Conservative sorting in a primitive plastid The cyanelle of Cyanophora paradoxa Juergen M. Steiner 1 , Juergen Bergho ¨ fer 2 , Fumie Yusa 1 , Johannes A. Pompe 1 , Ralf B. Klo ¨ sgen 2 and Wolfgang Lo ¨ ffelhardt 1 1 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Biochemistry and Molecular Cell Biology and Ludwig Boltzmann Research Unit for Biochemistry 2 Martin-Luther-Universitaet Halle-Wittenberg, Institute for Plant Physiology, Vienna, Austria The photosynthetic apparatus of cyanobacteria and chloroplasts are organized in an analogous way, with some differences in detail [1], and comprise stromal proteins that temporarily attach to the thylakoid surface, a number of integral thylakoid membrane proteins, and some soluble or loosely membrane-asso- ciated proteins of the thylakoid lumen. In addition, there are certain proteins that possess a thylakoid membrane anchor but expose the bulk of the poly- peptide chain and the prosthetic group involved in electron transfer to the lumen. In higher plants, the chloroplast and nuclear genomes both contribute to the complement of thylakoid proteins [2]. Nucleus- encoded precursors to integral proteins may contain a transit sequence only so that information for thylakoid integration is contained in the mature protein: the cab protein family is thought to use an exclusive mechan- ism, the post-translational signal recognition particle (SRP) pathway [3]. Others possess a bipartite pre- sequence consisting of a stroma-targeting peptide fol- lowed by a signal peptide-like hydrophobic domain. Such proteins as CF 0 -II integrate via the spontaneous (unassisted) pathway without the need for stroma fac- tors receptors, ATP, and DpH [4]. Members of the group of (predominately) lumenal proteins are passen- gers of the Sec and DpH-dependent (or Tat) pathways, corresponding to their relatively unfolded or folded state during translocation, respectively. Identified components of the chloroplast Sec machinery are SecY ⁄ SecE (forming the translocation pore) and the Keywords Cyanophora paradoxa; cyanelles; conservative sorting; Sec translocase; Tat translocase Correspondence W. Lo ¨ ffelhardt, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Biochemistry and Molecular Cell Biology and Ludwig Boltzmann Research Unit for Biochemistry, Dr Bohrgasse 9, 1030 Vienna, Austria Fax: +43 14277 9528 Tel: +43 14277 52811 E-mail: wolfgang.loeffelhardt@univie.ac.at (Received 6 September 2004, revised 11 November 2004, accepted 17 December 2004) doi:10.1111/j.1742-4658.2004.04533.x Higher plant chloroplasts possess at least four different pathways for pro- tein translocation across and protein integration into the thylakoid mem- branes. It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor (‘conserva- tive sorting’), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e. are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic pre- cursor proteins. In the absence of data on cyanobacterial protein transloca- tion, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a pepti- doglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria. We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein. Abbreviations GFP, green fluorescent protein; SRP, signal recognition particle. FEBS Journal 272 (2005) 987–998 ª 2005 FEBS 987 translocation ATPase SecA which is a stromal protein that binds to the pore during action. Energy source: ATP. Inhibitor: sodium azide, acting on SecA. Proteins that fold rather quickly or must acquire their pros- thetic groups in the stroma prior to translocation enter the Tat pathway, in most cases indicated by a ‘twin- arginine’ motif preceding the hydrophobic core of their signal sequences. The known components Tat A (Hcf106), TatB (Tha4) and TatC are all membrane- bound. Energy source is DpH at the thylakoid mem- brane. Inhibitor is nigericin, dissipating DpH [5]. All these data were collected using higher plant chloro- plasts, with the exception of fucoxanthin ⁄ chloro- phyll c-binding protein and the secondary plastids (derived from endosymbiotic red algae) of the diatom Odontella sinensis where indications for a SRP path- way were obtained [6]. Cyanelles are the peptidoglycan-surrounded plastids of glaucocystophyte algae [7], assumed to be the first phototrophic eukaryotes [8]. The protein import appar- atus of the cyanelle envelope seems to function in an analogous way as for rhodoplasts and chloroplasts [9]. Are all four pathways for further protein routing inside the chloroplast also operative in a primitive plastid? The meaning of ‘conservative sorting’ [10], i.e. the retainment of prokaryotic translocons in organelles of endosymbiotic origin as first exemplified by the Sec pathway, has changed during the past decade: