FEBS Letters 584 (2010) 4553–4558

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Comparing system-specific chaperone interactions with their Tat dependentredox enzyme substrates

Catherine S. Chan 1,2, Limei Chang 1, Tara M.L. Winstone 1, Raymond J. Turner ⇑,1

Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 August 2010Revised 16 September 2010Accepted 17 October 2010Available online 26 October 2010

Edited by Gianni Cesareni

Keywords:System specific chaperoneTwin-arginine translocase systemTwin arginine translocaseLeader sequenceBacterial two-hybridProtein maturation

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.10.043

Abbreviations: REMP, redox enzyme maturationtranslocase system; BACTH, bacterial two-hybrid⇑ Corresponding author. Address: Department of B

Science, University of Calgary, 2500 University Dr. N1N4. Fax: +1 403 298 9311.

E-mail address: turnerr@ucalgary.ca (R.J. Turner).1 These authors contribute equally to the first autho2 Present address: Department of Biochemistry and

Medicine, University of British Columbia, Vancouver, B

Redox enzyme substrates of the twin-arginine translocation (Tat) system contain a RR-motif in theirleader peptide and require the assistance of chaperones, redox enzyme maturation proteins(REMPs). Here various regions of the RR-containing oxidoreductase subunit (leader peptide, fullpreprotein with and without a leader cleavage site, mature protein) were assayed for interactionwith their REMPs. All REMPs bound their preprotein substrates independent of the cleavage site.Some showed binding to either the leader or mature region, whereas in one case only the preproteinbound its REMP. The absence of Tat also influenced the amount of chaperone–substrate interaction.

Structured summary:MINT-8047497: FdhE (uniprotkb:P13024) and FdoG (uniprotkb:P32176) physically interact (MI:0915) bytwo hybrid (MI:0018)MINT-8046441: HybO (uniprotkb:P69741) and HybE (uniprotkb:P0AAN1) physically interact (MI:0915) bytwo hybrid (MI:0018)MINT-8046375: DmsA (uniprotkb:P18775) and DmsD (uniprotkb:P69853) physically interact (MI:0915)by two hybrid (MI:0018)MINT-8046425: TorA (uniprotkb:P33225) and TorD (uniprotkb:P36662) physically interact (MI:0915) bytwo hybrid (MI:0018)MINT-8046393: NarJ (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) physically interact (MI:0915) bytwo hybrid (MI:0018)MINT-8046409: NapD (uniprotkb:P0A9I5) and NapA (uniprotkb:P33937) physically interact (MI:0915) bytwo hybrid (MI:0018)

� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

A subset of proteins in Escherichia coli are synthesized with aSRRxFLK twin-arginine (RR-) motif in their N-terminal signalpeptides [1]. They are targeted and translocated post-translation-ally by the twin-arginine translocation (Tat) system in a foldedstate [2]. The translocon is formed by the TatABC subunits in thecytoplasmic membrane.

chemical Societies. Published by E

protein; Tat, twin-arginine

iological Sciences, Faculty ofW, Calgary, AB, Canada T2N

rship.Molecular Biology, Faculty ofritish Columbia, Canada.

Of the growing list of Tat-substrates predicted and identified,we find that a number of redox enzymes also appear to have theirown system specific accessory chaperone protein [3,4]. The chaper-ones termed REMPs (redox enzyme maturation proteins) arerequired for assembly, protease protection, maturation, and target-ing to the translocon through a complex multi-step process [5,6].The REMPs are not part of the final active holoenzyme complexes,but instead seem to be important in monitoring assembly pro-cesses by mechanisms unknown.

The Tat dependent redox enzymes can be separated into twogroups based on their catalytic cofactors [5]. Here we investigatethe molybdopterin-containing enzymes dimethyl sulfoxide reduc-tase DmsABC, trimethylamine N-oxide reductase TorAC, formatedehydrogenases FdnGHI and FdoGHI, periplasmic nitrate reductaseNapABC, and cytoplasmic nitrate reductase NarGHI. The respectiveREMP chaperones for each of the enzymes are DmsD, TorD, NapD,NarJ, FdhD, and FdhE [5,7]. The other group consists of two hydrog-enases, HyaAB and HybOC, which have a Ni–Fe cofactor in their

lsevier B.V. All rights reserved.

