Oligomeric properties and signal peptide binding by Escherichia coli ...

1138 Escherichia coli Tat Protein Transport ComplexesABCT- T+ S MSMFt NiT- T+ S MSMFt NiT- T+ S MSMFt Ni66352420TatB/CTatATatB/CTatATatB/CTatA1 2 3 4 5 6 71 2 3 4 5 6 71 2 3 4 5 6 7TatATatATatATatBTatBTatBTatCTatCTatC1 2 3 4 5 6 71 2 3 4 5 6 7 1 2 3 4 5 6 7Figure 2. Affinity purification of Tat complexes. TatABCDE were co-ordinately overproduced with a hexahistidinetag on (a) TatA (b) TatB or (c) TatC. The proteins present at each step of the purifications were analysed by SDS-PAGE and visualised either by Coomassie brilliant blue staining (upper panel) or immunoblotting using antiseradirected against TatA, TatB or TatC as indicated (lower panels). Fractions are (Tþ) whole cells induced for TatABCDEoverproduction; (S) high speed supernatant and (M) total membranes following subcellular fractionation of the cells;(SM) the Chaps-solubilized membrane supernatant; the (Ft) unbound and (Ni) pooled Tat-protein-containing boundfractions following application of the solubilized membrane extract to a Ni 2þ affinity column. Lane (T2) showswhole cells that were not induced for TatABCDE overproduction. The molecular masses (kDa) of standard proteinsare given to the left of the Coomassie blue-stained gels.proteins isolated by affinity chromatography werepurified away from contaminating proteins by gelpermeation chromatography and were found in apeak of apparent molecular mass .650 kDa(Figure 3(b)). Thus a TatB-specific purification ledto the isolation of a high molecular mass Tat(A)BCcomplex similar to that obtained when the hexahistidinetag was located on the TatC componentAFr. Nr. 8 9 10 11 12 13 14TatBTatCTatAB9 10 11 1213CTatBTatCTatA8 9 10 11 12 13TatBTatCTatAFr. Nr.2 4 6 8 10 12 14 16 18 20 22 24 4 6 8 10 12 14 16 18 20 22 24 4 6 8 10 12 14 16 18 20 22 24Vo 669 200 66443 150Vo 669 200 66443 150Vo 669 200 66443 150Figure 3. Gel filtration purification of Tat complexes following subunit-specific affinity chromatography. The boundfractions from the Ni 2þ affinity chromatography separations shown in Figure 2 were each individually pooled, concentratedand subject to size exclusion chromatography. The samples analysed are those in which the histidine tag was on(a) TatA, (b) TatB or (c) TatC. The 280 nm absorbance of the column eluant is plotted in each case. Only the fractionsanalysed by SDS-PAGE and Coomassie staining at the top of the Figure contain Tat proteins. The lane numbers oneach SDS-PAGE gel correlate to the fraction numbers of the elution profile. In addition the positions of the eluted fractionsare shown by the comb above the elution profile. The elution positions of water-soluble standard proteins areshown below the column profile and labelled with the protein mass (kDa). The standards used were (V 0 ) blue dextran,(669 kDa) thyroglobulin, (443 kDa) apoferritin, (200 kDa) alcohol dehydrogenase, (150 kDa) carbonic anhydrase and(66 kDa) bovine serum albumin.

Escherichia coli Tat Protein Transport Complexes 1139(above). Taken together the results of the TatB andTatC-specific purification suggest that TatB preferentiallyassociates with TatC and/or that the interactionof TatA with TatBC is destabilised duringthe affinity chromatography step.Affinity-tagging of the TatA proteinNext we investigated the nature of Tat complexesthat could be purified when a hexahistidineaffinity tag was placed at the carboxy terminus ofTatA. Membranes were prepared from a strainexpressing the modified tat operon on pFAT75AH.All the TatA protein in the detergent-solubilisedextract was bound to the affinity matrix(Figure 2(a), lane 7). In addition, the columnbound almost the entire complement of TatB andTatC (Figure 2(a), lane 7), indicating that these proteinsare found in complexes containing TatA. Gelpermeation chromatography of the pooled boundfractions from the Ni 2þ column indicated that theTat proteins were again found in complexes withan apparent molecular mass of greater than650 kDa (Figure 3(a)).The material isolated with the TatA-specific tagis unlikely to represent a single type of complexsince the Tat proteins did not elute at a constantratio from the Ni 2þ column (data not shown butanalogous to Figure 4(a), see below). Specifically,while the ratio of TatB to TatC in successive fractionswas constant the ratio of TatA to TatBCincreased in fractions eluting at higher imidazoleconcentrations. Such behaviour would be consistentwith, for example, a mixture of Tat(A)BC andTatA(B) complexes since these complexes containdifferent numbers of tag-bearing TatA molecules(and therefore differing affinities for Ni 2þ ) butwould be difficult