the Tat pathway was considered as an achievement of higher plants until its occurrence in (cyano)bacteria was dem- onstrated [5]. On the other hand, the spontaneous pathway now seems to be restricted to chloroplasts since related insertion processes in bacteria were found to depend on the novel translocon component YidC [11,12]. In cyanobacteria, there are dual Sec translocons, in the thylakoid membrane as well as in the inner envel- ope membrane [13]. Tat translocase is assumed to be located in the inner envelope membrane of cyanobac- teria: numerous periplasmic proteins were found where the corresponding genes contained signal sequences with the twin-arginine motif [14] and Tat signal pep- tides directed green fluorescent protein (GFP) to the periplasmic space of transgenic cyanobacteria [15]. On the other hand, the Tat passengers known from chlo- roplasts are largely absent from several completely sequenced cyanobacterial genomes: the two extrinsic proteins from the oxygen evolving complex, PsbP and PsbQ, are replaced in cyanobacteria by the unrelated proteins PsbV and PsbU [16], respectively, that both lack the twin arginine motif in the precursors. Even when a conserved lumenal protein like Hcf136 was considered, the sorting signal appeared to have changed after gene transfer to the nuclear genome: the precursor ⁄ intermediate was shown to use the Sec pathway in cyanobacteria and the Tat pathway in higher plants [17]. One reason to investigate thylakoid transloca- tion ⁄ integration in cyanelles is the bridge position of these organelles between chloroplasts and free-living cyanobacteria. Cyanelle thylakoids resemble the cyano- bacterial ones in the composition of the OEC, the presence of phycobilisomes [18] as antenna system and the possibility of connections to the inner envelope membrane [19]. In freeze-fracture experiments, cyanelle thylakoids also behaved cyanobacteria-like and upon isolation did not readily form closed vesicles as chloro- plast thylakoids do [20]. In vitro experiments with cyanobacterial thylakoids are hampered through this problem: successful translocation is evidenced through protease protection of the mature (processed) protein which is not feasible when no tight vesicles can be pre- pared (C. Robinson, personal communication). Assays that cannot be done ‘in thylakoido’ with cyanobacteria can be performed ‘in organello’ using intact, isolated cyanelles. So phycobilisome assembly could be monit- ored via integration of imported, labeled core linker protein [18]. The Sec pathway in cyanelles, which was made likely by the first demonstration of a functional organellar-encoded secY gene [21], was corroborated by determining the energetic requirements for cyto- chrome c 6 , cytochrome c 550 and PsbO import. In this paper, we demonstrate Rieske Fe ⁄ S protein as a Tat passenger, and, for the first time with phycobilisome- bearing thylakoids, we show protease protection of translocated, processed thylakoid lumenal proteins. Another advantage of the cyanelle system is that pas- senger proteins can be studied that are different from the established chloroplast import systems (i.e. that are not imported in vivo into chloroplasts) as AtpI. Results Cytochrome c 6 uses the Sec pathway Using a modified cyanelle isolation procedure, the effi- ciency of homologous (envelope) import could be greatly increased [9,19]. Import-competent cyanelles from Cyanophora paradoxa efficiently took up the 15 kDa pre-apocytochrome c 6 and converted it into the protease-protected mature form of 9.2 kDa, comi- grating with the holoprotein (Fig. 1, left panel). The time-course showed that the import was largely com- pleted after 3 min. Prolonged incubation resulted in eventual degradation of the mature protein, presuma- bly through lumenal proteases [22]. No intermediate Conservative sorting in cyanelles J. M. Steiner et al. 988 FEBS Journal 272 (2005) 987–998 ª 2005 FEBS was observed under these assay conditions, indicating that envelope import is the rate-determining step. However, two-step processing, as reported for the homologue from Chlamydomonas reinhardtii [23], could be demonstrated when intermediate accumulated due to the addition of the SecA inhibitor, sodium azide (Fig. 1, right panel), whereas nigericin, the inhibitor of the DpH-dependent Tat pathway had no effect (data not shown). The effect of azide was most pronounced after 3 min compared with the control assay. After that time the intermediate is slowly imported and pro- cessed, maybe because of intracyanellar ATP genera- tion, as the azide-inhibition of SecA can be reverted by a higher amount of ATP competing for the same bind- ing sites [24]. As sodium azide might inhibit chloro- plast and cyanelle proteases (our own experiments and K. Cline, personal communication), it is somewhat dif- ficult to compare the posterior time points. Sodium azide also appeared to impede the overall import process of pre-apocytochrome c 6 to some extent, since a small amount of precursor protein was still bound to the envelope (Fig. 1, right panel). For a small protein- like cytochrome c 6 complete protease protection could not be achieved [19]. We therefore extended our experiments to a heterologous system where tight thylakoid vesicles can be obtained. In vitro assays with isolated spinach chloroplasts were performed including competition experiments. Saturating amounts of the OEC23 (PsbP) precursor from spinach were used to block the DpH-dependent Tat pathway [5] and – in parallel – saturating amounts of the spinach OEC33 (PsbO) precursor, a well known Sec-passenger [24] to inhibit the Sec-pathway (Fig. 2). It could be clearly shown that pre-apocytochrome c 6 from C. paradoxa was readily imported into chloroplasts, processed to an intermediate form in the stroma and then translocated into the thylakoid lumen, where it was processed again to its protease-protected mature form (Fig. 2). In the heterologous system, the stroma-processing protease cleavage site obviously was not properly recognized leading to an intermediate of higher MW than that observed in the cyanelle system (Fig. 1, right panel). When the OEC23 precursor was used as a competitor, the amount of mature protein was almost unchanged and its location in the thylakoid lumen could be pro- ven by protease protection (Fig. 2). In competition experiments with the OEC33 precursor, the intermedi- ate accumulated in the stroma fraction by a factor of two (ImageQuant) and the amount of mature protein was reduced by a factor of two compared to the con- trol assay (Fig. 2). The results of homologous and heterologous import experiments indicate that apocyto- chrome c 6 is a Sec passenger in cyanelles (as its func- tional homologue plastocyanin in higher plants) in spite of the lack of protease protection due to system- inherent problems. Protease protection of lumenal proteins in cyanobacterial-type thylakoid vesicles In order to identify additional Sec passengers and, eventually, to prove protease protection of a larger lumenal protein in cyanelle thylakoids, we cloned the psbO gene (GenBank accession number AJ784854) via a PCR approach based on N-terminal sequence infor- mation [25] and used the labeled precursor to study the function of the cyanelle thylakoid translocons. Figure 3 shows a typical import experiment in a time Fig. 1. Time-course of import of 35 S-labeled pre-apocytochrome c 6 into isolated cyanelles. T, translation mix; – ⁄ +, without ⁄ with addition of thermolysin; p, precursor; i, intermediate; m, mature protein. Left panel: control; right panel: plus 10 m M sodium azide. J. M. Steiner et al. Conservative sorting in cyanelles FEBS Journal 272 (2005) 987–998 ª 2005 FEBS 989 course from 3 to 15 min. Compared to pre-apocyto- chrome c 6 , thylakoid import was retarded in the stroma, whereas envelope translocation was almost completed after 3 min. The resulting intermediate form of the OEC33 protein (iOEC33) appeared to be trans- ported into the thylakoid lumen in a time-dependent manner (Fig. 3). When sodium azide was added, mat- uration of iOEC33 was completely stopped (Fig. 3). These data do not unequivocally show if the mature protein is internalized into the thylakoid lumen and thus we wanted to demonstrate protease protection, at least for larger proteins as OEC33. We established a method to isolate tight thylakoid vesicles, which was hitherto not achieved for phycobilisome-bearing, cyanobacterial-type membranes. When the novel cyanelle fractionation procedure was applied after an import experiment, the mature OEC33 localized to the thylakoid fraction (Fig. 4). Thermolysin treatment digested the residual amount of precursor bound to the peptidoglycan-containing envelope membranes, which co-sedimented with the thylakoids and therefore served as internal controls for protease activity, whereas the internalized mature protein was protected in the thylakoid lumen. Sodium azide decreased the amount of precursor imported into the cyanelles (Fig. 4), possibly due to inhibition of other ATP- dependent processes [26]. iOEC33 accumulated in the stroma and even a small proportion of mature PsbO fractionated with the thylakoid membranes, possibly due to the longer incubation time (25min.). When this membrane pellet (containing also envelope membranes) was treated with thermolysin, the bound precursor as Fig. 2. Import of 35 S-labeled pre-apocytochrome c 6 of C. paradoxa into isolated spinach chloroplasts. Tr, translation