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Curr Genet (2004) 44: 295–304 DOI 10.1007/s00294-003-0450-z R EV IE W A RT I C L E Danja Schünemann Structure and function of the chloroplast signal recognition particle Received: 16 July 2003 / Revised: 5 September 2003 / Accepted: 7 September 2003 / Published online: 21 October 2003 Springer-Verlag 2003 Abstract The targeting of proteins, including the insertion and translocation of proteins in or across membranes, is a fundamental process within a cell, and a variety of specialized mechanisms for protein transport have been developed during evolution. The signal recognition particle (SRP) is found in the cytoplasm of most, if not all, eukaryotes and prokaryotes where it plays a central role in the co-translational insertion of membrane proteins into the endoplasmic reticulum and plasma membrane, respectively. SRP is a ribonucleoprotein consisting of an RNA and at least one polypeptide of 54 kDa (SRP54). Interestingly, chloroplasts contain a specialized type of signal recognition particle. Chloroplast SRP (cpSRP) contains a SRP54 homologue but differs strikingly from cytosolic SRP in various aspects of structure and function. In contrast to cytosolic SRP, it contains a novel protein subunit (cpSRP43) and lacks RNA. CpSRP is also distinctive in its ability to interact with its substrate, light-harvesting chlorophyll a/b-binding protein, post-translationally. Furthermore, it is remarkable that the 54 kDa subunit of cpSRP is also involved in the co-translational transport of chloroplast-encoded thylakoid proteins, and is therefore able to switch between the co- and post-translational means of interaction with its respective substrate proteins. Keywords Chloroplast Æ Thylakoid Æ Signal recognition particle Æ Protein transport Æ LHCP Æ ALB3 Communicated by S. Hohmann D. Schünemann Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany E-mail: [email protected] Tel.: +49-234-3228561 Fax: +49-234-3214184 Introduction A mechanism of protein transport that has been extensively studied is the co-translational protein transport to the endoplasmic reticulum (ER) of eukaryotic cells. The cytosolic components, as well as the membrane components like the receptors and the translocation pore, have been characterized (reviewed in Martoglio and Dobberstein 1996; Rapoport et al. 1996; Johnson and van Waes 1999). The targeting process is initiated when the signal peptide of the protein emerges from the ribosome and is recognized by the signal recognition particle (SRP). SRP is a cytosolic ribonucleoprotein consisting of a 7S-RNA and six polypeptides. The 54-kDa subunit (SRP54) mediates the binding to the signal peptide, whereas the subcomplex SRP9/14 causes an elongation arrest. The complex of ribosome, SRP, and the nascent protein binds to the SRP receptor (SR) in the ER. The SRP receptor consists of an integral membrane protein (SRb) and a peripheral membrane protein (SRa). Upon dissociation of SRP from the signal peptide, translation resumes and the protein is translocated co-translationally through the translocation pore (Sec61p) into the ER, and SRP dissociates from the receptor in a GTP requiring step. The Escherichia coli SRP pathway is related to the eukaryotic SRP pathway and mediates primarily the cotranslational targeting of most plasma membrane proteins (reviewed in Bernstein 2000; de Gier and Luirink 2001; Valent 2001). Like the eukaryotic SRP, the E. coli SRP is also a ribonucleoprotein. It consists of an RNA component (4.5S-RNA) and one protein component [Ffh (fifty four homologue)]. Both components are homologous to the 7S-RNA and SRP54 of the eukaryotic SRP, respectively. The E. coli protein FtsY was identified as a SRP receptor based on sequence similarity to the a-subunit of the eukaryotic SRP receptor (SRa). In contrast to SRa, FtsY is localized as a peripheral protein at the plasma membrane as well as a soluble protein in the cytoplasm (Luirink et al. 1994). It was 296 shown that the cytoplasmic FtsY binds to the SRP/ ribosome/nascent protein complex and delivers this complex to the plasma membrane via interaction of the N-terminal domain of FtsY with membrane components (Zelazny et al. 1997; Valent et al. 1998; de Leeuw et al. 2000). The direct contact of FtsY with acidic phospholipids in the membrane might be the reason why no homologous protein for the b-subunit of the eukaryotic SRP receptor has yet been identified. SRP is released at the plasma membrane from the translating ribosome in a GTP-dependent reaction, and the