4554 C.S. Chan et al. / FEBS Letters 584 (2010) 4553–4558

catalytic sites, where HyaE and HybE assist their maturation,respectively.

Previously our group has shown that the different RR-contain-ing N-terminal peptides and/or preprotein enzymes subunits in-deed interact with specific REMPs [7]. Here using the in vivoadenylate cyclase based two-hybrid (BACTH) system, we presenta comparative study of the REMP interactions with different re-gions of the RR-leader containing subunits. Hybrid recombinantsof the full preprotein (EnzymeF, complete sequence including RR-peptide, peptidase cleavage site, and mature region), RR-leaderpeptides (EnzymeP, RR-peptide region before the peptidase cleav-age site), mature proteins (EnzymeM, mature region only), andthe peptidase cleavage site deleted (EnzymeX) forms of RR-con-taining subunit were generated (Fig. 1A). The interaction betweenthese constructs and their cognate REMPs were investigated inboth wildtype and Tat subunit(s) deletion strains. We find all

Fig. 1. Regions of RR-leader containing redox enzymes. (A) The different regions of the suN-terminal leader peptide (P), the leader cleavage site (triangle) within the full preproteinremains following leader cleavage and translocation (M). (B) An abbreviated model of thnascent polypeptide chain synthesized with a RR-motif in its amino terminal leader woula site(s) within the redox enzyme preprotein. Cofactor biosynthesis and insertion into thform. Following recruitment and assembly with any other enzyme subunit(s), the enzsubunits are moved across the membrane with cleavage of the RR-leader peptide.

REMPs interact with the full preprotein of RR motif-containingsubunit. However, not all REMPS interact with the RR-leader pep-tide or the mature protein alone. Many REMP interactions are alsoaffected by the absence of the Tat system.

2. Materials and methods

2.1. Plasmid constructions and TatABC/E deletion mutantconstructions

Strains and recombinants used and produced in this study aredescribed in Supplementary Table S1. Recombinants of REMPswith T25- and enzyme with T18-domains of the adenylate cyclasefused to their C-termini were generated as described in [7] usingprimers listed in Table S2. The peptidase I cleavage sites wereremoved from the preprotein sequences by site-directed

bstrate enzymes studied here. The translated sequence consists of the RR-containing(F), the full preprotein with the cleavage site deleted (X), and the mature region thate proposed Tat-dependent biogenesis of RR-leader containing redox enzymes. The

d be present during early translation. The corresponding REMP chaperone recognizese mature region of the enzyme as the mature polypeptide folds into its holoenzymeyme complex is targeted to the membrane and Tat translocon where the enzyme

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mutagenesis using a Quickchange II kit (Stratagene) with the T25fusion recombinants above as templates.

Gene deletions were performed according to the method of Dat-senko and Wanner [8] to generate strains CLBTH4B (tatA/E),CLBTH5B (tatBC) and CLBTH6B (tatABC/E) from BTH101 (Primers;Table S3).

2.2. BACTH interaction screening and b-galactosidase assays

Assays based on the reconstitution of functional adenylatecyclase was performed as described in [7,9,10] with the followingexceptions. Pre-screening of interactions was performed on LBplates containing 40 lg/ml bromo-chloro-indolyl-galactopyrano-side. Overnight cultures also contained 0.5 mM isopropyl-b-D-thiogalactopyranoside. Western blots were performed to verifyaccumulation of each fusion construct (not shown).

3. Results

3.1. Regions of the RR-containing subunits

To further investigate the differential interactions between theREMPs and the RR-peptides of Tat-specific redox enzymes ob-served in our previous study [7], further two-hybrid experimentswere undertaken. DmsD was initially identified by its ability tobind to the RR-leader peptide alone [11]. We now consider poten-tial interactions of REMPs with the mature protein. Specifically,constructs were generated to determine the interacting region(s)of the RR-signature containing substrate. The different enzymesubstrate forms investigated in this study are shown in Fig. 1A(EnzymeP, EnzymeF, EnzymeX, and EnzymeM). Note that the NarGsubunit contains an uncleaved vestige RR-peptide and thus noEnzymeX form was necessary. NarGP was constructed as the first50 amino acids in NarG, while the ‘‘mature” form (NarGM) startsat residue 51. This approach is aimed at providing a glimpse ofthe substrate form that the REMP interacts with along the transla-tion, assembly, and targeting process pathway (Fig. 1B).

