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Protein translocation and thylakoid biogenesis in cyanobacteria
BBABIO-47520; No. of pages: 8; 4C: 2, 3 Biochimica et Biophysica Acta xxx (2015) xxx–xxx
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Protein translocation and thylakoid biogenesis in cyanobacteria Kelly M. Frain 1, Doris Gangl 1, Alexander Jones 1, Julie A.Z. Zedler 1, Colin Robinson ⁎,1 Centre for Molecular Processing, School of Biosciences, University of Kent, Ingram Building, Canterbury, CT2 7NJ, United Kingdom
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Article history: Received 16 June 2015 Received in revised form 17 August 2015 Accepted 31 August 2015 Available online xxxx Keywords: Cyanobacteria Protein translocation Membrane biogenesis Sec Tat SRP
a b s t r a c t Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments — specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called ‘spontaneous’ pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. © 2015 Published by Elsevier B.V.
1. Introduction Cyanobacteria were the first organisms to perform oxygenic photosynthesis [1] and today they play a major role in global carbon fixation [2]. Cyanobacteria have long been used as model organisms for photosynthesis research, but they have also attracted interest for other reasons [3]: often they are associated with harmful algal blooms (reviewed in [4]), and they are also interesting candidates for biotechnological applications (reviewed in [5]). In structural terms, they are relatively complex microorganisms. A typical cyanobacterium contains an outer membrane and a plasma membrane which are separated by the periplasm-typical features of Gram-negative bacteria. However, key photosynthetic activities are carried out within an elaborate internal thylakoid membrane system, and cyanobacteria are thus unusual among Gram-negative bacteria in exhibiting a high degree of membrane differentiation [6]. Finally, the thylakoid membrane encloses a further soluble phase which is often termed the thylakoid lumen (Fig. 1). Many aspects of cyanobacterial cell biology remain poorly understood, and this applies particularly to the mechanisms involved in the generation and maintenance of the membrane systems in these organisms. Indeed, our knowledge of these processes in cyanobacteria is still very much in its infancy. This review article aims to review the processes of protein translocation and membrane biogenesis in cyanobacteria; Abbreviations: Tat, twin-arginine translocation; SRP, signal recognition particle. ⁎ Corresponding author. E-mail address: [email protected] (C. Robinson). 1 These authors contributed equally to this work.
we describe aspects that have been confirmed experimentally, and we will draw parallels from studies on chloroplasts and other bacteria, where we have a better understanding of membrane biogenesis in general. Chloroplasts are generally accepted to have arisen from endosymbiotic cyanobacteria, and they share many features with typical cyanobacteria; it is to be expected that there may be similarities in thylakoid protein translocation and sorting mechanisms, and these have been extensively studied in chloroplasts. Escherichia coli, on the other hand, is a model organism for the study of protein transport into and across bacterial plasma membranes, and it is more than likely that some of the established mechanisms are also used in cyanobacteria. We will therefore provide a general overview of the main chloroplast and bacterial translocation systems, including the general secretory (Sec) pathway, the twin arginine translocation (Tat) pathway and the signal recognition particle (SRP) pathway, and we discuss which mechanisms are likely to operate in cyanobacteria. We also highlight the potential importance of membrane organisation and the implications for the translocation, integration and targeting of proteins within the various membranes. The composition of cyanobacterial membranes and their potential interconnections may play a major role for protein sorting. 2. Membrane organisation in cyanobacteria Cyanobacteria contain three different membranes: the outer membrane, the plasma membrane and the thylakoid membrane. Each carries out distinct functions but there is still much debate about whether the plasma membrane and the thylakoid membrane are actually connected and form a continuous network (reviewed in [7–9]). In brief, three
http://dx.doi.org/10.1016/j.bbabio.2015.08.010 0005-2728/© 2015 Published by Elsevier B.V.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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Fig. 2. Organisation of the plasma membrane, thylakoid centres and thylakoid membranes. Evidence suggests that thylakoid membranes coalesce with the plasma membrane at specific points termed thylakoid centres. PratA appears to be an important component of these sites and it has been found in an intermediate fraction during sedimentation studies, denoted the PratA-defined membrane (PDM) sub-fraction. This may be the initial site of PSII and PSI biogenesis, and the site at which membrane lipids and proteins are transferred to developing thylakoids.
Fig. 1. Structure and essential features of a typical unicellular cyanobacterium. The majority of cyanobacteria contain outer and inner (plasma) membranes which are separated by a periplasmic space, together with internal thylakoid membranes which are believed to be attached to the plasma membrane at ‘thylakoid centres’. The thylakoid membrane encloses a further soluble compartment termed the thylakoid lumen. It is unclear how the plasma membrane and thylakoid membrane (and the periplasm/thylakoid lumen) are physically connected. Within the cytoplasm are the nucleoid, carboxysome and ribosomes.
different models have been proposed to explain how thylakoid membrane biogenesis occurs: (1) pre-existing thylakoid membrane structures are the sites of synthesis of proteins, lipids and pigments for that particular membrane. (2) Protein-pigment complexes are synthesised and assembled in the plasma membrane and then transferred to the thylakoids. (3) Thylakoid ‘centres’ are formed as specialised structures initiating the biosynthesis of components [8]. The latter is the currently favoured model, supported by several observations. In general, thylakoids appear as stacks of parallel sheets near the plasma membrane. However, in certain regions they converge near the plasma membrane forming so-called thylakoid centres [10]. The first observation of these thylakoid centres in cyanobacteria was reported by Kunkel et al. [11]. In Anabaena cylindrica, Dermocarpa violaceae, Gleocapsa alpicola and Pleurocapsa minor, thylakoid centres were observed as cylindrical structures of 30 nm by 20 nm situated close to the plasma membrane. These consisted of globular subunits orientated in nonparallel, stacked arrays and were seemingly attached to thylakoids (see Fig. 2). There is evidence that thylakoid centres are involved in the biogenesis of photosystem II. In the plasma membrane of Synechocystis sp. PCC 6803 the photosystem II assembly factor PratA was found enriched in certain areas and it was therefore termed PratA-defined membrane (PDM) [12]. PratA in turn accumulates near thylakoid centres. Semicircular membranous structures surrounding thylakoid centres were hypothesised to consist of PDMs and seemed to contact both the thylakoid and plasma membrane [13]. The ‘vesicle inducing protein in plastids’ (VIPP1) seems to be an important component found in thylakoid centres (reviewed in [14,15]). VIPP1 forms oligomers of 12–17 fold symmetry that associate into the same cylindrical structures observed at thylakoid centres. Interestingly, a recent report also implicates VIPP1 in the biogenesis of photosystem I [16]. The primordial cyanobacterium Gleobacter violaceus also shows signs of membrane differentiation although the organism does not possess thylakoid membranes. Instead, it harbours its photosynthetic and respiratory machinery in
the plasma membrane. Still, two distinct membrane domains are clearly distinguishable, which are similar in protein composition to the thylakoid and plasma membranes in other cyanobacteria [17]. Overall, it appears that thylakoid centres may play a key role in thylakoid membrane organisation. 3. Translocation into and across the plasma membrane and thylakoid membranes Whatever the mechanisms by which the plasma and thylakoid membranes arise in cyanobacteria are, proteins need to be targeted into and across these membranes. These processes have been studied to only a minor extent in cyanobacteria, for several reasons. First, the isolation of intact thylakoids from cyanobacteria is extremely difficult, and in vitro protein translocation assays have yet to be developed. Secondly, although genetic approaches are more feasible, there has been a tendency to focus on the much more tractable E. coli for detailed studies on bacterial protein transport systems. As a result, we have little direct information on cyanobacterial protein targeting pathways, and we are obliged to draw potential parallels from other systems. However, this is problematic; it is not clear whether cyanobacterial protein targeting pathways most closely resemble those in the well-studied E. coli (which does not of course contain thylakoids) or higher plant chloroplasts. Cyanobacteria are widely accepted to be the progenitors of higher plant chloroplasts, which suggests that parallels can be made between thylakoid protein translocation/insertion in cyanobacteria and chloroplasts [18]. However, chloroplasts differ from cyanobacteria in key respects. For example, plant thylakoids are separated from the cytoplasm by the stroma and chloroplast envelope, which means that protein transport into plant thylakoids is almost entirely posttranslational (since the vast majority of thylakoid proteins are imported from the cytosol). In contrast, some pathways may well operate cotranslationally in cyanobacteria (as is the case for some of the E. coli pathways). Otherwise, cyanobacteria target numerous proteins to the plasma membrane, outer membrane and periplasm, and it appears very likely that these proteins are translocated by mechanisms that resemble those operating in E. coli. Altogether, these factors suggest that cyanobacteria may utilise protein trafficking pathways that operate in both chloroplasts and other bacteria, making inferences difficult to finalise. In the following sections we review the pathways that are used to
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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target proteins into and across E. coli and chloroplast thylakoid membranes, and we discuss those mechanisms that most likely operate in cyanobacteria. In both chloroplasts and E. coli there are three largely independent protein translocation/insertion pathways, with a fourth functioning in chloroplasts [18–20]. Proteins are transported across the plasma membrane and thylakoid membrane via the Secretory (Sec) pathway and the Twin-Arginine Translocation (Tat) pathways, while membrane
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proteins are inserted by the SRP pathway in both systems. Additionally, membrane proteins are inserted into the thylakoid membrane by a novel pathway sometimes termed ‘spontaneous insertion’ (Fig. 3). Each of these pathways utilise individual precursor targeting determinants and require different energy sources. Indeed it was the requirement for different energy components in in vitro assays that gave rise to the identification of the four different pathways in the first place.
