Nuclear encoded thylakoid precursor proteins are imported across the chloroplast envelope into the chlo-roplast stroma by a common import apparatus, namely the Toc⁄ Tic translocon at the
Trang 1Protein transport in organelles: Protein transport into and across the thylakoid membrane
Cassie Aldridge*, Peter Cain* and Colin Robinson
Department of Biological Sciences, University of Warwick, Coventry, UK
Introduction
Chloroplasts are the site of photosynthesis and other
important biochemical processes that are vital for the
functioning of plant cells They are believed to have
arisen from a photosynthetic bacterium taken up by a
primitive eukaryotic cell Although some of the
chloro-plast proteome is encoded by the chlorochloro-plast genome,
during endosymbiosis, most of the original prokaryotic
genome was lost or transferred to the nuclear genome;
therefore, the vast majority of chloroplast proteins are
nuclear encoded and require transport into the
plast Whether synthesized in the cytosol or the
chloro-plast stroma, a sub-set of proteins require transport
into or across the thylakoid membranes to attain their
functional locations
Nuclear encoded thylakoid precursor proteins are imported across the chloroplast envelope into the chlo-roplast stroma by a common import apparatus, namely the Toc⁄ Tic (translocon at the outer ⁄ inner envelope membrane of chloroplasts) complex [1] By contrast, import into or across the thylakoid membrane is thought to occur through four independent precursor-specific thylakoid transport pathways that are descen-dent from membrane transport systems present in the original prokaryotic endosymbiont These pathways are characterized as being spontaneous, signal recogni-tion particle (SRP)-, secretory (Sec)- or twin-arginine translocase (Tat)-dependent The existence of several different thylakoid import pathways was first proposed when analysis of the energy requirements for thylakoid transport of several proteins showed them to be protein
Keywords
protein transport; secretory pathway; SRP;
Tat; thylakoid; twin-arginine
Correspondence
C Robinson, Department of Biological
Sciences, University of Warwick, Coventry
CV4 7AL, UK
Fax: +44 2476 523568
Tel: +44 2476 523557
E-mail: colin.robinson@warwick.ac.uk
*These authors contributed equally to this
work
(Received 5 August 2008, revised 25
November 2008, accepted 4 December
2008)
doi:10.1111/j.1742-4658.2009.06875.x
The chloroplast thylakoid is the most abundant membrane system in nat-ure, and is responsible for the critical processes of light captnat-ure, electron transport and photophosphorylation Most of the resident proteins are imported from the cytosol and then transported into or across the thyla-koid membrane This minireview describes the multitude of pathways used for these proteins We discuss the huge differences in the mechanisms involved in the secretory and twin-arginine translocase pathways used for the transport of proteins into the lumen, with an emphasis on the differing substrate conformations and energy requirements We also discuss the rationale for the use of two different systems for membrane protein inser-tion: the signal recognition particle pathway and the so-called spontaneous pathway The recent crystallization of a key chloroplast signal recognition particle component provides new insights into this rather unique form of signal recognition particle
Abbreviations
ALB3, albino 3; cp, chloroplast; EGFP, enhanced green fluorescent protein; LHCP, light-harvesting chlorophyll a ⁄ b-binding protein; OE, oxygen evolving; Sec, secretory; SRP, signal recognition particle; Tat, twin-arginine translocase; Tic, translocon at the inner envelope membrane of chloroplasts; Toc, translocon at the outer envelope membrane of chloroplasts; TPP, thylakoid processing peptidase.