to separate by gel permeationchromatography because they possess similarapparent molecular masses.We attempted to clarify the nature of the TatAcontainingcomplexes present in the detergentsolublisedextract using a double-tagging approachin which TatABC were co-overexpressed with ahexahistidine affinity tag fused to the carboxy terminusof TatA and a streptavidin affinity tagfused to the carboxy terminus of TatC (plasmidpFAT75AhisBCstrep). As a first purification stepthe soluble extract was subjected to Ni 2þ chromatography.The elution profile is similar to thatreported above for the TatA-tagged material withfractions of varying TatA to TatBC ratio (Figure4(a)). The fractions with a high TatA to TatBC ratiowere pooled and re-chromatographed on a Strep-Tactin affinity column to purify those complexesthat contained TatC. Most of the TatA proteinloaded onto the column did not bind and wasrecovered in the flow through fractions (Figure4(b), lane Ft). However, a small amount of TatAwas retained on the column and co-eluted togetherwith most of the TatB and TatC present in theloaded sample (Figure 4(b), lanes 3–6; note thatthese samples were TCA-precipitated and representFigure 4. Purification of a TatABC complex by adouble tagging strategy. TatABC were co-ordinatelyoverproduced with a hexahistidine tag on the carboxyterminus of TatA and a Strep tag at the carboxy terminusof TatC. Membranes from the overproducing strain weresolubilized in Chaps and subject to Ni 2þ affinity chromatography.(a) Fractions eluting from the Ni 2þ affinity columnwith an increasing imidazole gradient. Theproteins present in each successive fraction were analysedby SDS-PAGE and visualised by Coomassie brilliantblue staining. (b) The three right-hand fractionsfrom the Ni 2þ affinity column which contain a largeamount of TatA were pooled (lane L) and loaded on astreptactin column. Unbound proteins were retained(lane Ft). Bound material was then eluted with desthiobiotinand successive fractions collected (eluate, lanes1–6). The proteins present at each step were separatedby SDS-PAGE and visualised by Coomassie brilliantblue staining (upper panel) or immunoblotting usingantisera directed against TatA, TatB or TatC as indicated(lower panels). The molecular masses (kDa) of standardproteins are given to the left of the Coomassie bluestainedgels.material that is 100 times concentrated relative tothe unbound fraction in lane Ft). Detailed characterizationof the TatABC material purified in thisexperiment was precluded by the low yield; theentire material purified from a four litre bacterialculture is loaded on the gel shown in the righthandpanel of Figure 4(b). Nevertheless, it is apparentfrom the Coomassie blue staining intensities(cf. Figure 2(c), lane 7 and Figure 4(b), lane 3) thatthe TatABC complexes purified by the doubletaggingapproach have a considerably higher ratio

1140 Escherichia coli Tat Protein Transport ComplexesABFigure 5. Purification of a TatAcontainingcomplex from a strainT- T+ S M SM Ft Nilacking TatC. TatAB were co-ordinately66TatBoverproduced in a Dtat-ABCDDtatE background with a45hexahistidine tag on the carboxy29TatBTatAterminus of TatA. Membranes from24Fr. Nr. 10 11 12 13 14the overproducing strain were solubilizedin Chaps and subject to Ni20TatA2þaffinity chromatography. (a) The1 2 3 4 5 6 7proteins present at each step of thepurification were analysed by SDS-PAGE and visualised either by CoomassieTatAbrilliant blue staining(upper panel) or immunoblottingusing antisera directed againstTatBTatA or TatB as indicated (lowerFr. Nr. 4 6 8 10 12 14 16 18 20 22 24 261 2 3 4 5 6 7panels). Fractions are (Tþ) wholeVo cells induced for TatAB overproduction;(S) high speed supernatant669 200 66443 150and (M) total membranes followingsubcellular fractionation of thecells; (SM) the Chaps-solubilized membrane supernatant; the (Ft) unbound and (Ni) pooled Tat-protein-containingbound fractions following application of the solubilized membrane extract to a Ni 2þ affinity column. Lane (T2)shows whole cells that were not induced for TatAB overproduction. The molecular masses (kDa) of standard proteinsare given to the left of the Coomassie blue-stained gel. (b) Pooled fractions after the Ni 2þ affinity step were concentratedand further purified by gel permeation chromatography. The 280 nm absorbance of the column eluant is plotted.Proteins in the indicated peak fractions were separated by SDS-PAGE and visualised by Coomassie brilliant blue stainingwith the lane numbers on the SDS-PAGE gel at the top of the Figure corresponding to the fraction numbers of theelution profile. The positions of the fractions analysed by