mix; S, stroma; T–, thyl- akoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; i, intermediate; m, mature protein. Left panel, control; middle panel, saturation of the Tat-pathway by the 23-kDa protein; right panel, saturation of the Sec-pathway by the 33-kDa protein. Fig. 3. Time-course of import of 35 S-labeled pre-OEC33 into isolated cyanelles. ivT, translation mix; p, precursor; i, intermediate; m, mature protein. (A) control; (B) plus 10 m M sodium azide; control + TL, plus thermolysin. Conservative sorting in cyanelles J. M. Steiner et al. 990 FEBS Journal 272 (2005) 987–998 ª 2005 FEBS well as the putative mature protein band disappeared, indicating that PsbO protein generated in the presence of azide is only bound to the thylakoid surface (or rather arrested in a preliminary insertion stage that allowed processing) but not internalized and thus not protease-protected. Nigericin also caused a drop in over- all import efficiency, but did not lead to accumulation of the intermediate in the stroma. Furthermore, in con- trast to the azide experiment, mature protein fractionat- ing with the thylakoids was protease-protected (Fig. 4). All these data point towards a functional Sec translocase with an azide-sensitive SecA homologue in the cyanelle thylakoid membrane and that lumenal cytochrome c 6 as well as OEC33 protein had used this pathway. The Rieske Fe/S protein is inserted into cyanelle thylakoids via the DpH-dependent Tat pathway In chloroplasts, Rieske Fe ⁄ S protein, a nuclear-enco- ded subunit of the cytochrome b 6 ⁄ f complex, appeared in the stroma after in vitro import and only slowly translocated further into the thylakoid membrane sys- tem. It could also be shown, via competition experi- ments and the sensitivity to nigericin, that thylakoid translocation ⁄ integration of this protein in chloroplasts takes place through the DpH-dependent Tat pathway [27]. As the Rieske protein lacks a cleavable signal peptide, its transport is mediated by the N-terminal membrane anchor which does not contain the twin- arginine motif typical of Tat transport signals. Instead, higher plant as well as cyanelle Rieske proteins contain a lysin-arginine sequence at this position, whereas cyanobacterial Rieske proteins do possess the twin- arginine motif. This renders the Rieske protein an unusual Tat substrate and, concerning the evolutionary position of the cyanelles, it was interesting which pathway the authentic Rieske protein might use in the cyanelle system. For that reason we cloned two petC genes from C. paradoxa via a PCR approach (petC1, GenBank accession number AJ784852 and petC2, GenBank accession number AJ784853). We performed in organello experiments with isolated intact cyanelles and the precursor corresponding to petC1 in a time- course manner in the presence⁄ absence of specific translocase inhibitors (Fig. 5). A striking feature in the targeting process of Rieske protein is the remarkably slow sorting of the protein within the cyanelles to its final destination, the thylakoid membrane system. While the import of the Rieske precursor into the stroma proceeded within 5 min (Fig. 5), only a minor fraction (20%) reached the thylakoids and was cor- rectly integrated during a total incubation time of 25 min (Fig. 6). The majority of the processed mature protein of approximately 20 kDa accumulated in the Fig. 4. Fractionation of isolated cyanelles after import of 35 S-labeled pre-OEC33. ivT, translation mix; C, intact cyanelles; S, stroma; T–, thyla- koid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; i, intermediate; m, mature protein; azide, plus 10 m M sodium azide; nigericin, plus 2 lM nigericin. Fig. 5. Import of 35 S-labeled pre-Rieske-protein into isolated cya- nelles. ivT, translation mix; p, precursor; m, mature protein; azide, plus 10 m M sodium azide; nig, plus 2 lM nigericin. J. M. Steiner et al. Conservative sorting in cyanelles FEBS Journal 272 (2005) 987–998 ª 2005 FEBS 991 cyanelle stroma. Once the Rieske protein had reached the thylakoids, it was to a large extent protected against the activity of proteases that were added exter- nally to the thylakoid vesicles, indicating that the C-terminal hydrophilic domain had been completely translocated into the lumenal space (Fig. 6). Only the utmost N-terminal residues preceding the membrane anchor domain remained accessible to thermolysin resulting in a decrease in apparent molecular mass of approximately 0.5 kDa. In the presence of nigericin, thylakoid translocation of the Rieske protein was com- pletely abolished, and the bulk of intracyanellar mature protein localized with the stroma, while the thylakoid membrane fraction contained only a minor amount of loosely bound Rieske protein which disappeared after