protein uses the SecYEG translocon for insertion into the membrane (Valent et al. 1998). Plant cells are characterized by the presence of specific organelles: the plastids. Chloroplasts are the site of photosynthesis and of various essential metabolic pathways, e.g., fatty acid and amino acid biosynthesis. It is estimated that a chloroplast contains up to 3,500 Fig. 1 Targeting pathways for nuclear encoded proteins from the chloroplast stroma to the thylakoid lumen or thylakoid membrane. Transport of nuclear encoded proteins from the stroma to the thylakoid lumen is mediated via the cpTat pathway and the cpSec pathway (cp chloroplast, Tat twin arginine translocation, Sec secretory). Substrate proteins for these pathways (i.e., OE23 23kDa subunit of the oxygen-evolving complex, PC plastocyanin) require an N-terminal targeting domain (TD) that directs the protein to either pathway. After translocation, the signal peptide is cleaved off by a thylakoidal processing peptidase. A twin arginine motif (RR) is the main characteristic of the signal peptide for the Tat pathway. Insertion of nuclear encoded proteins into the thylakoid membrane is mediated via the cpSRP pathway (cpSRP chloroplast signal recognition particle) and by a spontaneous insertion process. Proteins that insert spontaneously into the membrane also require a specific signal peptide and are mostly single-span membrane proteins. As substrate proteins for the cpSRP pathway, only members of the light-harvesting chlorophyll a/b binding proteins (LHCPs) are known, whose targeting information resides within the mature protein. In contrast to the spontaneous insertion process, several soluble and membrane proteins (yellow boxes) in addition to specific energy conditions (white boxes) are required for the three assisted pathways. The different targeting pathways are reviewed in, for example, Eichacker and Henry 2001; Mori and Cline 2001; Robinson et al. 2001; Schleiff and Klösgen 2001, and this work proteins (Abdallah et al. 2000), the vast majority of which are encoded in the nucleus with only about 100 encoded in the plastid genome. Most of the nuclearencoded proteins must be imported post-translationally across the translocon of the inner and outer envelope into the organelle, and thereafter distributed to the different subcompartments within the plastid. Four different pathways have been described so far for the posttranslational translocation of proteins from the stroma to the thylakoid membrane or across the membrane into the thylakoid lumen: the Sec pathway and the Tat pathway are mainly involved in the transport of lumenal proteins, whereas the spontaneous pathway and the cpSRP pathway are used for the insertion of integral membrane proteins into the thylakoid membrane (reviewed in, e.g., Eichacker and Henry 2001; Mori and Cline 2001; Robinson et al. 2001; Schleiff and Klösgen 2001). The main characteristics of these different targeting mechanisms are summarized in Fig. 1. The post-translational cpSRP pathway The post-translational chloroplast SRP (cpSRP) pathway is involved in targeting of members of the nuclear encoded light-harvesting chlorophyll a/b-binding protein (LHCP) family from the stroma to the thylakoid membrane. LHCPs constitute approximately one-third of the total thylakoid protein, and therefore, are the major integral thylakoid membrane proteins (Yamamoto and Bassi 1996). The LHC super-gene family of Arabidopsis contains more than 30 members (Jansson 1999). The major LHCP (Lhcb1) belongs to photosystem II and represents perhaps the most abundant membrane protein in nature. LHCPs are encoded in the nucleus and synthesized in the cytosol with an N-terminal transit sequence that codes for the post-translational transport across the envelope membranes into the stroma of the chloroplast. In the stroma, the transit sequence is cleaved off and LHCP is bound by stromal 297 components. This complex was designated ‘‘transit complex’’ and maintains the solubility of the hydrophobic LHCPs as they are transported through the stroma (Payan and Cline 1991). The first stromal component of the transit complex was identified as a soluble 54 kDa GTPase (cpSRP54), which is a homologue of the 54-kDa subunit of the eukaryotic signal recognition particle (SRP54) and the Ffh subunit of the prokaryotic SRP (Franklin and Hoffman 1993; Li et al. 1995). Purification of the chloroplast signal recognition particle (cpSRP) by immunoprecipitation of stroma using anticpSRP54 antibodies (Schünemann et al. 1998) and a genetic approach using