Protein Translocons in Photosynthetic Organelles of Paulinella Chromatophora

Acta Societatis Botanicorum Poloniae

INVITED ORIGINAL RESEARCH PAPER Acta Soc Bot Pol 83(4):399–407 DOI: 10.5586/asbp.2014.053 Received: 2014-11-30 Accepted: 2014-12-18 Published electronically: 2014-12-31

Protein translocons in photosynthetic organelles of Paulinella chromatophora

Przemysław Gagat, Paweł Mackiewicz* Department of Genomics, Faculty of Biotechnology, University of Wrocław, Fryderyka Joliot-Curie 14a, 50-383 Wrocław, Poland

Abstract

The rhizarian amoeba Paulinella chromatophora harbors two photosynthetic cyanobacterial endosymbionts (chromatophores), acquired independently of primary plastids of glaucophytes, red algae and green plants. These endosymbionts have lost many essential genes, and transferred substantial number of genes to the host nuclear genome via endosymbiotic gene transfer (EGT), including those involved in photosynthesis. This indicates that, similar to primary plastids, Paulinella endosymbionts must have evolved a transport system to import their EGT-derived proteins. This system involves vesicular trafficking to the outer chromatophore membrane and presumably a simplified Tic-like complex at the inner chromatophore membrane. Since both sequenced Paulinella strains have been shown to undergo differential plastid gene losses, they do not have to possess the same set of Toc and Tic homologs. We searched the genome of Paulinella FK01 strain for potential Toc and Tic homologs, and compared the results with the data obtained for Paulinella CCAC 0185 strain, and 72 cyanobacteria, eight Archaeplastida as well as some other bacteria. Our studies revealed that chromatophore genomes from both Paulinella strains encode the same set of translocons that could potentially create a simplified but fully-functional Tic-like complex at the inner chromatophore membranes. The common maintenance of the same set of translocon proteins in two Paulinella strains suggests a similar import mechanism and/or supports the proposed model of protein import. Moreover, we have discovered a new putative Tic component, Tic62, a redox sensor protein not identified in previous comparative studies of Paulinella translocons.

Keywords: Paulinella chromatophora; chromatophores; endosymbiosis; primary plastids; protein import; translocons

Introduction rare evolutionary phenomenon [6]. For the second time, it took place in the case of Paulinella chromatophora, a testate The primary plastid endosymbiosis is one of the most filose amoeba, belonging to the supergroup Rhizaria, a important transitions in the evolution of life on our planet. lineage that is very distantly related to the primary plastid It took place sometime before 1.5 billion years ago, when a bearing lineages, i.e. glaucophytes, red algae and green plants, phagotrophic eukaryote, the ancestor of glaucophytes, red united in the supergroup Archaeplastida [7,8]. algae and green plants, enslaved a β-cyanobacterium, which Paulinella chromatophora harbors two photosynthetic was transformed into a two-membrane photosynthetic pri- cyanobacterial endosymbionts (chromatophores), acquired mary plastid [1,2]. In order to become a true cell organelle, independently of Archaeplastida primary plastids ~60 mil- the prokaryote underwent a tremendous transformation that lion years ago [8–11]. Similarly, to primary plastids, chro- involved two key processes: (i) endosymbiotic gene transfer matophores are surrounded by a two-membrane envelope (EGT), i.e. gene transfer from the endosymbiont to the host and are deeply integrated with their host cell. They divide nuclear genome, and (ii) origin of an import machinery in synchronously with Paulinella cell (after being distributed its envelope membranes for host genome-encoded proteins, to daughter cells), exchange metabolites with it and are including the products of EGT-derived genes [3–5]. incapable of independent life [9,12,13]. They are especially Interestingly, primary plastid endosymbiosis was not a similar in their structure to glaucophyte primary plastids singular event in the history of our biosphere, but has hap- (cyanelles) because they still retain the peptidoglycan wall pened at least twice, questioning thereby a paradigm that en- in the intermembrane space, a clear sign of their bacterial dosymbiont-to-organelle transformation is an exceptionally inheritance [12]. The intimacy between thePaulienlla host and its endosymbiotic bodies is well emphasized by substantial reduction of the latter genome, which was sequenced * Corresponding author. Email: [email protected] for two Paulinella strains CCAC 0185 [11] and FK01 [14]. Handling Editor: Andrzej Bodył They were reduced in size and coding capacity by a factor