Trang 2specific: transport of the 33 kDa protein of the
oxy-gen evolving complex (OE33) and plastocyanin
absolutely requires ATP [2–4]; light-harvesting
chloro-phyll a⁄ b-binding protein (LHCP) integration requires
GTP and is stimulated by ATP [5,6]; and transport of
the 23 kDa (OE23) and 17 kDa (OE17) proteins of the
oxygen evolving complex requires only the thylakoidal
DpH [3] Furthermore, competition studies revealed
dis-tinct precursor specific groups further demonstrating
the existence of several different pathways for thylakoid
import [1] In the present minireview, we describe the
components and mechanisms of these four different
thylakoid import pathways
Transport into the thylakoid lumen
Proteins destined for the thylakoid lumen are
trans-ported via the DpH⁄ Tat and Sec-dependent pathways,
although examples of thylakoid membrane proteins
have also been reported to be transported by these
pathways Imported Sec and Tat substrates are
synthe-sized in the cytosol with an N-terminal bipartite transit
peptide that carries two transport signals in tandem
The amino-proximal targeting domain mediates import
of precursor proteins into the chloroplast via the Toc⁄
Tic translocon After transportation into the
chloro-plast, this transit peptide is cleaved off by a processing
peptidase in the stroma exposing the second transport
signal, which then mediates transport across the
thyla-koid membrane Once across the thylathyla-koid membrane,
this signal peptide is also cleaved off, this time by the
thylakoid processing peptidase (TPP) [7]
Thylakoid signal peptides have a broadly similar
structure for both Sec and Tat protein substrates and
are similar to prokaryotic signal sequences They are
characterized by an N-terminal basic region, a
hydro-phobic central core and a polar C-terminal region
end-ing in an Ala-X-Ala terminal processend-ing site Proteins
destined to be transported by the Tat pathway contain
a characteristic pair of arginine residues in the
N-ter-minal region of the signal peptide, which gives the
pathway its name
The Sec pathway
The chloroplast Sec pathway evolved from the general
secretory pathway involved in export of Sec-dependent
proteins to the periplasm in bacteria In Escherichia
coli, the Sec translocon consists minimally of SecA,
SecE and SecY [8] In the bacterial system, the signal
peptide of the preprotein interacts post-translationally
with SecA in the cytoplasm The SecA–preprotein
com-plex associates with the Sec core components composed
of the integral membrane proteins SecY and SecE, which are thought to form the Sec protein conducting channel SecA is an ATPase and drives the transloca-tion of the protein through the Sec pore by multiple cycles of membrane insertion and deinsertion [9]
In chloroplasts, homologues to SecA (cpSecA), SecY (cpSecY) and SecE (cpSecE) have been identified [10–14] and there is strong evidence that the thylakoid membrane contains a SecAYE translocase that is func-tionally and structurally similar to the bacterial Sec complex: Sec transportation across thylakoid mem-branes is dependent on ATP and is sensitive to azide [11,15] and antibodies against cpSecY inhibit cpSecA-dependent protein translocation, suggesting that cpSecA and cpSecY work in concert, analogous to the situation
in bacteria [16] Additionally, cpSecE can functionally replace E coli SecE [17] and the chloroplast Sec translo-case is implicated in the co-translational insertion of SRP-dependant proteins into the thylakoid membrane,
as it is in bacterial plasma membranes Despite these similarities, homologues of several other bacterial Sec components (SecB, SecG and SecD⁄ F) have not been identified in chloroplasts Similar to bacteria, transport
by the chloroplast Sec translocon requires protein sub-strates to be in an unfolded state for transport [18,19],
as demonstrated by the inability of the chloroplast Sec translocon to transport dihydrofolate reductase fused to
a Sec signal peptide in the presence of folate analogues that stabilize dihydrofolate reductase in a tightly folded form [18] Transport of enhanced green fluorescent pro-tein (EGFP), which spontaneously and tightly folds, is also impossible through the chloroplast Sec translocon [19] In the bacterial system SecB, a cytosolic chaperone, binds post-translationally to the mature portion of Sec-dependent preproteins and stabilizes them in an unfolded conformation ready for transport Due to the absence of a SecB homologue in chloroplasts, the identi-ties of the stromal factors necessary to keep Sec preproteins in an unfolded state remain elusive
