Biogenesis of the Cyanobacterial Thylakoid Membrane System An

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MINIREVIEW Biogenesis ofthe cyanobacterial thylakoid membrane system ^ an update Jorg¨ Nickelsen1, Birgit Rengstl1, Anna Stengel1, Marco Schottkowski1,Jurgen¨ Soll2 & Elisabeth Ankele2

1Molekulare Pflanzenwissenschaften, Biozentrum LMU M ünchen, Planegg-Martinsried, Germany; and 2Biochemie und Physiologie der Pflanzen, Biozentrum LMU M ünchen,Planegg-Martinsried, Germany Downloaded from https://academic.oup.com/femsle/article/315/1/1/540911 by guest on 02 October 2021

Correspondence: Jorg¨ Nickelsen, Abstract Molekulare Pﬂanzenwissenschaften, Biozentrum LMU Munchen, ¨ Großhaderner Current molecular analyses suggest that initial steps of the biogenesis of cyano- Str. 2-4, 82152 Planegg-Martinsried, bacterial photosystems progress in a membrane subfraction representing a Germany. Tel.: 10049 89 2180 74773; fax: biosynthetic center with contact to both plasma and thylakoid membranes. This 10049 89 2180 997 4773; e-mail: special membrane fraction is deﬁned by the presence of the photosystem II [email protected] assembly factor PratA. The proposed model suggests that both biogenesis of protein complexes and insertion of chlorophyll molecules into the photosystems Received 7 July 2010; revised 30 July 2010; occur in this intermediate membrane system. accepted 2 August 2010. Final version published online 10 September 2010.

DOI:10.1111/j.1574-6968.2010.02096.x

Editor: Hermann Heipieper

Keywords Synechocystis; membrane biogenesis; photosystem II; PratA; Pitt.

scenarios can be envisioned. (1) Protein, lipid and pigment Introduction synthesis occurs directly on pre-existing TMs. (2) The Cyanobacteria represent the phylogenetic ancestors of chlo- components are synthesized and assembled in specialized roplasts from present-day plants and, similar to those, they thylakoid regions. (3) Initial production of polypeptides and contain three major differentiated membrane systems. These assembly of protein/pigment complexes occur at the PM, include the outer membrane and the inner or plasma and these precomplexes are transferred to the thylakoids via membrane (PM), which, together with the intervening an unknown way (Fig. 1). periplasm and the peptidoglycan layer, form the cellular Scenario 1 appears rather unlikely, because ultrastructural envelope. Interior to the PM is the thylakoid membrane cryo-electron microscopy data clearly show that TM layers are (TM) system representing the site of the photosynthetic essentially devoid of ribosomes (van de Meene et al., 2006). light reactions coupled to ATP and NADPH generation. All This suggests that protein synthesis, and thus biogenesis, does three membrane systems differ from one another with not occur in direct association with the photosynthetically regard to their pigment, lipid and protein composition active thylakoids. However, ribosome clusters are observed (Norling et al., 1998; Wada & Murata, 1998). This observa- close to the PM and near TM structures that extend into the tion provokes the following questions: Where is TM synth- central cytoplasm, favoring models 2 and/or 3 (van de Meene esis initiated in cyanobacteria? How is speciﬁcity between et al., 2006). Furthermore, TMs appear to converge on the the different membranes achieved and maintained? And PM at speciﬁc sites (Fig. 2). These convergence sites have been how are these processes organized at the molecular level? speculated to eventually mark so-called thylakoid centers,

MICROBIOLOGY LETTERS MICROBIOLOGY Two excellent reviews have recently summarized the possible where TM biogenesis is initiated (van de Meene et al., 2006). models and key questions of TM biogenesis, which are It is still under debate whether at these regions permanent or controversially discussed (Liberton & Pakrasi, 2008; Mulli- transient fusions between PM and TM occur. If so, these neaux, 2008 and references therein). In brief, three different would allow the transfer of lipids and proteins to the