Fig. 3. Comparison of different translocation mechanisms operating in (A) cyanobacteria, (B) bacteria and (C) plant chloroplasts. A. In cyanobacteria, proteins destined for the periplasm or thylakoid lumen are transported via the Sec or Tat pathways, while plasma membrane proteins are probably inserted using the SRP pathway (the default pathway for insertion of membrane proteins in E. coli — see B). It is not known whether thylakoid membrane proteins are inserted using the SRP pathway or the ‘spontaneous’ insertion pathway used for many chloroplast thylakoid proteins. B. in E. coli, soluble proteins are transported across the plasma membrane using either the Sec or Tat pathways. In the Sec pathway, the substrate is transported through a SecYEG channel in an unfolded state using energy derived from ATP hydrolysis by SecA. In the Tat pathway, the substrate is transported in a folded state through a translocon that is assembled from separate TatBC and TatA complexes. C. In chloroplasts, thylakoid proteins are mostly synthesised in the cytosol and first cross the chloroplast outer and inner membranes before being directed to the thylakoids. Soluble lumenal proteins are transported by either the Sec or Tat pathways; the Tat system appears to be similar to bacterial systems while the Sec system is simpler, lacking SecG and some ancillary Sec proteins such as SecDF (not shown). Only a subset of thylakoid membrane proteins — the light harvesting chlorophyll binding protein (LHCP) family — are inserted by the SRP pathway and this further differs from the E. coli model in that the Sec channel is not involved. The only known membrane-bound receptor element is Alb3. Other membrane proteins are inserted by a different mechanism termed the ‘spontaneous’ pathway (see text). PP, periplasm; PM, plasma membrane; CP, cytoplasm; TM, thylakoid membrane; TL, thylakoid lumen; S, stroma.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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3.1. Targeting of proteins to the periplasm or thylakoid lumen via the Sec pathway The Sec pathway is evolutionarily conserved across kingdoms — present in the bacterial plasma membrane, the endoplasmic reticulum and chloroplasts. Soluble proteins targeted by the Sec machinery are synthesised with an N-terminal signal peptide that contains a hydrophobic ‘core’ region with a charged N-terminal domain. The Sec pathway clearly operates in cyanobacteria and it has been estimated that 82% of translocated proteins (whether to the periplasm or thylakoid lumen) in Synechocystis sp. PCC6803 contain a Sec signal peptide [21]. Upon successful translocation of the substrate protein into the periplasm or lumen, this signal peptide is cleaved by signal peptidase at an Ala–Xaa–Ala consensus motif present in the C-domain of the signal peptide, releasing the mature protein. Representative cyanobacterial Sec signal peptides are listed in Fig. 4. The Sec machinery has been best characterised in E. coli where it is composed of the membrane-embedded multi-span protein SecY and single-span SecE, the non-essential SecG, SecD, SecF, and finally the cytoplasmic SecA protein [22]. YidC can also be defined as a component of the Sec apparatus as it sometimes co-purifies with the Sec translocase [23] although it plays a more important role in SRP transport (discussed below). Chaperones such as SecB act in the cytosol of E. coli to keep preprotein in an unfolded state and to inhibit unwanted reactions (such as aggregation and/or degradation) prior to translocation. SecA binds to the Sec substrate in the cytoplasm, forming a SecA-preprotein complex that next interacts with the core translocation apparatus, SecYEG, in the membrane. Repeated rounds of ATP hydrolysis by SecA drive the translocation of the substrate through the SecYEG channel. Homologues to SecA, SecY and SecE are found in chloroplasts of plants (cpSecA, cpSecY and cpSecE respectively, with cpSecE able to functionally replace E. coli SecE [24]). However, the chloroplast Sec system appears to be a ‘minimalist’ Sec system because SecB and SecG are absent (Fig. 3). The E. coli Sec system also includes non-essential SecDF subunits (not shown in Fig. 3), and these are also absent. The plant chloroplast Sec pathway transports a subset of lumenal proteins to the lumen, including plastocyanin and the 33 kDa component of the oxygen-evolving complex, PsbO [18]. Interestingly it has been demonstrated that the Sec systems of the chloroplast and E. coli require very different membrane lipid environments in order to optimally translocate substrate, and it was suggested that cpSecA has evolved to be specifically suited to the thylakoid membrane environment [25]. This is in keeping with the very unusual lipid composition (the thylakoid membrane is mostly galactolipid whereas bacterial plasma membranes are composed primarily of phospholipids). These differences in membrane composition could be a piece of the jigsaw to the successful sorting of proteins to their requisite membrane in cyanobacteria — either the thylakoid lumen or the periplasm — as discussed below.
In cyanobacteria the Sec translocation machinery seems to be broadly similar to that found in E. coli (Fig. 3 A, B). SecY has been localised in both the plasma and the thylakoid membrane of Synechocystis sp. PCC6803 [26] and SecA is present in the thylakoid membrane [27] and presumably the plasma membrane. This organism contains only a single set of sec genes (as do most, if not all cyanobacteria) which suggests that the same Sec components are present in both the plasma and thylakoid membrane. In turn, this raises the question of how protein targeting to a specific compartment (i.e. the periplasm or the thylakoid lumen) can be achieved. One of the limitations of the Sec pathway is the requirement of substrate proteins to be in an unfolded state. This is due to the fixed-width nature of the core SecYE pore (~12Ǻ, reflecting the width of a polypeptide chain) and is demonstrated by the classic example of the Sec pathway's inability to translocate dihydrofolate reductase ‘locked’ in a folded conformation through binding around methotrexate [28]. Despite the integrity and flexibility of substrate (in terms of size, shape and complexity of structure) this ‘one size fits all’ pore, and translocation of substrates in an unfolded state enables, it does preclude translocation of proteins that require cytoplasmically-inserted cofactors, or substrates that fold rapidly in the cytosol. 3.2. Protein targeting via the Tat pathway The Tat system operates in parallel with the Sec system in the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria, and there is evidence that it is present in both the thylakoid membrane and plasma membrane of cyanobacteria [29]. Initially it was referred to as the ΔpH-dependent pathway because the ΔpH was shown to be the sole energy requirement for translocation into thylakoids [30]. It has since been re-named the Twin-Arginine Translocation (Tat) pathway in view of the importance of a twin arginine motif in signal peptides of Tat-targeted proteins [19]. The Tat pathway is unique in its ability to translocate fully folded proteins across energy-transducing membranes; indeed its primary purpose in E. coli is the translocation of complex cofactor-containing proteins into the periplasm. The uniqueness of the Tat system does not end there though - not only are substrates folded, they are invariably correctly folded, with mounting evidence for an in-built proofreading and/or quality control mechanism that rejects proteins lacking their requisite cofactors or even substrates that are incorrectly folded [31–33]. Although the Tat machinery is present in both the plasma membrane and the thylakoid membrane of cyanobacteria the proofreading and quality control of cyanobacterial systems has yet to be studied in any detail. Tat signal peptides are surprisingly similar to those utilised by the Sec system — they are tripartite in nature, composed of an N-terminal domain, a hydrophobic domain (H-domain) and a C-terminal domain
Fig. 4. Sec- and Tat-specific signal peptides in Synechocystis sp. PCC6803. The Figure shows example signal peptides that direct transport by the Sec or Tat pathway in Synechocystis. The signal peptides show virtually no sequence homology apart from the presence of a conserved twin-arginine motif in Tat signal peptides and an Ala–Xaa–Ala consensus motif prior to the signal peptidase cleavage site (note, however, that the Rieske Fes proteins (PetC1/2/3) are believed to contain uncleavable signal peptides that serve as membrane anchors).
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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prior to the AxA cleavage site (Fig. 4). However, they do differ in several key respects. Tat signal peptides generally contain a higher proportion of acidic residues in the N-domain, have an extended H-domain (although Sec signal peptides are overall more hydrophobic in nature) and some contain basic residues in the C-domain that act as a ‘Sec avoidance’ motif when compared with Sec signal peptides [34]. The Tat signal peptide is thought to form an amphipathic helix with charged residues on one face and hydrophobic residues on the other aiding its association with the Tat translocation machinery. The first identified component of the Tat machinery was Hcf106 (High Chlorophyll Fluorescence 106) – found in the thylakoids of maize chloroplasts in the early 1990s [35], and this was followed by the identification of the remaining components, Tha4 and cpTatC. Homologues to all three of these components have been identified in bacteria including cyanobacteria. In E. coli the Tat components are named TatA, TatB and TatC (which correspond to Tha4, Hcf106 and cpTatC, respectively, in chloroplasts), and these proteins are found as a TatBC complex of 1:1 stoichiometric ratio and separate TatA homooligomers at steady state. A similar setup is found in thylakoid membranes where an Hcf106-cpTatC complex and separate Tha4 complexes are found. In E. coli, TatA and TatB are generally similar in structure and share 20% sequence identity. TatA is 9.6 kDa in size whereas TatB is 18.5 kDa; both have an N-out-C-in topology (i.e. the N-terminus is located in the periplasm whereas the C-terminus is located on the cytoplasmic side of the plasma membrane). They both contain a transmembrane helix linked to a cytoplasmic amphipathic helix (which lies parallel to the membrane), separated by a conserved “hinge” region that contains a glycine residue. The majority of the additional size of TatB can be attributed to a highly charged, unstructured C-terminal tail region. TatC is significantly larger at 28.9 kDa and is composed of six transmembrane helices with the N- and C-termini both located in the cytoplasm (for more detailed reviews see [19,33]). Despite their similarities the TatA/B components perform very different functions. The TatBC complex is roughly 370 kDa, corresponding to several copies of each subunit and this is the site of substrate recognition and docking [36] thus beginning the translocation process. Docking of substrate then triggers recruitment of TatA complexes to coalesce with the TatBC-substrate complex, thereby forming the functional translocase. Once the translocase is assembled, the substrate is translocated into the periplasm or lumen by a mechanism that remains very poorly understood. Finally the signal peptide is cleaved by a signal peptidase allowing substrate release into the periplasm. Native E. coli Tat substrates cover a wide range of sizes, from 30 kDa to 150 kDa with diameters of 30 Å to 70 Å, however chloroplast Tat substrates are only 4 kDa to 60 kDa [37]. To cope with this variation in size, the Tat system is thought to recruit TatA oligomers that form a putative translocation pore of the necessary size. Electron microscopy of purified TatA complexes shows a double-ring structure, ranging from 50 kDa to 500 kDa that spans the membrane surrounding an aqueous pore open at one end [38]. The varying size of these cylinders and the distinctive TatA “ladder” present on native, non-denaturing polyacrylamide gels, give credence to the hypothesis that TatA protomers are recruited to form the translocation channel tailored to fit the size of the substrate to be translocated. However, E. coli also contains a TatA paralog, TatE, which can substitute for TatA, yet this complex purifies as a much smaller, more homogeneous particle less than 100 kDa in size [39]. The true significance of the TatA complex size variability is thus unclear. The Synechocystis sp. PCC6803 genome appears to contain three tat genes: sll0194, which encodes a TatC homologue, and the slr1046 and ssl2823 genes, which encode TatA/B-like proteins. However, it is surprisingly difficult to identify the TatA and TatB homologues. In most Gram-negative bacteria, TatB subunits are usually larger than TatA proteins, but the slr1046 and ssl2823 genes encode proteins of a similar size. TatA and TatB family proteins can usually be distinguished by a different criterion; while all TatA/B proteins contain a critical Gly residue in
