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

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MINIREVIEWProtein transport in organelles: Protein transport into andacross the thylakoid membraneCassie Aldridge*, Peter Cain* and Colin RobinsonDepartment of Biological Sciences, University of Warwick, Coventry, UKIntroductionChloroplasts are the site of photosynthesis and otherimportant biochemical processes that are vital for thefunctioning of plant cells. They are believed to havearisen from a photosynthetic bacterium taken up by aprimitive eukaryotic cell. Although some of the chloro-plast proteome is encoded by the chloroplast genome,during endosymbiosis, most of the original prokaryoticgenome was lost or transferred to the nuclear genome;therefore, the vast majority of chloroplast proteins arenuclear encoded and require transport into the chloro-plast. Whether synthesized in the cytosol or the chloro-plast stroma, a sub-set of proteins require transportinto or across the thylakoid membranes to attain theirfunctional locations.Nuclear encoded thylakoid precursor proteins areimported across the chloroplast envelope into the chlo-roplast stroma by a common import apparatus, namelythe Toc ⁄ Tic (translocon at the outer ⁄ inner envelopemembrane of chloroplasts) complex [1]. By contrast,import into or across the thylakoid membrane isthought to occur through four independent precursor-specific thylakoid transport pathways that are descen-dent from membrane transport systems present in theoriginal prokaryotic endosymbiont. These pathwaysare characterized as being spontaneous, signal recogni-tion particle (SRP)-, secretory (Sec)- or twin-argininetranslocase (Tat)-dependent. The existence of severaldifferent thylakoid import pathways was first proposedwhen analysis of the energy requirements for thylakoidtransport of several proteins showed them to be proteinKeywordsprotein transport; secretory pathway; SRP;Tat; thylakoid; twin-arginineCorrespondenceC. Robinson, Department of BiologicalSciences, University of Warwick, CoventryCV4 7AL, UKFax: +44 2476 523568Tel: +44 2476 523557E-mail: colin.robinson@warwick.ac.uk*These authors contributed equally to thiswork(Received 5 August 2008, revised 25November 2008, accepted 4 December2008)doi:10.1111/j.1742-4658.2009.06875.xThe chloroplast thylakoid is the most abundant membrane system in nat-ure, and is responsible for the critical processes of light capture, electrontransport and photophosphorylation. Most of the resident proteins areimported from the cytosol and then transported into or across the thyla-koid membrane. This minireview describes the multitude of pathways usedfor these proteins. We discuss the huge differences in the mechanismsinvolved in the secretory and twin-arginine translocase pathways used forthe transport of proteins into the lumen, with an emphasis on the differingsubstrate conformations and energy requirements. We also discuss therationale for the use of two different systems for membrane protein inser-tion: the signal recognition particle pathway and the so-called spontaneouspathway. The recent crystallization of a key chloroplast signal recognitionparticle component provides new insights into this rather unique form ofsignal recognition particle.AbbreviationsALB3, 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 envelopemembrane 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 1177specific: transport of the 33 kDa protein of the oxy-gen evolving complex (OE33) and plastocyaninabsolutely requires ATP [2–4]; light-harvesting chloro-phyll a ⁄ b-binding protein (LHCP) integration requiresGTP and is stimulated by ATP [5,6]; and transport ofthe 23 kDa (OE23) and 17 kDa (OE17) proteins of theoxygen evolving complex requires only the thylakoidalDpH [3]. Furthermore, competition studies revealed dis-tinct precursor specific groups further demonstratingthe existence of several different pathways for thylakoidimport [1]. In the present minireview, we describe thecomponents and mechanisms of these four differentthylakoid import pathways.Transport into the thylakoid lumenProteins destined for the thylakoid lumen are trans-ported via the DpH ⁄ Tat and Sec-dependent pathways,although examples of thylakoid membrane proteinshave also been reported to be transported by thesepathways. Imported Sec and Tat substrates are synthe-sized in the cytosol with an N-terminal bipartite transitpeptide that carries two transport signals in tandem.The amino-proximal targeting domain mediates importof precursor proteins into the chloroplast via the Toc ⁄Tic translocon. After transportation into the chloro-plast, this transit peptide is cleaved off by a processingpeptidase in the stroma exposing the second transportsignal, which then mediates transport across the thyla-koid membrane. Once