SDS-PAGE are also indicted by the comb above the elutionprofile. The elution positions of water-soluble standard proteins are shown below the column profile and labelledwith the protein mass (kDa). The identities of the marker proteins are given in the legend to Figure 3.of TatA to TatBC than the Tat(A)BC complexes purifiedusing only a TatC-specific step. Indeed thelevels of the TatB and TatC proteins are so low inthe material purified by double tagging that theyare only clearly detected by Coomassie blue stainingin the peak eluted fraction (Figure 4(b), lane3). This experiment demonstrates that the Tat systemis capable of assembling complexes containingall three essential Tat proteins but with very highTatA to TatBC ratios. It also shows that most ofthe histidine-tagged TatA protein present in thedetergent-solubilised extract is found in complexesthat do not contain TatC. In a control experimentwe found that if the solubilized extract wasinitially chromatographed on Strep-Tactin agaroserather than a Ni 2þ affinity column, then a Tat(A)BCcomplex similar to that purified using the constructwith a hexahistidine tag on TatC was isolated (datanot shown). Thus the type of tag on TatC does notinfluence the type of complex purified.Direct interaction between TatA and TatBIt was reported 16 that it is possible to purify ahigh molecular mass TatA(B) complex from astrain that is co-ordinately overexpressingtatABCDE. To determine whether TatC, -D, and/or-E are required for the formation of the TatA(B)complex we undertook purification of TatAcontainingspecies from a DtatABCDDtatE strainco-overexpressing tatA and tatB only. In order tofacilitate purification of TatA-containing complexesthe TatA protein was provided with a carboxyterminalhexahistidine tag.Following overexpression of plasmid pFAT75AhisB,membranes were solubilized by Chapsunder our standard conditions and proteins subjectedto chromatography on Ni 2þ affinity resin.The TatA protein was specifically bound to thecolumn and was co-eluted with a small portion ofthe TatB protein found in the initial extract (Figure5(a), lane 7). The majority of the TatB protein was,however, found in the unbound fraction (Figure5(a), lane 6; note that as in earlier Figures, the totalvolume of the pooled unbound fraction is threetimes that of the pooled bound fraction). Gel permeationchromatography shows that the TatA(B)complexes that bind to the Ni 2þ column have anapparent molecular mass in excess of 650 kDa.In conclusion, although an earlier study 18 proposedthat TatA and TatB only interact via TatC,our data indicate that TatA and TatB are capableof direct interaction when expressed and purifiedin the absence of TatC.Isolated TatA forms large oligomersSince the TatA(B) complex is composed predominantlyof TatA we examined whether TatAalone is capable of forming a high molecular masscomplex. TatA was purified from a DtatABCDDtatEstrain overproducing hexahistidine-tagged TatAfrom plasmid pFAT584 12 using the same solubilisationand Ni 2þ affinity protocol as the TatA(B)

Escherichia coli Tat Protein Transport Complexes 1141AB663524201424203520Fr.Nr.T- T+ MSMFt Ni1 2 3 4 5 6TatA8 9 10 11 12 13 14 15 16 17 18 19 20V 0 669 443200150TatATatAFigure 6. Purification of a TatA-containing complexfrom a strain lacking both TatB and TatC. TatA with ahexahistidine tag on the carboxy terminus was overproducedin a DtatABCDDtatE background. Membranesfrom the overproducing strain were solubilized inChaps and subject to Ni 2þ affinity chromatography. (a)The proteins present at each step of the purificationwere analysed by SDS-PAGE and visualised by Coomassiebrilliant blue staining. Fractions are (Tþ) whole cellsinduced for TatA, (M) total membranes following subcellularfractionation of the cells; (SM) the Chaps-solubilizedmembrane supernatant; the (Ft) unbound and (Ni)pooled Tat-protein-containing bound fractions followingapplication of the solubilized membrane extract to aNi 2þ affinity column. Lane (T2) shows whole cells thatwere not induced for TatA overproduction. (b) Characterizationof TatA complexes by gel permeation chromatography.Separations are shown for the pooledconcentrated fractions from the Ni 2þ affinity column separationperformed in (a) (upper panel) or for the pooledunbound (Ft) fraction from the Ni 2þ affinity column inFigure 2(c) (lower panel). The proteins present in theeluted fractions were analysed by SDS-PAGE and visualisedby Coomassie brilliant blue staining. The molecularmasses in kDa of standard proteins are given to the leftof the Coomassie blue-stained gels. The elution positionsof water-soluble standard proteins are shown below thecolumn profile