University of GroningenThe 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in ThermosynechococcuselongatusWulfhorst, Hannes; Franken, Linda E.; Wessinghage, Thomas; Boekema, Egbert J.;Nowaczyk, Marc M.Published in:PLoS ONEDOI:10.1371/journal.pone.0103584IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2014Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Wulfhorst, H., Franken, L. E., Wessinghage, T., Boekema, E. J., & Nowaczyk, M. M. (2014). The 5 kDaProtein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus. PLoS ONE,9(8), [e103584]. ghtOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: ing-pure/taverneamendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

The 5 kDa Protein NdhP Is Essential for Stable NDH-1LAssembly in Thermosynechococcus elongatusHannes Wulfhorst1, Linda E. Franken2, Thomas Wessinghage1, Egbert J. Boekema2, Marc M. Nowaczyk1*1 Department of Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany, 2 Electron Microscopy Department, University of Groningen, Groningen, TheNetherlandsAbstractThe cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria andmitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in aunique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein compositionand the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in aNDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) withmolecular weights below 5 kDa. Here we show that NdhP is a unique component of the ,450 kDa NDH-1L complex, that isinvolved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS’ complexesthat play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequentanalysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1Lspecific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential forNDH-1L formation, as this type of NDH-1 was not detectable in a DndhP::Km mutant.Citation: Wulfhorst H, Franken LE, Wessinghage T, Boekema EJ, Nowaczyk MM (2014) The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly inThermosynechococcus elongatus. PLoS ONE 9(8): e103584. doi:10.1371/journal.pone.0103584Editor: Andrew Webber, Arizona State University, United States of AmericaReceived April 24, 2014; Accepted July 2, 2014; Published August 13, 2014Copyright: ß 2014 Wulfhorst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and itsSupporting Information files.Funding: This work was supported by the RUB Research School (HW) and by grants of the Deutsche Forschungsgemeinschaft (HW and MMN) and the BioSolarCells project (LEF and EJB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* Email: [email protected] ferredoxin might be a possible pathway to chloroplast NDHand cyanobacterial NDH-1 [11,12].Based on reverse genetics and proteomic studies, significantprogress has been made in resolving the subunit composition andfunction of the cyanobacterial complex [13,14] At least fourdifferent NDH-1 subtypes (NDH-1L, NDH-1L’, NDH-1MS,NDH-1MS’) have been identified or postulated for cyanobacteriaso far. NDH-1L and NDH-1L’ are involved in respiration whereasNDH1-MS and NDH-1MS’ link the Complex I specific andelectron flow dependent proton-pumping activity to uniquecarbon concentration mechanisms (CCM) in an unknown process.All NDH-1 variations have been shown to play a role in CET, inparticular under stress conditions [15]. They all share a commonNDH-1M core unit but they differ in the presence of subunits thatare unique for each complex type like NdhF1/D1 (NDH-1L),NdhF1/D2 (NDH-1L’), NdhF3/D3/CupA/CupS (NDH-1MS)and NdhF4/D4/CupB (NDH-1MS’). Two more isoforms of theNdhD subunit (NdhD5, NdhD6) were identified on genome levelbut they have not been assigned to specific NDH-1 complexes yet.Although reverse genetic and proteomic studies have alreadyrevealed a detailed picture of cyanobacterial NDH-1 complexes, itis still incomplete in many aspects. We have recently identified twonovel peptides – NdhP and NdhQ – in purified NDH-1 complexesfrom T. elongatus [16]. NdhP plays a