Jongbloed, Jan Douwe Harm

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   University of GroningenSpecifity determinants for protein secretion in Bacillus subtilis.Jongbloed, Jan Douwe HarmIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2002Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Jongbloed, J. D. H. (2002). Specifity determinants for protein secretion in Bacillus subtilis. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022$L;*r ir :ary,: ir i i  g*n*rai r{:nf lu$ionsChapter 10Summary and general conclusionsIn both prokaryotes and eukaryotes, a largenumber of proteins that are synthesised incytoplasmic ompartments, are transportedacross membranes in order to reach their Íinaldestination a d Íulfil their Íunction. Because oÍthe hydrophobic nature of membranes and thenecessity to maintain their integrity, complex andstrictly organised protein translocation machineryhave volved. Transpoded proteins are usuallysynthesised as precursors containing amino-terminal exporl signals. These signals containinformation about the destination of theseproleins and the transport machinery that shouldbe used to reach their destination. During orimmediately after synthesis of pre-proteins,soluble chaperones and/or targeting factorsrecognise such proteins and assist in targeting ofthe pre-protein to the cytoplasmic membrane.AÍter arriving at the membrane and associationwith the correct translocation machinery, theseproteins are transported through a proteinaceouschannel. lÍ the protein is translocated in anunfolded state, it folds into its Íinal conformationafter leaving the translocase. Depending on thenature oÍ the export signal, the protein is theneither retained in the membrane or releasedupon processing by specialised signalpeptidases (SPases).ln the Gram-positive bacterium Bacillussubtilis al least four pathways Íor protein exportare known. The largest number oÍ secretoryproteins appear to be transporled via the generalSec (protein secretion) pathway. In contrast tothe Sec machinery, which has a general functionin protein transporl, small numbers of proteinsare exported vra dedicated transport pathways.For example, proteins that are involved in theuptake of DNA during competence developmentuse a pseudopil in export machinery, generallyknown as the Com pathway. In addition, somepeptide antibiotics and pheromones are secretedva ATP-binding cassette (ABC) transporters.Finally, proteins can be exported vr,a the twin-arginine translocation (Tat) pathway. Thispathway has probably evolved to Íacilitate thetranslocation oÍ proteins that have to incorporatea cofactor in the cytoplasm, or proteins thatsimply Íold too rapidly or too tightly to be handledby the Sec machinery. Based on specif icfeatures of their respective signal peptides, thetransport route oÍ secretory pre-proteins can bepredicted. While the signal peptides of proteinstransported via the Com pathway or ABCtransporters difÍer significantly Írom Sec{ype ortwin-arginine (RR-) signal peptides, the latter twoshare a characteristic tripartite structure. Duringthe transport process via lhe Sec and/or Tatpathways, the recognition oÍ the respective pre-proteins by specific pathway components isimportant for the subsequent steps in proteintranslocation. Thus, specificity determinants playkey roles during protein export via thesetransport routes of B. subtilis.First, the recognition by variouschaperones and/or targeting Íactors is important.This determines whether the protein wil l fold, orwil l be kept in an unfolded conformation duringits sojourn in the cytoplasm. The latter is requiredfor transport via lhe Sec pathway. Second, thespeciÍic binding to chaperones and/or targetingÍactors, as well as the nature of the signa.peptides of the transported proteins, determinepathway choice. The majority of proteinssynthesised with Sec-type signal peptides wil l betargeted to the Sec machinery. However, asubset of proteins that have Sec{ype signalpeptides but, in addition, contain a so-called RR-or KR-motif have the ootential to travel via theTat pathway. Third, receptor-like components ofthe translocase select precursors for subsequenttranslocation. For example, the Ffh protein hasan important signal peptide receptor function inthe transport oÍ proteins that are exported fromthe cytoplasm vra the Sec pathway. Likewise, thesignal peptides of proteins that travel viathefalpathway may be recognised specifically by onet29- h - , ^ + ^ .  