This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 License (creativecommons.org/licenses/by/3.0/), which permits 399 redistribution, commercial and non-commercial, provided that the article is properly cited. © The Author(s) 2014 Published by Polish Botanical Society Gagat and Mackiewicz / Translocons in Paulinella chromatophores of three (to 1 Mb and ~900 genes), compared with their to show that PsaE, PsaK1 and PsaK2 from Paulinella CCAC closest free-living relative, the cyanobacterium Cyanobium 0185 strain are localized both in chromatophores and the gracile PCC 6307 (~3 Mb and ~3300 genes) [11,14] (Fig. 1). endomembrane system, including the Golgi apparatus. They The genome-size reduction involved loss of many genes, also indicated that these proteins are indeed PS I subunits as including those engaged in essential biosynthetic pathways they associate with PS I components. Interestingly, Nowack and endosymbiotic gene transfer [11,14]. Transcriptome et al. [21] revealed that the three proteins do not have typical analyses have identified more than 30 EGT-derived genes signal peptides at their N-termini. In the case of PsaE some in the Paulinella nuclear genome [14–16]; however, many internal targeting information was suggested by Mackiewicz more are expected; Nowack et al. [16] estimated their number et al. [20], whereas, for PsaK proteins algorithms recognized between 40 and 125. Because the lost genes are of the utmost uncleavable signal peptides in their N-terminal hydrophobic importance for cell functioning, and at least some of the domains, suggesting that their N-termini might fulfill a signal transferred ones are transcriptionally regulated by the host peptide role in the protein import after all [19]. and involved in photosynthesis or photosynthesis-related Both Paulinella strains have undergone differential plastid processes, Paulinella chromatophores are expected to have gene losses [14,22] and have even been suggested to represent evolved a protein import route, or routes. two different species on the basis of some morphological and To check if chromatophore protein import system is phylogenetic analyses [8]. Therefore it is interesting to check similar to the primary plastid translocons at the outer and if they possess the same set of potential Toc and Tic homo- inner envelope membrane, Toc and Tic, respectively, Bodył logs. The common maintenance of the same set of proteins et al. [17] searched for homologs of Toc and Tic genes in the would indicate a similar import mechanism and/or support completely sequenced chromatophore genome of Paulinella the model proposed by Mackiewicz et al. [20]. Therefore, CCAC 0185 strain. They found that thePaulinella chro- we searched Paulinella FK01 strain genome for Toc and Tic matophore genome encodes homologs to Toc12, Toc64, homologs, and compared the results with the data obtained Tic21 and Tic32, but lost those of Toc75, Tic20, Tic55 and for Paulinella CCAC 0185 strain, 72 cyanobacteria, eight Tic62. They suggested that the missing genes, which are still Archaeplastida, as well as some other bacteria. We carried present in the genomes of closely related cyanobacteria, were out more sensitive homology and conserved domain searches most probably relocated via the EGT to the host nuclear than in previous approaches, which is justified by a short genome, and that their products are now imported into the length and relatively high divergence of these proteins [17]. photosynthetic bodies’ membranes to create a Toc-Tic-like import machinery [17]. Mackiewicz and Bodył [18] also found that an EGT-derived gene encoding the subunit IV of Material and methods PS I reaction center (PsaE) form Paulinella FK01 strain has a clear signal peptide predicted from an alternative initiation We downloaded 867 and 841 sequences of chromato- site [18]. Since signal peptides are involved in protein import phore-encoded proteins for Paulinella CCAC 0185 strain into the endoplasmic reticulum, such a presequence in the from Genbank [23] and for Paulinella FK01 strain from case of PsaE indicates the possibility of endomembrane- Debashish Bhattacharya’s Laboratory website (http://dblab. dependent targeting to the outer chromatophore membrane. rutgers.edu/home/index.php), respectively. We also acquired Subsequent studies confirmed the presence of a signal proteomes of 72 completely sequenced cyanobacteria, eight peptide-like presequence in another four of nine investigated Archaeplastida and two reference bacteria for comparative EGT-derived proteins that evolved by modification of their studies from Genbank [23]. On the basis of the obtained N-terminal mature parts, but not the missing Toc and Tic sequences a local protein database was created. Sequences components in the nuclear genome [16,19]. of 16S and 23S rRNA for phylogenetic studies were extracted On the basis of their bioinformatics analyses, Mackiewicz from gbk files downloaded from GenBank (genome data- et al. [20] formulated a model for protein import into Pau- base) for appropriate organisms. This set included sequences, linella chromatophores. According to the model, Paulinella which were derived from two Paulinella chromatophora EGT-derived proteins established endomembrane-dependent genomes, 72 completely sequenced cyanobacterial genomes, mechanism, vesicular trafficking, to pass the outer chromato- eight primary plastid genomes and five genomes representing phore membrane, while the inner membrane is crossed by different bacterial lineages as an outgroup. a simplified Tic-like complex. All investigated EGT-derived To find potential Toc-Tic components, we searched the proteins, presumed to follow this pathway, characterize low database using Pisum sativum translocons as query sequences molecular weight and nearly neutral charge, which constitute with PsiBlast (E-value set to 0.01, word size to two, filtering good adaptations to their passage through the peptidogly- for low complexity regions on, five iterations) to achieve can wall still present in the chromatophores. Mackiewicz better sensitivity than with standard BLAST or FASTA [24]. et al. [20] also identified additional putative elements of In the case of Tic21 a homolog from Arabidopsis thaliana the system, for example chaperons in the intermembrane was used as Tic21 is absent from P. s ativ um [25]. The ob- space and components of the molecular motor responsible tained candidates were verified in terms of domain content for pulling the imported proteins into the chromatophore by searching CDD database with E-value < 0.01 (Tab. 1) stroma. It should be noted that part of the model, concern- [26]. Alignments of Toc12, Tic21, Tic32 and Tic62 from ing the vesicular trafficking to the outer chromatophore P. s ativ um or A. thaliana and two strains of P. chromatophora, membrane, has recently been confirmed by Nowack et al. CCAC0185 and FK01, were performed in PSI-Coffee [27], [21]. They used immunogold labeling electron microscopy slow and accurate algorithm dedicated to distantly related