role in NDH-1 mediatedelectron flow in Synechocystis sp. PCC 6803 [17] and shares aweak similarity with NDF6, which is important for NDH-1 activityIntroductionThe cyanobacterial and chloroplast type I NADPH dehydrogenase (NDH-1) complex is structurally and functionally related tothe energy-converting NAD(P)H:Quinone oxidoreductase (Complex I) – one key-enzyme of the energy metabolism in eubacteria(like Escherichia coli) and the respiratory chain of mitochondria[1–3]. This large membrane protein complex is composed of up to,45 subunits in mammals with a total molecular weightapproaching 1 MDa [4]. Remarkable improvements in the Xray structural analysis of eubacterial Complex I revealed detailedinsights into the electron transfer dependent proton-pumpingmechanism on the molecular level [5,6].Cyanobacterial NDH-1 and the closely related chloroplastNDH complex are located in the thylakoid membrane and play apivotal role in respiration (chlororespiration in chloroplasts) as wellas in cyclic electron transfer (CET) around PSI [7,8]. They sharethe so called oxygenic photosynthesis-specific domain of unknownfunction, which is composed of NdhL, -M, -N and -O [9] and thebasic subunits NdhA-NdhK, which are homologous to subunits ofthe eubacterial complex. Chloroplast NDH has a more intricatestructure and includes several additional proteins compared tocyanobacteria [10]. Most strikingly, the cyanobacterial andchloroplast type NDH-1 complex seem to lack homologues toNuoE/F/G of the eubacterial complex that are responsible forNADH oxidation. This led to the recent idea that electron transferPLOS ONE www.plosone.org1August 2014 Volume 9 Issue 8 e103584

NdhP Is Essential for Stable NDH-1L Assemblyin Arabidopsis thaliana [18]. Here we show by isolation andstructural characterization of individual NDH-1 complexes fromT. elongatus that the 5 kDa NdhP peptide is a unique componentof the NDH-1L subtype and essential for its assembly.benzenesulfonyl fluoride) and for streptavidin affinity chromatography Tris-buffer (100 mM Tris pH 8.0; 25 mM MgCl2; 1% (w/v) DDM; 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride) wasused. The thylakoid membranes were suspended in the respectivebuffer at a final chlorophyll concentration of 1 mg/ml andincubated under gentle agitation at 20uC for 60 min. To adjust thefinal DDM concentration to 0.5% an equal volume of dilutionbuffer (same composition but without DDM) was added. Insolublematerial was removed by centrifugation at 45,000 g.Materials and MethodsConstruction of T. elongatus mutantsTo delete the ndhP gene (genomic region: 1189596–1189465)the coding region was replaced by a kanamycin resistance cassette.Genomic DNA from T. elongatus was used as a template for theamplification of the upstream region of ndhP by PCR using thespecific oligonucleotide primers ndhP up for and ndhP upKO rev (Table S1). The ndhP downstream region was amplifiedusing the primer pair ndhP down for and ndhP down rev. TheDNA fragments were restricted with SacI and XbaI (upstream)and PstI and XhoI (downstream) and ligated into the vectorpBluescript SK ( ) (Stratagene). A kanamycin resistance cassettewas introduced via the XbaI and PstI restriction sites and theresulting plasmid pNdhP KO was used for transformation of T.elongatus according to [19]. Complete segregation of the mutantallele was confirmed by PCR with the primers SegCheckNdhP for and SegCheck NdhP rev.The NdhP-sfGFP-His6 mutant was generated based on amodified pNdhP KO plasmid. NdhP and the correspondingupstream region were replaced by a PCR product (primers:ndhP up for and ndhP up sfGFP rev) that introduces anEcoRV site upstream of the ndhP stop codon. The coding regionfor superfolder GFP [20] with a c-terminal His-Tag (sfGFP-His6)was cloned as a synthetic DNA construct (Life Technologies) andintroduced into the final plasmid pNdhP-sfGFP-His6 via theEcoRV restriction site. After transformation of T. elongatus withpNdhP-sfGFP-His6, segregation