Tat‐dependent targeting of Rieske iron‐sulphur proteins to both the plasma and thylakoid...

Tat-dependent targeting of Rieske iron-sulphur proteins toboth the plasma and thylakoid membranes in thecyanobacterium Synechocystis PCC6803

Cassie Aldridge,1 Edward Spence,1

Markus A. Kirkilionis,2 Lorenzo Frigerio1 andColin Robinson1*1Department of Biological Sciences and 2MathematicsInstitute, University of Warwick, Coventry CV4 7AL, UK.

Summary

Cyanobacteria possess a differentiated membranesystem and transport proteins into both the periplasmand thylakoid lumen. We have used green fluorescentprotein (GFP)-tagged constructs to study the Tatprotein transporter and Rieske Tat substrates in Syn-echocystis PCC6803. The Tat system has been shownto operate in the plasma membrane; we show herethat it is also relatively abundant in the thylakoidmembrane network, indicating that newly synthe-sized Tat substrates are targeted to both membranesystems. Synechocystis contains three Rieske iron-sulphur proteins, all of which contain typical twin-arginine signal-like sequences at their N-termini. Weshow that two of these proteins (PetC1 and PetC2) areobligate Tat substrates when expressed in Escheri-chia coli. The Rieske proteins exhibit differentiallocalization in Synechocystis 6803; PetC1 and PetC2are located in the thylakoid membrane, while PetC3 isprimarily targeted to the plasma membrane. The com-bined data show that Tat substrates are directed withhigh precision to both membrane systems in thiscyanobacterium, raising the question of how, andwhen, intracellular sorting to the correct membrane isachieved.

Introduction

Cyanobacteria comprise a numerous and diverse groupof photosynthetic prokaryotes that are presumed to be theprogenitors of higher plant chloroplasts. Like other Gram-negative eubacteria, cyanobacteria have a cell envelopeconsisting of an outer membrane, a periplasmic space

and a plasma membrane. In addition to this, cyanobacte-ria are virtually unique among these organisms in contain-ing a true internal membrane system: the thylakoidmembranes. Recent analyses have shown that the thyla-koid membranes are physically discontinuous from theplasma membrane, at least in Synechocystis PCC6803(Liberton et al., 2006; Schneider et al., 2007) and there-fore the thylakoid membranes represent a separate intra-cellular compartment. This additional membrane systemdemands a more complex system of protein sorting andtrafficking in Synechocystis and other cyanobacteria. InSynechocystis 6803 the targeting of newly synthesizedproteins across membranes is believed to occur via trans-port systems found in other eubacteria, chiefly the Secre-tory (Sec) and Twin-arginine translocation (Tat) pathways.The Tat translocon consists minimally of TatA, TatB andTatC in Escherichia coli (Bogsch et al., 1998; Sargentet al., 1998; 1999; Weiner et al., 1998) and is mechanis-tically related to the DpH-dependent pathway of plantthylakoid membranes. In contrast to the Sec pathway, theTat translocon transports folded proteins in an ATP-independent manner. In E. coli and chloroplasts, TatA andTatB share a degree of sequence homology but are func-tionally distinct. Synechocystis PCC6803 contains clearhomologues of these genes that encode two TatA/B-likeproteins (slr1046 and ssl2823) and a TatC homologue(sll0194). These components are more closely related tothe chloroplast homologues Tha4, Hcf106 and cpTatC,respectively, compared with other bacterial proteins(see below).

The Synechocystis PCC6803 genome contains onlysingle copies of the necessary subunits for both the Secand Tat machineries, and this raises the key question ofhow proteins are directed to specific compartments withinthe cell (in the case of translocated soluble proteins, thethylakoid lumen or periplasm). SecY and SecA of the Sectranslocon have been previously shown to be present inboth the plasma and thylakoid membranes of Synechoc-occus PCC7942 (Nakai et al., 1993; 1994), and SecA hasalso been detected in the thylakoid membrane of Syn-echocystis PCC6803 by MALDI-TOF analysis of proteinsfrom a 2D-gel of purified membranes (Srivastava et al.,2005). These findings suggest that Sec-dependent

Accepted 4 August, 2008. *For correspondence. [email protected]; Tel. (+44) 2476 523557; Fax(+44) 2476 523568.

Molecular Microbiology (2008) 70(1), 140–150 � doi:10.1111/j.1365-2958.2008.06401.xFirst published online 29 August 2008

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

proteins are targeted across both membranes, but thishas not been experimentally confirmed. Moreover, verylittle is known about the ‘sorting’ of proteins to the correctlocation, whether during or after translocation. One pos-sibility is that substrates are somehow recognized by Secapparatus in the appropriate membrane and thus targeteddirectly to the correct location; however, this would requireapparently identical Sec systems to function differently inthe thylakoid and plasma membranes. Another scenario isthat proteins are targeted randomly across both mem-branes and only subsequently sorted or targeted bydefault to the plasma membrane, with a subset of proteinsundergoing subsequent sorting to the thylakoid com-partment. Indeed, it has been reported that photosystem Ipartially assembles in the plasma membrane and is sub-sequently transported to the thylakoid membrane (Zaket al., 2001).

The operation of the Tat pathway in cyanobacteria hasbeen studied in even less detail. A chimeric protein com-prising green fluorescent protein (GFP) linked to a Tatsignal peptide (from TorA, an E. coli Tat substrate) is tar-geted almost exclusively to the periplasm (Spence et al.,2003). This restricted localization suggests that the Tatapparatus may only be located in the plasma membrane.To study how Tat substrates are targeted to the correctmembrane, we have used a GFP fusion to the slr1046Synechocystis Tat component to investigate whether theTat translocon is located solely on the plasma membraneor is present in the both the thylakoid and plasma mem-branes in a situation analogous to the Sec system. Wehave also used GFP fusions to the Synechocystis RieskeFeS proteins PetC1, PetC2 and PetC3 to demonstratethat the Tat apparatus can transport proteins across boththe thylakoid membranes and the plasma membrane. Thecombined data point to a complex system of intracellularprotein targeting and sorting, and may pave the way forstudies on the sorting processes involved.