across the thylakoid membrane,this signal peptide is also cleaved off, this time by thethylakoid processing peptidase (TPP) [7].Thylakoid signal peptides have a broadly similarstructure for both Sec and Tat protein substrates andare similar to prokaryotic signal sequences. They arecharacterized 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. Proteinsdestined to be transported by the Tat pathway containa characteristic pair of arginine residues in the N-ter-minal region of the signal peptide, which gives thepathway its name.The Sec pathwayThe chloroplast Sec pathway evolved from the generalsecretory pathway involved in export of Sec-dependentproteins to the periplasm in bacteria. In Escherichiacoli, the Sec translocon consists minimally of SecA,SecE and SecY [8]. In the bacterial system, the signalpeptide of the preprotein interacts post-translationallywith SecA in the cytoplasm. The SecA–preprotein com-plex associates with the Sec core components composedof the integral membrane proteins SecY and SecE,which are thought to form the Sec protein conductingchannel. SecA is an ATPase and drives the transloca-tion of the protein through the Sec pore by multiplecycles 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 thylakoidmembrane contains a SecAYE translocase that is func-tionally and structurally similar to the bacterial Seccomplex: 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 cpSecAand cpSecY work in concert, analogous to the situationin bacteria [16]. Additionally, cpSecE can functionallyreplace E. coli SecE [17] and the chloroplast Sec translo-case is implicated in the co-translational insertion ofSRP-dependant proteins into the thylakoid membrane,as it is in bacterial plasma membranes. Despite thesesimilarities, homologues of several other bacterial Seccomponents (SecB, SecG and SecD ⁄ F) have not beenidentified in chloroplasts. Similar to bacteria, transportby 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 Sectranslocon to transport dihydrofolate reductase fused toa Sec signal peptide in the presence of folate analoguesthat stabilize dihydrofolate reductase in a tightly foldedform [18]. Transport of enhanced green fluorescent pro-tein (EGFP), which spontaneously and tightly folds, isalso impossible through the chloroplast Sec translocon[19]. In the bacterial system SecB, a cytosolic chaperone,binds post-translationally to the mature portion ofSec-dependent preproteins and stabilizes them in anunfolded conformation ready for transport. Due to theabsence of a SecB homologue in chloroplasts, the identi-ties of the stromal factors necessary to keep Secpreproteins in an unfolded state remain elusive.Recently, it has been shown that cpSecA ATPaseactivity is stimulated by Sec-dependent thylakoid signalpeptides but not E. coli signal peptides, and that stimu-lation of cpSecA ATPase activity requires distinct lipidrequirements different to E. coli SecA [20]. These differ-ences suggest that cpSecA has evolved to be specificallysuited to the chloroplast thylakoid environment.The Tat pathwayUnlike the Sec pathway, the Tat pathway requires nostromal factors or ATP and, instead, is energized bythe trans-thylakoidal proton gradient [3,21,22]. Inaddition, protein substrates can be transported in aProtein transport across thylakoid membranes C. Aldridge et al.1178 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBSfolded conformation, allowing the transportation ofproteins that fold too quickly or tightly for the Secpathway, or proteins that require the insertion ofco-factors in the stroma before transport into the thy-lakoid lumen. This remarkable property of the Tatpathway was first recognized during in vitro importexperiments following the observation that the OE23,a Tat substrate, assumes a folded conformation duringits passage through the stroma [23]. Translocation ofchimeric proteins consisting of EGFP fused to thetransit peptides of the Tat substrates OE16 and OE23have shown that the Tat pathway can also transportfolded proteins in vivo because EGFP is known to foldquickly and spontaneously and cannot be transportedthrough the Sec pathway [19]. However, in contrast tobacterial Tat proteins where protein folding appears tobe a prerequisite to Tat transport, folding is notrequired 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] andcpTatC [26], which are closely related to their bacterialcounterparts, designated TatA, TatB and TatC, respec-tively. Tha4 and Hcf106 are single-span membraneproteins containing an N-terminal transmembranedomain followed by a short amphipathic helical regionand an unstructured stromal C-terminal domain. Stud-ies have shown that the C-terminal domain is dispens-able for Tha4 function but the transmembrane domainand amphipathic helix are essential for function [27].TatC is