and labelled with the protein mass inkDa. The identities of the marker proteins are given inthe legend to Figure 3.purification described above. TatA was recoveredat high purity after Ni 2þ chromatography (Figure6(a), lane 6). However, the isolated TatA proteinwas found over a broad range of apparent molecularmasses as judged by gel permeation chromatographywith a major peak centred at approximately400 kDa and a minor peak below 65 kDa (Figure6(b), upper panel). Thus TatA expressed in theabsence of other Tat components is apparently66more heterogeneous with respect to oligomericstate or molecular conformation than TatAexpressed in their presence. Nevertheless, giventhat the mass of a TatA protomer is 9.6 kDa, it isclear that isolated TatA protein is still mainly presentas higher order oligomers.In the TatC-specific affinity purificationdescribed above the majority of the TatA moleculespresent in the initial soluble extract were notretained on the Ni 2þ affinity column (Figure 2(c),lane 6). Interestingly, gel permeation chromatographydemonstrates that the apparent molecularmass of the TatA in these flow-through fractions issimilar to that of the peak fractions of TatAexpressed in the absence of other Tat components(compare lower and upper panels in Figure 6(b)).This indicates that Tat(A)BC and high apparentmolecular mass TatA complexes are present simultaneouslyin the detergent-solubilised extract.Binding of twin-arginine signal peptides byTatC-containing complexesIn the initial descriptions of E. coli Tat complexpurification 16,18 the functional integrity of the variouspreparations was not addressed. Here, weconsidered that an assay of the initial step of thetranslocation cycle, in which the substrate proteintwin-arginine signal peptide is recognised by oneor more components of the Tat translocationmachinery, might be tractable to in vitro analysis.We therefore decided to test whether our purifiedTat complexes contained a functional twin-argininesignal peptide binding site.We took a direct approach in which we monitoredchanges in the tryptophan fluorescence propertiesof the purified complexes upon addition of achemically synthesised Tat signal peptide. The fluorescenceproperties of tryptophan residues aresensitive, endogenous reporters of their localenvironment. TatA and TatB each contain a singletryptophan residue while TatC possesses twotryptophan side-chains. For these experiments theaffinity-tagged Tat complexes described abovewere purified in the detergent C 12 E 9 rather than inChaps in order to prevent minor backgroundabsorbance. The Tat protein complexes purified inC 12 E 9 were indistinguishable on the basis of proteincomposition, purity, or apparent molecular massby gel permeation chromatography from those preparedin Chaps, with the exception that isolatedTatA had a less heterogeneous gel permeation profile(data not shown). The signal peptide used inthese experiments was that of the SufI protein thathad been used in the in vitro binding and transportexperiments described above (Figure 1). The isolatedSufI twin-arginine signal peptide has beenshown to competitively inhibit Tat transport invitro. 15 Thus the signal peptide alone is capable offunctional interaction with the E. coli Tat apparatusdespite the absence of its cognate passenger protein.As a control for the specificity of any interactionbetween the SufI signal peptide and the

1142 Escherichia coli Tat Protein Transport Complexesa352b352350350emission nm348346344emission nm348346344342342340340338295 300 305 310excitation nm338295 300 305 310excitation nmc352d352350350emission nm348346344emission nm348346344342342340340338295 300 305 310excitation nm338295 300 305 310excitation nmFigure 7. Interaction of purified Tat complexes with signal peptides analysed by REES. Dependence of the positionof the tryptophan emission maximum on the excitation wavelength for Tat complexes purified using the detergentC 12 E 9 . The samples are (a) a Tat(A)BC complex analogous to the preparation shown in Figure 3(c); (b) a sample analogousto that shown in Figure 2(a) containing TatABC with a high molar excess of TatA; (c) a TatA only complex analogousto the preparation shown in Figure 6(b); (d) a TatA(B) complex analogous to that shown in Figure 5(b). Datashown are those for the isolated Tat complexes alone (V) or following addition of either the SufI signal peptide (B) orSufI signal peptide in which the twin arginine residues had been substituted by twin lysine residues (O). It was desirableto carry out the experiments under conditions where the concentration of peptides exceeds the number of signalpeptide binding sites present. Since