Results

The Synechocystis PCC6803 genome contains twotatA/tatB genes and a tatC gene

Chloroplast and E. coli Tat systems minimally comprisethree essential subunits: TatABC (in E. coli ) and Tha4,Hcf106 and cpTatC in plants. The SynechocystisPCC6803 genome appears to contain three tat genes:sll0194, which encodes a TatC homologue, and theslr1046 and ssl2823 genes, which encode TatA/B-likeproteins. Figure 1A shows an alignment of the TatA/Bproteins from Synechocystis, Arabidopsis thaliana andE. coli. Some features usually distinguish between TatAand TatB proteins in Gram-negative bacteria, especiallythe residues found at the hinge region between the single

transmembrane (TM) domain and predicted amphipathica-helix of TatA/B proteins. In TatA homologues, an invari-ant phenylalanine is present immediately preceding the‘hinge’ glycine at position 21 in E. coli TatA (Hicks et al.,2003), and a strictly conserved phenylalanine is present inthe amphipathic helix (residue 39 in the E. coli protein;Hicks et al., 2003). TatB homologues contain a conservedproline immediately after the hinge glycine (Weiner et al.,1998; Hicks et al., 2003) and an invariant glutamic acidresidue in the TM domain (Sargent et al., 1999). BothSynechocystis proteins (slr1046 and ssl2823) contain allof these residues, as do the Arabidopsis Hcf106/Tha4proteins. It is thus impossible to attribute a TatA or TatBfunction to slr1046 or ssl2823 by sequence analysisalone. TatB proteins usually have a longer cytosolicC-terminal region than TatA proteins but, although slr1046is longer than ssl2823, the additional residues are at theN-terminus of the protein (before the TM domain) andneither protein contains the extended C-terminal domaincommonly found in TatB proteins.

Slr1046 and Ssl2823 complement E. coli DtatAE andDtatB mutants

In order to experimentally determine whether slr1046 andssl2823 are TatA or TatB subunits, C-terminal Strep tagII-tagged versions of slr1046 and ssl2823 were tested fortheir ability to complement DtatAE and DtatB mutations inE. coli (TatE is a TatA paralogue that is expressed at lowlevels, hence the necessity to use a strain disrupted inboth tatA and tatE for studies on TatA complementation).slr1046-strep or ssl2823-strep constructs were clonedinto the arabinose-inducible promoter pBAD24 expres-sion vector and used to transform DtatAE and DtatBstrains of E. coli. Cells expressing these constructs weregrown under anaerobic conditions in the presence of ara-binose, and TMAO was added to induce expression ofTMAO reductase (TorA), a known Tat substrate commonlyused to monitor Tat export activity (Silvestro et al., 1989;Bolhuis et al., 2001). Cells were fractionated into peri-plasm, cytoplasm and membrane samples (Fig. 1B, lanesP, C and M respectively) and proteins were separated ona polyacrylamide gel under native conditions. An in-gelTMAO activity assay was used to identify fractions con-taining TorA activity (Fig. 1B). In wild-type MC4100 cells,the majority of TorA activity was present in the periplasm,as expected. In the DtatAE or DtatB E. coli strains, all ofthe TorA activity was present in the cytoplasm indicatingno transport of TorA, again as expected as the Tatpathway is inoperative in these strains. In DtatAE andDtatB cells expressing either slr1046 or ssl2823, a pro-portion of the TMAO reductase activity was present in theperiplasm, indicating that both Synechocystis proteinscan complement both of these mutations. These unex-

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 70, 140–150

pected results show that slr1046 and ssl2823 have char-acteristics of both TatA and TatB, in keeping with thepresence of determinants normally associated with thesesubunits in Gram-negative bacteria. The data also raisethe possibility that slr1046 and ssl2823 may be bifunc-tional in Synechocystis PCC6803 (see Discussion).

The Tat translocon is located in both the thylakoid andplasma membranes

Previous work had shown that TorA–GFP is targeted onlyto the periplasm and it was suggested that the Tat appa-ratus may be exclusively located in the plasma membrane

(Spence et al., 2003). To test this, a C-terminal GFPfusion was made to slr1046. Previous studies have shownthat GFP-tagged TatA and TatB are correctly targeted intothe membrane in E. coli and the TatB–GFP construct wasshown to be active in supporting translocation (the activityof the TatA–GFP could not be assessed due to significantclipping of the construct to mature TatA; Ray et al., 2005).The slr1046–GFP construct was used to transform Syn-echocystis PCC6803, with expression under the control ofthe psbA2 promoter. The localization of slr1046–GFP wasanalysed by laser scanning confocal microscopy asshown in Fig. 2. Figure 2A shows the emission recordedin the GFP channel of wild-type cells and transformants

Fig. 1. Characteristics of TatA/B proteins from Synechocystis PCC6803.A. Alignment of TatA/B proteins from Synechocystis (Slr1046, Ssl2823) with TatA proteins from E. coli and Arabidopsis (EcTatA and Tha4respectively) and TatB proteins from E. coli and Arabidopsis (EcTatB and Hcf106 respectively). The transit peptides of Tha4 and Hcf106and the extreme N-terminal residues of Slr1046 have been removed to aid alignment. Alignment was performed using ClustalW(http://www.ebi.ac.uk/Tools/clustalw/). Asterisks indicate conserved residues discussed in the text.B. Complementation of E. coli DTatA/E and DTatB strains with Slr1046-strep and Ssl2823-strep. Wild-type (MC4100), DTatA/E, DTatB andDTatA/E, and DTatB strains expressing Slr1046-strep or Ssl2823-strep were grown for 3 h at 37°C in the presence of TMAO and arabinose.Periplasm (P), cytoplasm (C) and membrane (M) fractions were electrophoresed on native polyacrylamide gels that were subsequently stainedfor TMAO reductase (TorA) activity. TorA: mature TMAO reductase; TorA*: more slowly migrating form that accumulates in the cytoplasm.