Recently, it has been shown that cpSecA ATPase activity is stimulated by Sec-dependent thylakoid signal peptides but not E coli signal peptides, and that stimu-lation of cpSecA ATPase activity requires distinct lipid requirements different to E coli SecA [20] These differ-ences suggest that cpSecA has evolved to be specifically suited to the chloroplast thylakoid environment
The Tat pathway
Unlike the Sec pathway, the Tat pathway requires no stromal factors or ATP and, instead, is energized by the trans-thylakoidal proton gradient [3,21,22] In addition, protein substrates can be transported in a
Trang 3folded conformation, allowing the transportation of
proteins that fold too quickly or tightly for the Sec
pathway, or proteins that require the insertion of
co-factors in the stroma before transport into the
thy-lakoid lumen This remarkable property of the Tat
pathway was first recognized during in vitro import
experiments following the observation that the OE23,
a Tat substrate, assumes a folded conformation during
its passage through the stroma [23] Translocation of
chimeric proteins consisting of EGFP fused to the
transit peptides of the Tat substrates OE16 and OE23
have shown that the Tat pathway can also transport
folded proteins in vivo because EGFP is known to fold
quickly and spontaneously and cannot be transported
through the Sec pathway [19] However, in contrast to
bacterial Tat proteins where protein folding appears to
be a prerequisite to Tat transport, folding is not
required for translocation of Tat substrates in
chlorop-lasts [18] Figure 1 summarizes the differing
mecha-nisms of the Sec and Tat pathways in chloroplasts
The Tat pathway in chloroplasts consists of the inte-gral membrane proteins Tha4 [16,24], Hcf106 [25] and cpTatC [26], which are closely related to their bacterial counterparts, designated TatA, TatB and TatC, respec-tively Tha4 and Hcf106 are single-span membrane proteins containing an N-terminal transmembrane domain followed by a short amphipathic helical region and an unstructured stromal C-terminal domain Stud-ies have shown that the C-terminal domain is dispens-able for Tha4 function but the transmembrane domain and amphipathic helix are essential for function [27] TatC is predicted to contain six transmembrane domains with both the amino and carboxyl termini protruding into the stroma Similar to their bacterial counterparts, Tha4, Hcf106 and cpTatC exist in the membrane as two sub-complexes: cpTatC and Hcf106 form an approximately 700 kDa receptor complex [28] and Tha4 oligomers form separate complexes that associate with the receptor complex under conditions
of protein transport (i.e in the presence of bound pre-cursor and a trans-thylakoidal proton gradient) [29,30] The transport of proteins by the Tat pathway can be divided approximately into several stages, as illustrated in Fig 2: (i) the precursor protein binds to
a cpTatC-Hcf106 receptor complex; (ii) precursor bind-ing stimulates assembly of Tha4 oligomers with the precursor–receptor complex and the putative
translo-Tat pathway Sec pathway
Stroma
Lumen
cpTatC Tha4
Hcf106
SecY
SecE
SecA
SecA
Fig 1 Basic features of the Sec and Tat pathways used for the
translocation of lumenal proteins across the thylakoid membrane.
Both types of substrate bear cleavable N-terminal signal peptides,
depicted as black rectangles The Tat pathway involves Hcf106 and
cpTatC, which are believed to form a receptor complex that
recog-nizes Tat signal peptides, and Tha4, which interacts transiently with
the precursor ⁄ receptor complex during transport and is thought to
form part of a pore for Tat protein transport Tat substrates are
transported in a fully folded form and use the thylakoid proton
gra-dient to provide energy for translocation By contrast, Sec substrate
proteins are transported in an unfolded conformation in a process
that requires ATP Sec transport minimally involves SecA (an
ATPase) and the membrane-bound SecE and SecY subunits SecA
ATPase activity provides the energy to drive the translocation of
proteins through the SecE⁄ Y pore After translocation, the signal
peptides of both Tat and Sec substrates are removed by the
thyla-koid processing peptidase (represented as scissors).