transposon tagging (Klimyuk et al. 1999) led to the identification of the second subunit of cpSRP, a stromal 43-kDa protein (cpSRP43). In vitro reconstitution experiments demonstrated that both cpSRP subunits, cpSRP54 and cpSRP43, are necessary and sufficient to form the transit complex with LHCP (Schünemann et al. 1998; Yuan et al. 2002). It should be noted that cpSRP43 shows no homologues in the databases and therefore represents a novel type of SRP subunit with an unclear evolutionary origin. From these results, it became evident that chloroplasts contain a specialized SRP that contains a homologue of SRP54, but differs strikingly from the cytosolic SRPs as it (1) lacks an RNA, (2) contains a novel 43kDa subunit, and (3) interacts with its substrate proteins post-translationally (Table 1). The transit complex is thought to be an intermediate in the targeting of LHCP to the thylakoid membrane, because it was shown that the transit complex contains a productive form of LHCP capable of integrating into the membrane upon addition of stroma and GTP (Payan and Cline 1991; Hoffman and Franklin 1994). Besides cpSRP43 and cpSRP54, the third stromal protein required for LHCP integration was identified as cpFtsY, a chloroplast homologue of the bacterial SRP receptor (FtsY). Initially, this finding was based on the observation that anti-cpFtsY antibodies inhibited LHCP integration into isolated thylakoid membranes (Kogata et al. 1999). Later it was demonstrated that the soluble phase required for LHCP integration could be reconstituted by cpSRP (cpSRP54 and cpSRP43), cpFtsY, and GTP (Tu et al. 1999; Yuan et al. 2002). CpFtsY is localized mainly in the stroma, but a significant fraction is associated with the thylakoid membrane (Tu et al. 1999). Currently, nothing is known about the precise role of cpFtsY in LHCP integration. By analogy, with the bacterial system it can be speculated that stromal cpFtsY binds to subcomponents of the transit complex and delivers it to the translocon of the thylakoid membrane. However, no experimental data exist to support this assumption and it will be an interesting question for the future to analyze the mode of interaction between transit complex, cpFtsY, and the translocon. In order to understand the integration mechanism of LHCP into the thylakoid membrane in detail, it will also be important to elucidate the role of the nucleotides involved in this process. So far, it is known that GTP hydrolysis is absolutely required for LHCP integration and that ATP stimulates this process if it is present in combination with GTP (Hoffman and Franklin 1994; Yuan et al. 2002). As GTP and ATP do not seem to have a significant influence on transit complex formation, they are apparently involved in the later stages of LHCP integration. By analogy, with the bacterial SRP pathway, it is feasible that GTP is required to regulate the possible interaction of the GTPases cpSRP54 and cpFtsY during delivery of LHCP to the translocon. The role of ATP in LHCP insertion is completely unresolved as no ATP-binding protein involved in this process has yet been identified. Structure of cpSRP and transit complex Database analysis of the cpSRP43 sequence identified two types of motifs, the ankyrin repeat domain and the Table 1 Subunit composition of cytosolic and chloroplast SRPs. The individual components of cytosolic and chloroplast SRPs and their main functional characteristics are listed Mammaliana SRP54 7S RNA – SRP9/14 SRP19 SRP68/72 Binding to substrate Elongation Elongation Assembly proteins, SRP-RNA and arrest; catalytic arrest of SRP, promotes SRP receptor; GTPase influence on SRP association and SR interaction of SRP54 a Bacterial Ffh 4.5S RNA – – – – Binding to substrate Catalytic proteins, SRP-RNA and influence on Ffh SRP receptor (FtsY); and FtsY interaction GTPase cpSRP54 – cpSRP43 – – – Chloroplastb post-translational Binding to Binding LHCPs, cpSRP43 to L18 domain of and cpFtsY; GTPase LHCP and cpSRP54 cpSRP54 ? – ? ? ? Chloroplastb co-translational Binding to TM1 of D1, GTPase a Reviewed in Keenan et al. 2001 Reviewed in Eichacker and Henry 2001 and this work b 298 chromodomain, which are known to mediate proteinprotein interactions. The ankyrin repeat, an approximately 33-amino acid long motif, is found in a very large number of proteins, where it generates the dimerization interface for a variety of different protein substrates (reviewed in Bork 1993; Sedgwick and Smerdon 1999). The chromodomain is a 30–70 amino acid motif found in various proteins involved in the regulation