Báo cáo khoa học: Protein transport in organelles: Protein transport into and across the thylakoid membrane pptx

MINIREVIEW Protein 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 chloroplast 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 chloro- 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 capture, 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. FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS 1177 specific: 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 thylakoid 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 processing 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 Protein transport across thylakoid membranes C. Aldridge et al. 1178 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS folded 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. C. Aldridge et al. Protein transport across thylakoid membranes FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS 1179 case 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 Protein transport across thylakoid membranes C. Aldridge et al. 1180 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS trans-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. C. Aldridge et al. Protein transport across thylakoid membranes FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS 1181 After 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 interaction 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 Protein transport across thylakoid membranes C. Aldridge et al. 1182 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS bacterial 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. 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J Biol Chem 274, 4059–4066. 78 Schunemann D (2007) Mechanisms of protein import into thylakoids of chloroplasts. Biol Chem 388, 907– 915. Protein transport across thylakoid membranes C. Aldridge et al. 1186 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS . MINIREVIEW Protein transport in organelles: Protein transport into and across the thylakoid membrane Cassie Aldridge*, Peter Cain* and Colin Robinson Department. and require transport into the chloro- plast. Whether synthesized in the cytosol or the chloro- plast stroma, a sub-set of proteins require transport into