142 C. Aldridge et al. �


expressing slr1046–GFP. A limited amount of thylakoidautofluorescence is detected in the GFP channel, but theslr1046–GFP transformants exhibit a much higher level offluorescence. Mixtures of transformed and wild-type cellswere used to confirm that the fluorescence in the GFPchannel originates from GFP and not from the photosyn-thetic fluorophores. Comparison of the GFP (green) andautofluorescence (magenta) images in Fig. 2B shows thatonly about half of the cells exhibit significant fluorescencein the 510–530 nm emission region, confirming that theseare transformed cells expressing slr1046–GFP. Repre-sentative wild-type cells (with lower green fluorescence)are indicated by arrows. Figure 2B shows that the GFPfluorescence closely overlaps the autofluorescence fromthe thylakoid pigments, indicating that the bulk of theslr1046–GFP is located in the thylakoids. No halo effectis apparent, suggesting that the levels of slr1046–GFP inthe plasma membrane are no higher than those in thethylakoids. Some punctate areas of GFP fluorescence areapparent, and these almost certainly represent aggre-gates of GFP as seen before when tagged Tat subunitswere expressed in E. coli (Ray et al., 2005). Immunoblot-ting was used to demonstrate that slr1046–GFP had beeninserted into the membrane (see below in Fig. 5). Thethylakoid localization shown here, coupled with the factthat TorA–GFP can be transported across the plasma

membrane into the periplasm (Spence et al., 2003), dem-onstrates that the Tat translocon is located in both theplasma and thylakoid membranes.

Evidence that Rieske iron-sulphur (PetC) proteins areTat substrates in Synechocystis and in E. coli

To date, no Synechocystis proteins have been experimen-tally confirmed as Tat substrates but there are strongindications that the Rieske FeS proteins of the cyto-chrome b6f complex fall into this category. The N-terminalsignal peptides of Rieske proteins from several cyanobac-teria are shown in Fig. 3A. All contain a twin arginine motifimmediately prior to the hydrophobic core region, stronglysuggesting that they are Tat substrates. The sequencesdo not conform particularly closely to the S/T-R-R-x-F-L-Kconsensus sequence identified for other bacterial Tatsignal peptides (Stanley et al., 2000), but the consensusPhe is present in most cases [in addition, it might beexpected that cyanobacterial Tat signals resemble chloro-plast Tat signals, which rarely (if ever) contain the +4 Lysresidue]. Moreover, the Rieske protein from the Gram-negative bacterium Paracoccus denitrificans and thespinach chloroplast Rieske protein have been shownto be targeted via the Tat pathway (Molik et al., 2001;Bachmann et al., 2006). The Synechocystis genome

Fig. 2. Confocal microscopy ofSynechocystis cells expressing Slr1046–GFP.A. Images of wild-type Synechocystis orSynechocystis expressing Slr1046–GFP takenat identical settings, only fluorescence in theGFP channel is shown.B. Mixtures of wild-type and transformed cellswere used to analyse the localization ofSlr1046–GFP. The 488 nm laser line wasused for excitation, GFP was detectedbetween 510 and 530 nm (GFP panel) andautofluorescence from phycocyanin wasdetected between 600 and 700 nm(Autofluorescence panel). The right panelshows the merged image. White arrowsindicate wild-type cells not visible in the GFPchannel. Bars = 2.5 mm.



encodes three Rieske proteins designated PetC1, PetC2and PetC3. Double deletion experiments point to an over-lapping role for PetC1 and PetC2, with PetC1 being themajor Rieske protein in the cytochrome b6f complex,although PetC2 is able to functionally replace PetC1 in theevent of a deficiency (Schneider et al., 2004). Furtherdouble deletion analyses suggested that PetC3 has adifferent function to PetC1 (Schneider et al., 2004), andPetC3 has an unusually low midpoint potential whichfurther indicates a different function to PetC1/2. Despitethis, PetC3 has been detected in isolated cytocrome b6fcomplexes along with PetC1 although the amount ofPetC3 detected was very low (Schneider et al., 2002).Unusually for a Tat substrate, the signal peptide of plantRieske proteins is not cleaved off following translocationand forms part of the N-terminal TM domain in the matureprotein (Karnauchov et al., 1997). Due to the lack of tatmutants in Synechocystis, PetC1, PetC2 and PetC3 wereGFP-tagged and transformed into E. coli MC4100 andDtatABCDE (Dtat) strains to ascertain whether they arebona fide Tat substrates in this organism. Periplasmic,cytoplasmic and membrane fractions were collected fromthe MC4100 and Dtat strains expressing the PetC fusionsand subjected to immunoblotting with antibodies againstGFP.

In both the MC4100 wild-type and Dtat strains, PetC1–GFP was found only in the membrane fraction (Fig. 3B).To test whether correct targeting of PetC1–GFP had infact taken place, spheroplasts were generated and sub-jected to proteolysis with proteinase K. The rationale wasthat correct translocation of this substrate would result intransfer of the soluble FeS-containing domain to the peri-plasmic side of the membrane where it would becomeprotease-accessible; the GFP domain would presumablybe translocated as well. When spheroplasts from MC4100cells expressing PetC1–GFP were protease-treated, all ofthe PetC1–GFP was degraded signifying that the PetC1–GFP had been successfully transported to the periplasmicside of the membrane (Fig. 3B). When spheroplasts fromthe Dtat strain expressing PetC1–GFP were protease-treated, all of the PetC1–GFP remained intact, indicatingthat the PetC1–GFP was on the cytoplasmic side of themembrane. These data show that PetC1 is a genuine Tatsubstrate when expressed in E. coli and this, togetherwith the highly conserved twin-arginine-type signalpeptide features and the observed Tat-dependence inchloroplasts, provides strong evidence that PetC1 is a Tatsubstrate in Synechocystis PCC6803.

PetC2 was studied in exactly the same manner and thedata are shown in the lower panel of Fig. 3B. A PetC2–

Fig. 3. Synechocystis Rieske iron-sulphur proteins are Tat substrates in E. coli.A. Alignment of PetC sequences from cyanobacteria; sll1182: PetC1 Synechocystis sp. PCC6803; slr1185: PetC2 Synechocystis sp.PCC6803; sll1182: PetC3 Synechocystis sp. PCC6803; all1512: PetC Nostoc sp. PCC7120; pmm1322: PetC Prochlorococcus marinusMED4; pmt1322: PetC Prochlorococcus marinus MIT9313; syc0318: PetC Synechococcus elongates sp. PCC6301; synw1841: PetCSynechococcus sp. WH8102; tlr0959: PetC thermosynechococcus elongates BP-1. All sequences were found in CyanoBase(http://bacteria.kazusa.or.jp/cyanobase/) and aligned using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). The twin-arginine motif is shown inbold.B. PetC1/2–GFP were expressed in E. coli wild-type (WT; MC4100) and DtatABCDE (DTat) strains. Cells were grown for 3 h at 37°C in thepresence of arabinose. Proteins in the periplasmic (P), cytoplasmic (C) and membrane (M) fractions were separated by SDS-PAGE.PetC–GFP fusion proteins were detected by immunoblotting with anti-GFP antibodies. In the right panel spheroplasts were untreated ortreated (K+) with proteinase K.