Fig. 1 MrBayes tree based on 16S and 23S rRNA alignment. An independent origin of Paulinella endosymbionts from primary plastids of red algae, glaucophytes and green plants is clearly visible. The former are descendants of α-cyanobacteria whereas the latter of β-cyanobacteria. Numbers at nodes, in the presented order, correspond to posterior probabilities estimated in MrBayes (MB), support values calculated by local rearrangements–expected likelihood weights method (LR), as well as bootstrap percentages calculated in TreeFinder (BP) and PAUP using minimum evolution method (ME). Values of the posterior probabilities and bootstrap percentages lower than 0.50 and 50%, respectively, were omitted or indicated by a dash “–”. The length of branches leading to Bacteria taxa was shortened by 50% in comparison to their original length because of very high substitution rate.

proteins using profile information. The alignments were BIC, HQ). In MrBayes analyses, we assumed two separate visualized and edited in Jalview 2.4.0.b2 [28]. The N-terminal mixed+I+Γ(5) models for each rRNA partition to sample transmembrane β-barrel regions were predicted with BOC- appropriate models across the substitution model space in the TOPUS [29], whereas α-helical transmembrane regions with Bayesian MCMC analysis itself avoiding the need for a priori TopPred [30] and TMpred [31]. model testing [35]. In TreeFinder detailed search depth set Phylogenetic trees for the concatenated alignment of 16S to 2 was applied whereas in PAUP the final tree was searched and 23S rRNA were inferred by maximum likelihood (ML) from 10 starting trees obtained by stepwise and random se- method in TreeFinder [32], Bayesian approach in MrBayes quence addition followed by the tree-bisection-reconnection 3.2.1 [33] and minimum evolution (ME) method based on (TBR) branch-swapping algorithm. To assess significance logDet/paralinear distance in PAUP [34]. In TreeFinder of particular branches, non-parametric bootstrap analyses and MrBayes approaches, we used separate models of were performed on 1000 replicates in these two methods. nucleotide substitutions for each type of rRNA. Two different Additionally, we applied the local rearrangements-expected GTR+Γ(5) models for each data partition were applied in likelihood weights (LR-ELW) method in TreeFinder. In the ML method as suggested by the Propose Model module MrBayes analyses, two independent runs starting from in Treefinder considering all criteria (−lnL, AIC, AICc, random trees were applied, each using eight Markov chains. Trees were sampled every 100 generations for 10 000 000 generations. In the final analysis, we selected trees from the Tab. 1 Domains characteristic of Toc and Tic proteins from Pisum last 2 984 000 generations that reached the stationary phase sativum and Arabidopsis thaliana, which were used to predicted and convergence (i.e. the standard deviation of split frequen- their homologs in Paulinella, cyanobacteria and bacteria proteomes. cies stabilized and was lower than the proposed threshold of 0.01). The temperature parameter for heating the chains Protein CDD was suitably adjusted to keep the proportions of successful name domain Additional information state exchanges between chains close to the suggested range Tic20 233225 Tic20, chloroplast protein import component, from 0.10 to 0.70. length = 267 Tic21 256942 DUF3611, protein of unknown function, length = 183 Results and discussion Tic22 233226 TIC22, chloroplast protein import component, length = 270 We found that both Paulinella chromatophore genomes 252499 Tic22-like family, length = 268 encode the same set of sequence homologs to Toc12, Toc34, Tic32 212492 Retinol dehydrogenase, length = 269 Toc64, Toc159, Tic21, Tic32 and Tic62 but lost those of 235736 Oxidoreductase, length = 315 Toc75, Tic20, Tic22 and Tic55 (Fig. 2). Interestingly, the Tic55 239830 Tic55, a 55 kDa LLS1-related non-heme iron oxygenase, length = 134 missing genes are still present in the α-cyanobacterial Tic62 178748 Translocon at the inner envelope of chloroplast, genomes (Cyanobium gracile, Prochlorococcus marinus and length = 576 Synechococccus CC9311, CC9605, CC9902 and WH 7803) Toc12 197617 DnaJ molecular chaperone homology domain, closely related to Paulinella (Fig. 1). This indicates that length = 60 they might have been lost during chromatophore genome 249696 DnaJ domain, length = 63 reduction or transferred to the nucleus. An interesting case Toc34 130064 GTP-binding protein (chloroplast envelope is Tic22 homolog, which is absent not only from Paulinella protein translocase, length = 313 but also from all α-cyanobacteria grouped with Pauli- 206652 Toc34 like, length = 248 nella except for Synechococcus RCC307 (Fig. 1). The latter Toc75 215598 Protein TOC75, length = 796 α-cyanobacterium is placed at the basal position to the clade 130065 Chloroplast envelope protein translocase, IAP75 family, length = 718 indicating that the gene encoding Tic22 must have been Toc64 166363 Indole-3-acetamide amidohydrolase, length = lost after the divergence of this basal lineage from the other 422 α-cyanobacteria. It should be mentioned that Tic22 homolog 181375 Amidase, length = 395 is present in all other, more than 50 studied cyanobacterial 238112 TPR, Tetratricopeptide repeat domain, length = taxa (Fig. 2). Independent losses concern also the gene for 100 Tic62 homolog in the cyanobacterium Atelocyanobacterium Toc159 233224 Chloroplast protein import component thalassa, Tic21 in the land plant Pisum sativum as well as Toc86/159, length = 763 Tic55 in the cyanobacterium Synechococcus sp. JA-3-3Ab and