was checked by PCR (primers:SegCheck NdhP for and SegCheck NdhP rev).To generate the NdhL-TS (TwinStrep-tag) mutant, ndhL(tsr0706) and the corresponding upstream region were amplifiedby PCR (primers: ndhL up strep for and OneSTrEP rev1.) andthe product was used as template for a second PCR (primers:ndhL up strep for and OneSTrEP XbaI rev2). The fragmentwas cloned into pBluescript SK ( ) via SacI and XbaI restrictionsites. The ndhL downstream region was amplified by PCR(primers: ndhL down for and ndhL down rev) and cloned viaEcoRI and ApaI. Finally, the kanamycin resistance cassette wasintroduced via XbaI and EcoRI and the resulting plasmid wasused for transformation of T. elongatus. Segregation was checkedvia PCR (primers: SegCheck NdhL-strep for and SegCheck NdhL-strep rev).Purification of NDH-1 complexesSolubilized thylakoid membranes were filtered through a0.45 mm membrane filter and samples containing His-taggedNDH-1 complexes were applied to a 5 ml FF crude IMACcolumn (GE healthcare) at a flow rate of 1 ml/min afterequilibration with IMAC-equilibration buffer (20 mM MESpH 6.5; 0.5 M mannitol; 150 mM NaCl; 0.03% (w/v) DDM).The column was washed with 5 column volumes (CV) IMACequilibration buffer containing 10 mM imidazole and His-taggedcomplexes were eluted with a 10–500 mM imidazole gradient (4CV) in IMAC-equilibration buffer.Samples containing TwinStrep-tagged NDH-1 complexes wereapplied to a 1 ml StrepTactin high capacity column (IBABiotechnologies) at a flow rate of 1 ml/min after equilibrationwith Strep-equilibration buffer (100 mM Tris pH 8.0; 0,5 Mmannitol; 150 mM NaCl; 0.03% (w/v) DDM). Unbound materialwas removed by washing with 7 CV Strep-equilibration buffer.Strep-tagged protein complexes were eluted with buffer containing2.5 mM desthiobiotin. The eluted proteins were concentratedusing a spin concentrator with 100 kDa cut off (Millipore) andstored at 280uC.ElectrophoresisBlue native (BN)-PAGE was performed according to [23].Purified proteins (5–10 mg) were mixed with 1/10 volume ofsample buffer, loaded on a blue native gradient gel (5–12.5%acrylamid) and electrophoresis was carried out at 4uC at increasingvoltage (50–200 V). For electrophoresis in the second dimension,the respective BN gel-lane was cut out, incubated in buffercontaining 5% b-mercaptoethanol and 6 M urea and loaded on a1 mm-thick 14% SDS-PAGE gel with 6 M urea. After electrophoresis (4uC, 13 mA) the proteins were visualized by silverstaining according to [24].Identification of proteins by 1D-nLC-ESI-MS/MS andMALDI-ToF MSProtein spots from BN-gels and silver-stained 2D-gels wereexcised and digested with trypsin as described earlier [16]. Thedigests were desalted by ZipTips (Millipore), resuspended in 0.1%formic acid in water and analyzed by 1D-nLC-ESI-MS/MS asdescribed in [16]. Intact NDH-1 subunits were examined byMALDI-ToF MS according to [25].Culture conditions – T. elongatusT. elongatus wild type strain and mutants (NdhP-sfGFP-His6,DndhP::Km and NdhL-TS) were grown in BG-11 liquid medium[21] at 45uC, bubbled with 5% CO2 under illumination ofincreasing intensity of 50–200 mmol photons (dependent on celldensity). The medium used for the cultivation of the mutants wassupplemented with 80 mg/ml kanamycin.Transmission electron microscopy and single particleanalysisCell harvest and solubilization of thylakoid membranesPurified samples were prepared for negative staining with 2%uranyl acetate on glow-discharged carbon-coated quantifoil grids.Electron microscopy was performed on a Tecnai G2 20 Twinelectron microscope (FEI, Eindhoven, the Netherlands) equippedwith a LaB6 cathode, operated at 200 kV. Images were recordedat 330 nm defocus with an UltraScan 4000 UHS CCD camera(Gatan, Pleasanton, CA, USA) at 100,000-fold nominal magnification with a pixel size of 0.224 nm at the specimen level. GRACEThe cells were harvested and thylakoids were prepared asdescribed earlier [22]. The buffer composition for n-dodecyl-betaD-maltoside (DDM, Glycon Biochemicals) solubilization ofthylakoid membranes depended on the following purificationmethod. For subsequent Ni2 affinity chromatography thethylakoids were suspended in MES-Buffer (20 mM MESpH 6.5; 25 mM MgCl2; 1% (w/v) DDM; 1 mM 4-(2-Aminoethyl)PLOS ONE www.plosone.org2August 2014 Volume 9 Issue 8 e103584

