University of Groningen Sec translocase in action Komarudin, Amalina Ghaisani
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(2) CHAPTER 4 Dynamics of the Interaction between the Two-Helix Finger of SecA ATPase and SecYEG "NBMJOB(IBJTBOJ,PNBSVEJO *OUBO5BVmL "SOPME+.%SJFTTFO.
(3) Chapter 4. ABSTRACT 5IF 4FD USBOTMPDPO JT UIF NBKPS SPVUF GPS QSPUFJO USBOTMPDBUJPO BDSPTT PS JOUP UIF DZUPQMBTNJDNFNCSBOFPGCBDUFSJB*OJUTNJOJNBMGPSN JUDPOTJTUTPGQSPUFJODPOEVDUJOH DIBOOFM 4FD:&( BOE UIF NPUPS QSPUFJO 4FD" #BTFE PO TUSVDUVSBM TUVEJFT PG UIF Thermotoga maritima SecA-SecYEG complex, SecA enters the SecYEG channel via UIFTPDBMMFEUXPIFMJYmOHFS )' XIJDIDPOTJTUTPGUXPTIPSUIFMJDFTQSFTFOUJOUIF ЃIFMJDBMXJOHEPNBJO5IFSPMFPGUIJTJOUFSBDUJPOXJUI4FD:&(JTOPUGVMMZVOEFSTUPPE BOEUXPIZQPUIFTFTQSFWBJM JF UIF)'BDUTBTBMFWFSUPQVTIQSPUFJOTBDSPTTUIF USBOTMPDBUJPOQPSF PSUIF)'GVODUJPOTJOUIFPQFOJOHPGUIF4FD:&(DIBOOFM)FSF XFIBWFBOBMZ[FEUIFEZOBNJDTPGJOUFSBDUJPOCFUXFFOUIF)'PG4FD"XJUI4FD:&( VTJOH'PSTUFS3FTPOBODF&OFSHZ5SBOTGFS '3&5 5IFEBUBEFNPOTUSBUFBTUSPOH'3&5 TJHOBMVQPOUIFJOJUJBMJOUFSBDUJPOCFUXFFOUIF)'PG4FD"BOEBDDFQUPSQPTJUJPOTBUUIF transmembrane domains 2-4 of SecY, that result in the pre-open state but this interaction JTOPUGVSUIFSDIBOHFEVOEFSDPOEJUJPOTPGUSBOTMPDBUJPO. 74.
(4) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. INTRODUCTION Cellular processes depend on many systems of which proteinaceous complexes DPOTUJUVUFBNBKPSSPMF.PSFUIBOPOFUIJSEPGUIFDFMMVMBSQSPUFJOTJOCBDUFSJB BSFMPDBMJ[FE BU UIF DZUPQMBTNJD NFNCSBOF PS PVUTJEF UIF DFMM "GUFS UIFJS TZOUIFTJT BU SJCPTPNFT JOUIFDZUPQMBTN UIFNBKPSJUZPGUIFTFQSPUFJOTBSFEJSFDUFEUPUIF4FDUSBOTMPDBTF and either inserted into the cytoplasmic membrane or translocated to the trans side of UIFDZUPQMBTNJDNFNCSBOF<>5IF4FDUSBOTMPDBTFJOJUTNJOJNBMGPSNDPOTJTUTPGB USBOTMPDBUJPOQPSF4FD:&(BOEBO"51ESJWFONPMFDVMBSNPUPS UIF4FD"QSPUFJO< > SecA is found in cells either as soluble cytosolic form or peripherally associated with cytoplasmic membrane [4] where it can bind to acidic phospholipids [5] and to SecYEG < >5IFFYBDUNFDIBOJTNPGUSBOTMPDBUJPOJTTUJMMOPUGVMMZVOEFSTUPPE1IPTQIPMJQJE bound SecA can diffuse along the membrane surface, and in this state it is triggered GPSUIFIJHIBGmOJUZCJOEJOHUP4FD:&(4FD"BDDFQUTVOGPMEFEQSFQSPUFJOTGSPNUIF NPMFDVMBSDIBQFSPOF4FD#< >4VCTFRVFOUMZ "51CJOEJOHJOJUJBUFTUIFJOTFSUJPOPG signal sequence of the preprotein into the SecYEG pore [10], and this is coupled to the