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the ‘hinge’ region separating the transmembrane and amphipathic helices, TatA proteins contain an invariant Phe–Gly motif while TatB proteins contain Gly-Pro. However, both Synechocystis proteins (slr1046 and ssl2823) contain all of these residues (in other words, Phe–Gly– Pro), and additionally, both genes are able to complement E. coli ΔtatAE and ΔtatB mutants [30]. It is thus impossible to attribute a TatA or TatB function to slr1046 or ssl2823 by sequence analysis alone. In vitro translocation assays have not been developed using cyanobacterial membranes, no sec or tat mutants have been isolated. Thus, it has not been possible to identify Sec- or Tat-translocated substrates in cyanobacteria, or indeed characterise the protein targeting pathways in any real detail. Aldridge et al. [30] did show that a set of Rieske iron–sulphur proteins (PetC) are almost certainly transported by Tat in cyanobacteria — they were shown to be transported only by Tat when expressed in E. coli. Moreover, PetC1 and PetC2 were shown to be located in the thylakoid membrane while PetC3 was localised to the plasma membrane, demonstrating that Tat substrates can be efficiently targeted to both membranes. Interestingly, expression of green fluorescent protein bearing an E. coli Tat signal peptide resulted in the protein being targeted exclusively to the periplasm [40]. This result suggested that this may be the default targeting pathway, with other determinants perhaps being required for targeting to the thylakoid lumen. 3.3. Protein insertion into the thylakoid membranes — the SRP pathway At least half of the proteins targeted to the thylakoids in chloroplasts are integral thylakoid membrane proteins [18,37]. There is evidence that a small number of these can be inserted into the membrane through the Tat or Sec pathways but the majority utilise the cpSRP pathway or an unusual ‘spontaneous insertion’ pathway [18]. However, the insertion of plasma membrane proteins in E. coli follows very different rules and this leads to a main area of uncertainty in cyanobacteria — does membrane protein insertion (particularly into thylakoids) most resemble the chloroplast model or the E. coli model? Possible scenarios are depicted in Fig. 3A. The SRP (Signal Recognition Pathway) system employed in the cytoplasm of prokaryotes (primarily studied in E. coli) functions cotranslationally, involving the action of an SRP comprising the Ffh protein and a 4.5S RNA molecule (reviewed in [20]). SRP binds to the nascent membrane protein as it emerges from the ribosome and mediates interaction with the Sec translocon. Thereafter, translation by the ribosome ‘pushes’ the nascent protein through/into the membrane via a SecYEG/YidC complex. YidC has been purified together with the Sec translocase [41] and there is evidence that it facilitates insertion of Sec-dependent membrane proteins. In this situation, YidC may stabilise transmembrane domains after they leave the SecYEG translocon channel and help mediate their partitioning into the lipid environment of the bacterial plasma membrane. A further protein, FtsY, is also involved in this process and it appears to play a key role in releasing SRP once insertion is underway. On its own, YidC is more critical for the insertion of a smaller subset of Sec-independent proteins, such as M13 procoat and Pf3 phage coat, as it alone can act as an insertase; one theory suggests that YidC binding to the substrate stabilises the inserted protein form and lowers the activation energy barrier for membrane insertion [20]. This pathway is depicted in Fig. 3B. Homologues of the SRP components, Ffh and FtsY are present in cyanobacteria and it appears likely that the insertion of plasma membrane proteins follows a similar pathway. However, it should be stressed that we have little information on this point. The insertion mechanism for thylakoid membrane proteins, on the other hand, may well resemble that shown for chloroplast thylakoid membrane proteins, where the situation is very different indeed. Chloroplasts also contain an SRP (cpSRP) but it is unique in several respects. Firstly, it has to be able to operate in a post-translational mode, since most of its substrates are imported from the cytosol. Secondly, it is unique in structural terms: it contains an Ffh homologue (cpSRP54) but lacks RNA [42] and contains
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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additionally an extra subunit, cpSRP43, that it is not found in other SRPs. Finally, it is involved in the insertion of a single class of proteins: the light-harvesting chlorophyll-binding protein (LHCP) family. The best studied example is Lhcb1, a three-α-helical thylakoid membranespanning protein that binds chlorophylls and carotenoids. Targeting of Lhcb1 via cpSRP does not require a cleavable signal peptide, rather the targeting sequence is formed by the mature protein [43]. Association of cpSRP54 and cpSRP43 is through the M-domain (methionine-rich) of cpSRP54 and the ankyrin repeats (ANK1-4) of cpSRP43 [44]. The cpSRP54 subunit also consists of a putative GTPase domain at its N-terminus [20]. LCHP proteins associate with cpSRP to form a substrate-cpSRP complex known as the ‘transit complex’. LCHP binding is mediated by a conserved hydrophobic 18 amino acid span between transmembrane segments 2 and 3 of LHCPs which is recognised by cpSRP43 [45,46]. The transit complex then interacts with the stromal pool of cpFtsY (a homologue of prokaryotic FtsY and the eukaryotic SRα). Chloroplast FtsY is thought to be responsible for targeting the transit complex to the thylakoid membrane, indeed cpFtsY is predominantly found on the stromal face of the thylakoid membrane albeit in an equilibrium with a smaller pool soluble in the stroma [47]. At the thylakoid membrane, cpFtsY targets the transit complex to the Albino3 (Alb3) membrane-insertion complex [20]. Although insertion of LCHP is probably one of the least characterised events in this process, Alb3 is known to play an important role in the mechanism. Alb3 binding to cpSRP43 of the transit complex is via the C-terminal domain [48]. Alb3 is essential for the insertion of SRP-dependent proteins in thylakoids, just as the E. coli homologue, YidC, is required for the efficient insertion of almost every E. coli membrane protein tested [49] and the eukaryotic homologue, Oxa1, plays an important role in the insertion of proteins into the inner mitochondrial membrane. Moreover, mutants deficient in Alb3 have an ‘albino phenotype’ and display clear deficiencies in thylakoid biogenesis and Lhcb1 was shown to directly require Alb3 for thylakoid insertion [50]. It is known that several LHCP family members, such as Lhcb4.1 and Lhcb5, are likewise Alb3 dependent [18]. Once the transit complex is associated with Alb3 at the thylakoid membrane, LHCP is inserted in a GTP-dependent manner. Hydrolysis of GTP is likely a function of cpSRP54 dissociating from the substrate which is now integrated into the thylakoid membrane. The available evidence suggests that this cpSRP-dependent pathway is used only for LHCP proteins, with the specificity dictated by the recognition of the conserved L18 peptide by cpSRP43. Another unusual feature is the lack of involvement of the Sec system — the only important membrane-bound component appears to be Alb3 (see Fig. 3). 3.4. Spontaneous or unassisted insertion of proteins into the chloroplast thylakoid membrane As previously mentioned, a baffling addition to the list of protein targeting pathways has been identified, which apparently requires no additional factors apart from the protein to be inserted and the thylakoid membrane. While LHCP family members have consistently been shown to require cpSRP, FtsY, Alb3 and GTP hydrolysis for insertion into thylakoids, studies on other thylakoid membrane proteins concluded that not all proteins require Alb3 for insertion into the thylakoid membrane. These proteins included CFoII, PsbS, PsbX, PsbY, PbsW, PsaK and PsaG, several of which contain more membrane spans than LHCP proteins. Even more surprisingly the insertion of these proteins did not require nucleoside triphosphates or the thylakoid proton motive force — ruling out an involvement of SRP, SecA or the Tat system [51,52]. In the absence of any identifiable insertion factors, a “spontaneous insertion” pathway was proposed, in which the protein simply partitions into the membrane. However, further research is certainly needed to characterise this insertion mechanism in more detail. For example, recent studies [53] have shown that E. coli YidC may assist the insertion of proteins using diverse mechanisms, and it remains possible that the
‘Alb3-independent’ mechanism does in fact involve Alb3 after all, and that this subset of proteins simply uses an Alb3-requiring mechanism that remains to be characterised in vitro. In the context of cyanobacterial cell biology, the major question is how proteins are inserted into the thylakoid membrane: by the Sec/ SRP route or the ‘spontaneous’ pathway. We have little hard evidence on these points, for reasons outlined above, but it is relevant that chlorophyll synthase was pulled down together with YidC/Alb3 from Synechocystis PCC6803 [54]. This would suggest that it may be targeted by the Alb3/SRP pathway, which would fit with the fact that this pathway is used by chlorophyll-binding proteins in plant thylakoids, as discussed above. What we need now are data on proteins that do not bind chlorophyll (or other cofactors), and clear evidence for or against the involvement of the major pathways identified in plants or other bacteria. So far, these data are sorely lacking.
4. Protein sorting mechanisms The presence of only single sets of tat and sec genes in well studied cyanobacteria such as Synechocystis sp. PCC6803 [18] raises the question: how do proteins reach their correct site of function in the thylakoid or plasma membranes, or in the thylakoid lumen/periplasm? This is a key aspect of cyanobacterial cell biology since many proteins reside in only one membrane system (for example, Rieske iron–sulphur proteins PetC1 and PetC2 reside in the thylakoid membrane), while PetC3 resides in the plasma membrane [30]. Given that they are both inserted by the Tat pathway, how does the cell ‘know’ which membrane a given protein should be targeted towards? This directionality and specific membrane tomography is an issue of protein sorting. There are currently two prevailing hypotheses for how this occurs: The simpler is named the ‘post-translocation’ sorting model — proteins are sorted after translocation and are initially targeted in an ad hoc manner across both the thylakoid and plasma membrane, regardless of their final destination. After translocation, they undergo subsequent sorting if they are not in the correct location. An example supporting this hypothesis is the assembly of photosystem II, which partially assembles in the plasma membrane before travelling to the thylakoid membrane for completion of assembly [55]. If both thylakoid and plasma membranes are connected, this model seems credible. The second model suggests that proteins are translocated only at their final site of function and it has thus been dubbed the ‘pre-translocation’ sorting model. Pre-translocation is currently the favoured method as it is more efficient, in terms of both energy and resources, for proteins to travel directly to the correct membrane. Raialahti et al. [56] have shown that cyanobacterial signal peptides along with the first 15 amino acids of mature protein (N15) have hetergeneous physiochemical properties depending on their final destination. At no point was signal peptide length a determining factor, but both amino acid volume and polarisability were deemed important. The various physiochemical properties, which they categorised as ‘dialects’, generate a predictable property profile for proteins destined to the same membrane. However, it is still unknown whether there is a specific component responsible for ‘sensing’ these dialects or if sorting is achieved through the individual protein's physiochemical properties alone. It is possible that differentially localised signal peptidases could play the sensing role — e.g. the slr1377 peptidase is found in the plasma membrane, while the sll0716 peptidase is localised to the thylakoid membrane [56] – and these may identify the signal peptides and their properties to either accept or reject their membrane incorporation. Nevertheless, a complete understanding of protein sorting in cyanobacteria is distinctly lacking. Alternatively, identical Sec/Tat systems could acquire different substrate selectivities in the plasma and thylakoid membranes by virtue of the different environments in which they reside — for example, the different lipid compositions could endow them with different properties.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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5. Conclusions and outlook Cyanobacteria exhibit a highly organised system of membrane differentiation and to a large extent the plasma and thylakoid membranes carry out very different activities. Numerous proteins are targeted into and across these membranes, and we have some idea of how this is achieved. However, a serious lack of biochemical and genetic tools means that this central area of cyanobacterial cell biology remains very poorly understood. In particular, we do not know whether the two membranes (and associated soluble phases) are truly connected. This is a key point because we do need to know whether proteins are randomly targeted into/across either membrane before migrating to their correct destination. In vitro translocation assays will always be a problem because cyanobacterial membranes tend to be highly fragmented after isolation, but genetic studies and time-resolved imaging studies should be able to shed light on these areas. Finally, although this review has focused primarily on protein targeting sorting mechanisms, it should be mentioned that our lack of understanding on this general area of cyanobacterial cell biology also extends to the sphere of membrane lipids. Thylakoids in plants and cyanobacteria are largely based galactolipids that account for more than 70% of total lipid content. As with proteins, the trafficking of these lipids, and hence the formation of bulk thylakoid membrane, is still poorly understood in cyanobacteria despite the identification of key lipid synthesis enzymes (reviewed in [9].) Acknowledgements D.G. and J.A.Z.Z. were supported by funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/under REA grant agreement no. 317184 (‘PHOTO.COMM’). This material reflects only the authors' views and the Union is not liable for any use that may be made of the information contained therein. References [1] R. Buick, When did oxygenic photosynthesis evolve? Philos. Trans. R. Soc. B Biol. Sci. 363 (2008) 2731–2743. [2] M. Hügler, S.M. Sievert, Beyond the calvin cycle: autotrophic carbon fixation in the ocean, Annu. Rev. Mar. Sci. 3 (2011) 261–289. [3] K. Sciuto, I. Moro, Cyanobacteria: the bright and dark sides of a charming group, Biodivers. Conserv. 24 (2015) 711–738. [4] H.W. Paerl, N.S. Hall, E.S. Calandrino, Controlling harmful cyanobacterial blooms in a world experiencing anthropogentic and climatic-induced change, Sci. Total Environ. 409 (2011) 1739–1745. [5] R.H. Wijffels, O. Kruse, K.J. Hellingwerf, Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae, Curr. Opin. Biotechnol. 24 (2013) 405–413. [6] S.A. Nierzwicki-Bauer, D.L. Balkwill, S.E. Stevens Jr., Three-dimensional ultrastructure of a unicellular cyanobacterium, J. Cell Biol. 97 (1983) 713–722. [7] U.C. Vothknecht, P. Westhoff, Biogenesis and origin of thylakoid membranes, Biochim. Biophys. Acta 1541 (2001) 91–101. [8] J. Nickelsen, B. Rengstl, A. Stengel, M. Schottkowski, J. Soll, E. Ankele, Biogenesis of the cyanobacterial thylakoid membrane system–an update, FEMS Microbiol. Lett. 315 (2011) 1–5. [9] A. Rast, S. Heinz, J. Nickelsen, Biogenesis of thylakoid membranes, Biochim. Biophys. Acta 1847 (2015) 821–830. [10] A.M. van de Meene, M.F. Hohmann-Marriott, W.F. Vermaas, R.W. Roberson, The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803, Arch. Microbiol. 184 (2006) 259–270. [11] D.D. Kunkel, Thylakoid centers: structures associated with the cyanobacterial photosynthetic membrane system, Arch. Microbiol. 133 (1982) 97–99. [12] M. Schottkowski, S. Gkalympoudis, N. Tzekova, C. Stelljes, D. Schunemann, E. Ankele, J. Nickelsen, Interaction of the periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp. PCC 6803, J. Biol. Chem. 284 (2009) 1813–1819. [13] A. Stengel, I.L. Gugel, D. Hilger, B. Rengstl, H. Jung, J. Nickelsen, Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis, Plant Cell 24 (2012) 660–675. [14] M. Rütgers, M. Schroda, A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystem biogenesis? Plant Signal. Behav. 8 (2013)http://dx.doi. org/10.4161/psb.27037. [15] U.C. Vothknecht, S. Otters, R. Raoul Hennig, D. Schneider, Vipp1: a very important protein in plastids?! J. Exp. Bot. 63 (2012) 1699–1712.