the number of signal peptide binding sites in each complex was not known, a peptideto protein molar ratio of 100 (based on a mass of 600 kDa for each complex) was employed. Samples were suspendedin a 500 ml final volume of 20 mM Mops (pH 7.2), 20 mM NaCl, 1 mM EDTA, 0.1% C 12 E 9 . Measurementswere carried out at 25 8C using a Perkin–Elmer LS50B fluorimeter. Each point was the average of at least four independentdeterminations and all spectra were corrected for the baseline spectrum of the water Raman peak contribution.purified Tat complexes we employed a second syntheticpeptide in which the twin-arginine residuesof the consensus motif were conservativelyreplaced with two lysine residues. These substitutionshave previously been shown to preventTat-dependent targeting and transport of the SufIprecursor both in vivo 21 and in vitro. 6,15 Furthermore,and in contrast to the authentic “RR”-containing twin-arginine signal peptide, the“KK”-variant signal peptide does not competitivelyinhibit in vitro Tat transport, stronglysuggesting that it has a very low affinity for the signalpeptide binding site of the Tat system. 15None of the purified Tat complexes exhibitedsignificant changes in tryptophan fluorescenceintensity upon addition of the SufI signal peptides(not shown). Instead, interactions were qualitativelymonitored using the so-called Red EdgeExcitation Shift (REES) effect which responds tochanges in the mobility of solvent molecules in theenvironment of the fluorophore. 22,23 A change intryptophan REES was detected for the Tat(A)BCcomplex (isolated via a hexahistidine tag on TatC)in the presence of the RR-containing signal peptide(Figure 7(a)). In contrast, the inactive KK-bearingsignal peptide failed to elicit a detectable changein the Tat(A)BC tryptophan REES (Figure 7(a)). Weconclude that the RR signal peptide interacts withthe Tat(A)BC complex, causing a conformationalchange, while the KK signal peptide either fails tobind or fails to elicit the same change in tryptophanenvironment as the RR signal peptide. Thefact that signal peptide binding occurs in detergentsolution indicates that neither a membraneenvironment nor the protonmotive force isrequired for this interaction. This is consistent

Escherichia coli Tat Protein Transport Complexes 1143with recent reports of energy-independent precursorbinding by the Tat systems of both bacteria15,24 – 27and thylakoids.A specific REES change in response to the RR,but not KK, signal peptide was also detected forTat proteins purified with a TatA-specific tag(Figure 7(b)), a preparation which is probably amixture of TatA(B), Tat(A)BC and TatABC complexes(above). However, no change in tryptophanREES was observed when either the TatA-only orTatA(B) complexes were exposed to either signalpeptide (Figure 7(c) and (d)).DiscussionHere, we report the purification by a double affinity-taggingmethod of a TatABC complex containinga high molar excess of the TatA protein. Thisdemonstrates for the first time that the three essentialTat components are capable of forming a complexin which the three proteins are present in aratio that reflects the high relative abundance ofTatA in the cytoplasmic membrane. 14,16 However,only a small proportion of the Tat proteins presentin the detergent-solublised extract were found insuch a complex. Instead, the major species purifiedunder the standardised expression, solubilisation,and chromatographic conditions utilised hereresemble the Tat(A)BC and TatA(B) complexesdescribed previously. 16,18 Tat(A)BC is based on aTatBC unit with some TatA present while TatA(B)is composed predominantly of TatA. Both types ofcomplex have a high apparent molecular massand will therefore contain multiple copies of atleast the major constituent subunits. Several linesof evidence suggest that these major species reflectthe complexes formed by the Tat proteins in theresting cell membrane when substrate proteinsand the protonmotive force are both absent. Tostart with, the same types of complex are isolatedif the purification protocol is varied. For example,the detergent C 12 E 9 can be substituted for Chapswithout affecting the species purified and othershave reported purification of a Tat(A)BC complexusing a different affinity tag, expression systemand yet another detergent. 