GFP fusion was expressed in the same E. coli strains andthe protein was found in both the membrane and cyto-plasmic fractions of MC4100, but only found in the cyto-plasmic fraction of the Dtat strain. This already indicatesthat the protein fails to be targeted into the membrane inthe Dtat strain, providing strong evidence that PetC2 isalso a Tat substrate. It is unclear why PetC1 associates sostrongly with the membrane in Dtat cells, whereas PetC2is purely cytoplasmic in this strain, but we have previouslynoted a strong membrane association with other Tat sub-strates under these conditions, possibly indicating non-specific association or aggregation tendencies. Proteasetreatment of spheroplasts results in a clear reduction ofPetC2–GFP signal in the case of wild-type cells, consis-tent with degradation of the targeted, membrane-boundfraction of PetC2–GFP (the cytoplasmic molecules wouldremain protected). We conclude that both PetC1–GFPand PetC2–GFP behave as obligate Tat substrates whenexpressed in E. coli.

A PetC3–GFP fusion failed to be expressed in E. coli,for unknown reasons. However, based on signal peptidesequence similarity and the presence of the twin argininemotif, PetC3 is likely to be a Tat substrate. The combineddata from sequence analysis and E. coli expressionstudies provide strong support that all three Rieske pro-teins are Tat substrates in Synechocystis.

PetC1 and PetC2 are located in the thylakoidmembrane whereas PetC3 is located in theplasma membrane

PetC1 has previously been detected in the thylakoidmembrane in Synechocystis PCC6803 (Srivastava et al.,2005) and more specifically in the isolated cytochrome b6fcomplex (Schneider et al., 2002). As PetC2 can partlyreplace the function of PetC1 (Schneider et al., 2004), athylakoid membrane localization of PetC2 is also implied.To confirm these reports, PetC1–GFP and PetC2–GFPwere expressed in Synechocystis and analysed by scan-ning confocal microscopy. As with the slr1046–GFP cells,transformed cells gave a higher GFP signal than wild-typecells at equivalent settings (Fig. 4A). Transformed cellswere mixed with wild-type cells to confirm that fluores-cence detected in the GFP channel is not from the photo-synthetic fluorophores (Fig. 4B, representative wild-typecells are arrowed). Comparison of images of the GFPchannel (green) and the autofluorescence channel(magenta) show that the localization of GFP closelymatches the autofluorescence from the thylakoid pig-ments, indicating that PetC1–GFP and PetC2–GFP arelocated in the thylakoid membrane. The thylakoid local-ization of PetC1 and PetC2 thus demonstrates that Tatsubstrates in Synechocystis can be transported acrossthe thylakoid membrane.

The PetC3–GFP coding region was transformed intoSynechocystis and the localization of PetC3–GFP wasalso analysed by scanning confocal microscopy. Again,transformed cells were mixed with wild-type cells to deter-mine the location of the protein (Fig. 4B). In transformedcells expressing PetC3–GFP, a clear halo of green fluo-rescence is apparent, with a weaker GFP signal present inthe thylakoid region (this overlaps with the magenta fluo-rescence in the autofluorescence image). The merge ofthe GFP and autofluoresence signals confirms that thegreen halo is on the periphery of the cell, and PetC3–GFPis thus exported to the plasma membrane (Fig. 4B).

With all of the transformants, we considered it importantto determine whether the proteins were efficiently tar-geted into the membrane, in order to exclude the possi-bility that some of the fluorescence signal from the cellinterior stemmed from cytoplasmic GFP-tagged proteinrather than thylakoid-inserted protein. In addition, wesought to confirm that PetC3–GFP was localized in theplasma membrane and not the periplasm. We lysedsamples of all of the transformants to generate solubleand membrane fractions, and immunoblots of theexpressed proteins are shown in Fig. 5A. The data showthat slr1046–GFP, PetC1–GFP, PetC2–GFP and PetC3–GFP are almost excusively found in the membrane frac-tion, with only a very small proportion of protein in thesoluble phase. This confirms that they have been effec-tively inserted into their cognate membrane. As controls,expression of TorA–GFP leads to the production of peri-plasmic GFP that is fully soluble, and we used a newtransformant expressing GFP-tagged slr1390 (an FtsHhomologue) to show that this protein is fully membranebound as expected.

We also analysed the periplasmic fraction of the PetC3–GFP transformant to confirm that this protein ismembrane-bound. Figure 5B shows that none of theprotein is detected in the periplasmic sample whereasGFP is readily detected in the corresponding sample fromTorA–GFP expressing cells (Spence et al., 2003). For anegative control, a plasma membrane protein (slr1390–GFP) was used.

In summary, the localization of PetC3 to the plasmamembrane and PetC1 and PetC2 to the thylakoid mem-brane provides strong evidence that Tat substrates inSynechocystis are transported across both membranes,and selectively targeted to specific locations either duringor after the translocation event.

Discussion

In this report we have addressed fundamental questionsregarding protein targeting and sorting in cyanobacteria.One of our aims was to understand the functioning of theTat system in these organisms, because very little is cur-



rently known about this system in these organisms, but amore general aim was to use GFP constructs to under-stand the targeting and sorting of proteins to the individualmembranes in these cells. We know very little about therouting of proteins within these cells, and the ability of theTat system to transport active GFP provides a powerfuland non-invasive tool to study these processes.

One of the first questions concerns the location of theTat apparatus. A TorA–GFP construct was previouslyshown to be directed primarily, if not exclusively, to theperiplasm in Synechocystis (Spence et al., 2003), sug-gesting that this may be the default route for Tat-dependent targeting. To address this key issue, a GFPfusion of the Synechocystis TatA/B protein slr1046–GFPwas created and tested. The majority of slr1046–GFP is

clearly present in the thylakoid membrane, providing thefirst evidence that Tat substrates are transported into thismembrane system. It is essentially certain that the Tatsystem is also present in the plasma membrane, but thisis difficult to prove using a bioimaging assay becauseslr1046–GFP in the plasma membrane is difficult to dis-tinguish from thylakoid-localized protein. These data thusprovide a clear indication that the Tat system is present inboth membranes, as found previously with the Sec appa-ratus in Synechococcus PCC7942 (Nakai et al., 1993;1994).