NdhP Is Essential for Stable NDH-1L Assemblysoftware [26] was used for semi-automated data acquisition.Particle picking was done manually in Eman2 [27]. Outputparticles were generated in image format and imported intoGroningen Image Processing software (GRIP), which was used forfurther analysis.A total of 9000 wild type and 15000 NdhP-GFP-labeled NDH1complexes were collected and processed using standard procedures [28]. Images were pretreated using a low frequency cut-offfilter based on the maximum size of the particle and a highfrequency cut-off filter based on the maximum resolution availablein negative stain (10 Å). The presented images were optimized byapplication of conditional summing with the correlation coefficientof the final alignment step as a quality parameter to select the mosthomogeneous images in each class (correlation decay was between0.1 and 0.2, depending on the number of particles in the class).Resolution of the final results was determined with a Fourier-ringcorrelation with a 3 s threshold criterion.Comparison of the X-ray structure of NDH-1 from T.thermophiles [6] (PDB code: 4HEA) to the electron density mapfrom NDH-1L that was retrieved in this study was done by usingUCSF Chimera [29] and Adobe Photoshop. All proteins werefitted separately.Figure 1. Construction of T. elongatus NdhL-TS and segregationcheck. Schematic representation of the ndhL mutant locus (left). TheTwin-StrepTag was fused to the NdhL c-terminus and a kanamycinresistance marker was used to force segregation of the mutant allele.Complete segregation was confirmed by PCR analysis formatic toolsSequence alignment was performed using the Clustal algorithmin the program JalView [30,31], visualized with the ClustalXresidue color code.Modelling of cyanobacterial subunits was performed using theSwissModel server (, [32]) with thestructure of NDH-1 from T. thermophilus [6] (PDB: 4HEA) astemplate.Results and DiscussionNdhP is a unique component of the NDH-1L complexWe have shown previously that the single-transmembraneprotein NdhP co-purifies with NDH-1L complexes, which wereisolated via the histidine-rich region of NdhF1 [16] but it remainedunclear whether NdhP is also a component of the NDH-1MS orNDH-1MS’ complex and the corresponding subcomplexes NDH1S or NDH-1S’. To answer this question a NdhL-TwinStrep-tag(NdhL-TS) mutant was constructed that enables purification of theother NDH-1 variants. The NdhL subunit is part of the basicNDH-1 unit called NDH-1M and it was shown by Zhang et al.that purification of NDH-1 complexes via NdhL-His resulted in amixture of NDH-1M, NDH-1L, NDH-1MS and NDH-1Scomplexes [33]. We used the Strep-tag/Streptactin affinitychromatography system due to its superior purity compared toHis-tag affinity chromatography [34].The TwinStrep-tag was fused to the c-terminus of NdhL andthe complete segregation of the NdhL-TS mutant was confirmedby PCR analysis (Fig. 1). Solubilized membranes of 5 L culturewere subjected to StrepTactin affinity chromatography and theconcentrated elution fractions were analyzed by BN- and 2Dgelelectrophoresis (Fig. 2). Four different NDH-1 complexes wereseparated by BN-PAGE and the identity of NDH-1L, NDH-1Mand NDH-1S was confirmed by high resolution LC-MS/MS(Tables S2, S3 and S4) and MALDI-ToF MS analysis (Fig. S1).Moreover, all specific subunits of NDH-1S’ (NdhD4, NdhF4,CupB) were identified by MS analysis (Table S5) which confirms –for the first time to our knowledge – its existence on protein level.The NdhP subunit was solely detected in the NDH-1L complexby identification of a specific peptide (Table S6, Fig. S2) that