FEMS Microbiol Lett 315 (2011) 1–5 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2 J. Nickelsen et al. developing TM resembling the situation found in purple convergence sites marking a membrane subfraction with bacteria such as Rhodospirillum rubrum (Collins & Remsen, contact to both the PM and the TM. These sites possibly 1991; Liberton et al., 2006; van de Meene et al., 2006). represent the regions at which protein/pigment complexes are Here, we aim at incorporating some very recent ﬁndings of assembled and incorporated into photosynthetic membranes. membrane fractionation studies of the model organism Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) Protein synthesis/assembly into the various abovementioned scenarios. We propose a novel working model combining scenarios 2 and 3 with TM Three major membrane complexes constitute the basic apparatus of TMs mediating photosynthetic electron ﬂow,

i.e. photosystem II (PSII), the cytochrome b6f complex and photosystem I (PSI). PSII functions as a water-plastoqui-

none oxidoreductase which, in cyanobacteria, consists of 20 Downloaded from https://academic.oup.com/femsle/article/315/1/1/540911 by guest on 02 October 2021 protein subunits, 35 chlorophyll a (chl a) molecules and several additional cofactors including the manganese cluster catalyzing photosynthetic water splitting (Nelson & Ben- Shem, 2004). PSI comprises only 12 subunits, approximately 80 chlorophylls as well as Fe–S clusters and phylloquinones (Nelson & Ben-Shem, 2004). While the structures of these molecular machines have recently been well established (Stroebel et al., 2003; Ferreira et al., 2004; Loll et al., 2005; Amunts et al., 2007), to date, only limited information is available on the molecular details of their biogenesis (Nixon et al., 2010). Earlier work based on membrane fractionation studies initially suggested that precomplexes of both photosystems are assembled within the PM and not the TM in the cyanobacterium Synechocystis 6803 (Zak et al., 2001). Using a combination of sucrose density centrifugation and aqu- eous two-phase partitioning, protein components of the

core reaction center of PSII (D1, D2, Cyt b559) as well as of PSI (PsaA and PsaB), were identiﬁed in the PM, whereas more extrinsic proteins such as the inner antenna protein CP47 of PSII were found in TM preparations only. In addition, PSII biogenesis factors, such as the D1 C-terminal protease CtpA or the PSI assembly factors Ycf3 and Ycf4, Fig. 1. Models for TM biogenesis. Synthesis and assembly of photo- were mainly or exclusively detected in the PM (Zak et al., system components occur on (a) pre-existing thylakoids or (b) in specialized TMs (STM). Alternatively, the PM represents the site of early 2001). Together with the ﬁnding that the PM-localized core photosystem biogenesis steps (c), subsequently precomplexes are trans- complexes contain chlorophyll molecules and can perform ported to the TM via (1) transient connecting regions or (2) vesicle single light-induced charge separations, these data strongly transport. suggest that the photosystem core complexes found in the

Fig. 2. Ultrastructure of Synechocystis 6803. Electron micrographs of a Synechocystis 6803 cell (A, scale bar = 200 nm). Sites of TM convergence are shown at higher magniﬁcation (a, scale bar = 100 nm).

