. 1999;
Jonas-Straube et al. 2001). Later, a third chromodomain
located near the N-terminus of the mature protein was
predicted (Eichacker and Henry 2001). It will be an
interesting quest for the future to get detailed information about the role of the different domains in the formation of the cpSRP and the transit complex, since only
a few studies have been undertaken to address this
question so far. Recently, it was shown by using the
yeast two-hybrid system that the C-terminal region of
cpSRP43, containing the two closely spaced chromodomains, mediates the binding of cpSRP54 (Jonas-Straube et al. 2001). The binding site of cpSRP54 for
cpSRP43 was determined to reside in the C-terminal half
of cpSRP54 containing the M-domain (Jonas-Straube
et al. 2001). Shortly after, it was shown that within this
region the last C-terminal 26 amino acids interact
strongly with cpSRP43 (Groves et al. 2001). Interestingly, these amino acids present a positively charged
region unique to cpSRP54. The absence of this region in
Ffh, the E. coli SRP54 homologue, explains the previous
finding that Ffh is not able to form a complex with
cpSRP43 (Schünemann et al. 1999).
A point of discussion is whether cpSRP43 can
dimerize. Initial data based on gel filtration and chemical
cross-linking experiments using recombinant cpSRP43
suggested that cpSRP43 is a dimer (Tu et al. 1999). A
dimerization of cpSRP43 was also observed in in vivo
experiments using the yeast two-hybrid system, and the
dimerization motif was confined to the third and fourth
ankyrin repeat (Jonas-Straube et al. 2001). These results,
together with the observation that cpSRP54 exists as a
monomer in cpSRP, led to the hypothesis that cpSRP is a
heterotrimer composed of one cpSRP54 and a cpSRP43
dimer (Tu et al. 1999). The molecular mass of this complex predicted from the cDNA sequence would be 123
kDa. This value differs significantly from a molecular
mass estimate of 200 kDa for stromal cpSRP obtained by
gel filtration (Schünemann et al. 1998, Tu et al. 1999).
Recent data indicate that cpSRP43 is not a globular
protein but exhibits an elongated shape (Groves et al.
2001). This structural feature of cpSRP43 is possibly the
reason for the overestimation of the apparent molecular
mass of cpSRP using gel filtration. In contrast to Tu et al.
(1999), no dimerization of recombinant 6-His-cpSRP43
could be observed by Groves et al. (2001), using equilibrium ultracentrifugation. By using the same method,
these authors showed that recombinant cpSRP has a
molecular mass of 98 kDa, which fits to the molecular
mass of 120 kDa for the transit complex that was
obtained by nondenaturing gel analysis (Payan and Cline
1991). Therefore, Groves et al. (2001) support the model
of cpSRP being a heterodimer. However, these authors
mention in their discussion initial data indicating that a
deletion construct of cpSRP43 lacking the C-terminal
chromodomains forms a stable dimer. These results
support the observation of Jonas-Straube et al. (2001)
that the ankyrin repeat containing domain of cpSRP43 is
able to dimerize. When we consider all of the evidence
together, it appears conceivable that a potential dimerization of cpSRP43 might occur only in a transient
functional state.
Post-translational cpSRP-mediated targeting to the
thylakoid membrane is restricted to LHCPs, and
therefore posed the question which structural characteristics of the LHCPs determine the transit complex
formation with cpSRP. By analogy, with the co-translational targeting system where binding of cytosolic
SRP54 to a substrate protein is mediated via its
hydrophobic signal sequence, it was shown that a
hydrophobic domain within LHCP is required for the
post-translational binding to cpSRP (DeLille et al.
2000; Tu et al. 2000). However, the presence of this
domain alone is not sufficient to promote transit
complex formation. A second unique recognition element present only in a subset of LHCPs was identified
to be an 18-amino acid hydrophilic domain (L18 domain) located between the second and third transmembrane domains of LHCP (DeLille et al. 2000). In
further studies, it was shown that the L18 domain of
LHCP binds to the cpSRP43 subunit of cpSRP (Tu
et al. 2000) and that this binding is mediated via the
first ankyrin repeat of cpSRP43 (Jonas-Straube et al.
2001). In vitro pull-down assays showed that the
binding between cpSRP43 and LHCP is much stronger
than the one between cpSRP54 and LHCP (Tu et al.