I  í \u '  r c l t J t t ; t  í  t ,of the two paralogous TatC proteins oÍ B. subtilis.Fourth, iÍ paralogous copies of translocationmachinery components are present, speciÍicrecognition of signal peptides and mature partsof the protein is important Íor effective transportof certain proteins. This does not apply to theSec machinery oÍ B. subtilis, since this organismdoes not contain paralogous copies of Sectranslocase components. In contrast, thepresence oÍ paralogous TatA and TatCcomponents that Íunction in Tat-dependenttranslocation in B. subtilis, suggests that thesecomponents might play an important role asspecif icity determinant at the level oÍ thetranslocase. Fifth, in organisms with multipleSPases, these specialised peptidases oftenshow differences in specificity Íor subsets ofprecursors. The availabil ity of the large numberoÍ five difÍerent SPases in B. subtilis suggeststhat at least some of the individual oroteins havespecialised f unctions. Sixth, the presence ofvarious Íolding catalysts implies that theiractivities towards difÍerent secreted proteins varywith the nature of the mature protein. Finally,when post-translocational modifications olsecreted proteins are needed, again componentswith different speciÍicit ies may be involved (r.e.disulphide bond{orming proteins).In this thesis, the role of specificity determinantsfor protein transport in B. subtilis is described.Especially, determinants that Íunction at the levelof pathway choice (signal peptides andtranslocase machinery components) andprecursor processing (SPases) were studied andare discussed.In Chapter 2, experiments are described toinvestigate whether the unique membraneanchor domain oÍ Bacillus SPases is required forin vitro activity. For this purpose, soluble forms ofSipS of B. subtilis, SipS of Bacillusamyloliquefaclens and SipC of the thermophileBacillus caldolyticus, which lack their uniqueamino-terminal membrane anchor domain, wereconstructed. Of these three proteins, only ahexa-histidine tagged soluble Íorm oÍ SipS of B.amyloliquefaciens could be isolated in significanl130quantities. This protein displayed optimal activityat pH 10, which is remarkable considering theÍact that the catalytic domain oÍ SPases islocated in an acidic environment at the outersurface of the membrane of living cells.Strikingly, in contrast to what has beenpreviously reported for the soluble Íorm oÍ theEscherichia coll SPase, soluble SipS was activein the absence of added detergents. Thisobservation can be explained by the Íact that ahighly hydrophobic sudace domain of the E collSPase, implicated in detergent-binding, is absentfrom SipS.High-level production of hexa-histidine taggedsoluble forms oÍ Bacillus SPases in E coll wasunsuccessÍul. Therefore, studies aiming atanswering the question whether suchoverproduction is precluded by proteolysis wereperÍormed and are presented in Chapter 3. Theresults show that the degradation of solubleÍorms oÍ lhe Bacillus SPase SipS is largely dueto self-cleavage. First, catalytically inactivesoluble Íorms oÍ this SPase were not prone t0degradation; in Íact, these mutant proteins wereproduced at very high levels in E. coli. Second,the purified active soluble form of SipS displayedself-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites oÍselÍ-cleavage (between Serl 5 and Met16 oÍ thetruncated enzyme) strongly resembles a typicalSPase cleavage site. Self-cleavage at the latterposition results in complete inactivation oÍ theenzyme, as Ser15 forms the SPase catalyticdyad with Lys55. lronically, selÍ-cleavagebetween Ser15 and Metl 6 can not be preventedby mutagenesis oÍ Gly13 and Ser15, whichconform to the "-1 , -3" rule for SPaserecognition, because these residues are criticalÍor SPase activitv.Since the in vitro characterisation oÍ solubleSPase variants lacking the membrane anchoÍdomain was severely hampered byautodegradation, intact hexa-histidine taggedvariants were overproduced, purified andcharacterised. Chaoter 4 describes ltpoverproduction of several intact Bacfl/us SPasegto high levels. InteresldiÍÍerences in the supuriÍied paralogous SFbe demonstrated ussubstrates. Moreover,role oÍ the membrisubstrate speciÍicitvobtained. Finalty, the nwas shown to be a nstability of Baciilus Spaautodegradation oÍ the cChapter 5 documenrsdiÍference in speciÍicrty tand SipT) and minor tSPases is, at least in rmembrane anchor domalSPases. Notably, the dmrnor Spases was baseocell viability. Because ilbetween major and minoreflected in seguencephylogeny was used to oSPases. The results werrÍrom various bacilli. As prebehaved as major orexpressed in B. subtitis.modelling indicated that thenot a critical parameter Íorand minor Bacfl/us Spastsubstrate-binding