PQFOJOHPGWFDUPSJBMBRVFPVTDIBOOFM<>"51CJOEJOHBMTPFGGFDUTUIFSFMFBTFPG4FD# UIBUDBOSFCJOEBOFXMZTZOUIFTJ[FEQSFQSPUFJOJOUIFDZUPTPM<>"51IZESPMZTJTDBVTFT a dissociation of SecA from the preprotein whereupon SecA can undergo cycles of ATP binding and hydrolysis that are associated with preprotein binding and release and TUFQXJTFUSBOTMPDBUJPO< >*OUIFBCTFODFPG4FD"BTTPDJBUJPO MBSHFSTFHNFOUTPG the preprotein can translocate by sliding [13,15] The structure of SecA has been solved either as a soluble protein as well as in a SecYEGCPVOE GPSN 5IJT IBT MFE UP NBKPS JOTJHIUT JO UIF QPUFOUJBM CJOEJOH NFDIBOJTN BOE DPOGPSNBUJPOBMDIBOHFTJOBTTPDJBUJPOXJUIUIFUSBOTMPDBUJPOQSPDFTT1SFQSPUFJOCJOEJOH PDDVSTBUQSFQSPUFJODSPTTMJOLJOHEPNBJO 119% PSQSFQSPUFJOCJOEJOHEPNBJO 1#% UIBU GPSNTBDMBNQMJLFTUSVDUVSFUPDBQUVSFUIFQPMZQFQUJEF<o>$POWFSTJPOPGFOFSHZ from ATP binding and hydrolysis is carried out by DEAD (Asp-Glu-Ala-Asp) motorEPNBJO<>0UIFSEPNBJOTBSFUIF$UFSNJOBMMJOLFS $5- BOEЃIFMJDBMTDBGGPME EPNBJO )4% PGUIF$EPNBJO<> XIFSFBTUIFAUXPIFMJYmOHFS )' PG)4%IBT CFFOQSPQPTFEUPEJSFDUUIFQPMZQFQUJEFBUUIFFOUSBODFPG4FD:&(QPSF<>'SPNUIF structure of Thermotoga maritima SecA-SecYEG complex, two possible mechanisms for QSPUFJOUSBOTMPDBUJPOJOWPMWJOHUIFUXPIFMJYmOHFSDBOCFEFSJWFE OBNFMZBQPXFSTUSPLF NFDIBOJTNJOXIJDIUIF)'GVODUJPOTBTBMFWFSUPQVTIQSPUFJOTJOUPUIFUSBOTMPDBUJPO QPSF< > PSB#SPXOJBOSBUDIFUNFDIBOJTNJOXIJDIUIF)'GBDJMJUBUFTPQFOJOH of the translocation pore to allow polypeptide segments to slide into the translocation QPSF< >. 75. 4.
(5) Chapter 4. *OQPXFSTUSPLFNFDIBOJTN NPWFNFOUPGUIF)'PG4FD"XPVMESFTVMUTBDIBOHFJO NPMFDVMBS EJTUBODF CFUXFFO UIF )' BOE TQFDJmD SFHJPOT PG 4FD: *O UIF #SPXOJBO SBUDIFU NFDIBOJTN UIF )' MJLFMZ SFNBJOT TJNJMBSMZ QPTJUJPOFE SFMBUJWF UP JUT TJUF PG JOUFSBDUJPOXJUI4FD: CVUUIFQPTJUJPONBZDIBOHFSFMBUJWFUPPUIFSTJUFTPO4FD:)FSF XFDPOEVDUFEBTZTUFNBUJDTUVEZUPNPOJUPSUIFEZOBNJDTPGUIFNPWFNFOUPGUIF)' PG4FD""51BTFCZNFBTVSJOH'3&5TJHOBMTPGBnVPSPQIPSFDPOKVHBUFEUPUIJTSFHJPOPG 4FD"SFMBUJWFUPQPTJUJPOTPO4FD:"EPOPSBOEBOBDDFQUPSnVPSPQIPSFXFSFDPOKVHBUFE UPBTFUPGDZTUFJOFNVUBOUTPG4FD"BOE4FD: SFTQFDUJWFMZ$POGPSNBUJPOBMDIBOHFT XFSFSFDPSEFEVTJOH'3&5UPFYQMPSFUIF)'4FD:JOUFSBDUJPOBUEJGGFSFOUTUBHFTPGUIF USBOTMPDBUJPOQSPDFTT. MATERIAL AND METHODS Chemical and reagents / /%JNFUIZM/ *PEPBDFUZM / /JUSPCFO[0YB %JB[PMZM FUIZMFOFEJBNJOF *"/#% BNJEFBOE5FYBT3FE$2NBMFJNJEFXFSFQVSDIBTFEGSPN*OWJUSPHFO$Z NBMFJNJEF BOE $ZNBMFJNJEF XFSF QVSDIBTFE GSPN (& )FBMUIDBSF -JGF 4DJFODFT 'MVPSFTDFJONBMFJNJEF XBT QVSDIBTFE GSPN .PMFDVMBS 1SPCFT $BUJPO FYDIBOHF