7
[16] S. Zhang, G. Shen, Z. Li, J.H. Golbeck, D.A. Bryant, Vipp1 is essential for the biogenesis of photosystem I but not thylakoid membranes in Synechococcus sp. PCC 7002, J, Biol. Chem. 289 (2014) 15904–15914. [17] S. Rexroth, C.W. Mullineaux, D. Ellinger, E. Sendtko, M. Rogner, F. Koenig, The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains, Plant Cell 23 (2011) 2379–2390. [18] C. Aldridge, P. Cain, C. Robinson, Protein transport into and across the thylakoid membrane. FEBS J. 276 (2009) 1177–1186. [19] T. Palmer, B.C. Berks, The twin-arginine translocation (Tat) protein export pathway, Nat. Rev. Microbiol. 10 (2012) 483–496. [20] S.W. Hennon, R. Soman, R.E. Dalbey, YidC/Alb3/Oxa1 family of insertases, J. Biol. Chem. 290 (2015) 14866–14874. [21] S. Fulda, F. Huang, F. Nilsson, M. Hagemann, B. Norling, Proteomics of Synechocystis sp. strain PCC 6803, identification of periplasmic proteins in cells grown at low and high salt concentrations, Eur. J. Biochem. 267 (2000) 5900–5907. [22] K.E. Chatzi, M.F. Sardis, A. Economou, S. Karamanou, SecA-mediated targeting and translocation of secretory proteins, Biochim. Biophys. Acta 1466-1474 (2014). [23] P.A. Scotti, M.L. Urbanus, J. Brunner, J.W. de Gier, G. von Heijne, C. van der Does, A.J.M. Driessen, B. Oudega, J. Luirink, YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase, EMBO J. 19 (2000) 542–549. [24] L. Fröderberg, T. Röhl, K.-J. van Wijk, J.-W.L. de Gier, Complementation of bacterial SecE by a chloroplast homologue, FEBS Lett. 498 (2001) 52–56. [25] C. Sun, S.L. Rusch, J. Kim, D.A. Kendall, Chloroplast SecA and Escherichia coli SecA have distinct lipid and signal peptide preferences, J. Bacteriol. 189 (2007) 1171–1175. [26] M. Nakai, D. Sugita, T. Omata, T. Endo, Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942, Biochem. Biophys. Res. Commun. 193 (1993) 228–234. [27] R. Srivastava, T. Pisareva, B. Norling, Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803, Proteomics 5 (2005) 4905–4916. [28] P.J. Hynds, D. Robinson, C. Robinson, The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane, J. Biol. Chem. 273 (1998) 34868–34874. [29] K. Cline, W.F. Ettinger, S.M. Theg, Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP, J. Biol. Chem. 267 (1992) 2688–2696. [30] C. Aldridge, E. Spence, M.A. Kirkilionis, L. Frigerio, C. Robinson, Tat-dependent targeting of rieske iron–sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803, Mol. Microbiol. 70 (2008) 140–150. [31] C.F.R.O. Matos, C. Robinson, A. Di Cola, The Tat system proofreads Fes protein substrates and directly initiates the disposal of rejected molecules, EMBO J. 27 (2008) 2055–2063. [32] M.P. DeLisa, D. Tullman, G. Georgiou, Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 6115–6120. [33] C. Robinson, C.F.R.O. Matos, D. Beck, C. Ren, J. Lawrence, N. Vasisht, S. Mendel, Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria, Biochim. Biophys. Acta 876-884 (2011). [34] E. Bogsch, S. Brink, C. Robinson, Pathway specificity for a ΔpH-dependent precursor thylakoid lumen protein is governed by a "Sec-avoidance" motif in the transfer peptide and a "Sec-incompatible" mature protein, EMBO J. 16 (1997) 3851–3859. [35] A.M. Settles, A. Yonetani, A. Baron, D.R. Bush, K. Cline, R. Martienssen, Secindependent protein translocation by the maize Hcf106 protein, Science 278 (1997) 1467–1470. [36] M.J. Tarry, E. Schäfer, S. Chen, G. Buchanan, N.P. Greene, S.M. Lea, T. Palmer, H.R. Saibil, B.C. Berks, Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 13284–13289. [37] K. Cline, S.M. Theg, The Sec and Tat protein translocation pathways in chloroplasts, Enzymes 25 (2006) 463–492. [38] U. Gohlke, L. Pullan, C.A. McDevitt, I. Porcelli, E. de Leeuw, T. Palmer, H.R. Saibil, B.C. Berks, The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 10482–10486. [39] J. Baglieri, D. Beck, N. Vasisht, C.J. Smith, C. Robinson, Structure of the TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter, J. Biol. Chem. 287 (2012) 7335–7344. [40] E. Spence, M. Sarcina, N. Ray, S.G. Møller, C.W. Mullineaux, C. Robinson, Membranespecific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803, Mol. Microbiol. 48 (2003) 1481–1489. [41] P.A. Scotti, M.L. Urbanus, J. Brunner, J.W. de Gier, G. von Heijne, C. van der Does, A.J.M. Driessen, B. Oudega, J. Luirink, YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase, EMBO J. 19 (2000) 542–549. [42] X. Li, R. Henry, J. Yuan, K. Cline, N.E. Hoffman, A chloroplast homologue of signal recognition particle unit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 3789–3793. [43] P.V. Viitanen, E.R. Doran, P. Dunsmuir, What is the role of the transit peptide in thylakoid integration of the light harvesting chlorophyll a/b protein? J. Biol. Chem. 263 (1988) 15000–15007. [44] R. Goforth, E. Peterson, J. Yuan, M. Moore, A. Kight, M. Lohse, J. Sakon, R. Henry, Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43, J. Biol. Chem. 279 (2004) 43077–43084.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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K.M. Frain et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx
[45] C.J. Tu, E.C. Peterson, R. Henry, N.E. Hoffman, The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43, J. Biol. Chem. 275 (2000) 13187–13190. [46] P. Cain, I. Holdermann, I. Sinning, A.E. Johnson, C. Robinson, Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain, Biochem. J. 437 (2011) 149–155. [47] N. Kogata, K. Nishio, T. Hirohashi, S. Kikuchi, M. Nakai, Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids, FEBS Lett. 447 (1999) 329–333. [48] S. Falk, S. Ravaud, J. Koch, I. Sinning, The C terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane, J. Biol. Chem. 285 (2010) 5954–5962. [49] J.C. Samuelson, M. Chen, F. Jiang, I. Möller, M. Wiedmann, A. Kuhn, G.J. Phillips, R.E. Dalbey, YidC mediates membrane protein insertion in bacteria, Nature 406 (2000) 637–641. [50] M. Moore, M.S. Harrison, E.C peterson, R. Henry, chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyllbinding protein into thylakoid membranes, J. Biol. Chem. 275 (2000) 1529–1532. [51] D. Michl, C. Robinson, J.B. Shackleton, R.G. Herrmann, R.B. Klösgen, Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway, EMBO J. 13 (1994) 1310–1317.
[52] C.A. Woolhead, S. J. Thompson, M. Moore, C. Tissier, A. Mant, A. Rodger, R. Henry, C. Robinson, Distinct Albino3-dependent and -independent pathways for thylakoid membrane protein insertion, J. Biol. Chem. 276 (2001) 40841-40846. [53] K. Kumazaki, S. Chiba, M. Takemoto, A. Furukawa, K. Nishiyama, Y. Sugano, T. Mori, N. Dohmae, K. Hirata, Y. Nakada-Nakura, A.D. Maturana, Y. Tanaka, H. Mori, Y. Sugita, F. Arisaka, K. Ito, R. Ishitani, T. Tsukazaki, O. Nureki, Structural basis of Secindependent membrane protein insertion by YidC, Nature 509 (2014) 516–520. [54] J.W. Chidgey, M. Linhartová, J. Komenda, P.J. Jackson, M.J. Dickman, D.P. Canniffe, P. Koník, J. Pilny, C.N. Hunter, R. Sobotka, A cyanobacterial chlorophyll synthase-hlid complex associates with the Ycf39 protein and the YidC/Alb3 insertase, Plant Cell 26 (2014) 1267–1279. [55] E. Zak, B. Norling, R. Maitra, F. Huang, B. Andersson, H.B. Pakrasi, The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13443–13448. [56] T. Rajalahti, F. Huang, M.R. Klement, T. Pisareva, M. Edman, M. Sjöström, A. Wieslander, B. Norling, Proteins in different synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis, J. Proteome Res. 6 (2007) 2420–2434.