thermolysin treatment. Sodium azide reduced the amount of membrane-integrated Rieske protein to about 30% of the control assay. This fraction of mature protein was protease-protected and fully integ- rated into the thylakoid membrane system through its single membrane span. A slight downward shift in polypeptide mobility was best noticeable here due to the removal of about four amino acids from the stroma-exposed N terminus by thermolysin treatment, indicating correct integration into the cytochrome b 6 f complex. In evaluating the azide effect on thylakoid translocation of Rieske protein one should consider its reported inhibitory action on numerous nucleotide- binding proteins [28]: the azide-sensitive steps occur very likely after the import of the apoprotein and prior to membrane integration of the holoprotein [27]. Thus our interpretation of the experiments shown in Fig. 6 is to name Rieske a bona fide Tat passenger: a similar conclusion was made in the chloroplast system [27]. The first precursor to a cyanelle lumenal protein (PsbU) containing the twin-arginine motif PrePsbU from the red alga Cyanidium caldarium was found to contain the twin-arginine motif in the thyla- koid transfer domain of the presequence [29]: this was the first incidence in algae containing ‘primitive’ plast- ids and prompted us to clone the counterpart from C. paradoxa based on sequence information from an EST collection (S. Burey and H. Bohnert, unpublished data). This completed the collection of cyanelle OEC component genes (GenBank accession number AJ784849) and presented another Tat passenger candi- date (Fig. 7). Cyanelle import of prePsbU occurred as readily as that of the other small cyanelle protein, pre- cytochrome c 6 , with almost no envelope-bound precur- sor or intermediate (Fig. 8). However, azide addition did not result in the accumulation of intermediate, excluding the Sec pathway. Nigericin did not produce a clear-cut effect either (except also increasing the amount of envelope-bound precursor). Small proteins obviously can escape from cyanelle thylakoid vesicles, thus mature PsbU localized to the stroma fraction in comparable amounts irrespective of any inhibitor used (Fig. 8). Considering these experimental difficulties with regard to protease protection and the reported DpH-independence of Tat translocation in C. reinhardtii chloroplasts [30] we propose that PsbU, i.e. one of the three cyanelle OEC proteins, is a Tat passenger. Fig. 6. Fractionation of isolated cyanelles after import of the 35 S-labeled pre-Rieske-protein. ivT, translation mix; C, intact cyanelles; S, stroma; T–, thylakoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; m, mature protein; azide, plus 10 m M sodium azide; nigericin, plus 2 lm nigericin. Conservative sorting in cyanelles J. M. Steiner et al. 992 FEBS Journal 272 (2005) 987–998 ª 2005 FEBS AtpI: SRP-dependent or spontaneous thylakoid integration? As other primitive plastids, cyanelles encode a higher number of ATP synthase subunits than higher plant chloroplasts, e.g. atpG and atpD [32]. However, in con- trast to all sequenced plastid genomes, atpI is a nuclear gene in C. paradoxa [7]. This means that a precursor exceeding Cab protein with respect to hydrophobicity has to be imported into cyanelles, must cross the stroma and insert into the thylakoid membranes. In the case of Cab, the solution is the post-translational SRP pathway involving a transit complex consisting of Cab, SRP54 and SRP43 [3]. We cloned two atpI genes via a PCR approach based on highly conserved domains of the protein. They are listed in under the GenBank accession numbers AJ784850 and AJ784851, respect- ively. The closely related sequences comprise four to five putative transmembrane regions as candidates for binding to SRP54 [33] and a hydrophilic loop with some resemblance to the ‘L18’ domain of Cab protein (Fig. 9) which was shown to interact with SRP 43 [3]. Highly hydrophobic precursor proteins pose problems upon in vitro import into isolated chloroplasts [34]. This also applies for cyanelle envelope translocation: only a small fraction of AtpI is processed and internal- ized (Fig. 10A) though a time course is noticeable. Cya- nelle fractionation after incubation resulted in recovery of substantial amounts of preAtpI in the thylakoid (and envelope) fraction. Low amounts of mature pro- tein were detected in the thylakoid fraction: here no influence of added azide or nigericin became apparent. Due to cleavage sites in the stromal loops thylakoid- inserted AtpI was degraded upon thermolysin treat- ment (Fig. 10B). With regard to the insertion pathway, Sec and Tat do not seem to be involved. In order to Domains: N H C Nucleus-encoded: pre-cytochrome c 6 SKKATFTAAATAAALLAASPVFA