DISPNBUPHSBQIZBOECVGGFSFYDIBOHFXFSFQFSGPSNFEPO)J5SBQ41BOE)J5SBQ%FTBMUJOH DPMVNOTXJUI4FQIBEFY(SFTJOGSPN(&)FBMUIDBSF SFTQFDUJWFMZ Bacterial strains and plasmids E. coli TUSBJOTBOEQMBTNJETBSFMJTUFEJO5BCMF%/"NBOJQVMBUJPOTXFSFQFSGPSNFE using E. coli%)Ѓ4JOHMFDZTUFJOFNVUBOUTPG4FD:XFSFDPOTUSVDUFEGSPNDZTUFJOFMFTT template, and resulting fragments were used to substitute the corresponding nucleotide SFHJPOPG4FD:JOQ&,*OUSPEVDUJPOPGDZTUFJOFSFTJEVFXFSFBDDPNQMJTIFEBDDPSEJOH UP 4USBUBHFOF 2VJDL$IBOHF¥ TJUFEJSFDUFE NVUBHFOFTJT LJU "MM TVCTUJUVUJPOT XFSF DPOmSNFECZTFRVFODFBOBMZTJT%/"SFTUSJDUJPOFO[ZNFTXFSFGSPN'FSNFOUBT XIJMF PUIFSDIFNJDBMTXFSFGSPN4JHNB. 76.
(6) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. Table 3 | Strains and plasmids used in this study Strains/plasmids. Description. Source. E. coli SF100. F- åMBD9 HBM& HBM, UIJ SQT- TUS" åQIP"(pvuII), DompT. [26]. E. coli %)B. TVQ& åMBD6 åMBD;@. ITE3 SFD" FOE" HZS" UIJSFM". [27]. &DPMJ#- %&. F-ompT hsdSB(rB-, mB-) gal dcm Ѝ%&. [28]. pAGK002. 8JMEUZQF4FD". This study. pET503. proOmpA C290S. [29]. pET80. QSP0NQ"$4%)'3. [30]. pEK20. Cysteine-less SecYEG. [31]. pEK20-100C. SecY(P100C)EG. This study. pEK20-109C. SecY(G109C)EG. This study. pEK20-179C. SecY(T179C)EG. This study. pT7SecA-Co. Cysteine-less SecA. This study. pT7SecA-792C. 4FD"3$. This study. pT7SecA-794C. SecA Y794C. This study. pT7SecA-795C. SecA A795C. This study. pT7SecA-797C. SecA K797C. This study. pT7SecA-801C. SecA Q801C. This study. 4. 2YHUH[SUHVVLRQSXULÀFDWLRQDQGODEHOLQJRI6HF$DQG6HF<PXWDQWV *OOFSNFNCSBOFWFTJDMFT *.7T DPOUBJOJOHPWFSFYQSFTTFE4FD:&(XFSFJTPMBUFEBT EFTDSJCFE<>4FD# QSP0NQ"$4BOEQSP0NQ"%)'3XFSFQVSJmFEBTEFTDSJCFE <>1SP0NQ"$4XBTMBCFMFEXJUInVPSFTDFJONBMFJNJEF<> -BCFMJOHPGTJOHMFDZTUFJOF4FD:&(XFSFBDDPNQMJTIFECZmSTUJODVCBUJOHNHPG*.7T containing designated overexpressed SecYEG with 1 mM Tris 2-carboxyethylphosphine 5$&1 GPSNJOVUFTGPMMPXFECZJODVCBUJPOJOCVGGFSDPOUBJOJOHFJUIFSç.PG$Z NBMFJNJEFPS5FYBT3FE$2NBMFJNJEFBU¡$POBSPMMJOHCBOLGPSIPVST5IFMBCFMFE *.7TXFSFTUSJQQFEVTJOHVSFBCVGGFS .VSFB N.)&1&4,0)Q) BOEUIFQFMMFU XFSFSFTVTQFOEFEXJUIHMZDFSPM N.5SJT)$MQ) BOEXFSFBOBMZ[FECZ 4%41"(&5IFnVPSFTDFODFXBTWJTVBMJ[FEVTJOH'VKJmMN-"4JNBHFBOBMZ[FS 4FD"XBTQVSJmFEBTEFTDSJCFEFMTFXIFSF<>E. coli BL21 (DE3) harboring wild type 4FD"PSJUTNVUBOUTXBTHSPXOBU¡$BOEJOEVDFECZBEEJUJPOPGN.*TPQSPQZMЄ% 1-thiogalactopyranoside at A600PGBOEGVSUIFSHSPXOGPSIPVST$FMMTXFSFIBSWFTUFE BUYHGPSNJOBU¡$ SFTVTQFOEFEJON.)&1&4,0) Q) BOETUPSFE BU¡$5IFDFMMGSFFFYUSBDUXBTTVCKFDUFEUP)J5SBQ41)1DPMVNOFRVJMJCSBUFEXJUI. 77.