Toc/Tic components Species name Tic32 Toc12 Tic21 Tic62 Tic55 Toc75 Tic20 Tic22 Toc159 Toc34 Toc64 Paulinella chromatophora CCAC 0185 9E-59 4E-33 1E-49 0.0003 11110.01 0.002 1 Paulinella chromatophora FK01 2E-57 6E-32 6E-50 0.005 11110.01 0.0004 1 Acaryochloris marina MBIC11017 2E-109 3E-32 2E-51 2E-41 3E-30 7E-149 4E-06 8E-64 0.013 1E-06 1 Anabaena cylindrica PCC 7122 2E-96 1E-33 2E-71 6E-43 1E-27 5E-162 4E-10 9E-43 2E-17 5E-36 1 Anabaena sp. 90 1E-71 5E-33 4E-68 6E-18 5E-31 1E-155 2E-09 3E-42 0.00002 7E-07 1 Anabaena variabilis ATCC 29413 3E-77 4E-34 5E-75 9E-44 1E-33 4E-169 1E-12 4E-43 0.0007 1E-07 1 Arthrospira platensis NIES39 3E-80 1E-35 2E-58 3E-45 2E-30 1E-150 1E-10 5E-78 0.14 4E-06 1 Calothrix sp. PCC 6303 3E-75 3E-34 1E-57 2E-42 7E-31 3E-163 0.00002 2E-38 0.005 6E-07 1 Calothrix sp. PCC 7507 8E-78 3E-34 6E-66 2E-42 2E-28 2E-166 1E-10 9E-45 3E-06 1E-09 1 Atelocyanobacterium thalassa ALOHA 3E-36 4E-29 1E-61 0.031 4E-15 0 0.0004 1E-55 0.047 0.00006 1 Chamaesiphon minutus PCC 6605 5E-67 2E-35 3E-57 2E-44 1E-33 1E-143 0.21 1E-38 1E-07 3E-10 1 Chroococcidiopsis thermalis PCC 7203 1E-72 1E-33 4E-67 6E-41 4E-27 2E-176 4E-10 1E-71 0.0002 2E-08 1 Crinalium epipsammum PCC 9333 3E-75 1E-33 2E-67 1E-44 5E-32 5E-180 9E-09 3E-73 0.0006 0.00002 1 Cyanobacterium aponinum PCC 10605 8E-74 3E-33 2E-51 5E-36 5E-29 0 2E-09 3E-62 0.00008 1E-08 1 Cyanobacterium stanieri PCC 7202 8E-71 8E-34 2E-49 3E-43 2E-28 0 1E-08 8E-64 0.0002 1E-10 1 Cyanobium gracile PCC 6307 5E-94 1E-31 6E-54 5E-17 5E-27 2E-85 2E-06 1 0.03 0.0003 1 Cyanothece sp. ATCC 51142 2E-78 1E-34 3E-62 2E-34 9E-29 0 0.001 1E-62 0.026 2E-06 1 Cyanothece sp. PCC 7424 6E-79 2E-34 2E-53 1E-42 2E-28 0 7E-11 2E-70 0.0002 5E-08 1 Cyanothece sp. PCC 7425 9E-71 3E-36 3E-46 8E-40 2E-30 5E-157 2E-09 1E-44 0.003 4E-06 1 Cyanothece sp. PCC 7822 2E-73 1E-34 3E-52 4E-44 9E-30 0 2E-07 3E-63 0.0008 3E-08 1 Cyanothece sp. PCC 8801 8E-76 1E-34 5E-50 1E-41 4E-68 0 2E-08 7E-59 0.003 0.00001 1 Cyanothece sp. PCC 8802 1E-75 1E-34 5E-50 4E-43 4E-68 0 2E-08 1E-58 0.003 0.00001 1 Cylindrospermum stagnale PCC 7417 9E-74 2E-34 1E-68 6E-47 2E-30 1E-161 3E-08 2E-43 0.002 9E-06 1 Dactylococcopsis salina PCC 8305 5E-74 2E-35 5E-57 9E-43 2E-27 0 8E-06 5E-70 0.31 0.002 1 Geitlerinema sp. PCC 7407 4E-69 7E-33 9E-64 5E-39 2E-22 2E-162 6E-09 9E-44 0.002 2E-07 1 Gloeobacter kilaueensis JS1 7E-50 8E-35 1 4E-27 8E-25 3E-132 1 6E-69 1 0.004 1 Gloeobacter violaceus PCC 7421 1E-98 4E-33 1 5E-32 6E-27 6E-130 0.011 1E-86 0.14 0.0005 1 Gloeocapsa sp. PCC 