18 Thus these two, andonly these two, types of complex are consistentlyisolated by different purification protocols. Indeed,both types of complex are apparently present simultaneouslyin the soluble extract since TatA isfound as a high molecular mass species followingremoval of Tat(A)BC by affinity chromatography(Figure 6(b)). The in vitro Tat transport data(Figure 1) strongly suggest that the major portionof the detergent-solublised Tat proteins are derivedfrom functional Tat complexes. Given that theminor TatABC complex is stable to purification(this work) then the TatA(B) and Tat(A)BC complexesare unlikely to be TatABC fragmentationproducts but rather distinct molecular species intheir own right. It should also be noted that the compositionsof the isolated complexes are consistentwith our current knowledge of the size and subunitinteractions in the E. coli Tat system: it has beenshown that TatA and TatB are present in high molecularmass complexes in detergent-solublisedextracts of membranes from wild-type E. coli cells, 28chemical crosslinking studies in wild-typemembranes 12 have demonstrated homo-oligomericinteractions for both TatA and TatB while there isalso evidence for direct interactions between TatBand TatC 11,18 as well as TatC self interactions. 17 Interestingly,we found that TatA expressed in theabsence of the other Tat proteins still forms high molecularmass complexes (Figure 6), reinforcing theview that TatA protomers predominantly form interactionswith other TatA protomers. 12Using intrinsic tryptophan fluorescence as ourspectroscopic probe we have been able to demonstratebinding of a twin-arginine signal peptide tothe Tat(A)BC complex (Figure 7(a)). This bindingis apparently of a specific nature since a responsewas not detected with the KK variant signal peptide.This is the first report of a functional propertyassociated with any purified Tat component andprovides further evidence for the physiologicalrelevance of this purified complex. A signal peptide-dependentchange in tryptophan fluorescenceproperties was only detected for those preparationsthat contain the TatC protein (Figure 7).This does not, however, allow us to conclude thatthe signal peptide binds to TatC since the particularfluorescence technique employed here onlydetects signal peptide binding indirectly throughchanges in tryptophan environment. Thus, to see afluorescence change the complex under studymust contain not only the subunit(s) that bind thesignal peptide but also the subunit(s) containingthe conformation-sensitive tryptophan residuesand any additional subunits that are required totransduce the effects of signal peptide bindinginto the conformational change that affects theresponsive tryptophan residues. All we can deduceis that TatC must carry out at least one of thesefunctions. Nevertheless our data do not contradictthe current view that Tat signal peptides bind toTatB, TatC or both. 3,26,29The Tat systems of bacteria and plant chloroplastthylakoid membranes are homologous 7,13 andwould therefore be expected to follow broadly thesame organisational and mechanistic principles.Blue native PAGE has been used to investigate theTat complexes found in resting, detergent-solubilisedthylakoids. 26 The major species are an approximately700 kDa complex containing TatC togetherwith the TatB homologue Hcf106, and separatehomo-oligomeric complexes containing the TatAequivalent, Tha4. Thus the types of Tat complexdetected in resting thylakoids are broadly consistentwith those deduced by protein purificationin the bacterium E. coli.In E. coli some TatB co-purifies with TatA whenco-expressed in the absence of TatC protein (Figure5). This demonstrates that TatA and TatB can interactdirectly. In this light it is interesting to note that

1144 Escherichia coli Tat Protein Transport Complexesin resting thylakoids not all the Hcf106 protein isassociated with TatC and that low yield crosslinkingcan be detected between Hcf106 and Tha4under certain conditions. 26Recently it was shown that Tha4 can be efficientlycrosslinked to both Hcf106 and TatC in thylakoidsbut only if a substrate protein and thetransmembrane protonmotive force are bothpresent. 30 These observations were taken to indicatethat an active translocation site is assembledfrom separate Hcf106-TatC and Tha4 components.If this model is correct then the E. coli TatABC complexpurified here could represent the assembledtranslocation site. The small proportion of Tat proteinsrecovered in this state in our experimentscan be rationalised by noting that the overproducedtransporter proteins are likely to be in vastexcess over the native Tat substrates and thereforeonly a low percentage of the transporters will beactively engaged in translocation at the time ofcell breakage. However, it is conceivable that theassembled thylakoid Tha4–Hcf106-TatC complexcould instead correspond to the E. coli Tat(A)BCcomplex, since this also contains TatA.When TatA was provided with a hexahistidinetag essentially all of the TatB and TatC proteinswere also retained on the Ni 2þ affinity column(Figure 2(a), lanes 6 and 7), indicating that allTatBC complexes bind at least one molecule ofTatA. This is a clear difference from the thylakoidTat system where Tha4 does not tightly associatewith Hcf106 or TatC in detergent solution underresting conditions. 