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Protein translocation and thylakoid biogenesis in cyanobacteria Kelly M. Frain 1, Doris Gangl 1, Alexander Jones 1, Julie A.Z. Zedler 1, Colin Robinson ⁎,1 Centre for Molecular Processing, School of Biosciences, University of Kent, Ingram Building, Canterbury, CT2 7NJ, United Kingdom
a r t i c l e
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Article history: Received 16 June 2015 Received in revised form 17 August 2015 Accepted 31 August 2015 Available online xxxx Keywords: Cyanobacteria Protein translocation Membrane biogenesis Sec Tat SRP
a b s t r a c t Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments — specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called ‘spontaneous’ pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. © 2015 Published by Elsevier B.V.
1. Introduction Cyanobacteria were the first organisms to perform oxygenic photosynthesis [1] and today they play a major role in global carbon fixation [2]. Cyanobacteria have long been used as model organisms for photosynthesis research, but they have also attracted interest for other reasons [3]: often they are associated with harmful algal blooms (reviewed in [4]), and they are also interesting candidates for biotechnological applications (reviewed in [5]). In structural terms, they are relatively complex microorganisms. A typical cyanobacterium contains an outer membrane and a plasma membrane which are separated by the periplasm-typical features of Gram-negative bacteria. However, key photosynthetic activities are carried out within an elaborate internal thylakoid membrane system, and cyanobacteria are thus unusual among Gram-negative bacteria in exhibiting a high degree of membrane differentiation [6]. Finally, the thylakoid membrane encloses a further soluble phase which is often termed the thylakoid lumen (Fig. 1). Many aspects of cyanobacterial cell biology remain poorly understood, and this applies particularly to the mechanisms involved in the generation and maintenance of the membrane systems in these organisms. Indeed, our knowledge of these processes in cyanobacteria is still very much in its infancy. This review article aims to review the processes of protein translocation and membrane biogenesis in cyanobacteria; Abbreviations: Tat, twin-arginine translocation; SRP, signal recognition particle. ⁎ Corresponding author. E-mail address: [email protected] (C. Robinson). 1 These authors contributed equally to this work.
we describe aspects that have been confirmed experimentally, and we will draw parallels from studies on chloroplasts and other bacteria, where we have a better understanding of membrane biogenesis in general. Chloroplasts are generally accepted to have arisen from endosymbiotic cyanobacteria, and they share many features with typical cyanobacteria; it is to be expected that there may be similarities in thylakoid protein translocation and sorting mechanisms, and these have been extensively studied in chloroplasts. Escherichia coli, on the other hand, is a model organism for the study of protein transport into and across bacterial plasma membranes, and it is more than likely that some of the established mechanisms are also used in cyanobacteria. We will therefore provide a general overview of the main chloroplast and bacterial translocation systems, including the general secretory (Sec) pathway, the twin arginine translocation (Tat) pathway and the signal recognition particle (SRP) pathway, and we discuss which mechanisms are likely to operate in cyanobacteria. We also highlight the potential importance of membrane organisation and the implications for the translocation, integration and targeting of proteins within the various membranes. The composition of cyanobacterial membranes and their potential interconnections may play a major role for protein sorting. 2. Membrane organisation in cyanobacteria Cyanobacteria contain three different membranes: the outer membrane, the plasma membrane and the thylakoid membrane. Each carries out distinct functions but there is still much debate about whether the plasma membrane and the thylakoid membrane are actually connected and form a continuous network (reviewed in [7–9]). In brief, three
http://dx.doi.org/10.1016/j.bbabio.2015.08.010 0005-2728/© 2015 Published by Elsevier B.V.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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Fig. 2. Organisation of the plasma membrane, thylakoid centres and thylakoid membranes. Evidence suggests that thylakoid membranes coalesce with the plasma membrane at specific points termed thylakoid centres. PratA appears to be an important component of these sites and it has been found in an intermediate fraction during sedimentation studies, denoted the PratA-defined membrane (PDM) sub-fraction. This may be the initial site of PSII and PSI biogenesis, and the site at which membrane lipids and proteins are transferred to developing thylakoids.
Fig. 1. Structure and essential features of a typical unicellular cyanobacterium. The majority of cyanobacteria contain outer and inner (plasma) membranes which are separated by a periplasmic space, together with internal thylakoid membranes which are believed to be attached to the plasma membrane at ‘thylakoid centres’. The thylakoid membrane encloses a further soluble compartment termed the thylakoid lumen. It is unclear how the plasma membrane and thylakoid membrane (and the periplasm/thylakoid lumen) are physically connected. Within the cytoplasm are the nucleoid, carboxysome and ribosomes.
different models have been proposed to explain how thylakoid membrane biogenesis occurs: (1) pre-existing thylakoid membrane structures are the sites of synthesis of proteins, lipids and pigments for that particular membrane. (2) Protein-pigment complexes are synthesised and assembled in the plasma membrane and then transferred to the thylakoids. (3) Thylakoid ‘centres’ are formed as specialised structures initiating the biosynthesis of components [8]. The latter is the currently favoured model, supported by several observations. In general, thylakoids appear as stacks of parallel sheets near the plasma membrane. However, in certain regions they converge near the plasma membrane forming so-called thylakoid centres [10]. The first observation of these thylakoid centres in cyanobacteria was reported by Kunkel et al. [11]. In Anabaena cylindrica, Dermocarpa violaceae, Gleocapsa alpicola and Pleurocapsa minor, thylakoid centres were observed as cylindrical structures of 30 nm by 20 nm situated close to the plasma membrane. These consisted of globular subunits orientated in nonparallel, stacked arrays and were seemingly attached to thylakoids (see Fig. 2). There is evidence that thylakoid centres are involved in the biogenesis of photosystem II. In the plasma membrane of Synechocystis sp. PCC 6803 the photosystem II assembly factor PratA was found enriched in certain areas and it was therefore termed PratA-defined membrane (PDM) [12]. PratA in turn accumulates near thylakoid centres. Semicircular membranous structures surrounding thylakoid centres were hypothesised to consist of PDMs and seemed to contact both the thylakoid and plasma membrane [13]. The ‘vesicle inducing protein in plastids’ (VIPP1) seems to be an important component found in thylakoid centres (reviewed in [14,15]). VIPP1 forms oligomers of 12–17 fold symmetry that associate into the same cylindrical structures observed at thylakoid centres. Interestingly, a recent report also implicates VIPP1 in the biogenesis of photosystem I [16]. The primordial cyanobacterium Gleobacter violaceus also shows signs of membrane differentiation although the organism does not possess thylakoid membranes. Instead, it harbours its photosynthetic and respiratory machinery in
the plasma membrane. Still, two distinct membrane domains are clearly distinguishable, which are similar in protein composition to the thylakoid and plasma membranes in other cyanobacteria [17]. Overall, it appears that thylakoid centres may play a key role in thylakoid membrane organisation. 3. Translocation into and across the plasma membrane and thylakoid membranes Whatever the mechanisms by which the plasma and thylakoid membranes arise in cyanobacteria are, proteins need to be targeted into and across these membranes. These processes have been studied to only a minor extent in cyanobacteria, for several reasons. First, the isolation of intact thylakoids from cyanobacteria is extremely difficult, and in vitro protein translocation assays have yet to be developed. Secondly, although genetic approaches are more feasible, there has been a tendency to focus on the much more tractable E. coli for detailed studies on bacterial protein transport systems. As a result, we have little direct information on cyanobacterial protein targeting pathways, and we are obliged to draw potential parallels from other systems. However, this is problematic; it is not clear whether cyanobacterial protein targeting pathways most closely resemble those in the well-studied E. coli (which does not of course contain thylakoids) or higher plant chloroplasts. Cyanobacteria are widely accepted to be the progenitors of higher plant chloroplasts, which suggests that parallels can be made between thylakoid protein translocation/insertion in cyanobacteria and chloroplasts [18]. However, chloroplasts differ from cyanobacteria in key respects. For example, plant thylakoids are separated from the cytoplasm by the stroma and chloroplast envelope, which means that protein transport into plant thylakoids is almost entirely posttranslational (since the vast majority of thylakoid proteins are imported from the cytosol). In contrast, some pathways may well operate cotranslationally in cyanobacteria (as is the case for some of the E. coli pathways). Otherwise, cyanobacteria target numerous proteins to the plasma membrane, outer membrane and periplasm, and it appears very likely that these proteins are translocated by mechanisms that resemble those operating in E. coli. Altogether, these factors suggest that cyanobacteria may utilise protein trafficking pathways that operate in both chloroplasts and other bacteria, making inferences difficult to finalise. In the following sections we review the pathways that are used to
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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target proteins into and across E. coli and chloroplast thylakoid membranes, and we discuss those mechanisms that most likely operate in cyanobacteria. In both chloroplasts and E. coli there are three largely independent protein translocation/insertion pathways, with a fourth functioning in chloroplasts [18–20]. Proteins are transported across the plasma membrane and thylakoid membrane via the Secretory (Sec) pathway and the Twin-Arginine Translocation (Tat) pathways, while membrane
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proteins are inserted by the SRP pathway in both systems. Additionally, membrane proteins are inserted into the thylakoid membrane by a novel pathway sometimes termed ‘spontaneous insertion’ (Fig. 3). Each of these pathways utilise individual precursor targeting determinants and require different energy sources. Indeed it was the requirement for different energy components in in vitro assays that gave rise to the identification of the four different pathways in the first place.