wasmissing in NDH-1M, NDH-1S and NDH-1S’ samples. AlthoughPLOS ONE www.plosone.orgFigure 2. Isolation of NDH-1 complexes via NdhL-TwinStrepTag and 2D-PAGE analysis of purified samples. A: 2D-PAGEanalysis isolated NDH-1 complexes. The identity of NDH-1L and NDH1M as well as NdhP was confirmed by MS/MS analysis (see Table S2, S3and S6). B: Identification of NDH-1S and NDH-1S’ by 2D-PAGE analysisand mass spectrometry (see Table S4 and S5). C: Tagged NDH-1complexes were purified by StrepTactin affinity 02no corresponding bands of the intact NDH-1MS or NDH-1MS’complex are visible in the BN-PAGE analysis, NdhP can beassigned to the NDH-1L complex – most likely inside the NdhD1/F1 unit - as it is not present in any of the correspondingsubcomplexes.Electron microscopy localizes NdhP in the distal NdhD1/F1 unit of NDH-1LIt was previously shown that fusion of several NDH-1 subunitswith YFP and the subsequent analysis by electron microscopy andsingle particle averaging revealed the position of the taggedsubunits inside the NDH-1 complex of Synechocystis sp. [9]. Toinvestigate the distinct localization of NdhP within the NDH-1Lcomplex, we fused the 27 kDa superfolder GFP (sfGFP) protein[20] with an additional His-tag to the NdhP c-terminus. A cterminal fusion was applied to ensure cytoplasmic localization of3August 2014 Volume 9 Issue 8 e103584

NdhP Is Essential for Stable NDH-1L Assemblythe sfGFP tag, as the n-terminus of the single transmembrane helixprotein NdhP was predicted to be oriented towards the lumen[16]. This construct should avoid misfolding as well as impairedmembrane insertion of NdhP caused by the sfGFP fusion. Thethermophilic target strain T. elongatus was cultured at elevatedtemperature (45u–50uC) for optimal growth. Superfolder GFP wasused for the tagging approach, as it shows enhanced thermostability compared to other GFP variants [35].The complete segregation of the T. elongatus NdhP-sfGFP-Hismutant was confirmed by PCR analysis (Fig. 3). His-sfGFP-taggedNDH-1 complexes were isolated via Ni-affinity chromatography(Fig. 4A) and analyzed by BN-PAGE (Fig. 4B). As expected, onlya single protein complex with a molecular weight of approximately450 kDa was isolated and mass spectrometry analysis revealed thepresence of the NDH-1L specific subunits NdhD1 and NdhF1(Table S7). The NdhP-sfGFP-fusion was verified by massspectrometry, as a specific peptide of the linker region thatconnects NdhP and sfGFP, was identified in the sample (Table S8,Fig. S3).Affinity purified NDH-1 complexes from both T. elongatusNdhP-sfGFP-His and NdhL-TS mutant were studied by electronmicroscopy. A total of 15000 NdhP-sfGFP-labeled and 9000NdhL-TS-labeled NDH-1L complexes were collected (see Materials and Methods) and the selected single particle projections wereanalyzed by single particle averaging and assigned to homogenousclasses, followed by subsequent averaging of class members into2D maps. From NDH-1L of T. elongatus NdhP-sfGFP-His twoclasses of differently oriented complexes were generated, resultingin a left- and right-handed side view (Fig. 5A B). Compared to theprojection maps of NDH-1L of T. elongatus NdhL-TS (Fig. 5C D) an extra density is visible in both side views of NDH-1L fromNdhP-sfGFP-His (red arrows), which has to be the sfGFP-His-tag.In conclusion, the previously predicted orientation of NdhP [16] isclearly confirmed by the results of the structural analysis. Based onthe asymmetric shape of NDH-1L, one can assign the c-terminallyfused sfGFP-tag to the cytoplasmic side of the membrane.Moreover, the size of the tag is optically different between sideview A and B. This is best explained by the tag pointing towardsthe viewer out of the stain-layer in side view A and away from theviewer into the stain-layer in side view B. This indicates that thetag is not exactly in the middle.By fitting the 2D projection map with the homologous part ofthe X-ray structure of NDH-1 from T. thermophilus [6] (Fig. 6A)the