7428 2E-76 3E-34 1E-58 2E-47 3E-24 2E-180 0.0002 1E-60 9E-06 2E-06 1 Halothece sp. PCC 7418 3E-74 1E-36 3E-58 6E-41 6E-31 1E-180 0.00002 6E-71 0.002 1E-07 1 Leptolyngbya sp. PCC 7376 2E-105 3E-35 4E-52 7E-42 2E-32 1E-166 6E-11 1E-21 0.0006 0.00004 1 Microcoleus sp. PCC 7113 3E-71 6E-34 3E-58 1E-42 1E-77 0 5E-14 3E-72 0.00002 2E-07 1 Microcystis aeruginosa NIES843 5E-104 2E-34 5E-56 6E-38 7E-56 0 1E-09 3E-50 1E-06 6E-08 1 Nostoc azollae' 0708 2E-74 7E-33 8E-70 5E-43 2E-27 1E-159 2E-10 3E-43 0.089 7E-06 1 Nostoc punctiforme PCC 73102 1E-111 2E-34 9E-67 3E-47 3E-87 6E-164 6E-10 2E-55 0.0005 4E-09 1 Nostoc sp. PCC 7107 4E-94 6E-33 7E-66 3E-45 7E-29 1E-160 6E-09 8E-45 0.00004 0.00001 1 Nostoc sp. PCC 7120 6E-97 1E-33 3E-73 2E-44 1E-87 1E-168 1E-11 2E-43 0.0002 1E-07 1 Nostoc sp. PCC 7524 8E-77 1E-34 9E-73 8E-46 5E-72 2E-164 1E-12 2E-42 0.0003 3E-06 1 Oscillatoria acuminata PCC 6304 8E-71 7E-34 1E-56 1E-43 2E-27 3E-155 1E-07 2E-66 0.015 0.00001 1 Oscillatoria nigroviridis PCC 7112 8E-76 3E-34 1E-60 5E-45 8E-26 7E-174 2E-13 2E-81 0.008 1E-07 1 Pleurocapsa sp. PCC 7327 1E-75 7E-36 4E-61 1E-45 4E-28 0 0.1 3E-64 0.002 2E-08 1 Prochlorococcus marinus AS9601 9E-81 3E-30 1E-58 6E-27 1E-20 2E-87 2E-06 1 0.008 0.00001 1 Prochlorococcus marinus MIT 9211 2E-93 1E-30 9E-65 1E-15 5E-22 4E-88 0.0002 1 0.002 0.001 1 Prochlorococcus marinus MIT 9215 1E-80 8E-30 1E-56 9E-24 6E-19 7E-87 9E-06 1 0.001 7E-06 1 Prochlorococcus marinus MIT 9301 1E-80 8E-30 2E-57 1E-26 6E-19 3E-86 2E-06 1 0.006 5E-06 1 Prochlorococcus marinus MIT 9303 5E-94 3E-33 8E-63 2E-15 2E-13 2E-92 1E-07 1 0.003 0.0005 1 Prochlorococcus marinus MIT 9312 2E-80 5E-30 2E-58 5E-26 5E-18 1E-89 2E-06 1 0.004 9E-06 1 Prochlorococcus marinus MIT 9313 4E-92 4E-33 2E-62 1E-15 3E-14 4E-92 5E-07 1 0.0003 0.0002 1 Prochlorococcus marinus MIT 9515 6E-85 1E-29 3E-55 3E-20 5E-17 2E-91 4E-08 1 0.018 0.0003 1 Prochlorococcus marinus NATL1A 7E-85 9E-30 6E-62 2E-20 2E-17 2E-75 0.002 1 0.006 2E-06 1 Prochlorococcus marinus NATL2A 1E-87 1E-29 6E-62 2E-20 8E-18 3E-76 0.005 1 0.01 7E-06 1 Prochlorococcus marinus marinus CCMP1375 6E-90 5E-30 8E-63 2E-14 6E-22 3E-84 0.004 1 0.015 0.00002 1 Prochlorococcus marinus pastoris CCMP1986 1E-81 7E-30 2E-56 1E-20 4E-18 5E-91 4E-10 1 0.015 0.00002 1 Pseudanabaena sp. PCC 7367 9E-73 2E-33 4E-45 2E-35 9E-26 1E-129 0.0001 1E-52 2E-08 2E-13 1 Rivularia sp. PCC 7116 3E-76 6E-35 1E-55 5E-43 4E-26 7E-161 3E-10 2E-39 0.007 0.00004 1 Stanieria cyanosphaera PCC 7437 9E-77 1E-35 1E-57 2E-39 0.00001 0 4E-09 5E-71 