PM, or a specialized section of it, exist in a preassembled acke & Zerges, 2007). This might indicate an evolutionary state (Keren et al., 2005; Srivastava et al., 2006). conservation of the molecular principles that underlie TM Further support for the idea that at least PSII biogenesis biogenesis. begins at non-TM sites was obtained during analysis of the PratA protein from Synechocystis 6803 (Klinkert et al., 2004). PratA consists of nine consecutive tetratricopeptide repeat Chlorophyll synthesis (TPR) units, a motif that is known to mediate protein–pro- Photosynthesis requires the absorption of light, which is tein interactions. Thereby, it could form a bridge connecting mediated by photoactive pigments, for example chloro- multiple proteins and serve as a scaffold factor for correct phylls. In chloroplasts of Arabidopsis thaliana, the synthesis assembly of PSII (Schottkowski et al., 2009a). PratA directly of chlorophyll was described to occur in several plastidic interacts with the C-terminus of the D1 reaction center subcompartments (Eckhardt et al., 2004). While early steps protein of PSII, and its inactivation affects the C-terminal Downloaded from https://academic.oup.com/femsle/article/315/1/1/540911 by guest on 02 October 2021 in synthesis, i.e. the conversion of glutamate to 5-aminole- processing of D1, an early step of PSII biogenesis. This D1 vulinic acid, occur in the chloroplast stroma, the enzymes maturation occurs in almost all photosynthetic organisms, required for later steps are associated with the inner and it is required for the subsequent docking of the subunits envelope membrane or the TM (Fig. 3). These membrane- of the oxygen-evolving complex to the lumenal side of PSII. attached enzymes include the NADPH-protochlorophyllide Most intriguingly, PratA was shown to be a soluble protein oxidoreductase (POR) and the chlorophyll synthase (CS), located in the periplasm, which forms part of a 200 kDa which catalyze the reduction of protochlorophyllide a complex of an as yet unknown composition and function (pchlide a) to chlorophyllide a (chlide a) and the subsequent (Fulda et al., 2000; Klinkert et al., 2004; Schottkowski et al., generation of chl a, respectively. Similar to the situation in 2009a). However, a minor fraction (10–20%) of PratA was higher plants, previous studies revealed that cyanobacterial found to associate with membranes in a D1-dependent chlorophyll biosynthesis also underlies a spatial organiza- manner. Cellular fractionation experiments using two con- tion (Peschek et al., 1989; Eckhardt et al., 2004). In secutive sucrose gradients revealed that the membrane- Synechococcus elongatus 7942 (formerly called Anacystis bound PratA is apparently not associated with either the nidulans), pchlide a and chlide a accumulate in PM pre- PM or TMs, but co-sediments with an intermediate mem- parations and cannot be detected in the TM (Peschek et al., brane subfraction, which was therefore named PratA- 1989). Moreover, in Synechocystis 6803, highest chlorophyll defined membrane (PDM) subfraction (Schottkowski et al., precursor concentrations were found in a membrane frac- 2009a). Albeit the different density of PDMs as compared tion suggested to represent the abovementioned thylakoid with that of PMs, it cannot be ruled out that PDMs might be center fraction resembling PDMs (Hinterstoisser et al., identical to previously described specialized PM subregions, 1993). As mentioned, photosynthetic precomplexes already in which PSII subunits tend to accumulate (Srivastava et al., contain chlorophyll molecules, suggesting that not only the 2006). Membrane fractions resembling PDMs with regard to later steps in chlorophyll synthesis but also the insertion of their density have already been observed in earlier studies, pigments occur at the protein assembly sites associated with where they have been postulated to be linked to so-called the PM or PDMs (Keren et al., 2005). thylakoid centers (Hinterstoisser et al., 1993). Based on electron microscopic analyses, thylakoid centers were initially described in some cyanobacteria as tubular structures found at the inner face of the PM, at points where thylakoids extend projections into the cytoplasm (Kunkel, 1982). Recently, this idea was revisited based on a more detailed cryo-electron tomography analysis in Synechocystis 6803 (van de Meene et al., 2006). Interestingly, PratA inactivation and, thus, defective PSII assembly leads to a significant accumulation of the pD1 precursor protein in PDM fractions (Schottkowski et al., 2009a). This suggests that PratA function is required for efficient membrane flow from PDMs to TMs, underlining the role of PDMs for PSII reaction-center assembly. Fig. 3. Scheme of chlorophyll synthesis in chloroplasts of higher plants [according to Eckhardt et al. (2004)]. Early steps in chlorophyll biosynth- Interestingly, related ‘biogenesis regions/centers’ have esis occur in the stroma, whereas later steps are performed by enzymes recently been observed in the eukaryotic green alga Chlamy- associated with the thylakoid or inner envelope membranes (EM). For domonas reinhardtii, where they are formed by membranes further details, see text. Glu, glutamate; ALA, 5-aminolevulinic acid; surrounding the pyrenoid structure of the chloroplast (Uni- Protogen, protoporphyrinogen IX; Proto, protoporphyrin IX.