position of the sfGFP-His-tag could be specified to be ‘‘above’’the subunits NdhB (Nqo13, red) and NdhD1 (Nqo14, yellow),possibly with its c-terminus pointing more into the direction ofNdhD1. Interestingly, the structures of Nqo7 (NdhC), Nqo8(NdhA) and the hydrophilic domain (Nqo4/NdhH, Nqo9/NdhI,Nqo5/NdhJ, Nqo6/NdhK) had to be rotated to make them fit tothe 2D projection map. This changes the direction of theseFigure 4. Isolation of NDH-1L via NdhP-sfGFP-His by Ni-affinitychromatography. A: His-tagged NDH-1L complexes were eluted froma Ni-NTA column with a linear gradient of 10–500 mM imidazole. Theabsorption of sfGFP at 485 nm was used to follow the purification. B:BN-PAGE of eluted proteins. The band with an apparent molecularweight of approximately 450 kDa was identified as NDH-1L by MS/MSanalysis (see Table S7 and S8).doi:10.1371/journal.pone.0103584.g004subunits from pointing to the right to pointing away from theviewer. A more detailed structural comparison of the Nqo13 andNqo14 subunits from eubacterial NDH-1 [6] with models of NdhBand NdhD1 from T. elongatus (Fig. 6B, 6C and S4) revealed thatthe general structure (position of transmembrane helices) seem tobe consistent. One striking difference between both complexesmight be an additional helix at the cytoplasmic side of NdhB. Atentative localization of NdhP in this part of the complex would bein good agreement with our data.Isolation of NDH-1L via NdhF1 is impaired in a DndhP::KmmutantTo investigate the role of NdhP in the NDH-1L complex, acomparative purification from T. elongatus wild type and aDndhP::Km mutant was conducted by Ni-affinity chromatography via the histidine rich region of the NdhF1 subunit [33]. TheFigure 5. Projection maps of NdhP-sfGFP-His-tagged (A and B)and NdhL-TS-tagged NDH-1L complexes (C and D). A is aconditional sum of 9158 images (correlation decay: 0.2) at 16 Åresolution. B contains 1326 images (corr. d.: 0.15) at 25 Å resolution.Projection map C is the sum of 4409 images (corr. d.: 0.15) at 26 Å. Dsums 1758 images (corr. d. 0.2) to a 27 Å resolution map. The positionof the sfGFP-tag is indicated by red arrows. The scale bar represents10 nm.doi:10.1371/journal.pone.0103584.g005Figure 3. Construction of T. elongatus NdhP-sfGFP-His andsegregation check. Schematic representation of the ndhP mutantlocus (left). The sfGFP-His6-Tag was fused to the NdhP c-terminus and akanamycin resistance marker was used to force segregation of themutant allele. Complete segregation was confirmed by PCR 003PLOS ONE www.plosone.org4August 2014 Volume 9 Issue 8 e103584

NdhP Is Essential for Stable NDH-1L AssemblyFigure 7. Construction of T. elongatus DndhP and segregationcheck. Schematic representation of the DndhP mutant locus (left). Akanamycin resistance marker was used to replace the entire ndhP gene.Complete segregation was confirmed by PCR analysis e 8. Comparative purification of NDH-1 complexes viaNdhF1 from wild type and DndhP::Km mutant cells by Niaffinity chromatography. A: BN-PAGE analysis of purified complexes.The 450 kDa complex in the wild type (WT) lane is indicated by a redasterisk. B: 2D-PAGE analysis. The band pattern of the respectivecomplex (red asterisk) is characteristic for n spot pattern that is visible in the SDS-PAGE dimension(Fig. 8B, red asterisk) confirms the presence of NDH-1L in thewild type sample and its absence in the DndhP::Km mutant.In a recent study Schwarz et al. analyzed a DndhP::Km mutantof Synechocystis sp. [17]. It showed reduced NDH-1 mediatedcyclic electron transport (CET) around PSI under high CO2 (HC)conditions, whereas under low CO2 (LC) no significant changewas observed. Interestingly, NDH-1L is the dominant complexunder HC conditions, whereas NDH-1MS is strongly inducedunder LC conditions. Since the latter complex also contributes toCET, the authors speculate that NdhP is a NDH-1L specificsubunit that mediates efficient CET under HC conditions. Herewe can clearly show that NdhP is