pre-PsbO TTFGR ALAAFVAAAGISFAGVSQANA pre-PsbU KKGRR EFVAAAGALFAAFAASPAAFA Plastid-encoded: pre-cytochrome c 550 MFNKNFWTSIIIGCLFCTITYSGVNA p re- p saF MRKLFLLMFCLSGLILTTDIRPVRA Fig. 7. Thylakoidal signal peptides of intermediates (precursors) to cyanelle proteins that are imported into or synthesized within the organelle, respectively. Charged residues are underlined. C, C-ter- minal domain; H, hydrophobic core (bold); N, N-terminal domain. The signal sequences of nucleus-encoded precursors start with the first amino acid after the putative SPP cleavage sites taken from predictions of the CHLOROP program [31]. Fig. 8. Fractionation of isolated cyanelles after import of the 35 S-labeled prepsbU-protein. ivT, translation mix; S, stroma; T–, thylakoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; m, mature protein; azide, plus 10 m M sodium azide; nigericin, plus 2 lm nigericin. Fig. 9. Sequence comparison of a hydrophilic loop between the putative TM helices 3 and 4 of AtpI from C. paradoxa to the ‘L18’ domain of pea LHCP. Similarities are indicated by bold letters. A B Fig. 10. (A) Time-course of import of the 35 S-labeled preatpI-protein into isolated cyanelles. T, translation mix; p, precursor; m, mature protein; 0°, incubation for 20 min on ice; –, control; +, plus thermo- lysin. (B) Fractionation of isolated cyanelles after import of the 35 S-labeled preatpI-protein. ivT, translation mix; S, stroma; T–, thylakoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; m, mature protein; azide, plus 10 m M sodium azide; nigericin, plus 2 lM nigericin. J. M. Steiner et al. Conservative sorting in cyanelles FEBS Journal 272 (2005) 987–998 ª 2005 FEBS 993 assess an SRP-based mechanism, the import efficiency will have to be increased first. Spontaneous insertion is less likely for a polytopic membrane protein as AtpI but cannot be excluded at present. Discussion Two of the four pathways operating in thylakoid translocation ⁄ integration in higher plant chloroplasts, i.e. the Sec and the Tat translocase could be demon- strated to function in the cyanelles of C. paradoxa. With respect to the hydrophobicity of the core domains signal sequences from cyanelle genes surpass those from nuclear genes (Fig. 7). This has also been observed with higher plants: the replacement (after gene transfer) of leucine and phenylalanine by alanine was interpreted as a measure to avoid interactions with cytosolic SRP [35]. Interestingly, there seem to be relatively more known Sec passengers in these primitive plastids than Tat passengers, whereas the opposite was found for chloroplasts [5]. There are several reasons for the observed prevalence of the Sec pathway in cyanelles: one of them is the replacement of evolutionary ancient, i.e. cyanobacterial proteins by unrelated counterparts in higher plants. The cyanelle-encoded cytochrome c 550 fulfills the function of the Tat passenger PsbP in the OEC. By analogy to the other c-type cytochromes it should use the Sec pathway. This was proven by homologous and heterologous import experiments of a construct containing the FNR transit sequence from C. paradoxa at the N terminus (T. Ko ¨ cher and J. Steiner, unpublished data). Second, Tat passengers like polyphenol oxidase, PsbT, PsaN and others (with- out a cyanobacterial homologue) might be absent from cyanelles. Third, C. paradoxa with its peculiar gene distribution between the nuclear and cyanelle genomes allows to test the hypothesis that rapidly folding (small) polypeptides without prosthetic groups can be Sec passengers in cyanobacteria and in primitive plast- ids (when they are plastom-encoded) but should be Tat passengers as nuclear gene products in higher plant chloroplasts. In the latter case, protein targeting to the thylakoid lumen is much more time-consuming and the intermediate should be rather tightly folded upon arrival at the membrane [17]. The cyanelle-enco- ded Hcf136 homolog resembles its cyanobacterial counterpart in the absence of the twin arginine motive, i.e. is a putative Sec passenger. PsbU, on the other hand, an OEC component of cyanobacteria and chlo- rophyll b-less algae, is nucleus-encoded in the latter. Consequently, PsbU contains a twin-arginine motif in its bipartite presequence and its translocation therefore most likely is Tat-dependent. In this case, evolutionary replacement through PsbQ in higher plants is not accompanied by a change in the translocase used. Isolated cyanobacterial thylakoid vesicles do not allow in thylakoido import experiments or protease protection of luminal proteins, in contrast to spinach thylakoids [5,34]. Therefore it was a considerable pro- gress to show protease protection at least for the 33 kDa PsbO protein after internalization