26 This raises the possibility thatthe E. coli Tat translocation site is at least partiallypre-assembled relative to that found in restingthylakoids.Materials and MethodsBacterial strains and growth conditionsE. coli strain M15 (F 2 , lac, ara, gal, mtl ) 31 or strainDADE (MC4100 32 DtatABCDDtatE ) 19 both harbouringpREP4 (Kan R , lacI þ , Roche Molecular Biochemicals)were used for expression of tat genes cloned in pQE60.Strains were grown aerobically at 37 8C in Luria-Bertanimedium supplemented with appropriate antibiotics asdescribed. 10Plasmid constructionConstruction of plasmids pFAT75AH and pFAT75CHemployed plasmid pFAT210, which contains the multicloningsite and C-terminal hexahistidine tag codingsequence from plasmid pQE60 (Qiagen) as a PCR-generatedSal I/Cla I fragment in pBluescript II KS þ (Stratagene).The plasmid pFAT75AH expresses tatABCDEwith a sequence encoding a hexahistidine tag at the 3 0end of tatA (a tatA his allele) and was constructed as follows.A 200 bp Cla I-Sac II fragment covering the 5 0 endof the tatB gene was excised from plasmid pFAT15 10 andcloned into pFAT210 to give pFAT581. The tatA genelacking the stop codon was then amplified by PCRusing primers TATA5 10 and TACHIS (5 0 -GCGCAGATCT-CACCTGCTCTTTATCGTGGCGC-3 0 ), digested withEco RI and Bgl II and cloned into pFAT581 to givepFAT582. Finally, a 600 bp Eco RI-Sac II fragment coveringthe tatA his allele was transferred from pFAT582 ontopFAT75 16 that had been predigested with Eco RI andSac II to give pFAT75AH. The plasmid pFAT75CHexpresses tatABCDE with a sequence encoding a carboxy-terminalhexahistidine tag at the 3 0 end of tatC (atatC his allele) and was constructed as follows. A 1200 bpfragment covering the 3 0 end of the tatB gene and all thetatC gene less the stop codon, was amplified withprimers TATCH1 (5 0 -GCGCGTCGACGCGTCGATGGAT-GAACTACGCCAG-3 0 and TATCH2 (5 0 -GCGCAGA TCT-CTTCAGTTTTTTCGCTTTCTGC-3 0 ), digested with Sal Iand Bgl II, and cloned into pFAT210 to give pFAT211.The tatD and tatE genes on plasmid pFAT75 (Sargentet al. 16 ) were then amplified by PCR using primersTATCH3B (5 0 -CGCTGAAGCAGAAAT CGATAAAACT-GAAG-3 0 ) and TATE8 (5 0 -GCGCAAGCTTGGATGGAA-GTTAAGTAA TCC-3 0 ), digested with Cla I/Pst I, andthen cloned into pFAT211 to give pFAT212. Finally, aSac II-Pst I fragment containing the tatC his allele wastransferred from pFAT212 onto pFAT75C that had beenpredigested with the same enzymes, yielding plasmidpFAT75CH.Plasmid pFAT575 expresses tatABCDE with asequence encoding an amino-terminal hexahistidine tagat the 5 0 end of the tatB gene and was constructed as follows.A primer TATBH1 (5 0 -GTGTAATCCGTGCATCAC-CATCACCATCACGTGTTTGATATCGGTTT TAGC-3 0 )covering the start of the tatB gene and including a hexahistidinecoding sequence was used with primerTATBH2 (5 0 -GGAACGCTTCATCGACTCCGCGGCCT-GGCG-3 0 ) to generate a 300 bp PCR product. This PCRproduct was used as a mega-primer together with primerTATA5 10 to generate a 600 bp PCR product that wasdigested with Eco RI and Sac II and then cloned intopFAT75 16 to give pFAT575.Plasmid pFAT75AhisBCstrep was constructed asfollows. The tatA his , tatB and tatC genes were amplifiedby PCR using primers QE1 and TATCSTREP (5 0 -GCGC-GGATCCTTATT TTTCAAACTGTGGGTGCGACCAAT-TCGATTCTTCAGTTTTTTCGCTTTCTGC-3 0 ) with pFA-T75AH as template. The product was digested withEco RI and Bam HI and cloned into pQE60 that had beenpreviously digested with the same enzymes.Plasmid pFAT75AhisB was constructed following PCRamplification of the tatA his and tatB genes from pFA-T75AH using primers QE1 (5 0 -GGCGTATCACGAGGCC-CTTTCG-3 0 ) and TATC2. 9 The product was digested withEco RI and Bam HI and cloned into pQE60 that had beenpreviously digested with the same enzymes.Plasmid pFAT584 expresses tatA his only and has beendescribed. 12In vitro binding and translocationTat wild-type IMVs were prepared from E. coli strainMC4100 and those for tatA his BCDE overexpression fromstrain M15 (pREP4) harbouring plasmid pFAT75AH.Coupled transcription/translation from a pBluescriptSKderivative of plasmid pNR14 21 encoding pre-SufI 15 orplasmid pDMB 33 encoding pro-OmpA were performedas described. 15 Reactions were incubated for 45 minutesat 37 8C. Where appropriate IMVs were added five minutesafter the start of the incubation to a final concentrationof one to two A 280 nm units ml 21 .