(7) Chapter 4. CVGGFS" N.)&1&4,0) Q) HMZDFSPM ./B$M $PMVNOXBTXBTIFE XJUICVGGFS"TVQQMFNFOUFEXJUI./B$MBOEQSPUFJOTXFSFFMVUFEXJUI./B$M HSBEJFOUJOCVGGFS" Labeling of SecA was accomplished by incubating the protein with a 10-fold molar FYDFTTPGFJUIFS$ZNBMFJNJEFPS*"/#%BU¡$POBSPMMJOHCBOLGPSIPVST5IFQSPUFJO XFSFUIFOTVCKFDUFEUPEFTBMUJOHDPMVNOVTJOHCVGGFS% N.5SJT)$MQ) HMZDFSPM N.,$M JOPSEFSUPSFNPWFGSFFEZFT1SPUFJODPODFOUSBUJPOXFSFFTUJNBUFE TQFDUSPQIPUPNFUSJDBMMZ DPSSFDUFEGPSnVPSPQIPSFBCTPSQUJPOBUON5IFFYUJODUJPO DPFGmDJFOUTVTFEXFSFЇ280 DN¦ M¦GPS4FD" Ї 550 DN¦ M¦ for $ZNBMFJNJEFBOEЇ 480 DN¦ M¦GPS*"/#% In vitro translocation of proOmpA In vitro USBOTMPDBUJPO PG QSP0NQ" XBT BTTBZFE CZ FNQMPZJOH nVPSFTDFJOMBCFMFE QSP0NQ"<>5SBOTMPDBUJPOSFBDUJPOTXFSFTUBSUFECZBEEJOH"51UPUIFUSBOTMPDBUJPO CVGGFSDPOUBJOJOH4FD" 4FD#BOE4FD:&(*.7TBOEJODVCBUFEBU¡$5SBOTMPDBUJPO SFBDUJPOT XFSF UFSNJOBUFE BGUFS NJOVUFT PO JDF CZ QSPUFJOBTF , USFBUNFOU 5IF translocation activity was analyzed by means of SDS PAGE as previously described <>'MVPSFTDFODFJNBHJOHXBTDBSSJFEPVUXJUIB'VKJmMN-"4JNBHFBOBMZ[FS BOERVBOUJmFEVTJOH*NBHF+TPGUXBSF FRET Measurements *OUFSBDUJPOPG4FD"BOE4FD:XBTNFBTVSFECZ'3&5BTTBZ'MVPSFTDFODFFNJTTJPO TQFDUSBXBTSFDPSEFEVTJOH2VBOUB.BTUFS4QFDUSPnVPSJNFUFS 1IPUPO5FDIOPMPHZ *OUFSOBUJPOBM 3FBDUJPOTXFSFDBSSJFEPVUBU$JON.)&1&4,0) Q) N. KCl, 2 mM MgCl2 N.%JUIJPUISFJUPMBOENHN-CPWJOFTFSVNBMCVNJO0CTFSWBUJPO JTTUBSUFEXJUIBEEJOHBQQSPYJNBUFMZO.PG4FD" GPMMPXFECZNHPG*.7TDPOUBJOJOH EFTJHOBUFEPWFSFYQSFTTFE4FD:&( N.OVDMFPUJEFBOEO.QSP0NQ"%)'3 XIFSF GPMEJOH%)'3EPNBJOXBTQFSGPSNFEBTEFTDSJCFE<>%POPSnVPSPQIPSFXBTFYDJUFE BUON BMMTQFDUSBXFSFDPSSFDUFEGPSCBDLHSPVOE'3&5XBTDBMDVMBUFEPOUIFCBTJT PGSBUJPCFUXFFOBDDFQUPSUPEPOPSJOUFOTJUZBTEFTDSJCFE<>. RESULTS Introduction of unique cysteines into SecA and SecY The crystal structure of SecA and protein translocation channel SecYEG from T.maritima TIPXTTFWFSBMSFHJPOTPGJOUFSBDUJPO<>$SJUJDBMJOUFSGBDFTPDDVSCFUXFFOUIFQPMZQFQUJEF DSPTTMJOLJOHEPNBJO 119% PG4FD"BOEUIFMBSHFDZUPQMBTNJD5.MPPQPG4FD:<>. 78.