0.001 0.00001 1 Synechococcus elongatus PCC 6301 1E-71 7E-33 7E-49 2E-33 6E-29 2E-138 8E-12 1E-24 0.033 0.00007 1 Synechococcus elongatus PCC 7942 3E-72 7E-33 7E-49 2E-33 6E-29 7E-139 8E-12 2E-24 0.033 0.00007 1 Synechococcus sp. CC9311 2E-90 8E-33 5E-60 5E-15 8E-27 1E-89 0.04 1 0.29 0.0002 1 Synechococcus sp. CC9605 7E-86 2E-32 4E-63 4E-18 1E-26 4E-76 0.007 1 0.047 0.0005 1 Synechococcus sp. CC9902 1E-85 2E-30 1E-62 4E-18 8E-26 9E-82 0.008 1 0.0006 2E-06 1 Synechococcus sp. JA23B'a(213) 1E-72 2E-37 3E-49 1E-38 2E-29 3E-118 0.00001 5E-83 4E-07 3E-09 1 Synechococcus sp. JA33Ab 6E-75 4E-38 2E-41 2E-39 1 2E-117 9E-07 1E-94 6E-08 2E-12 1 Synechococcus sp. PCC 6312 2E-76 4E-34 8E-53 2E-34 3E-19 3E-151 0.006 1E-29 0.00003 2E-08 1 Synechococcus sp. PCC 7002 3E-108 8E-36 8E-54 1E-32 1E-32 8E-166 3E-11 7E-20 0.0008 3E-06 1 Synechococcus sp. PCC 7502 7E-69 2E-33 6E-61 2E-40 6E-31 1E-137 2E-10 6E-53 0.035 0.00003 1 Synechococcus sp. RCC307 3E-94 5E-32 5E-59 1E-13 8E-09 5E-46 6E-11 5E-09 0.021 0.00001 1 Synechococcus sp. WH 7803 1E-88 2E-33 1E-58 3E-13 1E-27 3E-90 0.0003 1 0.17 0.0001 1 Synechocystis sp. PCC 6803 2E-69 1E-37 4E-62 6E-34 5E-29 0 4E-08 3E-124 0.0008 5E-07 1 Synechocystis sp. PCC 6803 GTI 2E-69 1E-37 4E-62 6E-34 5E-29 0 4E-08 3E-124 0.0008 5E-07 1 Synechocystis sp. PCC 6803 PCCN 2E-69 1E-37 4E-62 6E-34 5E-29 0 4E-08 3E-124 0.0008 5E-07 1 Synechocystis sp. PCC 6803 PCCP 2E-69 1E-37 4E-62 6E-34 5E-29 0 4E-08 3E-124 0.0008 5E-07 1 Thermosynechococcus elongatus BP1 2E-76 4E-35 8E-64 3E-32 3E-13 2E-142 0.002 5E-34 0.001 0.0004 1 Thermosynechococcus sp. NK55a 4E-66 6E-35 1E-62 1E-32 7E-22 1E-143 0.0008 2E-33 0.001 9E-06 1 Trichodesmium erythraeum IMS101 2E-65 1E-32 3E-61 1E-39 6E-29 8E-154 5E-10 7E-99 0.027 3E-08 1 Arabidopsis thaliana 5E-101 4E-32 0 0 4E-31 0 3E-149 1E-161 0 0 5E-155 Chlamydomonas reinhardtii 3E-97 7E-35 1E-22 2E-43 9E-33 6E-110 5E-12 2E-19 4E-33 5E-55 1 Chlorella variabilis 2E-104 7E-32 4E-43 9E-90 5E-40 2E-174 0.00005 5E-34 6E-112 7E-74 5E-16 Chondrus crispus 5E-64 9E-31 4E-30 6E-11 8E-31 1E-13 0.0002 5E-20 1E-07 4E-15 1 Cyanidioschyzon merolae 4E-65 3E-33 2E-36 5E-39 0.017 4E-19 0.00003 4E-13 5E-21 4E-34 1 Cyanophora paradoxa 6E-88 6E-31 4E-22 3E-42 4E-35 1E-15 4E-09 2E-25 1E-16 8E-25 1 Physcomitrella patens 3E-130 4E-33 4E-52 6E-133 3E-69 0 1E-87 1E-75 0 1E-141 9E-175 Pisum sativum 00 00000000 Bacillus subtilis 168 1E-34 6E-32 1 2E-07 7E-08 0.005 110.002 0.0006 1 Escherichia coli K12 MG1655 7E-32 2E-34 1 0.81 0.0001 0.001 1 1 0.1 0.0002 1