3.2. Investigating the REMP interaction sites within the catalyticsubunits

Using the four enzyme forms described in Fig. 1A, the enzymesNarG, DmsA, NapA, TorA, HybO and FdoG were targeted for

Fig. 2. Interactions between the chaperones and their cognate RR-containing subunitsdescribed in Fig. 1A. The bars from left to right are P, RR-leader peptide, white; X, fulsequence, dark grey; M, mature region, black. The degree of interaction is evaluated bindependent culture trials and assayed in each time in triplicate, indicated as standard

interaction with their respective REMPs: NarJ, DmsD, NapD, TorD,HybE and FdhE. b-Galactosidase activity (in Miller units) was usedto evaluate the degree of interaction between the two partners viathe reconstitution of the domains (T18 and T25) of adenylatecyclase (one fused to the enzyme substrate, while the other wasfused to the REMP). The results show that interactions were ob-served for all enzymes with their full preprotein (EnzymeF) and fullpreprotein cleavage site deleted (EnzymeX) forms (Fig. 2). Removalof the cleavage site had no effect on the interaction with any of thefull preprotein forms, which demonstrates that the interactionsobserved are independent of leader processing events.

The REMPs NarJ, DmsD and NapD interacted with the RR-leaderof their corresponding enzymes (NarGP, DmsAP and NapAP). How-ever, there was little to no interaction with the mature formsNarGM, DmsAM, and NapAM (Fig. 2) based on this assay approach.Investigation of the full preprotein (RR-leader still attached)showed weaker interaction between the REMP and NarGF/X andDmsAF/X, but was the same for NapAF/X (Fig. 2) when comparingto the interaction with the peptide form. This suggests that whilethe REMPs can bind the RR-peptide of NarG, DmsA and NapA,when the full preprotein is present the strength of interaction orfraction binding is reduced for NarG and DmsA only.

The REMPs TorD, HybE and FdhE show a different trend of inter-action. All four forms of TorA had approximately equivalent inter-action activity when tested with TorD, suggesting that equivalentbinding sites exist within both the leader and mature forms ofTorA. HybE interacts with the HybO mature enzyme (HybOM) aswell as the full preprotein forms (HybOX and HybOF), but not theRR-leader peptide (HybOP), suggesting the presence of one interac-tion site within the mature sequence of HybO, an observation alsoseen by Dubini and Sargent [12]. FdhE is only able to interact withthe full preprotein forms of FdoG (FdoGF and FdoGX) but does notinteract with either the RR-leader peptide (FdoGP) or mature form(FdoGM) alone (Fig. 2). This suggests that the interaction of FdhEwith the preprotein forms FdoGX/F is dependent on the presenceof both the RR-leader and mature sequence but not the cleavagesite.

3.3. Interaction dependence of the Tat subunits

Previous observations implied that DmsD localization to themembrane is dependent on the presence of the TatBC subunits[13]. This suggested that the holoenzyme DmsA may be guided

. The protein pairs were screened using the four forms of the substrate enzymesl preprotein sequence with the cleavage site deleted, light grey; F, full preproteiny cAMP-activated b-galactosidase activity that were calculated from at least threeerror bars.

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by DmsD to the translocon for Tat docking. To investigate therequirement of the individual Tat subunits for REMP–enzymeinteractions, the strain for BACTH, BTH101, was engineered withdeletions in tatA/E, tatBC, or tatABC/E. All REMP–enzyme interac-tion pairs described above were then assayed in these Dtat mutantstrains as well as in the BTH101 (‘‘wildtype”) containing the intacttat operon (Fig. 3).