(8) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. 5IFTUSVDUVSFBMTPTIPXTUIBUUIF)'PGUIF)4%PG4FD"JOTFSUTJOUPUIFUSBOTMPDBUJPO QPSFBOEDPOUBDUTUIFMPPQUIBUDPOOFDUT5.PG4FD:5PTUVEZUIFEZOBNJDTPGUIF JOUFSBDUJPOPGUIF)'PG4FD"XJUI4FD:JOUIFUSBOTMPDBUJPOQSPDFTT BTFUPG4FD" TJOHMFDZTUFJOFNVUBOUTXBTDSFBUFE6OJRVFDZTUFJOFSFTJEVFTXFSFJOUSPEVDFEPOUIF UJQSFHJPOPGUIF)' 'JH" WJBTJUFEJSFDUFENVUBHFOFTJTVTJOHUIFDZTUFJOFMFTT4FD" UFNQMBUFWFDUPSSFTVMUJOH4FD"3$, SecA Y794C, SecA A795C, SecA K797C, SecA 2$5IFTFQSPUFJOTXFSFFYQSFTTFEJOE. coli#- %& BOEQVSJmFE 'JH# . 4. Fig. 1. Cysteine mutation introduction to SecA and SecY. (A) Positions of cysteine mutations in SecY and SecA as mapped on Thermotoga maritima SecYEG and SecA co-structure (PDB: 3DIN) GSPNUXPBOHMFT*NBHFPOUIFMFGUJTWJFXPGUSBOTMPDPOGSPNAMBUFSBMHBUF XIFSFBTJNBHFPOUIF right is view from cisTJEFPGUIFNFNCSBOF*NBHFTIPXJOHUXPIBMWFTPG4FD:DPMPSFEJOSFE 5. 1-5) and blue (TM 6-10); SecE in light brown and SecG in pale green; SecA is only represented by JUTUXPIFMJYmOHFSDPMPSFEJOHSFFOGPSDMBSJUZ"TIPSUTFHNFOUPG5.BOEDZUPTPMJDMPPQPG4FD: JTPNJUUFEGSPNUIFJMMVTUSBUJPOUPQSPWJEFCFUUFSWJFXPGUIFDZTUFJOFQPTJUJPOT:FMMPXCBMMTSFQSFTFOUUIFQPTJUJPOPGTJOHMFDZTUFJOFNVUBOUT(B)$PPNBTTJFTUBJOFE4%41"(&PGQVSJmFE4FD" DZTUFJOFNVUBOUT(C)*.7TDPOUBJOJOHPWFSFYQSFTTFEPG4FD:1$ 4FD:($BOE5$ XIJDIDPSSFTQPOEUP5. 5.BOE5. SFTQFDUJWFMZ. 79.