This study has also provided very strong evidence forthe targeting of Tat substrates to the thylakoids in Syn-echocystis PCC6803. Expression of PetC1–GFP andPetC2–GFP in wild-type and DtatABCDE E. coli strains

Fig. 4. Localization of Synechocystis Rieskeiron-sulphur proteins.A. Images of wild-type Synechocystis andSynechocystis expressing PetC1–GFP orPetC2–GFP were taken using identicalsettings; only fluorescence in the GFPchannel is shown.B. Mixtures of wild-type and transformed cellswere used to analyse the localization ofPetC–GFP proteins. The 488 nm laser linewas used for excitation, GFP was detectedbetween 510 and 530 nm (GFP panel) andautofluorescence from phycocyanin wasdetected between 600 and 700 nm(Autofluorescence panel). The right panelshows the merged image. The top panelshows the localization of PetC1–GFP, themiddle panel is PetC2–GFP and the bottompanel is PetC3–GFP. White arrows indicatewild-type cells not visible in the GFP channel.Bars = 2.5 mm.



confirms that these proteins are likely SynechocystisTat substrates, and confocal imaging of SynechocystisPCC6803 cells expressing these proteins revealed a thy-lakoid membrane localization for both. This agrees withprevious studies that have detected PetC1 in the thylakoidmembrane and specifically in association with the cyto-chrome b6f complex (Schneider et al., 2002; Srivastavaet al., 2005).

In contrast, we have shown that PetC3–GFP is primarilylocalized in the plasma membrane (although it is possiblethat a small proportion of the PetC3–GFP is located in thethylakoid membrane). This localization is consistent withthe idea that PetC3 has a different function from PetC1/2,and there are indeed indications that PetC3 may notbe associated with the cytochrome b6f complex. PetC3has been shown to have a significantly lowered midpointpotential of +135 mV compared with +300 mV reported forPetC1 and PetC2 (Schneider et al., 2002). Plastoquinonecannot be oxidized efficiently at physiological pH if themidpoint potential of PetC3 is this low, strongly suggestingthat PetC3 does not function in the cytochrome b6fcomplex (Schneider et al., 2002; Schneider and Schmidt,2005). The exact role of PetC3 in Synechocystis has yetto be elucidated.

The combined data provide the first direct evidence forthe targeting of Tat substrates to both membrane systemsin Synechocystis PCC6803, and this focuses attention onthe means by which proteins are sorted to the correctmembrane system. One possibility is that the substratescontain signal peptides that somehow directly target themto the correct membrane system. Although difficult toenvisage (Tat systems would need to function very differ-ently in the two membranes, and recognize a subset of

substrates with high fidelity), this scenario would explainhow a heterologous substrate (TorA–GFP) could be trans-ported to only one membrane. The heterologous signalpeptide may be recognized only by a ‘default’ plasmamembrane Tat system, with the thylakoid version perhapsspecializing in the recognition of the small subset of pro-teins destined for this membrane. In support of thispremise, Rajalahti et al. (2007) reported recently thatcyanobacterial proteins apparently destined for differentextracytosolic compartments have different physico-chemical properties in their signal peptide and matureN-terminal segments.

Alternatively, proteins may be translocated acrosseither membrane and sorted afterwards. In support ofsuch a post-translocation sorting model, it has been sug-gested that elements of photosystem I may initially beassembled in the plasma membrane before transit to thethylakoids, possibly through vesicle transport (Zak et al.,2001; Nevo et al., 2007). Both models are currently fea-sible, but the identification of substrates and translocationapparatus in the thylakoids paves the way for experimentsto address these issues through attempts to re-route theproteins.

We have also studied the characteristics of the Tattranslocase itself, and these data also raise interestingpossibilities. The ability of slr1046 and ssl2823 to comple-ment both the DtatAE and DtatB E. coli mutants clearlyshows that slr1046 and ssl2823 have attributes of bothTatA and TatB proteins, at least when expressed in con-junction with the native E. coli TatC protein. Slr1046 andssl2823 could therefore be bifunctional in Synechocystis,in which case this organism would contain TatAC-type Tatsystems similar to those found in most Gram-positive

Fig. 5. The Synechocystis Rieskeiron-sulphur proteins and slr1046–GFP aremembrane-bound.A. Synechocystis cells expressingSlr1046–GFP, PetC1–GFP, PetC2–GFP orPetC3–GFP were fractionated into totalmembrane and soluble fractions. As controls,cells expressing a plasma membrane protein,slr1390–GFP, and TorA–GFP, which isprocessed to soluble GFP in the periplasmwere used.B. To investigate whether PetC3 is in theperiplasm, the periplasmic fraction of cellsexpressing slr1390–GFP, TorA–GFP or PetC3was isolated and 5 mg of each fraction wasanalysed. Proteins were separated bySDS-PAGE and GFP was detected usinganti-GFP antibodies.



bacteria, which lack a tatB gene altogether (discussed inMendel et al., 2008). In turn, this scenario raises the addi-tional possibility that slr1046 and ssl2823 operate in dif-ferent membranes to transport distinct subsets of Tatproteins. These possibilities require examination, andwe stress that we were not able to generate and testan ssl2823–GFP transformant, for unknown reasons.However, we presently believe that this scenario isunlikely. The Synechocystis TatA/B homologues are mostclosely related to plant Tha4/Hcf106 proteins, which carryout completely distinct functions that are comparableto the E. coli TatA and TatB subunits (Mori et al., 1999;Sargent et al., 1999; Bolhuis et al., 2001). The likelihoodis that Synechocystis PCC 6803 also uses a three-component Tat system, and that both slr1046 and ssl2823fortuitously contain characteristics that enable them tofunction as TatA or TatB in E. coli. Nevertheless, thisremains an open question that merits serious attention.