of chromatin structure by protein-protein interactions (Koonin et al. 1995; Eissenberg and Elgin 2000). Four ankyrin repeats were found in the N-terminal region of cpSRP43, and two closely spaced chromodomains were identified in the C-terminal region (Klimyuk et al. 1999; Jonas-Straube et al. 2001). Later, a third chromodomain located near the N-terminus of the mature protein was predicted (Eichacker and Henry 2001). It will be an interesting quest for the future to get detailed information about the role of the different domains in the formation of the cpSRP and the transit complex, since only a few studies have been undertaken to address this question so far. Recently, it was shown by using the yeast two-hybrid system that the C-terminal region of cpSRP43, containing the two closely spaced chromodomains, mediates the binding of cpSRP54 (Jonas-Straube et al. 2001). The binding site of cpSRP54 for cpSRP43 was determined to reside in the C-terminal half of cpSRP54 containing the M-domain (Jonas-Straube et al. 2001). Shortly after, it was shown that within this region the last C-terminal 26 amino acids interact strongly with cpSRP43 (Groves et al. 2001). Interestingly, these amino acids present a positively charged region unique to cpSRP54. The absence of this region in Ffh, the E. coli SRP54 homologue, explains the previous finding that Ffh is not able to form a complex with cpSRP43 (Schünemann et al. 1999). A point of discussion is whether cpSRP43 can dimerize. Initial data based on gel filtration and chemical cross-linking experiments using recombinant cpSRP43 suggested that cpSRP43 is a dimer (Tu et al. 1999). A dimerization of cpSRP43 was also observed in in vivo experiments using the yeast two-hybrid system, and the dimerization motif was confined to the third and fourth ankyrin repeat (Jonas-Straube et al. 2001). These results, together with the observation that cpSRP54 exists as a monomer in cpSRP, led to the hypothesis that cpSRP is a heterotrimer composed of one cpSRP54 and a cpSRP43 dimer (Tu et al. 1999). The molecular mass of this complex predicted from the cDNA sequence would be 123 kDa. This value differs significantly from a molecular mass estimate of 200 kDa for stromal cpSRP obtained by gel filtration (Schünemann et al. 1998, Tu et al. 1999). Recent data indicate that cpSRP43 is not a globular protein but exhibits an elongated shape (Groves et al. 2001). This structural feature of cpSRP43 is possibly the reason for the overestimation of the apparent molecular mass of cpSRP using gel filtration. In contrast to Tu et al. (1999), no dimerization of recombinant 6-His-cpSRP43 could be observed by Groves et al. (2001), using equilibrium ultracentrifugation. By using the same method, these authors showed that recombinant cpSRP has a molecular mass of 98 kDa, which fits to the molecular mass of 120 kDa for the transit complex that was obtained by nondenaturing gel analysis (Payan and Cline 1991). Therefore, Groves et al. (2001) support the model of cpSRP being a heterodimer. However, these authors mention in their discussion initial data indicating that a deletion construct of cpSRP43 lacking the C-terminal chromodomains forms a stable dimer. These results support the observation of Jonas-Straube et al. (2001) that the ankyrin repeat containing domain of cpSRP43 is able to dimerize. When we consider all of the evidence together, it appears conceivable that a potential dimerization of cpSRP43 might occur only in a transient functional state. Post-translational cpSRP-mediated targeting to the thylakoid membrane is restricted to LHCPs, and therefore posed the question which structural characteristics of the LHCPs determine the transit complex formation with cpSRP. By analogy, with the co-translational targeting system where binding of cytosolic SRP54 to a substrate protein is mediated via its hydrophobic signal sequence, it was shown that a hydrophobic domain within LHCP is required for the post-translational binding to cpSRP (DeLille et al. 2000; Tu et al. 2000). However, the presence of this domain alone is not sufficient to promote transit complex formation. A second unique recognition element present only in a subset of LHCPs was identified to be an 18-amino acid hydrophilic domain (L18 domain) located between the second and third transmembrane domains of LHCP (DeLille et al. 2000). In further studies, it was shown that the L18 domain of LHCP binds to the cpSRP43 subunit of cpSRP (Tu et al. 2000) and that this binding is mediated via the first ankyrin repeat of cpSRP43 (Jonas-Straube et al. 2001). In vitro pull-down assays showed that the binding between cpSRP43 and LHCP is much stronger than the one between cpSRP54 and LHCP (Tu et al. 2000). This observation together with the finding that cpSRP54 can be efficiently cross-linked to LHCP in the transit complex (Li et al. 1995) led to the assumption that the specific recruitment of LHCPs for transit complex formation is initiated by binding of L18 to the first ankyrin repeat of cpSRP43 followed by the contact formation between LHCP and cpSRP54. Experiments analyzing the interaction of cpSRP54 and LHCP by using a cross-linking or a pepscan approach demonstrated that cpSRP54 binds to the third transmembrane domain of LHCP, which represents the most hydrophobic of the three transmembrane domains of LHCP (High et al. 1997; Groves et al. 2001). However, the presence of the third transmembrane domain is not absolutely required for transit complex formation, as a deletion construct of LHCP lacking this domain is still able to form the transit complex with cpSRP (DeLille et al. 2000). These data indicate that cpSRP54 is also able to interact with the first and/or second transmembrane domain of LHCP in the transit complex. 299 Therefore, it is conceivable that the structure of the transit complex switches between different functional conformations that might be required for the delivery and consecutive release of LHCP to the thylakoid membrane. The co-translational cpSRP pathway In bacteria, the SRP pathway is mainly involved in the co-translational targeting of integral membrane proteins to the plasma membrane. The thylakoid membrane contains four major protein complexes involved in photosynthesis. Several of their membrane proteins are encoded in the chloroplast genome and inserted cotranslationally into the thylakoid membrane. The first evidence that a co-translational SRP pathway is functional in chloroplasts came from the observation that the stroma contains two different pools of cpSRP54. One pool is bound to cpSRP43 and active in transit complex formation with LHCP, whereas a second pool of cpSRP54 was found to be associated with 70S ribosomes in absence of cpSRP43 (Franklin and Hoffman 1993; Schünemann et al. 1998). The direct involvement of cpSRP54 in the co-translational targeting of a plastidencoded membrane protein was shown by cross-linking of stromal cpSRP54 to the ribosome bound nascent chain of D1, obtained by a homologous chloroplast in vitro translation system (Nilsson et al. 1999). Notably, no interaction of cpSRP43 with the D1 nascent chain was detected, supporting the specialized role of cpSRP43 in post-translational targeting (Nilsson et al. 1999). Further analysis of the interaction of cpSRP54 and the nascent chain of D1 revealed that cpSRP54 binds tightly, but transiently, to the first transmembrane domain of the elongating nascent chain. This interaction occurs only if the nascent chain is still attached to the ribosome, and it is gradually lost as elongation proceeds (Nilsson and van Wijk 2002). Furthermore, these authors demonstrated that the first transmembrane domain could be replaced by other hydrophobic domains of more than ten amino acids, indicating that the hydrophobicity of a nascent chain and not a specific sequence motif is the prerequisite for the binding of cpSRP54. This supports earlier results demonstrating that the efficiency of cross-linking of cpSRP54 to signal sequences correlates to their hydrophobicity (High et al. 1997). Therefore, the recognition mechanism between cpSRP54 and its substrate proteins seems to resemble the cytosolic system (SRP-dependent co-translational targeting to the ER or E. coli plasma membrane) where the signal sequence must possess a certain threshold level of hydrophobicity for SRP-mediated targeting (reviewed in Keenan et al. 2001). Recently, it was shown that the thylakoid membrane protein cpSecY, a homologue of the bacterial SecY subunit of the plasma membrane translocon, is involved in D1 protein insertion by transient interaction with D1 intermediates during elongation (Zhang et al. 2001). Currently, nothing is known about the targeting mechanism of the SRP-ribosome-nascent chain complex to the cpSecY translocon, but it seems very likely that by analogy with the bacterial SRP pathway cpFtsY, the chloroplast homologue of the SRP receptor is involved in this process. A model of the role of cpSRP in postand co-translational protein targeting to the thylakoid is shown in Fig. 2. Analysis of Arabidopsis mutants lacking either one or both