(9) Chapter 4. *O PSEFS UP OPU JOUFSGFSF XJUI UIF 4FD"4FD: JOUFSBDUJPOT '3&5 QBJST XFSF DSFBUFE CFUXFFO4FD"BOEUIFmSTUIBMWFPG4FD: 5.BXBZGSPNUIFDPOUBDUQPJOUCFUXFFO UIF )' PG 4FD" BOE 4FD: 'JH -JHBOE JOEVDFE DIBOOFM PQFOJOH NBZ UIVT CF SFDPSEFECZBSFEVDFE'3&5TJHOBMCFUXFFO4FD"BOE4FD:4JOHMFDZTUFJOFNVUBUJPOT XFSF HFOFSBUFE JOUP UIF DZTUFJOFMFTT 4FD: JF PO UIF cis interface of TM2b, TM3 BOE5.SFTJEVFTSFTVMUJOH4FD:1$ 4FD:($BOE4FD:5$ SFTQFDUJWFMZ Each of the aforementioned mutants of SecY was cloned into an expression vector and overproduced in E. coli 4'*OOFSNFNCSBOFWFTJDMFTDPOUBJOJOHUIFPWFSFYQSFTTFE 4FD:&(NVUBOUTXFSFJTPMBUFE 'JH$ /DEHOLQJRI6HF$DQG6HF<ZLWKÁXRUHVFHQFHG\HV 5IF VOJRVF DZTUFJOF SFTJEVFT BMMPXFE UIF DPOKVHBUJPO PG UIF EPOPS BOE BDDFQUPS nVPSPQIPSFTQFDJmDBMMZWJBNBMFJNJEFDIFNJTUSZUP4FD"BOE4FD: SFTQFDUJWFMZ1VSJmFE mutants of SecA and inner membrane vesicles harboring overproduced levels of SecY NVUBOUT XFSF MBCFMFE XJUI WBSJPVT EZFT 8F TFMFDUFE UIF $Z$ZNBMFJNJEF BOE *"/#%5FYBT3FE$2-maleimide pair since these two pair have been used successfully JOUIFQSFWJPVT'3&5TUVEJFT< >5IF$ZEZFTBSFSFMBUJWFMZCVMLZ XJUI*"/#%CFJOH relatively smaller than Cy dyes and Texas C2NBMFJNJEF 'JH"BOE" 5PFOTVSF TQFDJmDJUZPGUIF4FD"BOE4FD:DZTUFJOFMBCFMJOH XFUSFBUFE$ZTMFTT4FD" 4FD" 2$ *.7TDPOUBJOJOH$ZTMFTT4FD:BOE4FD:5$XJUIFBDIPGUIFnVPSFTDFOU QSPCFT 'JH#BOE# 5IFEPOPSBOEBDDFQUPSnVPSPQIPSFTDPVMETVDDFTTGVMMZMBCFM the SecA Q801C mutant and SecY T179C, respectively, but were unable to label the $ZTMFTT4FD"BOE$ZTMFTT4FD: 'JH#BOE# /FYU UIFPUIFS4FD"NVUBOUTXFSF UIFOMBCFMFEXJUI$ZNBMFJNJEF 'JH$ BOE*"/#% 'JH% . 80.
(10) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. 4. Fig. 2. Fluorescent labeling of SecA cysteine mutants (A)4USVDUVSFTPGEPOPSnVPSPQIPSFT $ZNBMFJNJEFBOE*"/#%(B)4QFDJmDJUZPG$ZNBMFJNJEFBOE*"/#%MBCFMJOH$ZTMFTT4FD" BOE2$4FD"NVUBOUXFSFMBCFMFEXJUIFJUIFS$ZNBMFJNJEFPS*"/#%(C) SecA was labeled XJUI$ZNBMFJNJEF(D)4FD"XBTMBCFMFEXJUI*"/#%4JNJMBSMBCFMJOHFGmDJFODZXFSFBDIJFWFE GPSBMMNVUBOUT"MMUIFTBNQMFXFSFSVOPOBO4%41"(&HFM GPMMPXFECZWJTVBMJ[BUJPOXJUI'VKJmMN -"4*NBHF"OBMZ[FSBOE$PPNBTTJF#SJMMJBOU#MVF3TUBJOJOH. 5IFUISFFDZTUFJOFNVUBOUTPG4FD:XFSFMBCFMFEXJUIFJUIFS$ZNBMFJNJEFPS5FYBT3FE C2NBMFJNJEF-BCFMJOHXBTEPOFCZJODVCBUJOHUIF*.7TDPOUBJOJOHUIF4FD:NVUBOUT with either dyes followed by urea treatment to inactivate and remove the endogenous 4FD"BOEUPSFNPWFMPPTFMZBTTPDJBUFEEZF5IF*.7TXFSFDPMMFDUFECZDFOUSJGVHBUJPO BOEFYUFOTJWFMZXBTIFEUPSFNPWFUIFSFNBJOJOHGSFFEZFT4FD:DZTUFJOFNVUBOUTIBWF BTJNJMBSEFHSFFPGMBCFMJOHFGmDJFODZXJUIFJUIFS$ZNBMFJNJEFBOE5FYBT3FE$2NBMFJNJEFBTBTTFTTFECZUIFnVPSFTDFODFPWFSDPPNBTTJFTUBJOJOHSBUJPBTTFTTFEJO HFM 'JH$BOE% 5IFTFEBUBTIPXUIBUUIF4FD"BOE4FD:NVUBOUTDBOCFFGGFDUJWFMZ MBCFMFEXJUIBSBOHFPGnVPSPQIPSFTGPS'3&5BOBMZTJT. 81.