a unique and essentialcomponent of the NDH-1L complex. NDH-1L is not detectableanymore in the DndhP::Km mutant and - as a direct consequence- this mutant shows the same phenotype [17] as DndhD1 andDndhF1, which also lack the NDH-1L complex [15,23].In conclusion, the present study corroborated the important roleof NdhP within the cyanobacterial NDH-1 complex. NdhP islocated in the distal unit of NDH-1 that is formed by the NdhBand NdhD1 subunits. It was allocated specifically to the NDH-1Lcomplex, which is involved in respiration and CET under highCO2 concentration (Fig. 9). And obviously NdhP is essential forthe stable assembly of this multisubunit membrane proteincomplex, as it is not detectable anymore in the DndhP::Kmmutant. A similar observation was published very recently for theNDH-1L complex of Synechocystis sp. PCC 6803 [37]. Although,they are challenging to identify and sometimes overlooked, singletransmembrane domain proteins seem to play a central role in theorganization of membrane protein complexes in general [38] andFigure 6. Localization of the NdhP subunit. A: Fitting of the NDH1L projection map with sfGFP [20] (PDB code: 2B3P) to the X-raystructure of NDH-1 from T. thermophilus [6] (PDB code: 4HEA). The scalebar represents 10 nm. Colour code: Nqo4 (NdhH): dark green, Nqo5(NdhJ): light purple, Nqo6 (NdhK): light red, Nqo7 (NdhC): light blue,Nqo8 (NdhA): orange, Nqo9 (NdhI): cyan, Nqo10 (NdhG): green, Nqo11(NdhE): grey, Nqo12 (NdhF1): purple, Nqo13 (NdhD1): yellow, Nqo14(NdhB): red. B and C: Structural comparison of the eubacterial NDH-1complex [6] with tentative models of the NDH-1 subunits NdhB andNdhD1 from T. elongatus (B: side view, C: top view). Green square:additional helix in NdhB of T. elongatus; NdhB is shown in red andNdhD1 in yellow. The subunits Nqo1-3 from T. thermophilus are missingin the cyanobacterial NDH-1 complex and were therefore t was generated by replacement of ndhP with a kanamycinresistance cassette and complete segregation was confirmed byPCR analysis (Fig. 7). Equivalent amounts (10 mg Chl) ofthylakoid membranes from wild type and mutant were solubilizedand applied to Ni-affinity chromatography. BN-PAGE analysis(Fig. 8A) of purified protein complexes revealed the presence of aputative NDH-1L complex (red asterisk) in the wild type samplethat is missing in the DndhP::Km mutant. Both samples containequal amounts of co-eluted trimeric PSI complexes that are typicalcontaminants for this type of purification [36]. The characteristicPLOS ONE www.plosone.org5August 2014 Volume 9 Issue 8 e103584

NdhP Is Essential for Stable NDH-1L AssemblyFigure S4 Sequence alignment of NdhB. Red square:position of the additional helix in NdhB of T. elongatus.(TIFF)Table S1 Primers used in this study.(DOCX)NDH-1L subunit analysis after in-gel digestionwith trypsin.(DOCX)Table S2Table S3 NDH-1M subunit analysis after in-gel diges-tion with trypsin.(DOCX)Table S4 NDH-1S subunit analysis after in-gel digestionwith trypsin.(DOCX)Table S5 NDH-1S’ subunit analysis after in-gel digestion with trypsin.(DOCX)Figure 9. Model of NDH-1 complexes that have been verifiedon protein level. The basic complex NDH-1M is combined withspecific domains to assemble the functional complexes NDH-1L, NDH1MS’ and NDH-1MS.doi:10.1371/journal.pone.0103584.g009Table S6 Identification of NdhP by specific peptide.(DOCX)Table S7 NDH-1L-sfGFP subunit analysis after in-geldigestion with trypsin.(DOCX)the 5 kDa NdhP single-transmembrane NDH-1 subunit is onestriking example for this often undervalued class of proteins.Table S8 Identification of NdhP-sfGFP-His by specificpeptides.(DOCX)Supporting InformationMALDI-TOF spectra of NDH-1 preparationsfrom A: NdhL-TS, B: NdhP-sfG

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus Hannes Wulfhorst1, Linda E. Franken2, Thomas Wessinghage1, Egbert J. Boekema2, Marc M. Nowaczyk1* 1Department of Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany, 2Electron Micro