predicted to contain six transmembranedomains with both the amino and carboxyl terminiprotruding into the stroma. Similar to their bacterialcounterparts, Tha4, Hcf106 and cpTatC exist in themembrane as two sub-complexes: cpTatC and Hcf106form an approximately 700 kDa receptor complex [28]and Tha4 oligomers form separate complexes thatassociate with the receptor complex under conditionsof 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 pathwaycan be divided approximately into several stages, asillustrated in Fig. 2: (i) the precursor protein binds toa cpTatC-Hcf106 receptor complex; (ii) precursor bind-ing stimulates assembly of Tha4 oligomers with theprecursor–receptor complex and the putative translo-Tat pathway Sec pathwayStromaLumencpTatCTha4Hcf106SecYSecESecASecAFig. 1. Basic features of the Sec and Tat pathways used for thetranslocation 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 andcpTatC, which are believed to form a receptor complex that recog-nizes Tat signal peptides, and Tha4, which interacts transiently withthe precursor ⁄ receptor complex during transport and is thought toform part of a pore for Tat protein transport. Tat substrates aretransported in a fully folded form and use the thylakoid proton gra-dient to provide energy for translocation. By contrast, Sec substrateproteins are transported in an unfolded conformation in a processthat requires ATP. Sec transport minimally involves SecA (anATPase) and the membrane-bound SecE and SecY subunits. SecAATPase activity provides the energy to drive the translocation ofproteins through the SecE ⁄ Y pore. After translocation, the signalpeptides of both Tat and Sec substrates are removed by the thyla-koid processing peptidase (represented as scissors).StromaLumenTha4cpTatC-Hcf106Precursor(ii) Tha4 assemblyTPP(iii) Protein translocationTha4TPP clevagedisassociation(i) Precursor bindingProton gradientFig. 2. Mechanism of the Tat system. (i) The precursor proteinbinds 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 theprecursor–receptor complex and the putative translocase is formed.(iii) The precursor protein is then transported in a process energizedby the DpH across the thylakoid membranes. The transported pro-tein is released from the translocase into the lipid bilayer, wherethe signal peptide is removed by the TPP and the mature protein isreleased into the lumen. After protein transport, Tha4 dissociatesfrom the receptor complex and the system is reset.C. Aldridge et al. Protein transport across thylakoid membranesFEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS 1179case is formed; and (iii), the precursor is transportedand released from the translocase into the lipid bilayerwhere the signal peptide is removed and the matureprotein is released into the lumen. After protein trans-port, Tha4 dissociates from the receptor complex andthe system is reset.It is believed that Tha4 forms at least part of a pro-tein conducting channel. Cross-linking studies haveshown that Tha4 undergoes conformational rearrange-ment during active protein transport, with the amphi-pathic helix and C-terminal tail interacting only inresponse to conditions leading to protein transport[30]. The Tat translocon needs to transport proteins ofvarying size without leakage of ions across the mem-brane and therefore some degree of flexibility isrequired to form adaptable pores to accommodate dif-ferent proteins. Analysis of E. coli TatA using single-particle electron microscopy reveals that TatA formsring-shaped structures of variable diameter [31], sup-porting a model in which Tha4 ⁄ TatA form a pore-likechannel and Tha4 oligomerization and recruitment ofTha4 can be tailored to the size of the protein to betransported.The cpTatC-Hcf106 complex forms the receptor forTat substrates and both Tat subunits were found tointeract with the protein precursor [28]; cross-linkingstudies found that cpTatC and Hcf106 interact withdifferent regions of the signal peptide. cpTatC cross-links strongly to residues in the immediate vicinity ofthe twin arginine motif, whereas Hcf106 cross-linksless strongly to residues in the hydrophobic core andthe early mature protein [32]. Binding of the precursorcan occur in the absence of DpH [33] but the thylakoidproton gradient induces a tighter interaction betweenthe signal peptide and cpTatC and Hcf106 such that,during transport, the signal peptide is bound deepwithin the Tat receptor complex [34]. Although thecpTatC-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 bilayerand subsequent interaction with the Tat translocasemay take place only in later stages of the translocationprocess [35]. Analysis of the chimeric 16 ⁄ 23 precursorpolypeptide, which consists of the transit peptide fromOE16 fused to the mature OE23 protein, presents analternative model for