Experimental procedures

Bacterial strains and growth conditions

Synechocystis sp. PCC6803 was grown either on solid BG11(Rippka, 1988) agar plates or in liquid BG11 medium withconstant shaking at 30°C under white light. The E. coli strainsused in this work are the wild-type strain MC4100, DTatA/E,DTatB and DTatABCDE, which have all been described pre-viously (Casadaban and Cohen, 1979; Bogsch et al., 1998;Sargent et al., 1998; 1999). E. coli was grown aerobically at37°C in Luria broth or anaerobically in minimal TMAO/glycerol medium (Bolhuis et al., 2000). Medium supplementswere used at the following final concentrations: ampicillin100 mg ml-1, spectinomycin 50 mg ml-1, apramycin 50 mg ml-1

and arabinose 100 mM.

Preparation of GFP fusions andSynechocystis transformation

To generate the slr1046–GFP fusion, the plasmid describedin Spence et al. (2003) (L38i plasmid) was used as atemplate. This plasmid encodes a TorA–GFP fusion betweenregions of homology to the psbA2 gene. The TorA signalpeptide sequence was removed by digestion with BbvCIand SacII and replaced with slr1046 sequence amplifiedfrom purified Synechocystis DNA using the primers F1046,5′-CTCGAGCCCTCAGCATGGCATTGACCTTGGTT-3′(BbvC1 site is underlined) and R1046, 5′-CTCGAGGCCGCGGCATAATTATTATTATTATTATTATTATTATTATTGCTGGCCTTTTCTGAGC-3′ (SacII site is underlined); removingthe stop codon and inserting a 10¥ Asparagine linkerbetween slr1046 and GFP. To generate PetC1/2–GFPfusions the L38i plasmid was again used as template. PetC1was amplified using the primers petC1f; 5′-CTCGAGCCCTCAGCATGACACAGATTTCTGGC-3′ (BbvCI site isunderlined) and petC1r; 5′-CTCGAGGCCGCGGCATAATTATTATTATTATTATTATTATTATTATTAGCCCACCAGGGATCTT-3′ (SacII site is underlined) removing the stop codon and

inserting a 10¥ Asparagine linker. The petC1 PCR productwas ligated into the BbvCI and SacII sites of the L38i plasmid,replacing the TorA signal peptide and creating a PetC1–GFPfusion. PetC2 was amplified using the primers petC2f:5′-CTCGAGCCCTCAGCATGGATAACACACAGGCGATC-3′(BbvCI site is underlined), and petC2r: 5′-CTCGAGGCCGCGGCATAATTATTATTATTATTATTATTATTATTATTAACCCACCAGGGTTTCTCTC-3′ (SacII site is underlined) andligated into the BbvCI and SacII sites of the L38i plasmid.GFP fusion to PetC3 was crated by generation of a knock incassette to replace the stop codon of petC3 with GFP andthe apramycin resistance gene using the principals of lambdared recombination of PCR products as demonstrated byDatsenko and Wanner (2000) and according to the methodof Gust et al. (2004). The primer pair Fsll1182: 5′-ACACGGTGGTGGAAGTAGAC-3′ and Rsll1182: 5′-GTAACACCTGCCAATGTTCG-3′ was used to amplify a2518 bp region approximately 1000 bp upstream and down-stream of the petC3 stop codon. This PCR product wascloned using the pGEM-T Easy cloning system (Promega)generating pGEM1182 which was used to transform theE. coli strain BW25112 that also contained the modifiedlambda red recombination plasmid pIJ790 (Datsenko andWanner, 2000). PCR primers Fsll1182GFP: 5′-GCTACCCAGGTGAGCGGCAACAACATTCTGGTCAAAGCCATTCCGGGGATCCGTCGACC-3′ and Rsll1182GFP: 5′-CCAATTATCCAAGCGGCTTTGAAAAACCTTGGGTGGCCCTGTAGGCTGGAGCTGCTTC-3′ were designed to amplify theGFP/apramycin region from the plasmid pIJ786. Theseprimers included 39 nucleotides of homologous sequence tothe upstream and downstream regions of the stop codon ofpetC3. This PCR product was then electroporated into theBW25113 E. coli strain containing pGEM1182 and thelambda red plasmid pIJ790. The expression of the lambdared pIJ790 plasmid allows the recombination of the GFP/apramycin resistance cassette PCR product into thepGEM1182 plasmid effectively removing the stop codon ofpetC3 and replacing it with the GFP/apramycin resistancecassette. The Synechocystis TorA–GFP construct has beenpreviously described (Spence et al., 2003). The slr1390–GFPSynechocystis construct was created by amplifying thecoding region of slr1390 from purified Synechocystis DNAusing the primers 1390f: 5′-CTCGAGCCTCAGCATGGGTTTACTGGTAGCTGG-3′(BbvCI site is underlined) and1390r: 5′-CTCGAGGCCGCGGCATACTTACCGGCTAGAGCAGGC-3′ (SacII site is underlined) and ligated into theBbvCI and SacII sites of the L38i plasmid. These constructswere used to transform Synechocystis using the methoddescribed in Spence et al. (2003). Fractionation of Syn-echocystis into total membranes and soluble fractions wasperformed according to the method of Norling et al. (1998).After fractionation the soluble fraction was subjected to addi-tional centrifugation at 70 000 r.p.m. for 30 min to remove anycontaminating membranes. The periplasmic fractions of Syn-echocystis cells was isolated according to the method ofFulda et al. (1999). Five micrograms of protein was loadedonto SDS poly acrylamide gels. After electrophoresis, pro-teins were blotted onto Hybond-P PVDF membrane (Amer-sham Biosciences) and GFP fusion proteins were detectedby probing the membrane using a anti-GFP antibody (SantaCruz Biotechnology) followed by goat anti-rabbit IgG horse-



radish peroxidize conjugate. An ECL detection kit (AmershamBiosciences) was used to visualize proteins.

Confocal microscopy and image analysis

Cells of Synechocystis were immobilized by drying a dropletof cell suspension onto BG11 agar plates and exposed to 2 hof high light to increase expression of GFP fusion proteinsfrom the psbA2 promoter. Cells were subsequently coveredwith a coverslip and placed under the microscope. Imageswere obtained using a Leica DMRE microscope equippedwith a Leica TCS SP2 confocal unit, an acousto-optical beamsplitter (AOBS) and a 100 mW argon laser for excitation. A63¥ oil-immersion objective was used and images were takenby averaging every scan line four times over an area of512 ¥ 512 pixels. The 488 nm laser line was used for excita-tion and GFP fluorescence was detected between 510 and530 nm. Phycocyanin was used as a marker for thylakoidmembranes. Excited with 488 nm it emits between 600 and700 nm.