subunits of cpSRP Studies on Arabidopsis mutants lacking cpSRP43 (chaos mutant, Klimyuk et al. 1999), cpSRP54 (ffc mutant, Amin et al. 1999) or both cpSRP subunits (chaos/ffc double mutant, Hutin et al. 2002) support the important role of cpSRP in the post-translational targeting of members of the LHCP family to the thylakoid membrane and the additional involvement of cpSRP54 in the co-translational targeting of chloroplast encoded thylakoid membrane proteins. The chaos mutant is characterized by a specific defect in LHCP biogenesis. The plant has a chlorotic phenotype, the chlorophyll content is reduced by 50%, and the chlorophyll a/b ratio is elevated. Western-blot analysis revealed that the amount of most analyzed LHCPs is significantly reduced, whereas no reduction of other proteins could be detected (a detailed description of the phenotype is given in Table 2). These findings are in agreement with the specialized role of cpSRP43 in LHCP targeting. Interestingly, plants lacking functional cpSRP54 (Pilgrim et al. 1998; Amin et al. 1999) differ significantly from the chaos mutant in two major aspects of their phenotype. Firstly, the phenotype of these plants is most visible at the young seedling stage when the first true leaves are yellow. In contrast to the chaos mutant, the phenotype becomes less severe when the plant matures, as seen by a recovery of the first true leaves and by the observation that the older ffc leaves are greener than the young ffc leaves. In addition, the subset of photosystem proteins that is reduced in the young leaves is present at wild-type level in the older leaves (for details see Table 2). Secondly, the young leaves of the ffc mutant show a reduced level of the chloroplast encoded photosystem I and II reaction center proteins in addition to a reduction of the same subset of LHCPs as the chaos mutant. These data support the overlapping roles of cpSRP43 and cpSRP54 in LHCP targeting and the additional involvement of cpSRP54 in targeting of chloroplast encoded thylakoid membrane proteins. However, it should be noted that the chaos and ffc mutants still contain a significant amount of LHCPs ( 50%), which was an unexpected outcome since the cpSRP subunits are inactive individually in vitro (Schünemann et al. 1998; Groves et al. 2001; Yuan et al. 2002). Therefore, it was speculated that the single subunits might be active in vivo, or that a fraction of the LHCPs are targeted by one or more alternative targeting pathways. To address this question, 300 Fig. 2 Role of the cpSRP subunits in the post- and co-translational targeting of nuclear or chloroplast encoded proteins to the thylakoid membrane. CpSRP consists of the stromal subunits cpSRP54 and cpSRP43. A point of discussion is whether cpSRP43 can dimerize (see text for further information). Upon import of the nuclear encoded LHCP into the stroma, cpSRP binds to LHCP to form the transit complex, a soluble intermediate of LHCP. Besides cpSRP, integration of LHCP into the thylakoid membrane requires GTP, cpFtsY (the chloroplast homologue of the bacterial SRP receptor) and the integral membrane protein Alb3. Current evidence suggests that insertion of LHCP into the thylakoid membrane is independent of the cpSecY/E translocase. In contrast to cpSRP43, cpSRP54 is also involved in the co-translational targeting of the chloroplast encoded D1 to the thylakoid membrane. Insertion of D1 into the membrane is mediated via the cpSecY/E translocase possibly in concert with Alb3. Alb3 was found to be at least partially associated with the cpSecY/E translocase a double mutant containing no functional cpSRP54 and cpSRP43 was created (Hutin et al. 2002). The phenotype of these plants is more severe than that of the single mutants. The double mutant has pale yellow leaves at all stages of growth, and the levels of all LHCPs (and ELIPs), except Lhcb4, are significantly more reduced than in the single mutants (Table 2). As expected, the double mutant exhibits also a reduction of the chloroplast encoded photosystem I and II reaction center proteins and the inner antennae proteins CP43 and CP47. As in the single mutants, no reduction was observed for psbS, which can insert spontaneously into the thylakoid membrane (Kim et al. 1999). Taken together, the stronger phenotype of the double mutant suggests that the individual cpSRP subunits are partially active in LHCP targeting in vivo. However, it should be noted that even the double mutant contains significant amounts of Lhcb1, Lhcb6, and Lhca2, and that the level of Lhcb4 in the