(11) Chapter 4. Fig. 3. Fluorescent Labeling of SecY Cysteine mutants. (A)4USVDUVSFTPGBDDFQUPSnVPSPQIPSFT $ZNBMFJNJEFBOE5FYBT3FE$2NBMFJNJEF(B)4QFDJmDJUZPG$ZNBMFJNJEFBOE5FYBT3FE MBCFMJOH*.7TDPOUBJOJOHUIFPWFSFYQSFTTFE$ZTMFTTPS5$4FD:NVUBOUXFSFMBCFMFEXJUI FJUIFS$ZNBMFJNJEFPS5FYBT3FE$2NBMFJNJEF(C)$ZNBMFJNJEFBOE5FYBT3FE$2-maleimJEFMBCFMJOHPGEJGGFSFOUTJOHMFDZTUFJOF4FD:NVUBOUT5IFSFEVDFE$ZNBMFJNJEFnVPSFTDFODF for SecY P100C is explained by lower protein loaded on the gel as seen as from the Coomassie TUBJOJOH(D)5FYBT3FE$2NBMFJNJEFMBCFMJOHPGEJGGFSFOUTJOHMFDZTUFJOF4FD:NVUBOUT. Translocation activity of labeled SecA and SecY variants 4JODFUIFDZTUFJOFNVUBUJPOTBOEUIFnVPSPQIPSFBUUBDINFOUBEESFTTFTUIFGVODUJPOBM )'EPNBJOPG4FD" FBDINVUBOUXBTUFTUFEGPSJUTBCJMJUZJOTVQQPSUJOHUSBOTMPDBUJPO by means of in vitroUSBOTMPDBUJPOBTTBZ5IFBDUJWJUJFTPGVOMBCFMFEBOEMBCFMFE4FD" NVUBOUTXFSFBTTFTTFEVTJOHXJMEUZQF4FD:5IFDZTUFJOFMFTT4FD"NVUBOUTIPXFE TJNJMBSUSBOTMPDBUJPOBDUJWJUZBTXJMEUZQF4FD"4FD"3$ 4FD""$ 4FD",$ BOE4FD"2$BSFBMMBDUJWF CVUUIFJSBDUJWJUZJTMPXFSBTDPNQBSFEUPXJMEUZQF4FD" Substitution of the tyrosine residue in position 794 for a cysteine severely perturbed the QSPUFJOGVODUJPO 'JH" CMBDLCBS 0VSEBUBTIPXTUIBUJOUSPEVDUJPOPGBDZTUFJOFBUUIF $UFSNJOBMFOEPGUIF)'JTNPSFUPMFSBCMFUIBOBUUIF/UFSNJOBMFOE8IFOUIF4FD" NVUBOUTXFSFMBCFMFEXJUI$ZNBMFJNJEF BMM4FD"NVUBOUTMPTUPGJUTBDUJWJUZ 'JH. 82.
(12) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. " HSBZCBS )PXFWFS UIFWBSJBOUTMBCFMFEXJUI*"/#%SFUBJOFEIJHIMFWFMTPGBDUJWJUZ 'JH" XIJUFCBS 3FNBSLBCMZ MBCFMJOHPG4FD":$XJUI*"/#%FWFOSFTUPSFE TPNFPGUIFBDUJWJUZPGUIJTNVUBOUUIBUJTFTTFOUJBMMZJOBDUJWFBGUFSNVUBUJPO5IF4FD" mutants K797C and Q801C show substantial translocation activity after IANBD labeling BOEXFSFGVSUIFSVTFEGPSUIF'3&5BOBMZTJT. 4 Fig. 4. Translocation activity of SecA and SecY cysteine mutants. (A) Normalized translocation BDUJWJUZPGUIFXJMEUZQF DZTUFJOFMFTTBOEDZTUFJOFNVUBOUTPG4FD"UIBUXFSFVOMBCFMFE CMBDL CBST MBCFMFEXJUI$Z HSFZCBST PS*"/#% XIJUFCBST (B) Translocation activity of wild-type BOEDZTUFJOFNVUBOUTPG4FD:UIBUXFSFVOMBCFMFE CMBDLCBST MBCFMFEXJUI$Z HSFZCBST PS 5FYBT3FE XIJUFCBST . 