the interaction of the preproteinwith the receptor. The 16 ⁄ 23 chimera is retarded dur-ing translocation; early in the process, the proteinassumes a structure within the membrane in which theN-terminus and C-terminus are both exposed to thestroma. The formation of this early intermediate doesnot depend on a functional Tat translocase [36]. Subse-quently, the C-terminal domain is fully translocated ina Tat dependent manner and the signal peptide isremoved by the TPP and the mature polypeptide isreleased into the thylakoid lumen.Although several studies have demonstrated therequirement for DpH in Tat transport in vitro, Finazziet al. [37] demonstrated that elimination of the trans-thylakoidal DpH in vivo in Chlamydomonas reinhardtiihad no effect on thylakoid targeting of Tat passengerproteins. It was suggested that, in vivo, the chloroplastTat pathway may also utilize the transmembrane elec-tric potential as an energy source [38]; however, theefficiency of translocation of OE23 is undiminished inthe 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]. Itwas suggested that the thylakoid proton motive forceis present long after actinic illumination of the thylak-oids ceases and this may be achieved through a poolof protons in the thylakoid held out of equilibriumwith those in the bulk aqueous phase. Clearly, the dif-ferences in energetic requirements between in vitro andin vivo experiments require further study and mayresult from unknown factors present in vivo but miss-ing from in vitro experiments.Transport into the thylakoid membraneNuclear encoded proteins destined to be inserted intothe thylakoid membrane are transported by either anassisted, SRP-dependent pathway or by an unassisted,possibly spontaneous insertion route (Fig. 3). Traffick-ing of proteins to the thylakoid membrane occurs ona substantial scale and is essential for thylakoidbiogenesis.The cpSRP pathwayClassical SRP systems can be found in the cytoplasmof both prokaryotes and eukaryotes. These systems areco-translational and rely on the presence of the ribo-some and a highly conserved RNA component [41]. Inhigher-plant chloroplasts, a unique post-translationalSRP pathway has been identified in a system that tar-gets proteins into the thylakoid membrane but has noRNA requirement [42].The post-translational cpSRP transport pathway hasa narrow range of closely-related substrates that are allmembers of the abundant LHCP family [43]. Thesepigment-binding proteins are found in the thylakoidmembrane system of chloroplasts and form compo-nents of the light-harvesting antenna complexes. LHCP(Lhcb1) is the most studied of the cpSRP transportsubstrates. It is highly hydrophobic, composed of threeProtein transport across thylakoid membranes C. Aldridge et al.1180 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBStrans-membrane a-helices (TM1-3) that bind bothchlorophylls and carotenoid pigments [44]. LHCP issynthesized in the cytoplasm as a precursor protein,which includes an N-terminal transit peptide thatmediates chloroplast targeting [45]. After chloroplastimport, LHCP is targeted to the thylakoid membrane.Unlike other chloroplast routing pathways, such asTat and Sec that require a bipartite signal peptide, thethylakoid targeting sequence of cpSRP substrates islocated within the mature span of the protein [46].In the stroma, LHCP associates with cpSRP toform the ‘transit complex’ [47]. Within the transitcomplex, two SRP subunits (cpSRP54 and cpSRP43)are present in addition to LHCP. cpSRP54 hasstrong homology to both the fifty-four homologueSRP subunit of prokaryotes and the SRP54 subunitof the eukaryotic SRP system [42,48]. However,although homologous, cpSRP54 is not functionallyequivalent to these cytoplasmic forms in complemen-tation studies [49]. The second subunit, cpSRP43, hasno known homologues. This novel subunit was con-firmed by peptide analysis to be the Cao (CHAOS)gene product [47]. Closer analysis of cpSRP54 revealsthat it has GTPase activity, which suggests a role inthylakoid insertion events [42]. This GTPase activityis due to an N-terminal domain called the GTPase-containing domain (G-domain). CpSRP54 also has asecond domain designated the methionine-richdomain (M-domain) [48].Within cpSRP43, two domain structures have beendefined. The first of these are chromo (chromosomeorganization modifier) domains, of which three havebeen identified in cpSRP43. The first chromodomain(CD1) is located in the N-terminal region [50]. Theremaining two chromodomains (CD2 and CD3) arelocated 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 sequentialankyrin repeats that are located between CD1 andCD2 ⁄ CD3 [51]. These ankyrin repeats (ANK 1–4) havebeen implicated in protein–protein interactions and arelikely to be involved in complex formation. Recently, ahigh resolution crystal structure of cpSRP43 has beensolved [53]. Formation of the stromal transit complexrequires a series of specific recognition and interactionevents between the LHCP substrate and the cpSRPsubunits. 