PetC protein expression in E. coli

For expression of PetC1–GFP in E. coli, PetC1–GFP wasamplified from the L38i/PetC1–GFP plasmid using theprimers, petC1pBADf: 5′-CTCGAGCGAATTCACCATGACACAGATTTCTGGCTC-3′ (EcoRI site is underlined) andpetC1pBADr: 5′-GCTCGAGTCTAGATTATTTGTATAGTTCATCCATGC-3′ (XbaI site is underlined) and ligated into theEcoRI and XbaI sites of the pBAD24 expression vector. Forexpression of PetC2–GFP in E. coli, PetC2–GFP was ampli-fied from the L38i/PetC2–GFP plasmid using the primers:petC2pBADf: 5′-CTCGAGCGAATTCACCATGGATAACACACAGGCGATC-3′ (EcoRI site is underlined) and petC1pBADrand also ligated into the EcoRI and XbaI sites of pBAD24.The resultant plasmids were used to transform a wild-typestrain of E. coli (MC4100) and also a DTatABCDE strain.E. coli was grown in LB media in the presence of 100 mM

arabinose at 37°C with shaking until an OD600 of 0.6 wasreached. Spheroplasts were prepared and the periplasmicfraction was isolated according to Randal and Hardy (1986).Subsequently, the spheroplast sample was split and halfwas treated with 15 ml of 3.5 mg ml-1 proteinase K on ice for20 min. Samples were then TCA-precipitated and equallyloaded for separation by SDS-PAGE. Separately preparedspheroplasts were further fractionated into cytoplasmic andmembrane fractions; spheroplasts were collected by centrifu-gation and re-suspended in 50 mM Tris-acetate (pH 8.2),2.5 mM EDTA and disrupted by sonication. Membranes werecollected using an ultracentrifuge at 70 000 r.p.m. for 30 min,and the supernatant was saved as the cytoplasmic fraction.The membranes were washed with 50 mM Tris-acetate(pH 8.2) 2.5 mM EDTA and solubilized in 50 mM Tris-acetate(pH 8.2) 2.5 mM EDTA plus 1% SDS. GFP fusion proteinswere detected by probing the membrane using an anti-GFPantibody.

Slr1046 and ssl2823 complementation

To investigate whether slr1046 and ssl2823 can complementa DTatA/E or DTatB deficiency in E. coli, slr1046 and ssl2823

were strep-tagged and inserted into the pBAD24 vector underthe control of the arabinose inducible promoter. The slr1046sequence was amplified from purified SynechocystisDNA using the primers f1046pBAD: 5′-CTCGAGCGAATTCACCATGGCATTGACCTTGGTTATGGG-3′ (EcoRI site isunderlined) and r1046pBAD: 5′-CTCGAGTCTAGATTATTTTTCAAACTGTGGGTGCGACCAATTCGAGCTGGCCTTTTCTGAGC-3′ (XbaI site is underlined; Step-tag II is in italics)and ligated into the EcoRI and XbaI sites of pBAD24.The ssl2823 sequence was amplified using the primers2823pBADf: 5′-CTCGAGCCCATGGCAGTGACCCTCCAGGAGCAAAAAATG-3′ (NcoI site is underlined) and 2823pBADr:5′-CTCGAGTCTAGATTATTTTTCAAACTGTGGGTGCGACCAATTCGATTTTTTGCCGGCCGCAGGATC-3′ (XbaI site isunderlined; Step-tag II is in italics) and ligated into the NcoIand XbaI sites of pBAD24. These constructs were used totransform DTatA/E and DTatB E. coli strains. Overnight cul-tures were used to inoculate anaerobic media and weregrown for 3 h in the presence of arabinose. Cells were frac-tionated as above, and protein fractions were separated on a10% non-denaturing polyacrylamide gel. TMAO reductaseactivity was detected via a gel-based methyl viologen-linkedassay (Silvestro et al., 1989).

Acknowledgements

Work was supported by grant BB/C00437X from the Biotech-nology and Biological Sciences Research Council (BBSRC).

References

Bachmann, J., Bauer, B., Zwicker, K., Ludwig, B., andAnderka, O. (2006) The Rieske protein from Paracoccusdenitrificans is inserted into the cytoplasmic membrane bythe twin-arginine translocase. FEBS J 273: 4817–4830.

Bogsch, E.G., Sargent, F., Stanley, N.R., Berks, B.C.,Robinson, C., and Palmer, T. (1998) An essential compo-nent of a novel bacterial protein export system with homo-logues in plastids and mitochondria. J Biol Chem 273:18003–18006.

Bolhuis, A., Bogsch, E.G., and Robinson, C. (2000) Subunitinteractions in the twin-arginine translocase complex ofEscherichia coli. FEBS Lett 472: 88–92.

Bolhuis, A., Mathers, J.E., Thomas, J.D., Barrett, C.M., andRobinson, C. (2001) TatB and TatC form a functionaland structural unit of the twin-arginine translocase fromEscherichia coli. J Biol Chem 276: 20213–20219.

Casadaban, M.J., and Cohen, S.N. (1979) Lactose genesfused to exogenous promoters in one step using a Mu-lacbacteriophage: in vivo probe for transcriptional controlsequences. Proc Natl Acad Sci USA 76: 4530–4533.

Datsenko, K.A., and Wanner, B.L. (2000) One-step inactiva-tion of chromosomal genes in Escherichia coli K-12 usingPCR products. Proc Natl Acad Sci USA 97: 6640–6645.

Fulda, S., Mikkat, S., Schröder, W., and Hagemann, M.(1999) Isolation of salt-induced periplasmic proteins fromSynechocystis sp. strain PCC 6803. Arch Microbiol 171:214–217.

Gust, B., Chandra, G., Jakimowicz, D., Yuqing, T., Bruton,C.J., and Chater, K.F. (2004) Lambda red-mediated



genetic manipulation of antibiotic-producing Streptomyces.Adv Appl Microbiol 54: 107–128.

Hicks, M.G., de Leeuw, E., Porcelli, I., Buchanan, G., Berks,B.C., and Palmer, T. (2003) The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatBfamily components involved in protein transport. FEBS Lett539: 61–67.