2000). This observation together with the finding that
cpSRP54 can be efficiently cross-linked to LHCP in the
transit complex (Li et al. 1995) led to the assumption
that the specific recruitment of LHCPs for transit
complex formation is initiated by binding of L18 to the
first ankyrin repeat of cpSRP43 followed by the contact
formation between LHCP and cpSRP54. Experiments
analyzing the interaction of cpSRP54 and LHCP by
using a cross-linking or a pepscan approach demonstrated that cpSRP54 binds to the third transmembrane
domain of LHCP, which represents the most hydrophobic of the three transmembrane domains of LHCP
(High et al. 1997; Groves et al. 2001). However, the
presence of the third transmembrane domain is not
absolutely required for transit complex formation, as a
deletion construct of LHCP lacking this domain is still
able to form the transit complex with cpSRP (DeLille
et al. 2000). These data indicate that cpSRP54 is also
able to interact with the first and/or second transmembrane domain of LHCP in the transit complex.
299
Therefore, it is conceivable that the structure of the
transit complex switches between different functional
conformations that might be required for the delivery
and consecutive release of LHCP to the thylakoid
membrane.
The co-translational cpSRP pathway
In bacteria, the SRP pathway is mainly involved in the
co-translational targeting of integral membrane proteins
to the plasma membrane. The thylakoid membrane
contains four major protein complexes involved in
photosynthesis. Several of their membrane proteins are
encoded in the chloroplast genome and inserted cotranslationally into the thylakoid membrane. The first
evidence that a co-translational SRP pathway is functional in chloroplasts came from the observation that the
stroma contains two different pools of cpSRP54. One
pool is bound to cpSRP43 and active in transit complex
formation with LHCP, whereas a second pool of
cpSRP54 was found to be associated with 70S ribosomes
in absence of cpSRP43 (Franklin and Hoffman 1993;
Schünemann et al. 1998). The direct involvement of
cpSRP54 in the co-translational targeting of a plastidencoded membrane protein was shown by cross-linking
of stromal cpSRP54 to the ribosome bound nascent
chain of D1, obtained by a homologous chloroplast in
vitro translation system (Nilsson et al. 1999). Notably,
no interaction of cpSRP43 with the D1 nascent chain
was detected, supporting the specialized role of cpSRP43
in post-translational targeting (Nilsson et al. 1999).
Further analysis of the interaction of cpSRP54 and the
nascent chain of D1 revealed that cpSRP54 binds
tightly, but transiently, to the first transmembrane domain of the elongating nascent chain. This interaction
occurs only if the nascent chain is still attached to the
ribosome, and it is gradually lost as elongation proceeds
(Nilsson and van Wijk 2002). Furthermore, these authors demonstrated that the first transmembrane domain could be replaced by other hydrophobic domains
of more than ten amino acids, indicating that the
hydrophobicity of a nascent chain and not a specific
sequence motif is the prerequisite for the binding of
cpSRP54. This supports earlier results demonstrating
that the efficiency of cross-linking of cpSRP54 to signal
sequences correlates to their hydrophobicity (High et al.
1997). Therefore, the recognition mechanism between
cpSRP54 and its substrate proteins seems to resemble
the cytosolic system (SRP-dependent co-translational
targeting to the ER or E. coli plasma membrane) where
the signal sequence must possess a certain threshold level
of hydrophobicity for SRP-mediated targeting (reviewed
in Keenan et al. 2001).
Recently, it was shown that the thylakoid membrane
protein cpSecY, a homologue of the bacterial SecY
subunit of the plasma membrane translocon, is involved
in D1 protein insertion by transient interaction with D1
intermediates during elongation (Zhang et al. 2001).
Currently, nothing is known about the targeting mechanism of the SRP-ribosome-nascent chain complex to
the cpSecY translocon, but it seems very likely that by
analogy with the bacterial SRP pathway cpFtsY, the
chloroplast homologue of the SRP receptor is involved
in this process. A model of the role of cpSRP in postand co-translational protein targeting to the thylakoid is
shown in Fig. 2.