Stroma
Lumen Tha4
cpTatC-Hcf106 Precursor
(ii) Tha4 assembly
TPP (iii) Protein translocation Tha4
TPP clevage disassociation
(i) Precursor binding
Proton gradient
Fig 2 Mechanism of the Tat system (i) The precursor protein binds through the signal peptide to a cpTatC-Hcf106 receptor com-plex in the thylakoid membrane (ii) Precursor binding in the pres-ence of DpH stimulates assembly of Tha4 oligomers with the precursor–receptor complex and the putative translocase is formed (iii) The precursor protein is then transported in a process energized
by the DpH across the thylakoid membranes The transported pro-tein is released from the translocase into the lipid bilayer, where the signal peptide is removed by the TPP and the mature protein is released into the lumen After protein transport, Tha4 dissociates from the receptor complex and the system is reset.
Trang 4case is formed; and (iii), the precursor is transported
and released from the translocase into the lipid bilayer
where the signal peptide is removed and the mature
protein is released into the lumen After protein
trans-port, Tha4 dissociates from the receptor complex and
the system is reset
It is believed that Tha4 forms at least part of a
pro-tein conducting channel Cross-linking studies have
shown that Tha4 undergoes conformational
rearrange-ment during active protein transport, with the
amphi-pathic helix and C-terminal tail interacting only in
response to conditions leading to protein transport
[30] The Tat translocon needs to transport proteins of
varying size without leakage of ions across the
mem-brane and therefore some degree of flexibility is
required to form adaptable pores to accommodate
dif-ferent proteins Analysis of E coli TatA using
single-particle electron microscopy reveals that TatA forms
ring-shaped structures of variable diameter [31],
sup-porting a model in which Tha4⁄ TatA form a pore-like
channel and Tha4 oligomerization and recruitment of
Tha4 can be tailored to the size of the protein to be
transported
The cpTatC-Hcf106 complex forms the receptor for
Tat substrates and both Tat subunits were found to
interact with the protein precursor [28]; cross-linking
studies found that cpTatC and Hcf106 interact with
different regions of the signal peptide cpTatC
cross-links strongly to residues in the immediate vicinity of
the twin arginine motif, whereas Hcf106 cross-links
less strongly to residues in the hydrophobic core and
the early mature protein [32] Binding of the precursor
can occur in the absence of DpH [33] but the thylakoid
proton gradient induces a tighter interaction between
the signal peptide and cpTatC and Hcf106 such that,
during transport, the signal peptide is bound deep
within the Tat receptor complex [34] Although the
cpTatC-Hcf106 acts as a receptor for the Tat complex,
Tat-dependent transport may be initiated by the
unas-sisted insertion of the substrate into the lipid bilayer
and subsequent interaction with the Tat translocase
may take place only in later stages of the translocation
process [35] Analysis of the chimeric 16⁄ 23 precursor
polypeptide, which consists of the transit peptide from
OE16 fused to the mature OE23 protein, presents an
alternative model for the interaction of the preprotein
with the receptor The 16⁄ 23 chimera is retarded
dur-ing translocation; early in the process, the protein
assumes a structure within the membrane in which the
N-terminus and C-terminus are both exposed to the
stroma The formation of this early intermediate does
not depend on a functional Tat translocase [36]
Subse-quently, the C-terminal domain is fully translocated in
a Tat dependent manner and the signal peptide is removed by the TPP and the mature polypeptide is released into the thylakoid lumen
Although several studies have demonstrated the requirement for DpH in Tat transport in vitro, Finazzi
et al [37] demonstrated that elimination of the trans-thylakoidal DpH in vivo in Chlamydomonas reinhardtii had no effect on thylakoid targeting of Tat passenger proteins It was suggested that, in vivo, the chloroplast Tat pathway may also utilize the transmembrane elec-tric potential as an energy source [38]; however, the efficiency of translocation of OE23 is undiminished in the absence of DpH and⁄ or DW in tobacco protoplasts [39] It has recently been reported that the Tat path-way can also transport substrates in the dark [40] It was suggested that the thylakoid proton motive force