site of thesmaller than that of otherminor SipV could be convenwíthout changing this site. lramino-terminal residuescorresponding residues of tlwas sufficient for conversuggests that ditferencesmrnor Spases are baseq orsubstrate cleavage site selerChapter 6 reports the exislTat pathway in B. sdemonstrated by studyingsecretion oÍ phoD, abelonging to a novel proteirKnown members are syntlRR-signal peptides. UntikeSurnrri lry and gener*1 ui:*clusicnsn displayed optimal activityemarkable considering the:ic domain of Spases isenvtronment at the outerlmbrane oÍ living cells.lst to what has beentr the soluble Íorm of the;e, soluble SipS was activeadded detergents. Thisxplained by the Íact that arrface domain oÍ the E. cotiletergent-binding, is absentoÍ hexa-histidine taggedí/us SPases in E coll wasfore, studies aiming atuestion whether suchcluded by proteolysis wereesented in Chapter 3. Thee degradation oÍ solubleSPase SipS is largely duerst, catalytically inactrveSPase were not prone to'lese mutant proteins werelevels in E. coti. Second,ble form oÍ SipS displayeol-hird, as determined by N-rt least one oÍ the sites ofr Ser15 and Metl 6 of theongty resembles a typicalSelÍ-cleavage at the latternplete inactivation oÍ therms the SPase catalyticlronically, self-cleavagert16 can not be preventedi ly13 and Ser15,  which-3" rule Íor Spaserese residues are criticalaracterisation of soluble; the membrane anchor:rely hampered byI hexa-histidine taggedroduced, purif ied and>r 4 describes theal intact Bacillus Spasesto high levels. Interestingly, both similarit ies anddiÍferences in the substrate specificity oÍ thepuriÍ ied paralogous SPases SipS and SipT couldbe demonstrated using diÍÍerent pre-proteinsubstrates. Moreover, evidence for a possiblerole of the membrane anchor domain insubstrate specif icity or accessibility wasobtained. Finally, the membrane anchor domaint,tlas shown to be a major determinant Íor thestability of Bacillus SPases in vitro by preventingautodegradation Í the catalytic domain.Chapter 5 documents the conclusion that thedifference in specificity between the maTbr (SipSand SipT) and minor (SipU, SipV, and SipW)SPases is, at least in part, determined by themembrane anchor domain oÍ these two groups ofSPases. Notably, the distinction in major andmrnor SPases was based on their imoortance Íorcell viability. Because the Íunctional difÍerencebetween major and rnlnor SPases is not clearlyreÍlected in sequence alignments, molecularphylogeny was used to predict major and minorSPases. The results were verified with SPasesÍrom various bacilli. As predicted, these enzymesbehaved as major or mlnor SPases whenexpressed in B. subtilis. Strikingly, molecularmodelling indicated that the active site geometry isnot a critical parameter for classiÍication ol majorand minor Bacillus SPases. Even though thesubstrate-binding site oï the minor SPase SipV issmaller than that of other known SPases, themlnor SipV could be converted into a maybr SPasewithout changing this site. Instead, replacement oÍamino{erminal residues oÍ SioV withcorresponding residues oÍ lhe major SPase SipSwas sufÍicient for conversion of SioV. Thissuggests that differences between major andmrnor SPases are based on activities other thansubstrate cleavage site selection.Chapter 6 reports the existence of a functionalTat pathway in B. subtilis. This wasdemonstrated by studying the TatC-dependentsecretion oÍ PhoD, a phosphodiesterasebelonging to a novel protein Íamily oÍ which allknown members are synthesised with typicalRR-signal peptides. Unlike most organisms oÍwhich the genome has been sequencedcompletely, the Gram-positive bacterium B.subtilis contains two ÍaÍC-like genes, denotedtatCd and tatCy. The Íact that TatCd was shownto be oÍ major importance for the secretion ofPhoD, whereas TatCy is not required Íor thisorocess. indicated for the first time that TatC is aspecificity determinant Íor protein secretion vrathe Tat pathway. Based on these observations, itis hypothesised that the TatC-determineopathway specificity is based on speciÍicinteractions between TatC-like Droteins and otherpathway components, such as TatA, of whichthree paralogs are present in B. subtilis.The studies described in Chapter 7 were aimedat determining the number of extracellular E.subtilis proteins that follow the Tat pathway byapplying proteomic techniques. While mostexported proteins appear to use the Secpathway, 69 oÍ these proteins could potentiallyuse the Tat pathway, as their signal peptidescontain RR- or KR-motifs. The results show thatonly the phosphodiesterase PhoD was