into cyanelle thylakoids. It is unknown, why these membranes are still leaky for smaller proteins as cytochrome c 6 and PsbU. Sodium azide appeared to be the diagnostic inhibitor of choice for in cyanello experiments. Thyla- koid import was retarded for small Sec passenger proteins and completely blocked for larger ones, respectively. This indicates high azide-sensitivity of cyanelle SecA. Thus the Sec pathway can be excluded when no significant azide effect is observed, e.g. in the case of PsbU. Nigericin completely abolished protease protection of membrane-inserted Rieske protein. However, azide addition resulted in a reduction of the amount of correctly assembled cyanelle Rieske protein as was observed with the chloroplast in organello system [27]. There it was shown that although the Rieske protein is targeted exclusively by the DpH ⁄ Tat pathway, some azide-sensitive stromal factors, such as the Cpn60 chaperonin [36] might play a role in correct folding and ⁄ or attachment of the Fe ⁄ S cluster to the Rieske mature (apo)protein. Recently, an interaction and ⁄ or regulation partner for the Rieske protein has been identified in Arabidopsis thaliana [37]. A thylakoid lum- enal FKBP (immuno-suppressant FK506 binding pro- tein) was isolated, whereof only the precursor, but not the mature form, interacted with Rieske protein. AtFKBP13 might serve as an ‘anchor’ chaperone that holds the Rieske protein in the cytoplasm or in the stroma so that excessive Rieske protein is not targeted to the thylakoid, since its integration into the cyto- chrome b6f complex underlies complex regulation and coordination events. It might well be that sodium azide blocks either one of those chaperones and ⁄ or other factors necessary for the build-up of a functional cyto- chrome b 6 f complex, and the resulting malfunctioning or only partially moulded unit fails to be translocated via the Tat-pathway. The effects of nigericin on thyla- koid translocation of the other candidate Tat passen- ger, PsbU, were not clear-cut. In this context it should be noted that the Tat translocase in the alga C. reinhardtii appeared to operate in the absence of a DpH [30], whereas proton efflux at the expense of the pH gradient was a prerequisite for Tat-dependent translocation in higher plant chloroplasts [38]. Conservative sorting in cyanelles J. M. Steiner et al. 994 FEBS Journal 272 (2005) 987–998 ª 2005 FEBS AtpI would be a candidate for the post-translational SRP pathway in cyanelles. In Escherichia coli, its homologue, subunit a, needs the assistance of YidC for integration into the inner membrane [39]. Mitochondrial Atp6 is also partially dependent upon Oxa1 in that respect [40]. In chloroplasts, by analogy, the cotransla- tional SRP pathway and the Albino3 translocon might be involved. However, in the ac29 mutant of C. reinhardtii, where one out of the two albino3 genes is inactivated, no effect on ATP synthase (at least on the a subunit) could be observed [41]. Thus far, there is no evidence for ALB3 in the plastids of chlorophyll b-less algae. In C. paradoxa, SecY is a component of two different high molecular mass thylakoid-bound protein complexes (F. Yusa and W. Lo ¨ ffelhardt, unpublished data), a parallel to A. thaliana where ALB3 is also contained [42]. Another crucial point will be to identify SRP43 in nonchlorophytes, e.g. diatoms that integrate fucoxanthin chlorophyll a ⁄ c-binding protein into their thylakoid membranes [6]. On the other hand, reports on vesicle transport of cab protein from the inner envelope membrane to the thylakoid membrane in C. reinhardtii [43] render the function of the transit complex and of ALB3 questionable in this alga. However this vesicle transport must be different from that described for higher plant chloroplasts that can be visualized upon lowering the temperature and is sensitive to microcystin [44]. The fourth pathway might also exist in cyanelles: SecE from C. paradoxa is not yet identified; in chloro- plasts it was shown to use the spontaneous pathway [11,12]. Chloroplast and cyanelle thylakoids are both rich in galactolipids which obviously support unas- sisted integration of proteins in contrast to the phos- pholipids of the E. coli cytoplasmic membrane [12]. Experimental procedures Materials Cyanophora paradoxa LB555UTEX was grown as previ- ously described [45]. In general, cells were harvested in the exponential growth phase. Nucleic acids were isolated according to published methods [45]. Spinach (Spinacia oleracea) was purchased from the local market and kept overnight at 4 °C before isolating chloroplasts. Pea seed- lings (Pisum sativum) were grown for 8–10 days under a 16 h photoperiod. Protein import into isolated chloroplasts Precursor proteins