Escherichia coli Tat Protein Transport Complexes 1145For the binding experiments the reaction mixture wassubjected to floatation gradient centrifugation 34 adaptedas described. 20 Four fractions were sequentially withdrawnfrom the top of the gradient and subjected toTCA-precipitation. The pelleted material was also recoveredand directly resuspended into SDS-PAGE loadingbuffer.For the translocation experiments, samples weredivided into two halves following the precursor synthesisand incubation reactions. One half was directly subjectedto TCA-precipitation. To the other half was added anequal volume of 1 mg ml 21 Proteinase K and this samplewas incubated for 20 minutes at 25 8C. The Proteinase Kdigestion was stopped by addition of an equal volume ofa 10% TCA solution. This sample was heated for ten minutesat 56 8C to inactivate the Proteinase K and then placedon ice for TCA-precipitation of the proteins.Purification of recombinant Tat complexesE. coli strain M15 was used for purifications involvingplasmids pFAT75AH, pFAT575, pFAT75CH or pFAT75AhisBCstrepwhile strain DADE was used for purificationsinvolving plasmids pFAT584 12 or pFAT75AhisB. Freshlytransformed cells were grown to mid-log phase andexpression was induced by the addition of IPTG to afinal concentration of 2 mM. After three hours cellswere harvested by centrifugation (7000g; 20 minutes)and resuspended in 20 mM Mops (pH 7.2), 200 mMNaCl (buffer A) supplemented with 10 mgml 21 DNaseIand 50 mgml 21 lysozyme. Cells were disrupted by twopassages through a French pressure cell at 5000 psi inbuffer A supplemented with protease inhibitors (Completee,Roche Molecular Biochemicals) at the manufacturer’srecommended concentration. Crude membranefractions were obtained by differential centrifugation asdescribed. 10 These membrane fractions were solubilisedat a protein concentration of 5 mg ml 21 for one hour at4 8C in buffer A containing 50 mM final concentrationChaps. Insoluble material was removed by ultracentrifugationat 150,000g for 30 minutes and the cleared supernatantwas applied to a Ni 2þ -loaded 5 ml HiTrapChelating HP column (Amersham-Pharmacia Biotech)equilibrated in buffer A containing 5 mM Chaps and30 mM imidazole. The column was washed with 25 mlof buffer A containing 5 mM Chaps, 30 mM imidazoleand then developed in 20 ml of the same buffer with alinear gradient of imidazole to a final concentration of750 mM. Fractions containing the purified Tat proteinsas judged by SDS-PAGE and Coomassie brilliant bluestaining were pooled and concentrated by ultrafiltration.In most cases the concentrated sample was then appliedto a superose-6 HR 10/30 gel filtration column (Amersham-PharmaciaBiotech) equilibrated in buffer A containing5 mM Chaps and 1 mM Na 2 EDTA. Alternatively,for the preparation employing pTatAhisBCstrep, thepooled and concentrated samples were supplementedwith 10 mgml 21 avidin and applied to a 1.7 ml gravityflow Streptactin–Sepharose column (Institut fürBioanalytik, Göttingen, Germany) that had been preequilibratedin 2 ml of buffer B (100 mM Tris (pH 8.0),150 mM NaCl, 1 mM Na 2 EDTA, 5 mM Chaps). Thecolumn was washed with 5 ml of buffer B and boundproteins eluted by 3 ml of buffer B containing 2.5 mMdesthiobiotin (Sigma).Tat proteins for use in the REES experiments werepurified in C 12 E 9 rather than Chaps. Membranes weredispersed at a final C 12 E 9 concentration of 1% (v/v) and0.1% (v/v) C 12 E 9 replaced Chaps in the column chromatographysteps.General analytical methodsSDS-PAGE and immunoblotting were carried out asdescribed 35,36 and immunoreactive bands were visualisedwith the ECL detection system (Amersham-PharmaciaBiotech). Immunoaffinity purified anti-TatA and anti-TatB antibodies were prepared as described. 16 The anti-TatC antibody was raised against a synthetic peptide. 15The SufI signal peptides used here were chemicallysynthesised by Affiniti Research Products Ltd. (Mamhead,UK) and had the sequences SLSRRQFIQA SGIALAAG-AVPLKASA (SufI-RR) and SLSKKQFIQASGIALAAGAV-PLKASA (SufI-KK). In these peptides, alanine wassubstituted for the cysteine residue found at position 16in the native SufI signal peptide in order to avoid oxidationproblems during synthesis. This substitution hasno effect on the in vivo transport kinetics of the SufI protein(N. R. Stanley & T.P., unpublished results).AcknowledgementsWe thank Dr Christopher McDevitt for providinga sample of C 12 E 9 -purified Tat(A)BC complexand Jason Southgate and Verity Lyall for technicalassistance. This work was supported by the Commissionof the European Community through theprogramme “ExporteRRs”, by a grant from theWellcome Trust and by the Biotechnology and BiologicalSciences Research Council through grant88/P09634. I.P. is the recipient of a graduate studentshipfrom the Department of Biochemistry,University of Oxford. F.S. and T.P. are RoyalSociety University Research Fellows and B.C.B. isR. J. P. Williams Senior Research Fellow at WadhamCollege, Oxford.References1. Pugsley, A. P. (1993). The complete general-secretorypathway in Gram-negative bacteria. Microbiol. Rev.57, 50–108.2. Manting, E. H. & Driessen, A. J. (2000). Escherichiacoli translocase: the unravelling of a molecularmachine. Mol. Microbiol. 37, 226–238.3. Berks, B. C., Sargent, F. & Palmer, T. (2000). 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USA, 76,4350–4354.Edited by G. von Heijne(Received 1 July 2002; received in revised form 2 August 2002; accepted 2 August 2002)