Analysis of Arabidopsis mutants lacking either one
or both subunits of cpSRP
Studies on Arabidopsis mutants lacking cpSRP43 (chaos
mutant, Klimyuk et al. 1999), cpSRP54 (ffc mutant,
Amin et al. 1999) or both cpSRP subunits (chaos/ffc
double mutant, Hutin et al. 2002) support the important
role of cpSRP in the post-translational targeting of
members of the LHCP family to the thylakoid membrane and the additional involvement of cpSRP54 in the
co-translational targeting of chloroplast encoded thylakoid membrane proteins. The chaos mutant is characterized by a specific defect in LHCP biogenesis. The
plant has a chlorotic phenotype, the chlorophyll content
is reduced by 50%, and the chlorophyll a/b ratio is elevated. Western-blot analysis revealed that the amount of
most analyzed LHCPs is significantly reduced, whereas
no reduction of other proteins could be detected (a detailed description of the phenotype is given in Table 2).
These findings are in agreement with the specialized role
of cpSRP43 in LHCP targeting. Interestingly, plants
lacking functional cpSRP54 (Pilgrim et al. 1998; Amin
et al. 1999) differ significantly from the chaos mutant in
two major aspects of their phenotype. Firstly, the phenotype of these plants is most visible at the young
seedling stage when the first true leaves are yellow. In
contrast to the chaos mutant, the phenotype becomes
less severe when the plant matures, as seen by a recovery
of the first true leaves and by the observation that the
older ffc leaves are greener than the young ffc leaves. In
addition, the subset of photosystem proteins that is reduced in the young leaves is present at wild-type level in
the older leaves (for details see Table 2). Secondly, the
young leaves of the ffc mutant show a reduced level of
the chloroplast encoded photosystem I and II reaction
center proteins in addition to a reduction of the same
subset of LHCPs as the chaos mutant. These data support the overlapping roles of cpSRP43 and cpSRP54 in
LHCP targeting and the additional involvement of
cpSRP54 in targeting of chloroplast encoded thylakoid
membrane proteins. However, it should be noted that
the chaos and ffc mutants still contain a significant
amount of LHCPs ( 50%), which was an unexpected
outcome since the cpSRP subunits are inactive individually in vitro (Schünemann et al. 1998; Groves et al.
2001; Yuan et al. 2002). Therefore, it was speculated that
the single subunits might be active in vivo, or that a
fraction of the LHCPs are targeted by one or more
alternative targeting pathways. To address this question,
300
Fig. 2 Role of the cpSRP subunits in the post- and co-translational
targeting of nuclear or chloroplast encoded proteins to the
thylakoid membrane. CpSRP consists of the stromal subunits
cpSRP54 and cpSRP43. A point of discussion is whether cpSRP43
can dimerize (see text for further information). Upon import of the
nuclear encoded LHCP into the stroma, cpSRP binds to LHCP to
form the transit complex, a soluble intermediate of LHCP. Besides
cpSRP, integration of LHCP into the thylakoid membrane requires
GTP, cpFtsY (the chloroplast homologue of the bacterial SRP
receptor) and the integral membrane protein Alb3. Current
evidence suggests that insertion of LHCP into the thylakoid
membrane is independent of the cpSecY/E translocase. In contrast
to cpSRP43, cpSRP54 is also involved in the co-translational
targeting of the chloroplast encoded D1 to the thylakoid
membrane. Insertion of D1 into the membrane is mediated via
the cpSecY/E translocase possibly in concert with Alb3. Alb3 was
found to be at least partially associated with the cpSecY/E
translocase
a double mutant containing no functional cpSRP54 and
cpSRP43 was created (Hutin et al. 2002). The phenotype
of these plants is more severe than that of the single
mutants. The double mutant has pale yellow leaves at all
stages of growth, and the levels of all LHCPs (and
ELIPs), except Lhcb4, are significantly more reduced
than in the single mutants (Table 2). As expected, the
double mutant exhibits also a reduction of the chloroplast encoded photosystem I and II reaction center
proteins and the inner antennae proteins CP43 and
CP47. As in the single mutants, no reduction was observed for psbS, which can insert spontaneously into the
thylakoid membrane (Kim et al. 1999). Taken together,
the stronger phenotype of the double mutant suggests
that the individual cpSRP subunits are partially active in
LHCP targeting in vivo. However, it should be noted
that even the double mutant contains significant
amounts of Lhcb1, Lhcb6, and Lhca2, and that the level
of Lhcb4 in the double mutant is equal to, or even higher
than, the wild-type level, although in vitro experiments
point to a SRP-dependent targeting of Lhcb4 (Woolhead et al. 2001). Based on these observations, the
hypothesis that at least some members of the LHCP
family can be partially transported to the thylakoid
membrane by a SRP-independent alternative pathway
cannot be ruled out. Several studies analyzing the biogenesis of thylakoid membranes provide growing evidence that chloroplasts contain a transport system via
vesicles that bud from the envelope membrane and fuse
with growing thylakoids (reviewed in Hoober and
Eggink 1999; Vothknecht and Westhoff 2001). It may be
possible that such a pathway moves LHCPs from the
envelope to the thylakoid membrane. Alternatively, it
might be possible that other mechanisms like the
induction of heat shock proteins partially compensate
the lack of cpSRP as observed in yeast mutants lacking
SRP (Mutka and Walter 2001).