Fig. 3. Comparison of different translocation mechanisms operating in (A) cyanobacteria, (B) bacteria and (C) plant chloroplasts. A. In cyanobacteria, proteins destined for the periplasm or thylakoid lumen are transported via the Sec or Tat pathways, while plasma membrane proteins are probably inserted using the SRP pathway (the default pathway for insertion of membrane proteins in E. coli — see B). It is not known whether thylakoid membrane proteins are inserted using the SRP pathway or the ‘spontaneous’ insertion pathway used for many chloroplast thylakoid proteins. B. in E. coli, soluble proteins are transported across the plasma membrane using either the Sec or Tat pathways. In the Sec pathway, the substrate is transported through a SecYEG channel in an unfolded state using energy derived from ATP hydrolysis by SecA. In the Tat pathway, the substrate is transported in a folded state through a translocon that is assembled from separate TatBC and TatA complexes. C. In chloroplasts, thylakoid proteins are mostly synthesised in the cytosol and first cross the chloroplast outer and inner membranes before being directed to the thylakoids. Soluble lumenal proteins are transported by either the Sec or Tat pathways; the Tat system appears to be similar to bacterial systems while the Sec system is simpler, lacking SecG and some ancillary Sec proteins such as SecDF (not shown). Only a subset of thylakoid membrane proteins — the light harvesting chlorophyll binding protein (LHCP) family — are inserted by the SRP pathway and this further differs from the E. coli model in that the Sec channel is not involved. The only known membrane-bound receptor element is Alb3. Other membrane proteins are inserted by a different mechanism termed the ‘spontaneous’ pathway (see text). PP, periplasm; PM, plasma membrane; CP, cytoplasm; TM, thylakoid membrane; TL, thylakoid lumen; S, stroma.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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3.1. Targeting of proteins to the periplasm or thylakoid lumen via the Sec pathway The Sec pathway is evolutionarily conserved across kingdoms — present in the bacterial plasma membrane, the endoplasmic reticulum and chloroplasts. Soluble proteins targeted by the Sec machinery are synthesised with an N-terminal signal peptide that contains a hydrophobic ‘core’ region with a charged N-terminal domain. The Sec pathway clearly operates in cyanobacteria and it has been estimated that 82% of translocated proteins (whether to the periplasm or thylakoid lumen) in Synechocystis sp. PCC6803 contain a Sec signal peptide [21]. Upon successful translocation of the substrate protein into the periplasm or lumen, this signal peptide is cleaved by signal peptidase at an Ala–Xaa–Ala consensus motif present in the C-domain of the signal peptide, releasing the mature protein. Representative cyanobacterial Sec signal peptides are listed in Fig. 4. The Sec machinery has been best characterised in E. coli where it is composed of the membrane-embedded multi-span protein SecY and single-span SecE, the non-essential SecG, SecD, SecF, and finally the cytoplasmic SecA protein [22]. YidC can also be defined as a component of the Sec apparatus as it sometimes co-purifies with the Sec translocase [23] although it plays a more important role in SRP transport (discussed below). Chaperones such as SecB act in the cytosol of E. coli to keep preprotein in an unfolded state and to inhibit unwanted reactions (such as aggregation and/or degradation) prior to translocation. SecA binds to the Sec substrate in the cytoplasm, forming a SecA-preprotein complex that next interacts with the core translocation apparatus, SecYEG, in the membrane. Repeated rounds of ATP hydrolysis by SecA drive the translocation of the substrate through the SecYEG channel. Homologues to SecA, SecY and SecE are found in chloroplasts of plants (cpSecA, cpSecY and cpSecE respectively, with cpSecE able to functionally replace E. coli SecE [24]). However, the chloroplast Sec system appears to be a ‘minimalist’ Sec system because SecB and SecG are absent (Fig. 3). The E. coli Sec system also includes non-essential SecDF subunits (not shown in Fig. 3), and these are also absent. The plant chloroplast Sec pathway transports a subset of lumenal proteins to the lumen, including plastocyanin and the 33 kDa component of the oxygen-evolving complex, PsbO [18]. Interestingly it has been demonstrated that the Sec systems of the chloroplast and E. coli require very different membrane lipid environments in order to optimally translocate substrate, and it was suggested that cpSecA has evolved to be specifically suited to the thylakoid membrane environment [25]. This is in keeping with the very unusual lipid composition (the thylakoid membrane is mostly galactolipid whereas bacterial plasma membranes are composed primarily of phospholipids). These differences in membrane composition could be a piece of the jigsaw to the successful sorting of proteins to their requisite membrane in cyanobacteria — either the thylakoid lumen or the periplasm — as discussed below.
In cyanobacteria the Sec translocation machinery seems to be broadly similar to that found in E. coli (Fig. 3 A, B). SecY has been localised in both the plasma and the thylakoid membrane of Synechocystis sp. PCC6803 [26] and SecA is present in the thylakoid membrane [27] and presumably the plasma membrane. This organism contains only a single set of sec genes (as do most, if not all cyanobacteria) which suggests that the same Sec components are present in both the plasma and thylakoid membrane. In turn, this raises the question of how protein targeting to a specific compartment (i.e. the periplasm or the thylakoid lumen) can be achieved. One of the limitations of the Sec pathway is the requirement of substrate proteins to be in an unfolded state. This is due to the fixed-width nature of the core SecYE pore (~12Ǻ, reflecting the width of a polypeptide chain) and is demonstrated by the classic example of the Sec pathway's inability to translocate dihydrofolate reductase ‘locked’ in a folded conformation through binding around methotrexate [28]. Despite the integrity and flexibility of substrate (in terms of size, shape and complexity of structure) this ‘one size fits all’ pore, and translocation of substrates in an unfolded state enables, it does preclude translocation of proteins that require cytoplasmically-inserted cofactors, or substrates that fold rapidly in the cytosol. 3.2. Protein targeting via the Tat pathway The Tat system operates in parallel with the Sec system in the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria, and there is evidence that it is present in both the thylakoid membrane and plasma membrane of cyanobacteria [29]. Initially it was referred to as the ΔpH-dependent pathway because the ΔpH was shown to be the sole energy requirement for translocation into thylakoids [30]. It has since been re-named the Twin-Arginine Translocation (Tat) pathway in view of the importance of a twin arginine motif in signal peptides of Tat-targeted proteins [19]. The Tat pathway is unique in its ability to translocate fully folded proteins across energy-transducing membranes; indeed its primary purpose in E. coli is the translocation of complex cofactor-containing proteins into the periplasm. The uniqueness of the Tat system does not end there though - not only are substrates folded, they are invariably correctly folded, with mounting evidence for an in-built proofreading and/or quality control mechanism that rejects proteins lacking their requisite cofactors or even substrates that are incorrectly folded [31–33]. Although the Tat machinery is present in both the plasma membrane and the thylakoid membrane of cyanobacteria the proofreading and quality control of cyanobacterial systems has yet to be studied in any detail. Tat signal peptides are surprisingly similar to those utilised by the Sec system — they are tripartite in nature, composed of an N-terminal domain, a hydrophobic domain (H-domain) and a C-terminal domain
Fig. 4. Sec- and Tat-specific signal peptides in Synechocystis sp. PCC6803. The Figure shows example signal peptides that direct transport by the Sec or Tat pathway in Synechocystis. The signal peptides show virtually no sequence homology apart from the presence of a conserved twin-arginine motif in Tat signal peptides and an Ala–Xaa–Ala consensus motif prior to the signal peptidase cleavage site (note, however, that the Rieske Fes proteins (PetC1/2/3) are believed to contain uncleavable signal peptides that serve as membrane anchors).
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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prior to the AxA cleavage site (Fig. 4). However, they do differ in several key respects. Tat signal peptides generally contain a higher proportion of acidic residues in the N-domain, have an extended H-domain (although Sec signal peptides are overall more hydrophobic in nature) and some contain basic residues in the C-domain that act as a ‘Sec avoidance’ motif when compared with Sec signal peptides [34]. The Tat signal peptide is thought to form an amphipathic helix with charged residues on one face and hydrophobic residues on the other aiding its association with the Tat translocation machinery. The first identified component of the Tat machinery was Hcf106 (High Chlorophyll Fluorescence 106) – found in the thylakoids of maize chloroplasts in the early 1990s [35], and this was followed by the identification of the remaining components, Tha4 and cpTatC. Homologues to all three of these components have been identified in bacteria including cyanobacteria. In E. coli the Tat components are named TatA, TatB and TatC (which correspond to Tha4, Hcf106 and cpTatC, respectively, in chloroplasts), and these proteins are found as a TatBC complex of 1:1 stoichiometric ratio and separate TatA homooligomers at steady state. A similar setup is found in thylakoid membranes where an Hcf106-cpTatC complex and separate Tha4 complexes are found. In E. coli, TatA and TatB are generally similar in structure and share 20% sequence identity. TatA is 9.6 kDa in size whereas TatB is 18.5 kDa; both have an N-out-C-in topology (i.e. the N-terminus is located in the periplasm whereas the C-terminus is located on the cytoplasmic side of the plasma membrane). They both contain a transmembrane helix linked to a cytoplasmic amphipathic helix (which lies parallel to the membrane), separated by a conserved “hinge” region that contains a glycine residue. The majority of the additional size of TatB can be attributed to a highly charged, unstructured C-terminal tail region. TatC is significantly larger at 28.9 kDa and is composed of six transmembrane helices with the N- and C-termini both located in the cytoplasm (for more detailed reviews see [19,33]). Despite their similarities the TatA/B components perform very different functions. The TatBC complex is roughly 370 kDa, corresponding to several copies of each subunit and this is the site of substrate recognition and docking [36] thus beginning the translocation process. Docking of substrate then triggers recruitment of TatA complexes to coalesce with the TatBC-substrate complex, thereby forming the functional translocase. Once the translocase is assembled, the substrate is translocated into the periplasm or lumen by a mechanism that remains very poorly understood. Finally the signal peptide is cleaved by a signal peptidase allowing substrate release into the periplasm. Native E. coli Tat substrates cover a wide range of sizes, from 30 kDa to 150 kDa with diameters of 30 Å to 70 Å, however chloroplast Tat substrates are only 4 kDa to 60 kDa [37]. To cope with this variation in size, the Tat system is thought to recruit TatA oligomers that form a putative translocation pore of the necessary size. Electron microscopy of purified TatA complexes shows a double-ring structure, ranging from 50 kDa to 500 kDa that spans the membrane surrounding an aqueous pore open at one end [38]. The varying size of these cylinders and the distinctive TatA “ladder” present on native, non-denaturing polyacrylamide gels, give credence to the hypothesis that TatA protomers are recruited to form the translocation channel tailored to fit the size of the substrate to be translocated. However, E. coli also contains a TatA paralog, TatE, which can substitute for TatA, yet this complex purifies as a much smaller, more homogeneous particle less than 100 kDa in size [39]. The true significance of the TatA complex size variability is thus unclear. The Synechocystis sp. PCC6803 genome appears to contain three tat genes: sll0194, which encodes a TatC homologue, and the slr1046 and ssl2823 genes, which encode TatA/B-like proteins. However, it is surprisingly difficult to identify the TatA and TatB homologues. In most Gram-negative bacteria, TatB subunits are usually larger than TatA proteins, but the slr1046 and ssl2823 genes encode proteins of a similar size. TatA and TatB family proteins can usually be distinguished by a different criterion; while all TatA/B proteins contain a critical Gly residue in