secretedin a strictly Tat-dependent manner, whereas 13other proteins are secreted Tat-independently,showing that their RR/KR-motifs are notrecognised by the Tat machinery. In Íact, theextracellular accumulation of three oÍ these 13proteins was shown to be SecA-deoendent. Theobservation that the exoort oÍ LioA is SecA- andnot Tat-dependent is particularly remarkable,because its signal peptide conforms to the moststringent criteria for the prediction oÍ Tat-dependent export in E. coli. Taken together,these observations show that the Tat pathwaymakes a very selective contribution to theextracellular proteome ol B. subtilis.Chapter 8 was aimed at answering the questionwhether certain SPases oÍ B. subtilis have adedicated f unction in the secretion oÍ thephosphodiesterase PhoD, which is translocatedvia lhe Tat pathway oÍ this organism. By usingproteomic techniques it was shown that none ofthe SPases oÍ B. subtilis is specifically requiredÍor PhoD secretion. Furthermore, the resultssuggest that the presence of either SipS or SipTC;hepïsr 1Cis suÍficient Íor this process. Taken together, theobservations presented in this Chapter supportthe view that the type I SPases oÍ B. subtilis arefunctionally redundant and act in a secretionpathway-independent manner.Notably, the secretion of only 14 of the 69 B.subtilis proteins with predicted RR/KR-signapeptides could be visualised by analysis of theextracellular proteome of this organism. Chapter9 documents the use oÍ epitope-tagging andcontrolled gene expression to identify additionaTat substrates of B. subtilis. Using thesetechniques, the YwbN protein was shown to besecreted Tat-dependently. This observation isconsistent with the Íact that YwbN is synthesisedwith an RR-signal peptide that conforms to themost stringent criteria for the prediction of Tat-dependency as defined for known RR-signa.peptides oÍ E. coli. Notably, YwbN is the firstprotein shown to require the TatCy component oÍthe B. subtilis Tat machinery Íor its secretion. Incontrast, the Tatcd component, which is critica,Íor the secretion oÍ PhoD, is not required ÍorYwbN secretion. These findings support he viewthat TatC is a specificity determinant Íor proteinsecretion in B. subtilis.In conclusion, the studies described in this thesisprovide more insights in the role of specificitydeterminants in protein export processes in 8.subtilis. First, the membrane anchor domain oÍSPases was shown to be an imoortantdeterminant for diÍferences in activity, speciÍicityand stability oÍ these enzymes in vitro (Chapters2-4\. Moreover, the diÍference in specificitybetween lhe major (SipS and SipT) and mlnor(SipU, SipV, and SipW) SPases was shown tobe, at least in part, determined by this membraneanchor domain (Chapter 5). Second, it wasshown that signal peptides determine pathwaychoice, but that the Tat pathway is very selectivein the transport oÍ proteins containing potentialRR/KR-motiÍs in their signal peptides (Chapters6 and 7). Third, by studying the secretion of twodiÍferent Tat substrates. the TatC component ofB. subfllrs was shown to be a specificitydeterminant Íor protein secretion (Chapters 6132and 9). Finally, no Tat-dedicated B. subtilisSPase could be identif ied. as the presence ofeither the maJbr SipS or SipT protein was shownto be sufÍicient for the processing oÍ PhoD(Chapter 8).SiIn zowel prokaryotewordt een deel vancytoplasmatischegesynthetiseerd zijn, gmembranen. Dit is nooIn staat te stellen hun Ibereiken en hun Íunktithet hydrofobe karakterde noodzaak hun intrcomplexe en strikt gecmachines geëvolueer,membranen getranspodoorgaans gesynthetiseexpon signalen aan hevan het eiwit, Deze srcniet alleen informatiebestemming van hetinÍormatie aangaande hnodig is om die bestemmoÍ direkt na hun synthesrherkend door cytop(cnaperones en/oÍ targeprecursors begeleiden ccytoplasma_membraan. Nzn aangekomen en (associatie met het correwordt het eiwit ovrgetransporteerd door eengevormd export kanaal. Inongevouwen toestand wrvindt vouwing in zijn ufieinrpassage van het transpor.Y1.1 ", type expon sisvervoÍgens met de memlwordt het mature eiwit (hetPeptide) gesecreteerd als gr(processing) van gespecílsrgnaal peptidases; Spases. In de Gram_positiersubtilis zijn tenminste viereiwit transport over de memmeeste gesecreteerde eivrmembraan via de algeeiwitsecretíe) export route. Inuec-apparaat, met een

Specifity determinants for protein secretion in Bacillus subtilis.

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Specifity determinants for protein secretion in Bacillus subtilis.

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