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]. Asseen from the crystal structure, L18 fits a grooveformed 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 tocpSRP43 occurs through the first ankyrin repeat [55].As with cpSRP43, cpSRP54 also binds directly toLHCP within the transit complex [42]. TM3 has beenshown to be particularly important in this binding butit is not clear whether functional interactions alsooccur with the other TM spans [42,56].Between the cpSRP subunits, the C-terminal locatedM-domain of cpSRP54 was identified as the cpSRP43binding site [55]. Interaction between cpSRP54 andcpSRP43 was localized to a highly positively chargedsegment of ten amino acids of cpSRP54. Furthermore,the cpSRP43 binding site was found to be conservedin all cpSRP54 proteins and absent from cytoplasmichomologues [57]. Mutational analysis of cpSRP43reveals that CD2 is responsible for cpSRP54 binding[52,58]. When this interaction was examined quantita-tively by surface plasmon resonance, binding ofcpSRP54 to the CD2 region alone was less efficientthan binding to the full-length cpSRP43, suggestingthat other regions of interaction remain uncharacter-ized [59]. Within CD2, the potential role of the nega-tively charged C-terminal a-helix in cpSRP54interactions has been highlighted [52,59]. Further stud-ies suggest that CD2 undergoes a conformationalchange upon binding cpSRP54 [60].StromaLumencpSRP pathway Spontaneous pathwayAlb3cpFtsYcpSRP54cpSRP43Fig. 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 thechloroplast where they bind to cpSRP (a heterodimer of SRP43 andSRP54 subunits) in the stroma. This complex then interacts withcpFtsY and the LHCP is inserted into the thylakoid membrane bya mechanism that requires ALB3, a member of the YidC ⁄ Oxa1family. Other thylakoid membrane proteins use an alternative inser-tion pathway that does not require any source of free energy orany of the known targeting apparatus. These proteins may there-fore insert spontaneously, although the possible involvement ofother, as yet unidentified factors cannot be excluded at present.C. Aldridge et al. Protein transport across thylakoid membranesFEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBS 1181After transit complex assembly, a third protein,cpFtsY, has a role in the cpSRP pathway where cpFtsYis assumed to target the transit complex to the thyla-koid membrane. CpFtsY was discovered in an attemptto find homologues of the eukaryotic SRP receptor,SRa, and the prokaryotic FtsY [61]. The exact parti-tioning of cpFtsY between the stroma and thylakoidmembrane is unclear and may be transient in nature,which could reflect its predicted role in membranetargeting, but the majority of cpFtsY is found on thestromal face of the thylakoid membrane [61]. Withinthe cpFtsY NG domain, the three domains for GTPbinding are conserved [61]. The crystal structure ofcpFtsY has been determined and demonstrates how theNG domain arrangement may contribute to efficientcpSRP54 ⁄ cpFtsY interactions in the absence of anRNA component [62,63]. In addition, a membrane tar-geting sequence has been defined in an extended regionof the NG domain [63]. A combination of cpSRP43,cpSRP54 and cpFtsY reconstitute the stromal activityin LHCP membrane insertion, hence confirming thatno other stromal components are required [6,64].The insertion of LHCP into the thylakoid membraneis probably one of the least well characterized stages inthe cpSRP pathway. An integral, multi-spanning pro-tein termed Albino 3 (ALB3) is involved and is a chlo-roplast homologue of the mitochondrial transloconcomponent, Oxa1p. Mutants that are deficient inALB3 have an albino phenotype and display clear defi-ciencies in thylakoid biosynthesis [65,66]. Evidenceexists of an interaction between ALB3 and the cpSecYtranslocase and, furthermore, this interaction has beenattributed to interactions by the C-terminal region ofALB3 [67]. It is not known whether this finding isrelated to a functional interaction, and hence a poten-tial role for cpSecY in cpSRP-mediated LHCP inser-tion [68]. This cpSecY interaction is perhaps anindication that the role of ALB3 extends beyondcpSRP substrate insertion to a wider role involvingthylakoid membrane proteins.In addition to the proteinatious requirements for thecpSRP pathway, there is also a less well understoodnucleotide requirement. This is likely to occur duringinsertion events because the formation of the transitcomplex 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 involvingdissociation of the cpSRP complex from the mem-brane-bound state [68,70]. ATP has been shown