is present long after actinic illumination of the thylak-oids ceases and this may be achieved through a pool
of protons in the thylakoid held out of equilibrium with those in the bulk aqueous phase Clearly, the dif-ferences in energetic requirements between in vitro and
in vivo experiments require further study and may result from unknown factors present in vivo but miss-ing from in vitro experiments
Transport into the thylakoid membrane
Nuclear encoded proteins destined to be inserted into the thylakoid membrane are transported by either an assisted, SRP-dependent pathway or by an unassisted, possibly spontaneous insertion route (Fig 3) Traffick-ing of proteins to the thylakoid membrane occurs on
a substantial scale and is essential for thylakoid biogenesis
The cpSRP pathway
Classical SRP systems can be found in the cytoplasm
of both prokaryotes and eukaryotes These systems are co-translational and rely on the presence of the ribo-some and a highly conserved RNA component [41] In higher-plant chloroplasts, a unique post-translational SRP pathway has been identified in a system that tar-gets proteins into the thylakoid membrane but has no RNA requirement [42]
The post-translational cpSRP transport pathway has
a narrow range of closely-related substrates that are all members of the abundant LHCP family [43] These pigment-binding proteins are found in the thylakoid membrane system of chloroplasts and form compo-nents of the light-harvesting antenna complexes LHCP (Lhcb1) is the most studied of the cpSRP transport substrates It is highly hydrophobic, composed of three
Trang 5trans-membrane a-helices (TM1-3) that bind both
chlorophylls and carotenoid pigments [44] LHCP is
synthesized in the cytoplasm as a precursor protein,
which includes an N-terminal transit peptide that
mediates chloroplast targeting [45] After chloroplast
import, LHCP is targeted to the thylakoid membrane
Unlike other chloroplast routing pathways, such as
Tat and Sec that require a bipartite signal peptide, the
thylakoid targeting sequence of cpSRP substrates is
located within the mature span of the protein [46]
In the stroma, LHCP associates with cpSRP to
form the ‘transit complex’ [47] Within the transit
complex, two SRP subunits (cpSRP54 and cpSRP43)
are present in addition to LHCP cpSRP54 has
strong homology to both the fifty-four homologue
SRP subunit of prokaryotes and the SRP54 subunit
of the eukaryotic SRP system [42,48] However,
although homologous, cpSRP54 is not functionally
equivalent to these cytoplasmic forms in
complemen-tation studies [49] The second subunit, cpSRP43, has
no known homologues This novel subunit was
con-firmed by peptide analysis to be the Cao (CHAOS)
gene product [47] Closer analysis of cpSRP54 reveals
that it has GTPase activity, which suggests a role in
thylakoid insertion events [42] This GTPase activity
is due to an N-terminal domain called the
GTPase-containing domain (G-domain) CpSRP54 also has a
second domain designated the methionine-rich domain (M-domain) [48]
Within cpSRP43, two domain structures have been defined The first of these are chromo (chromosome organization modifier) domains, of which three have been identified in cpSRP43 The first chromodomain (CD1) is located in the N-terminal region [50] The remaining two chromodomains (CD2 and CD3) are located at the C-terminus of cpSRP43 [51] The struc-tures of all three chromodomains have been deter-mined using triple resonance NMR experiments [52] The second domain structures are four sequential ankyrin repeats that are located between CD1 and CD2⁄ CD3 [51] These ankyrin repeats (ANK 1–4) have been implicated in protein–protein interactions and are likely to be involved in complex formation Recently, a high resolution crystal structure of cpSRP43 has been solved [53] Formation of the stromal transit complex requires a series of specific recognition and interaction events between the LHCP substrate and the cpSRP subunits Binding between LHCP and cpSRP43 is med-iated by a conserved 18 amino acid span, termed L18, positioned between TM2 and TM3 of LHCP [54] As seen from the crystal structure, L18 fits a groove formed by ANK2-4 of cpSRP43 An essential ‘DPLG’ motif within L18 is critically important in this interac-tion where it interacts with a tyrosine of ANK3 [53] Previously, it had been suggested that L18 binding to cpSRP43 occurs through the first ankyrin repeat [55]