were synthesized by in vitro transcription of the corresponding cDNA clones and subsequent in vitro translation in cell-free wheat germ lysates in the presence of [ 35 S]methionine. Intact chloroplasts were isolated from pea or spinach leaves by Percoll gradient centrifugation and were used in protein import experiments essentially as des- cribed [46]. Competition experiments were performed with precursor proteins that were obtained by overexpression in Escherichia coli [47] and recovered from inclusion bodies by solubilization in a buffer containing 7 m urea, 30 mm Hepes, pH 8.0 and 2 mm EDTA. The solubilized proteins were included in the import assays at concentrations up to 4 lm, taking care that the concentration of urea in the assays never exceeded 300 mm. Control assays contained the same amount of buffer lacking any such solubilized protein. Protein import into isolated cyanelles Import-competent cyanelles were isolated as described in [19]. They were used in protein transport experiments as described in [18]. Isolation of cyanelle thylakoid membranes The import reaction was stopped by the addition of 1 mL ice-cold sorbitol resuspension medium (SRM) buffer (50 mm Hepes, 0.33 m sorbitol, pH 8.0) followed by centri- fugation at 800 g and 4 °C for 2 min. The cyanelle pellet was washed in SRM and resuspended in 500 l L2· SRM and incubated for 25 min at room temperature with 15 lL of a 10 mgÆmL )1 lysozyme stock solution in the presence of protease inhibitors (Complete, Roche), which led to diges- tion of the peptidoglycan wall, cyanelle lysis and release of the phycobilisomes from the thylakoid membrane. After centrifugation for 5 min at 9300 g in an Eppendorf centri- fuge at 4 °C an aliquot of the deep-blue stromal superna- tant was precipitated with 100% (v ⁄ v) acetone, the pellet containing the thylakoids and the peptidoglycan-linked outer envelope membrane was washed in 2· SRM and finally resuspended in 500 lL2· SRM. An aliquot (250 lL) was treated with thermolysin plus 10 mm CaCl 2 for 30 min on ice. After stopping the reaction with EDTA all aliquots were pelleted by centrifugation. Miscellaneous Gel electrophoresis of proteins under denaturing conditions was carried out according to [48]. Import data were ana- lyzed using a PhosphoImager and the molecular dynam- ics imagequant program (version 3.3), such that all the signals remained in the linear detection range. PCR, gene isolation The nucleotide sequences determined via reverse translation of highly conserved regions of the ATP synthase subunit J. M. Steiner et al. Conservative sorting in cyanelles FEBS Journal 272 (2005) 987–998 ª 2005 FEBS 995 CF0-IV (AtpI) and of the Rieske iron–sulfur protein (PetC) were used to design degenerate primers. AtpI: forward primer 5¢-GCNTAYTTYTAYGCNGG-3¢, reverse primer 5¢-GGYTTNGTRAARTCYTC-3¢ (product size: 111 bp); PetC: forward primer 5¢-CARGGNYTNAA RGGNGAYCCNACNTA-3¢, reverse primer 5¢-TAYTGN WSNCCRTGRCANGGRCA-3¢ (product size: 156 bp). The PCR reaction mixture (50 lL) included 100 ng of DNA from C. paradoxa, 0.1 lm concentration of each primer species, 10 mm Tris ⁄ HCl (pH 8.3), 50 mm KCl, 1mm MgCl 2 , 0.2 mm dNTPs, and 1 U of Taq DNA polymerase (Dynazyme, Finnzymes Oy, Espoo, Finland). The following thermal cycle was used: Step 1, 96 °C for 5 min; Step 2, 94 ° C for 1 min; Step 3, 50 °C for 2 min; Step 4, 72 °C for 3 min; Step 5, repeat steps 2–4 35 times; Step 6, 72 °C for 7 min. The predominant PCR product was cloned into pGEM-T and sequenced. After identifi- cation of the correct products the fragments were labe- led with the Digoxigenin Labeling ⁄ Detection System (Boehringer Mannheim, Mannheim, Germany) and used for screening of a C. paradoxa cDNA library in the vec- tor k-ZAP II (Stratagene, La Jolla, CA, USA). Plaque hybridization was performed under high stringency conditions [49]. Full-length cDNAs were cloned into the vector pBAT [50] to allow sufficient translation efficiency. psbO: forward primer: 5¢-GARGGNYTNACNTAYGAYCA-3¢, reverse primer: 5¢-AANGGNACNCKYTCNCCNCC-3¢. The for- ward primer was designed using a peptide sequence (EGLTYDQ) obtained via Edman-sequencing [25]. psbU: forward primer: 5¢-AAGAATTCACGAGGCAGAAATG GCGTTC-3¢, reverse primer: 5¢-AAGGATCCTGGGGAC AGCAGAAACTTGG-3¢. The psbU gene was isolated by PCR with a proof-reading polymerase (Pfu) using data from a C. paradoxa EST lib- rary (S. Burey and H. J. Bohnert, unpublished data) and directly cloned into the pBAT-vector via its EcoRI and BamHI sites. Acknowledgements We appreciate support from the Austrian Research Fund (P15438-MOB, to W.L.). We thank Hans Bohnert (Urbana, IL, USA) and Suzanne Burey for providing EST data from Cyanophora paradoxa. 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