The interaction of NarJ with the RR-vestige peptide region fromNarG (NarGP) was affected in such that absence of any of the Tatcomponents reduced the activity by almost 50% (Fig. 3). However,the interaction of NarJ with either NarGF or NarGM showed no tatdependence. Tat dependence of DmsD interactions were not ob-served for any substrate form. Similarly interactions between

Fig. 3. Tat-dependence of the chaperone–substrate interactions. The protein pairs as desscreened using the four forms of the substrate enzymes described in Fig. 1A. The bars fromDtatBC, dark grey; DtatABC/E, black.

NapD and NapA had little Tat dependence, with the exception thatthe interaction with the NapAF form was much weaker in a Dta-tABC/E background (Fig. 3).

TorD bound all four forms of TorA regardless of Dtat strain (Figs.2 and 3). However, the TorD interaction with the TorAP and TorAM

forms was weaker in all deletion backgrounds, with the largestreduction in DtatBC or DtatABC/E (Fig. 3), implying that TatBCmay play a role in the TorD–TorA interaction pathway.

An interaction could only be observed between FdhE and FdoGin the full preprotein forms (FdoGX and FdoGF) and this interactionwas reduced in both DtatBC and DtatABC/E strains (Fig. 3). HybEinteracts with HybOM as well as both preprotein forms (Figs. 2and 3). Experiments performed with the different Dtat strains

cribed in Fig. 2 were assayed in various tat gene deletion strains. Interactions wereleft to right are WT, wildtype containing all Tat subunits, white; DtatA/E, light grey;

C.S. Chan et al. / FEBS Letters 584 (2010) 4553–4558 4557

show a similar pattern with the exception that the interaction withHybOF was almost completely abolished in the DtatABC/E strain,suggesting a dependence on the complete translocon for this inter-action (Fig. 3).

4. Discussion

REMP chaperones have many proposed roles in aiding the mat-uration of Tat-specific redox enzymes, which includes proteaseprotection, Sec-avoidance, targeting, and as an escort through theenzyme maturation pathway. Here we have explored the interac-tions between RR-motif leader containing substrates of Tat-depen-dent redox enzymes and the REMPs of E. coli. Given the uniqueprocessing of redox enzymes during maturation, the various re-gions of the enzymes were subjected to investigation using anin vivo two-hybrid assay. Distinct differences for the REMP interac-tions were observed, suggesting that the maturation pathways ofredox enzymes involving REMPs are more unique than previouslyassumed. The dependence of Tat subunits for these interactionsfurther illustrates this uniqueness.

Six different REMP–enzyme interaction pairs were investigatedin this study. All REMPs were shown to interact with the fullpreprotein forms of their respective enzymes (Figs. 2 and 3). How-ever, key differences suggest either sites or modes of interactionfor different REMPS are different. FdhE was only able to bind topreprotein forms of FdoG. Contrary to this, the TorD REMP was ableto bind to all four forms TorA at equivalent levels. NarJ, DmsD andNapD bound to the RR-leader peptide of their substrates (NarG,DmsA and NapA) but not to the mature forms. And finally, HybEbound to the mature form of its substrate (HybO) but not theRR-leader peptide. The interactions are summarized in Fig. 4.

Fig. 4. Summary of interaction sites of enzyme substrate forms and their degree ofdependence on Tat. (A) The REMP interaction site for each enzyme as determinedfrom BACTH results in Fig. 2. (B) The effect of various tat deletions on theinteractions between the REMP and enzyme interaction site as compared tointeraction in wildtype. Darker the shading the greater the interaction activity oreffect.

The complete lack of an interaction between the mature regionsof DmsA and NapA with DmsD and NapD, respectively, suggestthat the only interaction site is located within the RR motif-con-taining peptide region (Fig. 4A). Despite this similarity, the interac-tion between NapD and the full preprotein form of NapA (NapAF)was dependent on the presence of the entire Tat complex, whilethe DmsD interaction with DmsAF showed no Tat dependence(Fig. 4B). Recently the idea that DmsD interacts with the TatBand/or TatC proteins was confirmed [13,14]. The data here showthat interaction between DmsD and the DmsAP occurs independentof TatBC and the other Tat subunits (Fig. 3), suggesting that theinteraction likely occurs prior to targeting to the Tat components.