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the ‘hinge’ region separating the transmembrane and amphipathic helices, TatA proteins contain an invariant Phe–Gly motif while TatB proteins contain Gly-Pro. However, both Synechocystis proteins (slr1046 and ssl2823) contain all of these residues (in other words, Phe–Gly– Pro), and additionally, both genes are able to complement E. coli ΔtatAE and ΔtatB mutants [30]. It is thus impossible to attribute a TatA or TatB function to slr1046 or ssl2823 by sequence analysis alone. In vitro translocation assays have not been developed using cyanobacterial membranes, no sec or tat mutants have been isolated. Thus, it has not been possible to identify Sec- or Tat-translocated substrates in cyanobacteria, or indeed characterise the protein targeting pathways in any real detail. Aldridge et al. [30] did show that a set of Rieske iron–sulphur proteins (PetC) are almost certainly transported by Tat in cyanobacteria — they were shown to be transported only by Tat when expressed in E. coli. Moreover, PetC1 and PetC2 were shown to be located in the thylakoid membrane while PetC3 was localised to the plasma membrane, demonstrating that Tat substrates can be efficiently targeted to both membranes. Interestingly, expression of green fluorescent protein bearing an E. coli Tat signal peptide resulted in the protein being targeted exclusively to the periplasm [40]. This result suggested that this may be the default targeting pathway, with other determinants perhaps being required for targeting to the thylakoid lumen. 3.3. Protein insertion into the thylakoid membranes — the SRP pathway At least half of the proteins targeted to the thylakoids in chloroplasts are integral thylakoid membrane proteins [18,37]. There is evidence that a small number of these can be inserted into the membrane through the Tat or Sec pathways but the majority utilise the cpSRP pathway or an unusual ‘spontaneous insertion’ pathway [18]. However, the insertion of plasma membrane proteins in E. coli follows very different rules and this leads to a main area of uncertainty in cyanobacteria — does membrane protein insertion (particularly into thylakoids) most resemble the chloroplast model or the E. coli model? Possible scenarios are depicted in Fig. 3A. The SRP (Signal Recognition Pathway) system employed in the cytoplasm of prokaryotes (primarily studied in E. coli) functions cotranslationally, involving the action of an SRP comprising the Ffh protein and a 4.5S RNA molecule (reviewed in [20]). SRP binds to the nascent membrane protein as it emerges from the ribosome and mediates interaction with the Sec translocon. Thereafter, translation by the ribosome ‘pushes’ the nascent protein through/into the membrane via a SecYEG/YidC complex. YidC has been purified together with the Sec translocase [41] and there is evidence that it facilitates insertion of Sec-dependent membrane proteins. In this situation, YidC may stabilise transmembrane domains after they leave the SecYEG translocon channel and help mediate their partitioning into the lipid environment of the bacterial plasma membrane. A further protein, FtsY, is also involved in this process and it appears to play a key role in releasing SRP once insertion is underway. On its own, YidC is more critical for the insertion of a smaller subset of Sec-independent proteins, such as M13 procoat and Pf3 phage coat, as it alone can act as an insertase; one theory suggests that YidC binding to the substrate stabilises the inserted protein form and lowers the activation energy barrier for membrane insertion [20]. This pathway is depicted in Fig. 3B. Homologues of the SRP components, Ffh and FtsY are present in cyanobacteria and it appears likely that the insertion of plasma membrane proteins follows a similar pathway. However, it should be stressed that we have little information on this point. The insertion mechanism for thylakoid membrane proteins, on the other hand, may well resemble that shown for chloroplast thylakoid membrane proteins, where the situation is very different indeed. Chloroplasts also contain an SRP (cpSRP) but it is unique in several respects. Firstly, it has to be able to operate in a post-translational mode, since most of its substrates are imported from the cytosol. Secondly, it is unique in structural terms: it contains an Ffh homologue (cpSRP54) but lacks RNA [42] and contains
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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additionally an extra subunit, cpSRP43, that it is not found in other SRPs. Finally, it is involved in the insertion of a single class of proteins: the light-harvesting chlorophyll-binding protein (LHCP) family. The best studied example is Lhcb1, a three-α-helical thylakoid membranespanning protein that binds chlorophylls and carotenoids. Targeting of Lhcb1 via cpSRP does not require a cleavable signal peptide, rather the targeting sequence is formed by the mature protein [43]. Association of cpSRP54 and cpSRP43 is through the M-domain (methionine-rich) of cpSRP54 and the ankyrin repeats (ANK1-4) of cpSRP43 [44]. The cpSRP54 subunit also consists of a putative GTPase domain at its N-terminus [20]. LCHP proteins associate with cpSRP to form a substrate-cpSRP complex known as the ‘transit complex’. LCHP binding is mediated by a conserved hydrophobic 18 amino acid span between transmembrane segments 2 and 3 of LHCPs which is recognised by cpSRP43 [45,46]. The transit complex then interacts with the stromal pool of cpFtsY (a homologue of prokaryotic FtsY and the eukaryotic SRα). Chloroplast FtsY is thought to be responsible for targeting the transit complex to the thylakoid membrane, indeed cpFtsY is predominantly found on the stromal face of the thylakoid membrane albeit in an equilibrium with a smaller pool soluble in the stroma [47]. At the thylakoid membrane, cpFtsY targets the transit complex to the Albino3 (Alb3) membrane-insertion complex [20]. Although insertion of LCHP is probably one of the least characterised events in this process, Alb3 is known to play an important role in the mechanism. Alb3 binding to cpSRP43 of the transit complex is via the C-terminal domain [48]. Alb3 is essential for the insertion of SRP-dependent proteins in thylakoids, just as the E. coli homologue, YidC, is required for the efficient insertion of almost every E. coli membrane protein tested [49] and the eukaryotic homologue, Oxa1, plays an important role in the insertion of proteins into the inner mitochondrial membrane. Moreover, mutants deficient in Alb3 have an ‘albino phenotype’ and display clear deficiencies in thylakoid biogenesis and Lhcb1 was shown to directly require Alb3 for thylakoid insertion [50]. It is known that several LHCP family members, such as Lhcb4.1 and Lhcb5, are likewise Alb3 dependent [18]. Once the transit complex is associated with Alb3 at the thylakoid membrane, LHCP is inserted in a GTP-dependent manner. Hydrolysis of GTP is likely a function of cpSRP54 dissociating from the substrate which is now integrated into the thylakoid membrane. The available evidence suggests that this cpSRP-dependent pathway is used only for LHCP proteins, with the specificity dictated by the recognition of the conserved L18 peptide by cpSRP43. Another unusual feature is the lack of involvement of the Sec system — the only important membrane-bound component appears to be Alb3 (see Fig. 3). 3.4. Spontaneous or unassisted insertion of proteins into the chloroplast thylakoid membrane As previously mentioned, a baffling addition to the list of protein targeting pathways has been identified, which apparently requires no additional factors apart from the protein to be inserted and the thylakoid membrane. While LHCP family members have consistently been shown to require cpSRP, FtsY, Alb3 and GTP hydrolysis for insertion into thylakoids, studies on other thylakoid membrane proteins concluded that not all proteins require Alb3 for insertion into the thylakoid membrane. These proteins included CFoII, PsbS, PsbX, PsbY, PbsW, PsaK and PsaG, several of which contain more membrane spans than LHCP proteins. Even more surprisingly the insertion of these proteins did not require nucleoside triphosphates or the thylakoid proton motive force — ruling out an involvement of SRP, SecA or the Tat system [51,52]. In the absence of any identifiable insertion factors, a “spontaneous insertion” pathway was proposed, in which the protein simply partitions into the membrane. However, further research is certainly needed to characterise this insertion mechanism in more detail. For example, recent studies [53] have shown that E. coli YidC may assist the insertion of proteins using diverse mechanisms, and it remains possible that the
‘Alb3-independent’ mechanism does in fact involve Alb3 after all, and that this subset of proteins simply uses an Alb3-requiring mechanism that remains to be characterised in vitro. In the context of cyanobacterial cell biology, the major question is how proteins are inserted into the thylakoid membrane: by the Sec/ SRP route or the ‘spontaneous’ pathway. We have little hard evidence on these points, for reasons outlined above, but it is relevant that chlorophyll synthase was pulled down together with YidC/Alb3 from Synechocystis PCC6803 [54]. This would suggest that it may be targeted by the Alb3/SRP pathway, which would fit with the fact that this pathway is used by chlorophyll-binding proteins in plant thylakoids, as discussed above. What we need now are data on proteins that do not bind chlorophyll (or other cofactors), and clear evidence for or against the involvement of the major pathways identified in plants or other bacteria. So far, these data are sorely lacking.
4. Protein sorting mechanisms The presence of only single sets of tat and sec genes in well studied cyanobacteria such as Synechocystis sp. PCC6803 [18] raises the question: how do proteins reach their correct site of function in the thylakoid or plasma membranes, or in the thylakoid lumen/periplasm? This is a key aspect of cyanobacterial cell biology since many proteins reside in only one membrane system (for example, Rieske iron–sulphur proteins PetC1 and PetC2 reside in the thylakoid membrane), while PetC3 resides in the plasma membrane [30]. Given that they are both inserted by the Tat pathway, how does the cell ‘know’ which membrane a given protein should be targeted towards? This directionality and specific membrane tomography is an issue of protein sorting. There are currently two prevailing hypotheses for how this occurs: The simpler is named the ‘post-translocation’ sorting model — proteins are sorted after translocation and are initially targeted in an ad hoc manner across both the thylakoid and plasma membrane, regardless of their final destination. After translocation, they undergo subsequent sorting if they are not in the correct location. An example supporting this hypothesis is the assembly of photosystem II, which partially assembles in the plasma membrane before travelling to the thylakoid membrane for completion of assembly [55]. If both thylakoid and plasma membranes are connected, this model seems credible. The second model suggests that proteins are translocated only at their final site of function and it has thus been dubbed the ‘pre-translocation’ sorting model. Pre-translocation is currently the favoured method as it is more efficient, in terms of both energy and resources, for proteins to travel directly to the correct membrane. Raialahti et al. [56] have shown that cyanobacterial signal peptides along with the first 15 amino acids of mature protein (N15) have hetergeneous physiochemical properties depending on their final destination. At no point was signal peptide length a determining factor, but both amino acid volume and polarisability were deemed important. The various physiochemical properties, which they categorised as ‘dialects’, generate a predictable property profile for proteins destined to the same membrane. However, it is still unknown whether there is a specific component responsible for ‘sensing’ these dialects or if sorting is achieved through the individual protein's physiochemical properties alone. It is possible that differentially localised signal peptidases could play the sensing role — e.g. the slr1377 peptidase is found in the plasma membrane, while the sll0716 peptidase is localised to the thylakoid membrane [56] – and these may identify the signal peptides and their properties to either accept or reject their membrane incorporation. Nevertheless, a complete understanding of protein sorting in cyanobacteria is distinctly lacking. Alternatively, identical Sec/Tat systems could acquire different substrate selectivities in the plasma and thylakoid membranes by virtue of the different environments in which they reside — for example, the different lipid compositions could endow them with different properties.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
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5. Conclusions and outlook Cyanobacteria exhibit a highly organised system of membrane differentiation and to a large extent the plasma and thylakoid membranes carry out very different activities. Numerous proteins are targeted into and across these membranes, and we have some idea of how this is achieved. However, a serious lack of biochemical and genetic tools means that this central area of cyanobacterial cell biology remains very poorly understood. In particular, we do not know whether the two membranes (and associated soluble phases) are truly connected. This is a key point because we do need to know whether proteins are randomly targeted into/across either membrane before migrating to their correct destination. In vitro translocation assays will always be a problem because cyanobacterial membranes tend to be highly fragmented after isolation, but genetic studies and time-resolved imaging studies should be able to shed light on these areas. Finally, although this review has focused primarily on protein targeting sorting mechanisms, it should be mentioned that our lack of understanding on this general area of cyanobacterial cell biology also extends to the sphere of membrane lipids. Thylakoids in plants and cyanobacteria are largely based galactolipids that account for more than 70% of total lipid content. As with proteins, the trafficking of these lipids, and hence the formation of bulk thylakoid membrane, is still poorly understood in cyanobacteria despite the identification of key lipid synthesis enzymes (reviewed in [9].) Acknowledgements D.G. and J.A.Z.Z. were supported by funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/under REA grant agreement no. 317184 (‘PHOTO.COMM’). This material reflects only the authors' views and the Union is not liable for any use that may be made of the information contained therein. References [1] R. Buick, When did oxygenic photosynthesis evolve? Philos. Trans. R. Soc. B Biol. Sci. 363 (2008) 2731–2743. [2] M. Hügler, S.M. Sievert, Beyond the calvin cycle: autotrophic carbon fixation in the ocean, Annu. Rev. Mar. Sci. 3 (2011) 261–289. [3] K. Sciuto, I. Moro, Cyanobacteria: the bright and dark sides of a charming group, Biodivers. Conserv. 24 (2015) 711–738. [4] H.W. Paerl, N.S. Hall, E.S. Calandrino, Controlling harmful cyanobacterial blooms in a world experiencing anthropogentic and climatic-induced change, Sci. Total Environ. 409 (2011) 1739–1745. [5] R.H. Wijffels, O. Kruse, K.J. Hellingwerf, Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae, Curr. Opin. Biotechnol. 24 (2013) 405–413. [6] S.A. Nierzwicki-Bauer, D.L. Balkwill, S.E. Stevens Jr., Three-dimensional ultrastructure of a unicellular cyanobacterium, J. Cell Biol. 97 (1983) 713–722. [7] U.C. Vothknecht, P. Westhoff, Biogenesis and origin of thylakoid membranes, Biochim. Biophys. Acta 1541 (2001) 91–101. [8] J. Nickelsen, B. Rengstl, A. Stengel, M. Schottkowski, J. Soll, E. Ankele, Biogenesis of the cyanobacterial thylakoid membrane system–an update, FEMS Microbiol. Lett. 315 (2011) 1–5. [9] A. Rast, S. Heinz, J. Nickelsen, Biogenesis of thylakoid membranes, Biochim. Biophys. Acta 1847 (2015) 821–830. [10] A.M. van de Meene, M.F. Hohmann-Marriott, W.F. Vermaas, R.W. Roberson, The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803, Arch. Microbiol. 184 (2006) 259–270. [11] D.D. Kunkel, Thylakoid centers: structures associated with the cyanobacterial photosynthetic membrane system, Arch. Microbiol. 133 (1982) 97–99. [12] M. Schottkowski, S. Gkalympoudis, N. Tzekova, C. Stelljes, D. Schunemann, E. Ankele, J. Nickelsen, Interaction of the periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp. PCC 6803, J. Biol. Chem. 284 (2009) 1813–1819. [13] A. Stengel, I.L. Gugel, D. Hilger, B. Rengstl, H. Jung, J. Nickelsen, Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis, Plant Cell 24 (2012) 660–675. [14] M. Rütgers, M. Schroda, A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystem biogenesis? Plant Signal. Behav. 8 (2013)http://dx.doi. org/10.4161/psb.27037. [15] U.C. Vothknecht, S. Otters, R. Raoul Hennig, D. Schneider, Vipp1: a very important protein in plastids?! J. Exp. Bot. 63 (2012) 1699–1712.