double mutant is equal to, or even higher than, the wild-type level, although in vitro experiments point to a SRP-dependent targeting of Lhcb4 (Woolhead et al. 2001). Based on these observations, the hypothesis that at least some members of the LHCP family can be partially transported to the thylakoid membrane by a SRP-independent alternative pathway cannot be ruled out. Several studies analyzing the biogenesis of thylakoid membranes provide growing evidence that chloroplasts contain a transport system via vesicles that bud from the envelope membrane and fuse with growing thylakoids (reviewed in Hoober and Eggink 1999; Vothknecht and Westhoff 2001). It may be possible that such a pathway moves LHCPs from the envelope to the thylakoid membrane. Alternatively, it might be possible that other mechanisms like the induction of heat shock proteins partially compensate the lack of cpSRP as observed in yeast mutants lacking SRP (Mutka and Walter 2001). Role of the integral thylakoid membrane protein ALB3 in cpSRP-dependent protein targeting As described in the previous sections, the soluble components involved in LHCP targeting are well-defined; however, little is known about the translocase mediating the integration of LHCP into the thylakoid membrane. Recently, the integral thylakoid membrane protein ALB3 was identified as the first integral membrane protein required for the insertion of LHCP into the thylakoid membrane (Sundberg et al. 1997; Moore et al. 2000). The participation of ALB3 in LHCP insertion has been demonstrated by the observation that preincuba- 301 Table 2 Summary of the main characteristics of Arabidopsis mutants lacking functional cpSRP54 (T115 N line: Pilgrim et al. 1998, ffc1–2: Amin et al. 1999), cpSRP43 (chaos: Amin et al. 1999; Klimyuk et al. 1999), or both subunits of cpSRP (Hutin et al. 2002). fl, fi , ›: lower, equal, higher amount compared to wild-type (WT),10d 10-day old plants, 24d 24-day old plants, - not determined Appearance Chlorophyll content Chlorophyll a/b ratio Plastids Protein level: Lhca1 Lhca2 Lhca3 Lhca4 Lhcb1 Lhcb2 Lhcb3 Lhcb4 Lhcb5 Lhcb6 psbS Elips D1/D2 psaA/B CP43/CP47 DcpSRP54 DcpSRP43 Double mutant Yellow first true leaves that become green within a week; older leaves almost like WT Reduction by 75% in first true leaves WT level Chlorotic, all leaves pale green no recovery 50% reduction All leaves more yellow than chaos no recovery 85% reduction Elevated WT level Fewer thylakoids (first true leaves) 10 d 24 d fl fi fi fi fl fi fl fi fl fi fl fi fl fi › › fl fi fi fi › › – – fl fi fl fi fl fi Normal Drastic reduction of thylakoids tion of thylakoid membranes with anti-ALB3 antibodies inhibited this process (Moore et al. 2000). Later, it was shown that ALB3 is also required for the insertion of Lhcb5 and Lhcb4.1, two other LHC proteins whose insertion is dependent on the presence of NTPs and stromal factors (Kim et al. 1999; Woolhead et al. 2001). Although it has not been tested, it is highly likely that these stromal factors comprise cpSRP and cpFtsY. Therefore, it can be assumed that ALB3 plays a general role in the insertion of SRP-dependent LHC proteins. ALB3 is a homologue of the bacterial membrane protein YidC and the mitochondrial membrane protein Oxa1p that mediate the protein insertion into the inner membrane of bacteria and mitochondria, respectively. In E. coli, most inner membrane proteins are targeted co-translationally to the cytoplasmic membrane using SRP and the SRP-receptor FtsY. The insertion of these proteins into the inner membrane is mediated via the SecY-translocase. Recently, the inner membrane protein YidC was identified as a novel component of the SecY-translocase (Scotti et al. 2000). Several studies indicate that YidC facilitates the lateral diffusion of the transmembrane domains of the Secdependent proteins from the translocase into the lipid bilayer (Houben et al. 2000; Beck et al. 2001; van der Laan et al. 2001; Urbanus et al. 2001). Interestingly, other studies demonstrated that YidC can also function independently from the SecY-translocase by showing that YidC is essential for the insertion of a subset of E. coli inner membrane proteins whose insertion mechanism is Sec-independent (Samuelson et al. 2000, 2001; Chen et al. 2002). Taken together, these results indicate that YidC can function in concert fl fi fl fl fl fl fl › fl fi › – fi fi fi fl fl fl fl fl fl fl › fl fl › fl fl fl fl (0%) (<50%) (0%) (<50%) (<50%) (<10%) (0%) (<130%) (<10%) (<50%) with the SecY-translocase and can also function independently of