Further experimental evidence for an important role of PDMs in chlorophyll synthesis and insertion was recently provided by the analysis of another TPR protein from Synechocystis 6803, named Pitt (POR-interacting TPR protein). This TM protein was found to interact directly with and stabilize the light-dependent POR enzyme (Schottkows- ki et al., 2009b). Intriguingly, in a pratA mutant, a large proportion of both Pitt and POR was localized in PDM fractions. This is in contrast to wild-type cells, where only minor amounts are found in PDMs and the majority is TM associated (Schottkowski et al., 2009b). Hence, these two Fig. 4. Model of PSII biogenesis in Synechocystis 6803. PSII – and proteins are affected by the absence of PratA in the same way probably also PSI – biogenesis is hypothesized to initiate in a membrane Downloaded from https://academic.oup.com/femsle/article/315/1/1/540911 by guest on 02 October 2021 as the pD1 precursor protein. Apparently, a defective PSII subfraction (PDM subfraction) at thylakoid convergence sites close to the assembly and perturbation of membrane flow from PDMs PM (compare Fig. 2). Here, the assembly of PSII precomplexes containing to TMs causes the retardation of additional PSII biogenesis the reaction center proteins D1 and D2 as well as Cyt b599 is coordinated factors, including Pitt and POR, at the site of early PSII as indicated by the presence of the assembly factor PratA in its D1-bound assembly, i.e. the PDMs. form. In addition, PDMs harbor the chlorophyll synthesis enzyme POR and its interaction partner Pitt, supporting the idea that chlorophyll However, the question arises as to why in wild-type cells synthesis and insertion into precomplexes occur at the same site. chlide a is mainly localized in the PM and/or in the Subsequently, precomplexes move to thylakoids in an as yet unknown thylakoid centers (Peschek et al., 1989; Hinterstoisser et al., way, where their assembly is completed. The question mark refers to as 1993), whereas the chlide a-synthesizing enzyme POR is yet unidentified additional components of the assembly machinery. OM, mainly – but not exclusively – detected in the TM (Schott- outer membrane; Chlide, chlorophyllide a; Chl, chlorophyll a;pD1, kowski et al., 2009b). One explanation for this discrepancy precursor of D1. could be provided by the localization of CS, which catalyzes the final esterification of chlide a to chl a. In chloroplasts, fact that non-D1-bound PratA is a soluble periplasmic this enzyme has been exclusively localized to TMs (Soll et al., protein strongly argues for at least temporary contacts of 1983; Eckhardt et al., 2004; Fig. 3). If this TM localization of PDMs with the PM. These areas of contact are likely to be CS also holds for cyanobacteria, TM-synthesized chlide a identical to the previously described thylakoid centers, could be rapidly converted to chl a, whereas chlide a which are located at the cell periphery, between PM and synthesized by the minor POR fraction in PDMs would TMs (Hinterstoisser et al., 1993; van de Meene et al., 2006). accumulate due to scarce further processing. However, Hence, the existence of such structures close to both the PM previous CS activity measurements in Synechocystis 6803 and the TM could easily explain the involvement of the suggested the presence of CS in both the – putatively PDM- periplasmic PratA factor in TM biogenesis. Furthermore, related – thylakoid centers and TMs (Hinterstoisser et al., the finding that pD1, Pitt and POR are all localized to a 1993). Hence, higher chlide a synthesis rates in PDMs must higher amount in PDMs upon inactivation of PratA also be considered. These might be due to the activity of the strongly suggests an essential role of PratA in the functional second, light-independent, POR enzyme (LiPOR) from and/or structural organization of these biogenesis centers Synechocystis 6803, whose localization is still elusive (Arm- and, thus, membrane flow from PDMs to TMs. strong, 1998). Taken together and despite several open Although the described model seems to apply to PSII questions, the facts presented draw a picture of PDMs as a biogenesis, less evidence is available concerning the spatial subcompartment, in which not only protein complex bio- organization of the PSI assembly process. Nevertheless, the genesis but also the later steps in chlorophyll synthesis and detection of the PSI reaction center proteins PsaA and PsaB its insertion into polypeptides occur. in PM or PM-related fractions suggests that also PSI biogenesis is initiated in the PM or even in PDMs similar to PSII (Zak et al., 2001). Conclusion Future work will be directed toward the visualization of In conclusion, we propose the following working model for the biogenesis process, for instance by time-resolved studies the biogenesis of TMs in the model organism Synechocystis with green fluorescent protein-tagged proteins. The ultra- 6803 (Fig. 4): both protein synthesis/assembly and chlor- structural localization of the various factors involved, espe- ophyll synthesis/insertion are subject to tight spatial organi- cially the PratA protein, will unambiguously answer the zation. These two processes are localized in a specialized question whether, indeed, PDMs and thylakoid centers are membrane region, here termed PDMs, which is marked by directly linked. The identification of additional PDM-mar- the D1-bound form of the PSII biogenesis factor PratA. The ker proteins will enable one to elucidate the molecular

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