Role of the integral thylakoid membrane protein ALB3
in cpSRP-dependent protein targeting
As described in the previous sections, the soluble components involved in LHCP targeting are well-defined;
however, little is known about the translocase mediating
the integration of LHCP into the thylakoid membrane.
Recently, the integral thylakoid membrane protein
ALB3 was identified as the first integral membrane
protein required for the insertion of LHCP into the
thylakoid membrane (Sundberg et al. 1997; Moore et al.
2000). The participation of ALB3 in LHCP insertion has
been demonstrated by the observation that preincuba-
301
Table 2 Summary of the main
characteristics of Arabidopsis
mutants lacking functional
cpSRP54 (T115 N line: Pilgrim
et al. 1998, ffc1–2: Amin et al.
1999), cpSRP43 (chaos: Amin
et al. 1999; Klimyuk et al.
1999), or both subunits of
cpSRP (Hutin et al. 2002). fl,
fi , ›: lower, equal, higher
amount compared to wild-type
(WT),10d 10-day old plants,
24d 24-day old plants, - not
determined
Appearance
Chlorophyll
content
Chlorophyll
a/b ratio
Plastids
Protein level:
Lhca1
Lhca2
Lhca3
Lhca4
Lhcb1
Lhcb2
Lhcb3
Lhcb4
Lhcb5
Lhcb6
psbS
Elips
D1/D2
psaA/B
CP43/CP47
DcpSRP54
DcpSRP43
Double mutant
Yellow first true leaves that
become green within a
week; older leaves almost
like WT
Reduction by 75% in first
true leaves
WT level
Chlorotic, all
leaves pale
green no
recovery
50% reduction
All leaves more
yellow than
chaos no
recovery
85% reduction
Elevated
WT level
Fewer thylakoids (first true
leaves)
10 d
24 d
fl
fi
fi
fi
fl
fi
fl
fi
fl
fi
fl
fi
fl
fi
›
›
fl
fi
fi
fi
›
›
–
–
fl
fi
fl
fi
fl
fi
Normal
Drastic reduction
of thylakoids
tion of thylakoid membranes with anti-ALB3 antibodies
inhibited this process (Moore et al. 2000). Later, it was
shown that ALB3 is also required for the insertion of
Lhcb5 and Lhcb4.1, two other LHC proteins whose
insertion is dependent on the presence of NTPs and
stromal factors (Kim et al. 1999; Woolhead et al. 2001).
Although it has not been tested, it is highly likely that
these stromal factors comprise cpSRP and cpFtsY.
Therefore, it can be assumed that ALB3 plays a general
role in the insertion of SRP-dependent LHC proteins.
ALB3 is a homologue of the bacterial membrane
protein YidC and the mitochondrial membrane protein
Oxa1p that mediate the protein insertion into the inner
membrane of bacteria and mitochondria, respectively.
In E. coli, most inner membrane proteins are targeted
co-translationally to the cytoplasmic membrane using
SRP and the SRP-receptor FtsY. The insertion of
these proteins into the inner membrane is mediated via
the SecY-translocase. Recently, the inner membrane
protein YidC was identified as a novel component of
the SecY-translocase (Scotti et al. 2000). Several
studies indicate that YidC facilitates the lateral diffusion of the transmembrane domains of the Secdependent proteins from the translocase into the lipid
bilayer (Houben et al. 2000; Beck et al. 2001; van der
Laan et al. 2001; Urbanus et al. 2001). Interestingly,
other studies demonstrated that YidC can also function independently from the SecY-translocase by
showing that YidC is essential for the insertion of a
subset of E. coli inner membrane proteins whose
insertion mechanism is Sec-independent (Samuelson
et al. 2000, 2001; Chen et al. 2002). Taken together,
these results indicate that YidC can function in concert
fl
fi
fl
fl
fl
fl
fl
›
fl
fi
›
–
fi
fi
fi
fl
fl
fl
fl
fl
fl
fl
›
fl
fl
›
fl
fl
fl
fl
(0%)
(<50%)
(0%)
(<50%)
(<50%)
(<10%)
(0%)
(<130%)
(<10%)
(<50%)
with the SecY-translocase and can also function independently of the SecY-translocase.