the SecY-translocase. The mitochondrial homologue of YidC is the inner membrane protein Oxa1p that is involved in the insertion of nuclear and mitochondrial encoded inner membrane proteins (Kermorgant et al. 1997; Hell et al. 1998, 2001). Based on the observation that yeast mitochondria do not contain proteins with homology to the subunits of the bacterial-type Sec translocase, it is most likely that Oxa1p operates exclusively in a Sec-independent manner (Glick and von Heijne 1996). Currently, only little is known about the oligomeric state of ALB3 and its precise function in protein insertion into the thylakoid membrane. Work from Mori et al. (1999) suggests that ALB3 functions independently from the cpSecY-translocase in LHCP integration. This was concluded from the observation that the insertion of LHCP into the thylakoid membrane could not be inhibited by pre-incubation of the membrane with antibodies directed against cpSecY, the chloroplast homologue of bacterial SecY, whereas the translocation of a Sec-dependent protein was inhibited under these conditions. These results indicated for the first time that an inner membrane protein might be inserted using a SRP/ALB3-dependent but SecYindependent mechanism. Interestingly, recent work analyzing the biogenesis of a set of model inner membrane proteins in a variety of SRP, Sec, and YidC mutant E. coli strains demonstrated that a SRP/YidCdependent and SecY-independent pathway is operational in E. coli (Fröderberg et al. 2003) However, it is discussed in the literature that the cpSecY-independence of LHCP integration has not yet been demonstrated 302 the involvement of cpSRP54 in the co-translational transport of chloroplast-encoded thylakoid proteins. Despite considerable progress concerning mainly the interaction of cpSRP54 with nascent D1 during elongation, many major questions remain to be resolved. So far, cpSRP54 is the only identified component of the cotranslational cpSRP, and it will be very important to analyze whether other components associate with cpSRP54 during co-translational protein targeting. For the post-translational SRP pathway, it was unequivocally demonstrated that no RNA is required for a functional cpSRP. But it is not known yet whether the co-translational SRP pathway requires an SRP-RNA, as is the case for all known eukaryotic and prokaryotic cytosolic SRPs. Another interesting aspect that needs to be addressed in the future concerns the question of how cpSRP54 is recruited for functioning in either the posttranslational or co-translational cpSRP pathway. For instance, do the ribosome nascent chains and cpSRP43 or the different substrate proteins compete for binding cpSRP54? The analysis of these questions will provide more insight in the precise molecular details, the dynamics and the regulatory aspects of the cpSRP dependent protein targeting mechanism. unequivocally, as it might be possible that the anticpSecY antibodies bind to a region of cpSecY that is not required to mediate LHCP insertion. Furthermore, contradicting a previous assumption (Cline and Mori 2001), recent experimental data demonstrated that ALB3 is at least partially associated with the cpSecYtranslocase (Klostermann et al. 2002). Therefore, the possibility that the cpSecY/E translocase is involved in LHCP integration cannot be ruled out and further work is required to test this. By analogy with the role of Sec-dependent YidC in co-translational membrane protein insertion in bacteria, it seems likely that cpSecY-associated ALB3 is involved in the insertion of chloroplast encoded thylakoid membrane proteins. This hypothesis is supported by the recent observation that ALB3, cpSecE, and cpSecY co-purify with ribosome attached nascent chains of D1 that are detergent extracted from the thylakoid membrane (Klaas Jan van Wijk, personal communication). In addition, it was shown that ALB3 functionally complements a YidC depletion strain of E. coli and promotes the insertion of leader peptidase, a YidC/SRP/Sec-dependent integral membrane protein of E. coli (Jiang et al. 2002). Taken together, these experiments demonstrate that ALB3 is a functional homolog of YidC and can function in concert with a Sec-translocase in the SRP-dependent cotranslational insertion of integral membrane proteins. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft. Conclusions and outlook References During recent years, substantial progress has been made to elucidate the structure and function of the chloroplast SRP. 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