tohave an alternate and possibly regulatory role becauseit stimulates integration of LHCP into the membranein a mechanism that is independent of the DpH [6].Intriguingly, some interesting phenotypes haveemerged in studies on cpSRP mutants. It has been sug-gested that cpSRP43 can function alone, in LHCPinsertion, if both cpSRP54 and cpFtsY are absent [71].It is clear that additional studies are required in thisarea to resolve these findings.Spontaneous insertion pathwayThe spontaneous (unassisted) pathway for thylakoidmembrane proteins was first suggested to describe theinsertion of the single-membrane-spanning CFoII sub-unit of the ATP synthase [72]. The insertion of CFoIIwas described as having no requirement for nucleotidesor for proteinaceous insertion machinery. Other single-spanning proteins have also been suggested to use thismembrane integration route, including the photosys-tem II subunits, PsbW and PsbX. In describing theseinsertion characteristics, parallels were drawn to theinsertion of the M13 procoat protein in E. coli, whichwas also supposedly spontaneous in insertion. How-ever, it was subsequently shown that an integral mem-brane insertase, YidC, was actually important in itsinsertion, hence questioning a truly spontaneous mech-anism in bacteria [73]. Inactivation of the chloroplastYidC homologue ALB3 did not affect the thylakoidmembrane insertion of PsbW and PsbX; therefore, itappears that insertion of these proteins may be trulyindependent of any form of translocation apparatus[74].Spontaneous insertion has also been attributed tomore topologically complex proteins. The closely-related photosystem I components, PsaK and PsaG,are both observed to insert into the membrane withtwo 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 analysedand it was found they are essential for insertion andfunction [75]. In the case of PsbY, a complex series ofproteolytic events occurs as the precursor is convertedinto two individual membrane spans, A1 and A2 [77].Other multi-spanning proteins have also been sug-gested to insert spontaneously, including PsbS andELIP2. In addition, the SecE subunit of the Sectranslocase and the Hcf106 ⁄ Tha4 subunits of the Tattranslocase appear to use this spontaneous insertionmechanism [78].ConclusionsIt is clear that protein import into thylakoids occurs,through a variety mechanisms, via functionally inde-pendent pathways that have significant similarity toProtein transport across thylakoid membranes C. Aldridge et al.1182 FEBS Journal 276 (2009) 1177–1186 ª 2009 The Authors Journal compilation ª 2009 FEBSbacterial transport systems. These pathways have beentermed spontaneous, cpSRP, Sec and Tat. Although itis probable that all of the essential components of thecpSec, cpTat and cpSRP pathways have been identi-fied, the exact mechanism for each of these pathwaysremains largely unknown and clearly requires furtherinvestigation.In bacteria, much work has been performed aimingto characterize the Sec pathway, whereas, in chlorop-lasts, our knowledge of the cpSec pathway is limited,with current models being mainly based on homologyto the bacterial Sec system. Although there are obviousparallels 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 systemsoperate in the same manner, and further experimentalstudies are required to elucidate the exact mechanisticdetails of the chloroplast Sec pathway.The mechanism of the Tat pathway still remains tobe determined in both bacteria and chloroplasts.Although evidence indicates that Tha4 ⁄ TatA oligomersform a pore for protein conveyance, this remains to beconfirmed. 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 beenrecently made with respect to crystallizing variouscpSRP components and defining their interactiondomains. However, the exact method of thylakoidmembrane insertion is not well understood. The inser-tion process is believed to involve ALB3; however, theprecise role of ALB3 remains unclear. 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J Biol Chem 276,36200–36206.77 Thompson SJ, Robinson C & Mant A (1999) Dualsignal peptides mediate the signal recognitionparticle ⁄ Sec-independent insertion of a thylakoidmembrane polyprotein, PsbY. J Biol Chem 274,4059–4066.78 Schunemann D (2007) Mechanisms of protein importinto 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 RobinsonDepartment. 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
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Xem thêm: Báo cáo khoa học: Protein transport in organelles: Protein transport into and across the thylakoid membrane pptx, Báo cáo khoa học: Protein transport in organelles: Protein transport into and across the thylakoid membrane pptx, Báo cáo khoa học: Protein transport in organelles: Protein transport into and across the thylakoid membrane pptx