The mitochondrial homologue of YidC is the inner
membrane protein Oxa1p that is involved in the insertion of nuclear and mitochondrial encoded inner membrane proteins (Kermorgant et al. 1997; Hell et al. 1998,
2001). Based on the observation that yeast mitochondria
do not contain proteins with homology to the subunits
of the bacterial-type Sec translocase, it is most likely that
Oxa1p operates exclusively in a Sec-independent manner
(Glick and von Heijne 1996).
Currently, only little is known about the oligomeric
state of ALB3 and its precise function in protein
insertion into the thylakoid membrane. Work from
Mori et al. (1999) suggests that ALB3 functions independently from the cpSecY-translocase in LHCP integration. This was concluded from the observation that
the insertion of LHCP into the thylakoid membrane
could not be inhibited by pre-incubation of the membrane with antibodies directed against cpSecY, the
chloroplast homologue of bacterial SecY, whereas the
translocation of a Sec-dependent protein was inhibited
under these conditions. These results indicated for the
first time that an inner membrane protein might be
inserted using a SRP/ALB3-dependent but SecYindependent mechanism. Interestingly, recent work
analyzing the biogenesis of a set of model inner membrane proteins in a variety of SRP, Sec, and YidC
mutant E. coli strains demonstrated that a SRP/YidCdependent and SecY-independent pathway is operational in E. coli (Fröderberg et al. 2003) However, it is
discussed in the literature that the cpSecY-independence
of LHCP integration has not yet been demonstrated
302
the involvement of cpSRP54 in the co-translational
transport of chloroplast-encoded thylakoid proteins.
Despite considerable progress concerning mainly the
interaction of cpSRP54 with nascent D1 during elongation, many major questions remain to be resolved. So
far, cpSRP54 is the only identified component of the cotranslational cpSRP, and it will be very important to
analyze whether other components associate with
cpSRP54 during co-translational protein targeting. For
the post-translational SRP pathway, it was unequivocally demonstrated that no RNA is required for a
functional cpSRP. But it is not known yet whether the
co-translational SRP pathway requires an SRP-RNA, as
is the case for all known eukaryotic and prokaryotic
cytosolic SRPs. Another interesting aspect that needs to
be addressed in the future concerns the question of how
cpSRP54 is recruited for functioning in either the posttranslational or co-translational cpSRP pathway. For
instance, do the ribosome nascent chains and cpSRP43
or the different substrate proteins compete for binding
cpSRP54? The analysis of these questions will provide
more insight in the precise molecular details, the
dynamics and the regulatory aspects of the cpSRP
dependent protein targeting mechanism.
unequivocally, as it might be possible that the anticpSecY antibodies bind to a region of cpSecY that is
not required to mediate LHCP insertion. Furthermore,
contradicting a previous assumption (Cline and Mori
2001), recent experimental data demonstrated that
ALB3 is at least partially associated with the cpSecYtranslocase (Klostermann et al. 2002). Therefore, the
possibility that the cpSecY/E translocase is involved in
LHCP integration cannot be ruled out and further
work is required to test this. By analogy with the role of
Sec-dependent YidC in co-translational membrane
protein insertion in bacteria, it seems likely that cpSecY-associated ALB3 is involved in the insertion of
chloroplast encoded thylakoid membrane proteins. This
hypothesis is supported by the recent observation that
ALB3, cpSecE, and cpSecY co-purify with ribosome
attached nascent chains of D1 that are detergent extracted from the thylakoid membrane (Klaas Jan van
Wijk, personal communication). In addition, it was
shown that ALB3 functionally complements a YidC
depletion strain of E. coli and promotes the insertion of
leader peptidase, a YidC/SRP/Sec-dependent integral
membrane protein of E. coli (Jiang et al. 2002). Taken
together, these experiments demonstrate that ALB3 is a
functional homolog of YidC and can function in concert with a Sec-translocase in the SRP-dependent cotranslational insertion of integral membrane proteins.
Acknowledgements This work was supported by the Deutsche
Forschungsgemeinschaft.
Conclusions and outlook
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