7
[16] S. Zhang, G. Shen, Z. Li, J.H. Golbeck, D.A. Bryant, Vipp1 is essential for the biogenesis of photosystem I but not thylakoid membranes in Synechococcus sp. PCC 7002, J, Biol. Chem. 289 (2014) 15904–15914. [17] S. Rexroth, C.W. Mullineaux, D. Ellinger, E. Sendtko, M. Rogner, F. Koenig, The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains, Plant Cell 23 (2011) 2379–2390. [18] C. Aldridge, P. Cain, C. Robinson, Protein transport into and across the thylakoid membrane. FEBS J. 276 (2009) 1177–1186. [19] T. Palmer, B.C. Berks, The twin-arginine translocation (Tat) protein export pathway, Nat. Rev. Microbiol. 10 (2012) 483–496. [20] S.W. Hennon, R. Soman, R.E. Dalbey, YidC/Alb3/Oxa1 family of insertases, J. Biol. Chem. 290 (2015) 14866–14874. [21] S. Fulda, F. Huang, F. Nilsson, M. Hagemann, B. Norling, Proteomics of Synechocystis sp. strain PCC 6803, identification of periplasmic proteins in cells grown at low and high salt concentrations, Eur. J. Biochem. 267 (2000) 5900–5907. [22] K.E. Chatzi, M.F. Sardis, A. Economou, S. Karamanou, SecA-mediated targeting and translocation of secretory proteins, Biochim. Biophys. Acta 1466-1474 (2014). [23] P.A. Scotti, M.L. Urbanus, J. Brunner, J.W. de Gier, G. von Heijne, C. van der Does, A.J.M. Driessen, B. Oudega, J. Luirink, YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase, EMBO J. 19 (2000) 542–549. [24] L. Fröderberg, T. Röhl, K.-J. van Wijk, J.-W.L. de Gier, Complementation of bacterial SecE by a chloroplast homologue, FEBS Lett. 498 (2001) 52–56. [25] C. Sun, S.L. Rusch, J. Kim, D.A. Kendall, Chloroplast SecA and Escherichia coli SecA have distinct lipid and signal peptide preferences, J. Bacteriol. 189 (2007) 1171–1175. [26] M. Nakai, D. Sugita, T. Omata, T. Endo, Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942, Biochem. Biophys. Res. Commun. 193 (1993) 228–234. [27] R. Srivastava, T. Pisareva, B. Norling, Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803, Proteomics 5 (2005) 4905–4916. [28] P.J. Hynds, D. Robinson, C. Robinson, The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane, J. Biol. Chem. 273 (1998) 34868–34874. [29] K. Cline, W.F. Ettinger, S.M. Theg, Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP, J. Biol. Chem. 267 (1992) 2688–2696. [30] C. Aldridge, E. Spence, M.A. Kirkilionis, L. Frigerio, C. Robinson, Tat-dependent targeting of rieske iron–sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803, Mol. Microbiol. 70 (2008) 140–150. [31] C.F.R.O. Matos, C. Robinson, A. Di Cola, The Tat system proofreads Fes protein substrates and directly initiates the disposal of rejected molecules, EMBO J. 27 (2008) 2055–2063. [32] M.P. DeLisa, D. Tullman, G. Georgiou, Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 6115–6120. [33] C. Robinson, C.F.R.O. Matos, D. Beck, C. Ren, J. Lawrence, N. Vasisht, S. Mendel, Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria, Biochim. Biophys. Acta 876-884 (2011). [34] E. Bogsch, S. Brink, C. Robinson, Pathway specificity for a ΔpH-dependent precursor thylakoid lumen protein is governed by a "Sec-avoidance" motif in the transfer peptide and a "Sec-incompatible" mature protein, EMBO J. 16 (1997) 3851–3859. [35] A.M. Settles, A. Yonetani, A. Baron, D.R. Bush, K. Cline, R. Martienssen, Secindependent protein translocation by the maize Hcf106 protein, Science 278 (1997) 1467–1470. [36] M.J. Tarry, E. Schäfer, S. Chen, G. Buchanan, N.P. Greene, S.M. Lea, T. Palmer, H.R. Saibil, B.C. Berks, Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 13284–13289. [37] K. Cline, S.M. Theg, The Sec and Tat protein translocation pathways in chloroplasts, Enzymes 25 (2006) 463–492. [38] U. Gohlke, L. Pullan, C.A. McDevitt, I. Porcelli, E. de Leeuw, T. Palmer, H.R. Saibil, B.C. Berks, The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 10482–10486. [39] J. Baglieri, D. Beck, N. Vasisht, C.J. Smith, C. Robinson, Structure of the TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter, J. Biol. Chem. 287 (2012) 7335–7344. [40] E. Spence, M. Sarcina, N. Ray, S.G. Møller, C.W. Mullineaux, C. Robinson, Membranespecific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803, Mol. Microbiol. 48 (2003) 1481–1489. [41] P.A. Scotti, M.L. Urbanus, J. Brunner, J.W. de Gier, G. von Heijne, C. van der Does, A.J.M. Driessen, B. Oudega, J. Luirink, YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase, EMBO J. 19 (2000) 542–549. [42] X. Li, R. Henry, J. Yuan, K. Cline, N.E. Hoffman, A chloroplast homologue of signal recognition particle unit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 3789–3793. [43] P.V. Viitanen, E.R. Doran, P. Dunsmuir, What is the role of the transit peptide in thylakoid integration of the light harvesting chlorophyll a/b protein? J. Biol. Chem. 263 (1988) 15000–15007. [44] R. Goforth, E. Peterson, J. Yuan, M. Moore, A. Kight, M. Lohse, J. Sakon, R. Henry, Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43, J. Biol. Chem. 279 (2004) 43077–43084.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010
8
K.M. Frain et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx
[45] C.J. Tu, E.C. Peterson, R. Henry, N.E. Hoffman, The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43, J. Biol. Chem. 275 (2000) 13187–13190. [46] P. Cain, I. Holdermann, I. Sinning, A.E. Johnson, C. Robinson, Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain, Biochem. J. 437 (2011) 149–155. [47] N. Kogata, K. Nishio, T. Hirohashi, S. Kikuchi, M. Nakai, Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids, FEBS Lett. 447 (1999) 329–333. [48] S. Falk, S. Ravaud, J. Koch, I. Sinning, The C terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane, J. Biol. Chem. 285 (2010) 5954–5962. [49] J.C. Samuelson, M. Chen, F. Jiang, I. Möller, M. Wiedmann, A. Kuhn, G.J. Phillips, R.E. Dalbey, YidC mediates membrane protein insertion in bacteria, Nature 406 (2000) 637–641. [50] M. Moore, M.S. Harrison, E.C peterson, R. Henry, chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyllbinding protein into thylakoid membranes, J. Biol. Chem. 275 (2000) 1529–1532. [51] D. Michl, C. Robinson, J.B. Shackleton, R.G. Herrmann, R.B. Klösgen, Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway, EMBO J. 13 (1994) 1310–1317.
[52] C.A. Woolhead, S. J. Thompson, M. Moore, C. Tissier, A. Mant, A. Rodger, R. Henry, C. Robinson, Distinct Albino3-dependent and -independent pathways for thylakoid membrane protein insertion, J. Biol. Chem. 276 (2001) 40841-40846. [53] K. Kumazaki, S. Chiba, M. Takemoto, A. Furukawa, K. Nishiyama, Y. Sugano, T. Mori, N. Dohmae, K. Hirata, Y. Nakada-Nakura, A.D. Maturana, Y. Tanaka, H. Mori, Y. Sugita, F. Arisaka, K. Ito, R. Ishitani, T. Tsukazaki, O. Nureki, Structural basis of Secindependent membrane protein insertion by YidC, Nature 509 (2014) 516–520. [54] J.W. Chidgey, M. Linhartová, J. Komenda, P.J. Jackson, M.J. Dickman, D.P. Canniffe, P. Koník, J. Pilny, C.N. Hunter, R. Sobotka, A cyanobacterial chlorophyll synthase-hlid complex associates with the Ycf39 protein and the YidC/Alb3 insertase, Plant Cell 26 (2014) 1267–1279. [55] E. Zak, B. Norling, R. Maitra, F. Huang, B. Andersson, H.B. Pakrasi, The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13443–13448. [56] T. Rajalahti, F. Huang, M.R. Klement, T. Pisareva, M. Edman, M. Sjöström, A. Wieslander, B. Norling, Proteins in different synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis, J. Proteome Res. 6 (2007) 2420–2434.
Please cite this article as: K.M. Frain, et al., Protein translocation and thylakoid biogenesis in cyanobacteria, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbabio.2015.08.010