E-value 0.001 0.00099 0.000099 0.0000099 0.00000099 0.000000099 9.9E-09 <10-2 ≤10-3 ≤10-4 ≤10-5 ≤10-6 ≤10-7 ≤10-8 ≤10-9

Fig. 2 Distribution of Toc and Tic homologs in Paulinella chromatophora, cyanobacteria, Archaeplastida and two bacteria. The homologs were found by PsiBlast searches and verified for domain content by searching conserved domain database (CDD). Only best hits with E-values obtained from CDD equal or lower than 0.01 are indicated. The color scheme corresponds toE -value range. Please note that the genomes of Paulinella chromatophores encode homologs of Toc12,Toc34, Toc159, Tic21, Tic32 and Tic62 but those of, Toc75, Tic20, Tic22, Tic55 and Toc64 were lost. In the case of Toc64 significant homologous sequences were found; however, they did not contain a tetratrico-peptide domain, responsible for Hsp90 docking, and therefore we left this column blank.

the red alga Cyanidioshyzon merolae. The gene for Tic21 is α-helical regions. Toc12, is one of the four chromatophore- also absent from two studied Gleobacter species, which often encoded Hsp40 (DnaJ) proteins and a putative component of take basal position to the rest cyanobacteria in phylogenetic molecular motor responsible for pulling imported proteins trees [36–38], suggesting that lack of this gene might have into the chromatophore stroma [20]. characterised the ancestor of all cyanobacteria. The new discovery of the undertaken analyses was iden- Because no homolog to the crucial outer membrane trans- tification of potential homologs to Tic62 inPaulinella . In locon Toc75 was found in the two chromatophore genomes Pisum sativum Tic62 consists of two characteristic regions: and the Paulinella nuclear genome [16], the discovery of the N-terminal part with a highly conserved NAD(P)- homologs to its two receptors, Toc34 and Toc159, was much binding domain and C-terminal part with biding sites for unexpected (Fig. 2). However, we do not consider these ferredoxin-NAD(P)-oxido-reductase (FNR; Fig. 3) [45]. proteins to be components of protein import machinery Similar N-terminal organization was shown for Paulinella without Toc75, especially taking into account their relatively Tic62 homologs, but the C-terminal region with FNR in- low E-values, in both PsiBlast and CDD analyses (Fig. 2). teracting repeats is missing from both Paulinella proteins They may be distantly related to the plastid Toc components (Fig. 3). However, the C-terminal region, which apparently but they do not seem to be their functional homologs. evolved only in vascular plants, is not necessary for binding Interestingly, quite significant E-values obtained for some to the Tic translocon/inner membrane. The region respon- cyanobacterial Toc34/Toc159 homologs suggest that these sible for mediating binding to the Tic complex is localized proteins might be of cyanobacterial origin. This contrasts in the central part of Pisum sativum Tic62 [46], whereas the with the common prevailing idea that they are derived from C-terminal region allows for specific and strong binding of an ancient eukaryotic GTPase, Toc159 as the result of Toc34 ferredoxin-NAD(P)-oxido-reductase molecules, especially duplication [39–41]. A thorough phylogenetic investigation when Tic62 is present at the stroma lamellae of the thylakoid is, however, necessary to clarify the issue, which should be membrane [47]. The reversible interaction with Tic complex/ at present considered only a hypothesis. inner membrane is supposed to be mediated by some hydro- We also found sequences significantly similar to Pisum phobic contacts [48]. One or two transmembrane domains sativum Toc64 in both Paulinella chromatophore genomes were proposed in Pisum sativum Tic62 by Küchler et al. [45]. and bacterial genomes (Fig. 2). However, they should not In agreement with that, we predicted two such domains be considered its true functional homologs because all of using TopPred [30] and TMpred [31]. Interestingly, we also them did not have an important tetratrico-peptide (TPR) identified one potential transmembrane domains in each of domain, responsible for Hsp90 docking, and therefore cannot Paulinella Tic62 proteins using these two programs (Fig. 3). fulfil Toc64 function. The gene for Toc64 encoding the TPR We suggest that Paulinella Tic62 homolog is a new compo- domain is also absent from glaucophytes, rhodophytes and nent of the simplified but fully-functional Tic-like translocon the green alga Chlamydomonas reinhardtii but present in the at the inner chromatophore membrane, composed, beside other green alga Chlorella variabilis, some prasinophytes and Tic62, of Tic21, Tic32 and Toc12 (Hsp40; Fig. 4). The fact land plants (Fig. 2 and data not shown). It indicates that the that these proteins possess all the essential domains to fulfil gene is a late acquisition in the green lineage. the task further strengthens the reliability of a Tic complex In contrast to Toc64, homologs of Toc12, Tic21 and Tic32 at the inner chromatophore membrane. Because Tic55, the in both Paulinella strains contain all the essential domains third redox sensing protein [49–51], has not been discovered present in plant counterparts (Fig. 3). Paulinella Tic21 with in the chromatophore genome in previous [17] and present its four well predicted α-helical transmembrane regions study, as well as in the Paulinella nuclear genome [16], we is suggested to create a protein conducting channel at the suggest Tic32 and Tic62 to perform the task of redox sensing inner chromatophore membrane and can be involved in on their own or with some unknown partner. protein import. Such function of plant Tic21 was recently An interesting argument for functioning of a simplified reported by Kikuchi et al. [42] and Hirabayashi et al. [43], Tic system provided Kikuchi et al. [52]. They suggested and Lv et al. [44] proved functional compatibility of plant that the primordial Archaeplastida Tic complex was prob- and cyanobacterial Tic21 by showing that the knockout ably based on Tic20. Similarly to Tic21 and mitochondrial mutant of Arabidopsis tic21 was rescued by Synechocystis translocon at the inner membrane Tim23/Tim17, Tic20 ortholog. Tic32 homologs, which possess dehydrogenase contains four well predicted α-helical transmembrane do- and calmodulin-binding domains, probably regulate the mains capable of forming a protein conducting channel, and import via redox sensing [20]. Interestingly, Toc12 homologs, therefore together with Tic21 and Tim23/Tim17 represent instead of N-terminal transmembrane β-barrel domains typi- a good example of parallel evolution of import functions cal of plant proteins, comprise C-terminal transmembrane [42]. Since a Tic20-based system might have functioned