TorD was the only REMP to demonstrate an equivalent level ofinteraction signal with both the mature and leader peptide formsof TorA, suggesting that two equivalent yet independent interac-tion sites exist within the full preprotein sequence of TorA – onewithin TorAP and another in TorAM. Previous research also showedthat TorD was capable of interacting with the mature region ofTorA [15].

NarJ was also shown to interact with both the RR-vestige pep-tide and mature forms of NarG (Figs. 2 and 3) however the interac-tion with the NarGM was much weaker than with the NarGP. Thisresult indicated that a strong binding site responsible for NarGinteracting with NarJ was located in the RR motif-region of NarG,while a second possible binding site responsible for NarG interact-ing with NarJ is located further into the sequence of NarG. Directinteractions between the mature region of NarG and NarJ have pre-viously been suggested supporting that NarG may have two bindingsites for NarJ. The first site somewhere within the first 15 residues[16,17] while the second site is somewhere after residue 40 (Fig. 2and [17,18]). Although there was an effect of absence of Tat on NarJinteracting with NarGP, the effect on NarJ interacting with full andmature forms were minor. According to Blasco et al. [19], the abilityof NarGH to associate with the membrane is reduced in the absenceof NarJ and NarJ is also required for the membrane-bound enzymeto be activated. Reconciling our findings suggest that the interac-tions between NarJ and the premature form of NarG may happenprior to NarGH associating with NarI in the membrane whichmay be assisted by Tat. It is the NarJ–NarG vestige RR-peptide re-gion interaction that depends on Tat as found here and [20].

The investigations here with FdhE and the mature form of FdoGrule out the possibility that the binding site previously observed[7] is within the mature region (Fig. 2). Furthermore, the resultssuggest that the FdoG binding site may be comprised of a short se-quence from the leader and the mature region that immediatelysurrounds, but does not depend on the presence of the cleavagesite (Fig. 4A).

No interaction was observed between the RR-leader peptide ofHybO with HybE (Fig. 2 and [7]); thus the HybE binding site islikely located within the mature region of HybO. Although, theindividual absence of TatA/E or TatBC did not impair HybE bindingwith any HybO forms. The absence of TatABC/E resulted in the lossof about 90% of the binding ability of HybE with HybOF (Fig. 3). Theresults suggest a different maturation pathway for this NiFe en-zyme, where perhaps Tat interaction stabilizes a processing-com-petent enzyme.

The interaction with the four substrate forms pose interestingconsequences in terms of processing of these enzymes. In accor-dance with the maturation model presented in Fig. 1, an interac-tion with form P likely occurs immediately following translationand exit from the ribosome, when the leader peptide is exposedand potentially unfolded. Docking simulations of DmsD with theDmsA leader peptide shows the leader bound to DmsD in an ex-tended conformation without any observable secondary structure[21]. However, it is clear that this is REMP dependent, as fromNMR work; NarG leader region binds to NarJ as an a-helix [17].

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Forms F and X is present when translation is complete althoughlikely different structurally at every stage prior to translocation.Form F is cleaved to remove the leader peptide resulting in formM representing the final assembled enzyme, which occurs for mostenzymes during translocation across the membrane. In accordanceto this, any interaction site in the M region would be present informs F and X and have equal affinities for their REMP and process-ing events at the membrane was expected to have no effect on theinteraction with the REMP in the cytoplasm.

This work illustrates the specificity of these REMPs and theirsubtle differences in substrate binding. Our study further supportsthe idea that each REMP is indeed a system-specific chaperone andthat the nature of the substrate interaction can be more than theRR-motif peptide. Further the findings suggest that each redox en-zyme system follows a different interaction/maturation pathwayand degree of Tat assistance, requiring its own chaperone to facil-itate its pathway toward a folded, targeted, assembled, and func-tional enzyme.

Acknowledgements

We thank Dr. H. Li for molecular biology suggestions and Dr. T.Palmer for initial discussions on approaches. C.S.C. and T.M.L.W.were supported by NSERC graduate scholarships. This work wassupported by a Canadian Institutes of Health Research grant toR.J.T.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2010.10.043.

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