As with cpSRP43, cpSRP54 also binds directly to LHCP within the transit complex [42] TM3 has been shown to be particularly important in this binding but
it is not clear whether functional interactions also occur with the other TM spans [42,56]
Between the cpSRP subunits, the C-terminal located M-domain of cpSRP54 was identified as the cpSRP43 binding site [55] Interaction between cpSRP54 and cpSRP43 was localized to a highly positively charged segment of ten amino acids of cpSRP54 Furthermore, the cpSRP43 binding site was found to be conserved
in all cpSRP54 proteins and absent from cytoplasmic homologues [57] Mutational analysis of cpSRP43 reveals that CD2 is responsible for cpSRP54 binding [52,58] When this interaction was examined quantita-tively by surface plasmon resonance, binding of cpSRP54 to the CD2 region alone was less efficient than binding to the full-length cpSRP43, suggesting that other regions of interaction remain uncharacter-ized [59] Within CD2, the potential role of the nega-tively charged C-terminal a-helix in cpSRP54 interactions has been highlighted [52,59] Further stud-ies suggest that CD2 undergoes a conformational change upon binding cpSRP54 [60]
Stroma
Lumen
cpSRP pathway Spontaneous pathway
Alb3
cpFtsY cpSRP54 cpSRP43
Fig 3 SRP-dependent and ‘spontaneous’ pathways for the
inser-tion of thylakoid membrane proteins In the cpSRP-dependent
path-way, members of the LHCP family are imported into the
chloroplast where they bind to cpSRP (a heterodimer of SRP43 and
SRP54 subunits) in the stroma This complex then interacts with
cpFtsY and the LHCP is inserted into the thylakoid membrane by
a mechanism that requires ALB3, a member of the YidC ⁄ Oxa1
family Other thylakoid membrane proteins use an alternative
inser-tion pathway that does not require any source of free energy or
any of the known targeting apparatus These proteins may
there-fore insert spontaneously, although the possible involvement of
other, as yet unidentified factors cannot be excluded at present.
Trang 6After transit complex assembly, a third protein,
cpFtsY, has a role in the cpSRP pathway where cpFtsY
is assumed to target the transit complex to the
thyla-koid membrane CpFtsY was discovered in an attempt
to find homologues of the eukaryotic SRP receptor,
SRa, and the prokaryotic FtsY [61] The exact
parti-tioning of cpFtsY between the stroma and thylakoid
membrane is unclear and may be transient in nature,
which could reflect its predicted role in membrane
targeting, but the majority of cpFtsY is found on the
stromal face of the thylakoid membrane [61] Within
the cpFtsY NG domain, the three domains for GTP
binding are conserved [61] The crystal structure of
cpFtsY has been determined and demonstrates how the
NG domain arrangement may contribute to efficient
cpSRP54⁄ cpFtsY interactions in the absence of an
RNA component [62,63] In addition, a membrane
tar-geting sequence has been defined in an extended region
of the NG domain [63] A combination of cpSRP43,
cpSRP54 and cpFtsY reconstitute the stromal activity
in LHCP membrane insertion, hence confirming that
no other stromal components are required [6,64]
The insertion of LHCP into the thylakoid membrane
is probably one of the least well characterized stages in
the cpSRP pathway An integral, multi-spanning
pro-tein termed Albino 3 (ALB3) is involved and is a
chlo-roplast homologue of the mitochondrial translocon
component, Oxa1p Mutants that are deficient in
ALB3 have an albino phenotype and display clear
defi-ciencies in thylakoid biosynthesis [65,66] Evidence
exists of an interaction between ALB3 and the cpSecY
translocase and, furthermore, this interaction has been
attributed to interactions by the C-terminal region of
ALB3 [67] It is not known whether this finding is
related to a functional interaction, and hence a
poten-tial role for cpSecY in cpSRP-mediated LHCP
inser-tion [68] This cpSecY interacinser-tion is perhaps an
indication that the role of ALB3 extends beyond
cpSRP substrate insertion to a wider role involving
thylakoid membrane proteins
In addition to the proteinatious requirements for the
cpSRP pathway, there is also a less well understood
nucleotide requirement This is likely to occur during
insertion events because the formation of the transit
complex can take place in the absence of nucleotides
[69] For successful membrane insertion of LHCP,
GTP hydrolysis is essential [5] A role for GTP
hydro-lysis is likely in steps preceding or directly involving