Fig. 3 Alignment of Toc12, Tic21, Tic32 and Tic62 from Pisum sativum and two strains of Paulinella chromatophora CCAC0185 and FK01. The range of domains characteristic of a given protein was indicated according to CDD database prediction. A characteristic motif of calmodulin binding domain in Tic32 was also marked [20], as well as NADH(P) binding domain (CDD 257784) and FNR-interacting repeats in Tic62 [46]. The N-terminal transmembrane β-barrel regions were predicted with BOCTOPUS [29], α-helical transmembrane regions with TopPred [30] and TMpred [31]. The average range of the regions was calculated based on the two latter predictions.

in the ancestor of Archaeplastida, it is easy to imagine the Tic apparatus is responsible for protein import into primary possibility of analogous system based on Tic21 in the case plastids of glaucophytes and red algae and probably Paulinella of Paulinella. In support of this protein import via Tic21 chromatophora as well. Kikuchi et al. [52] also showed that was experimentally proved in plants [42,43]. Although eukaryote-derived Tic110, previously considered the main additional core translocon subunits of eukaryotic origin, translocation pore, is not a component of the complex such as Tic214, Tic100 and Tic56 were added to Tic20 in the consisting of Tic20, Tic214, Tic100 and Tic56, but plays a ‘green’ lineage, they are missing from basal Archaeplastida, role of a scaffold for stromal molecular chaperons at a later glaucophytes and red algae [52]. This suggests that a simple stage during protein import [49,50].

Fig. 4 Model for a protein translocon in inner membrane of Paulinella photosynthetic chromatophores. The translocon is a simplified Tic-like apparatus and is responsible for final import step of nuclear-encoded proteins across the inner chromatophore membrane. It is composed of Tic21 (protein-conducting channel), Tic32 and Tic62 (calcium and redox-sensing regulatory proteins) as well as a molecular motor responsible for pulling imported proteins into the organelle stroma. The latter could consist of Hsp93, Hsp70, Hsp40 (Toc12) and GrpE.

Conclusion in previous comparative studies of Paulinella translocons [17]. The common maintenance of the same set of Toc/Tic Our studies reveal that chromatophore genomes from proteins indicates a similar import mechanism in the two both Paulinella strains encode the same set of translocons investigated Paulinella strains and supports the proposed that could potentially create a simplified but fully-functional Tic-like translocon at the inner chromatophore membranes. model. We also suggest a possibility that Toc34 and Toc159, Moreover, we have discovered a new putative Tic compo- GTPases of presumed eukaryotic origin [39–41], might in nent, namely Tic62, a redox sensor protein not identified fact be derived from cyanobacterial proteins.

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