Karnauchov, I., Herrmann, R.G., and Klösgen, R.B. (1997)Transmembrane topology of the Rieske Fe/S protein of thecytochrome b6/f complex from spinach chloroplasts. FEBSLett 408: 206–210.

Liberton, M., Howard Berg, R., Heuser, J., Roth, R., andPakrasi, H.B. (2006) Ultrastructure of the membranesystems in the unicellular cyanobacterium Synechocystissp. strain PCC 6803. Protoplasma 227: 129–138.

Mendel, S., McCarthy, A., Barnett, J.P., Eijlander, R.T.,Nenninger, A., Kuipers, O.P., and Robinson, C. (2008) TheEscherichia coli TatABC system and a Bacillus subtilisTatAC-type system recognise three distinct targeting deter-minants in twin-arginine signal peptides. J Mol Biol 375:661–672.

Molik, S., Karnauchov, I., Weidlich, C., Herrmann, R.G., andKlösgen, R.B. (2001) The Rieske Fe/S protein of the cyto-chrome b6/f complex in chloroplasts: missing link in theevolution of protein transport pathways in chloroplasts?J Biol Chem 276: 42761–42766.

Mori, H., Summer, E.J., Ma, X., and Cline, K.J. (1999) Com-ponent specificity for the thylakoidal Sec and DeltapH-dependent protein transport pathways. Cell Biol 146:45–56.

Nakai, M., Sugita, D., Omata, T., and Endo, T. (1993) Sec-Yprotein is localized in both the cytoplasmic and thylakoidmembranes in the cyanobacterium SynechococcusPCC7942. Biochem Biophys Res Commun 193: 228–234.

Nakai, M., Goto, A., Nohara, T., Sugita, D., and Endo, T.(1994) Identification of the SecA protein homolog in peachloroplasts and its possible involvement in thylakoidalprotein transport. J Biol Chem 269: 31338–31341.

Nevo, R., Charuvi, D., Shimoni, E., Schwarz, R., Kaplan, A.,Ohad, I., and Reich, Z. (2007) Thylakoid membraneperforations and connectivity enable intracellular traffic incyanobacteria. EMBO J 26: 1467–1473.

Norling, B., Zak, E., Andersson, B., and Pakrasi, H. (1998)2D-isolation of pure plasma and thylakoid membranes fromthe cyanobacterium Synechocystis sp. PCC 6803. FEBSLett 436: 189–192.

Rajalahti, T., Huang, F., Klement, M.R., Pisareva, T., Edman,M., Sjöström, M., et al. (2007) Proteins in different Syn-echocystis compartments have distinguishing N-terminalfeatures: a combined proteomics and multivariate se-quence analysis. J Proteome Res 6: 2420–2434.

Randall, L.L., and Hardy, S.J. (1986) Correlation of compe-tence for export with lack of tertiary structure of the maturespecies: a study in vivo of maltose-binding protein inE. coli. Cell 46: 921–928.

Ray, N., Nenninger, A., Mullineaux, C.W., and Robinson, C.(2005) Location and mobility of twin-arginine translocasesubunits in the Escherichia coli plasma membrane. J BiolChem 280: 17961–17968.

Rippka, R. (1988) Isolation and purification of cyanobacteria.Methods Enzymol 167: 3–27.

Sargent, F., Bogsch, E.G., Stanley, N.R., Wexler, M.,Robinson, C., Berks, B.C., and Palmer, T. (1998) Overlap-ping functions of components of a bacterial Sec-independent protein export pathway. EMBO J 17: 3640–3650.

Sargent, F., Stanley, N.R., Berks, B.C., and Palmer, T. (1999)Sec-independent protein translocation in Escherichia coli.A distinct and pivotal role for the TatB protein. J Biol Chem274: 36073–36082.

Schneider, D., and Schmidt, C.L. (2005) Multiple Rieske pro-teins in prokaryotes: where and why? Biochim BiophysActa 171: 1–12.

Schneider, D., Skrzypczak, S., Anemüller, S., Schmidt, C.L.,Seidler, A., and Rögner, M. (2002) Heterogeneous Rieskeproteins in the cytochrome b6f complex of SynechocystisPCC6803? J Biol Chem 277: 10949–10954.

Schneider, D., Berry, S., Volkmer, T., Seidler, A., andRögner, M. (2004) PetC1 is the major Rieske iron-sulfurprotein in the cytochrome b6f complex of Synechocystissp. PCC 6803. J Biol Chem 279: 39383–39388.

Schneider, D., Fuhrmann, E., Scholz, I., Hess, W.R., andGraumann, P.L. (2007) Fluorescence staining of livecyanobacterial cells suggest non-stringent chromosomesegregation and absence of a connection between cyto-plasmic and thylakoid membranes. BMC Cell Biol 3: 8–39.

Silvestro, A., Pommier, J., Pascal, M.C., and Giordano, G.(1989) The inducible trimethylamine N-oxide reductase ofEscherichia coli K12: its localization and inducers. BiochimBiophys Acta 999: 208–216.

Spence, E., Sarcina, M., Ray, N., Møller, S.G., Mullineaux,C.W., and Robinson, C. (2003) Membrane-specific target-ing of green fluorescent protein by the Tat pathway in thecyanobacterium Synechocystis PCC6803. Mol Microbiol48: 1481–1489.

Srivastava, R., Pisareva, T., and Norling, B. (2005) Proteomicstudies of the thylakoid membrane of Synechocystis sp.PCC 6803. Proteomics 5: 4905–4916.

Stanley, N.R., Palmer, T., and Berks, B.C. (2000) The twinarginine consensus motif of Tat signal peptides is involvedin Sec-independent protein targeting in Escherichia coli.J Biol Chem 275: 11591–11596.

Weiner, J.H., Bilous, P.T., Shaw, G.M., Lubitz, S.P., Frost, L.,Thomas, G.H., et al. (1998) A novel and ubiquitous systemfor membrane targeting and secretion of cofactor-containing proteins. Cell 93: 93–101.

Zak, E., Norling, B., Maitra, R., Huang, F., Andersson, B., andPakrasi, H.B. (2001) The initial steps of biogenesis ofcyanobacterial photosystems occur in plasma membranes.Proc Natl Acad Sci USA 98: 13443–13448.



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