dissociation of the cpSRP complex from the
mem-brane-bound state [68,70] ATP has been shown to
have an alternate and possibly regulatory role because
it stimulates integration of LHCP into the membrane
in a mechanism that is independent of the DpH [6]
Intriguingly, some interesting phenotypes have emerged in studies on cpSRP mutants It has been sug-gested that cpSRP43 can function alone, in LHCP insertion, if both cpSRP54 and cpFtsY are absent [71]
It is clear that additional studies are required in this area to resolve these findings
Spontaneous insertion pathway
The spontaneous (unassisted) pathway for thylakoid membrane proteins was first suggested to describe the insertion of the single-membrane-spanning CFoII sub-unit of the ATP synthase [72] The insertion of CFoII was described as having no requirement for nucleotides
or for proteinaceous insertion machinery Other single-spanning proteins have also been suggested to use this membrane integration route, including the photosys-tem II subunits, PsbW and PsbX In describing these insertion characteristics, parallels were drawn to the insertion of the M13 procoat protein in E coli, which was also supposedly spontaneous in insertion How-ever, it was subsequently shown that an integral mem-brane insertase, YidC, was actually important in its insertion, hence questioning a truly spontaneous mech-anism in bacteria [73] Inactivation of the chloroplast YidC homologue ALB3 did not affect the thylakoid membrane insertion of PsbW and PsbX; therefore, it appears that insertion of these proteins may be truly independent of any form of translocation apparatus [74]
Spontaneous insertion has also been attributed to more topologically complex proteins The closely-related photosystem I components, PsaK and PsaG, are both observed to insert into the membrane with two trans-membrane spans, connected by a stroma-exposed loop [75,76] For PsaG, the influence of posi-tive charges in the loop region was further analysed and it was found they are essential for insertion and function [75] In the case of PsbY, a complex series of proteolytic events occurs as the precursor is converted into two individual membrane spans, A1 and A2 [77] Other multi-spanning proteins have also been sug-gested to insert spontaneously, including PsbS and ELIP2 In addition, the SecE subunit of the Sec translocase and the Hcf106⁄ Tha4 subunits of the Tat translocase appear to use this spontaneous insertion mechanism [78]
Conclusions
It is clear that protein import into thylakoids occurs, through a variety mechanisms, via functionally inde-pendent pathways that have significant similarity to
Trang 7bacterial transport systems These pathways have been
termed spontaneous, cpSRP, Sec and Tat Although it
is probable that all of the essential components of the
cpSec, cpTat and cpSRP pathways have been
identi-fied, the exact mechanism for each of these pathways
remains largely unknown and clearly requires further
investigation
In bacteria, much work has been performed aiming
to characterize the Sec pathway, whereas, in
chlorop-lasts, our knowledge of the cpSec pathway is limited,
with current models being mainly based on homology
to the bacterial Sec system Although there are obvious
parallels between bacterial and chloroplast Sec
sys-tems, several components of the bacterial Sec
appara-tus have not been identified in chloroplasts Therefore,
caution is warranted in assuming that these systems
operate in the same manner, and further experimental
studies are required to elucidate the exact mechanistic
details of the chloroplast Sec pathway
The mechanism of the Tat pathway still remains to
be determined in both bacteria and chloroplasts
Although evidence indicates that Tha4⁄ TatA oligomers
form a pore for protein conveyance, this remains to be
confirmed Clearly, in this situation, structural
infor-mation about the Tat complex and its individual
com-ponents will prove invaluable
In the field of cpSRP, much progress has been
recently made with respect to crystallizing various
cpSRP components and defining their interaction
domains However, the exact method of thylakoid
membrane insertion is not well understood The
inser-tion process is believed to involve ALB3; however, the
precise role of ALB3 remains unclear Investigation of
the role of ALB3 would allow a more complete picture
of SRP-dependent thylakoid import
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