5IFFGGFDUPGDZTUFJOFNVUBUJPOTBOETVCTFRVFOUnVPSPQIPSFMBCFMMJOHJO4FD:XBTBMTP UFTUFEGPSUSBOTMPDBUJPOBDUJWJUZXJUIXJMEUZQF4FD""MM4FD:NVUBOUTXFSFBDUJWFBTXJME UZQF4FD:JOUSBOTMPDBUJPO 'JH# CMBDLCBS BEEJUJPOBMMZJUTBDUJWJUZXBTOPUBGGFDUFE BGUFSMBCFMJOHXJUI$ZNBMFJNJEFEZF 'JH# HSBZCBS 5IFDPOKVHBUJPOPG5FYBT3FE C2-maleimide to SecY still allows translocation, although the activity is slightly reduced BTDPNQBSFEUPUIFVOMBCFMFE4FD: 'JH# XIJUFCBS FRET analysis of the SecA-SecY interaction To examine the interaction between SecA and SecY, and associated dynamical changes, '3&5XBTSFDPSEFECFUXFFOUIF*"/#%EPOPSnVPSPQIPSFJOUSPEVDFEJOUPUIF)'PG 4FD"BOEUIF5FYBT3FEBDDFQUPSnVPSPQIPSFQSFTFOUBUUIFcisJOUFSGBDFPG4FD:&( 'MVPSFTDFODFFNJTTJPOTQFDUSBXFSFSFDPSEFETIPXJOHBDMFBS'3&5TJHOBMCFUXFFO 4FD",*"/#%PS2$*"/#%BOEUIFWBSJPVT5FYBT3FEMBCFMFE4FD:QSPUFJOT &YDJUBUJPOBUONPG*"/#%DPOKVHBUFE4FD"JOTPMVUJPOHFOFSBUFEBTQFDUSVNXJUI NBYJNVNFNJTTJPOBUON"TTIPXOGPSUIF4FD:($5FYBT3FEBOE*"/#% MBCFMFE4FD"QSPUFJOT 'JH" BEEJUJPOPG5FYBT3FE$2NBMFJNJEFDPOKVHBUFE4FD: MPXFSFEUIFnVPSFTDFODFJOUFOTJUZBUONDPODPNJUBOUMZXJUIBOFNJTTJPOQFBLBU. 83.
(13) Chapter 4. ON"MMNFBTVSFNFOUTXFSFDPSSFDUFEGPSCBDLHSPVOEFNJTTJPOPG5FYBT3FEMBCFMFE 4FD:FYDJUFEBUON#JOEJOHPGVOMBCFMFE4FD"UPUIF5FYBT3FEMBCFMFE4FD:EJE OPUSFTVMUJOBOZDIBOHFJOUIF5FYBTSFEnVPSFTDFODF EBUBOPUTIPXO *"/#%JTLOPXOUPDIBOHFJUTnVPSFTDFODFEFQFOEJOHPOUIFQPMBSJUZPGUIFFOWJSPONFOU XIFSFJUSFTJEFT<>0VSNFBTVSFNFOUJOEJDBUFBEJGGFSFODFJOUIFnVPSFTDFODFJOUFOTJUZ PG*"/#%MBCFMFE,$WFSTVT2$4FD"NVUBOU 'JH" CMBDLMJOF 5IJTEJGGFSFODF BMTPSFTVMUFEJOBMPXFS'3&5TJHOBMGPS*"/#%MBCFMFE4FD",$BOEUIFSFTQFDUJWF 4FD:5FYBT3FENVUBOUT5IF'3&5TJHOBMGSPNBMMQBJSTXFSFNFBTVSFEBTSBUJPPGUIF nVPSFTDFODFJOUFOTJUZBUONUPUIFnVPSFTDFODFJOUFOTJUZBUON *611*537 'JH # 5IFJOJUJBMWBMVFPGUIF4FD"2$*"/#%BMPOFJTXIFSFBTUIFBEEJUJPOPG *.7TDPOUBJOJOH4FD:5FYBT3FEJODSFBTFTUIFSBUJPUPBSPVOEJOEJDBUJOHBCJOEJOH FWFOUCFUXFFO4FD"BOE4FD:5IF4FD",*"/#%BOE4FD:5FYBT3FEQBJSZJFMEFE BSBUJPPGBCPVU. 84.
(14) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. 4. )LJ'\QDPLFVRIWZRKHOL[ÀQJHULQELQGLQJDQGLQLQLWLDOVXEVWUDWHSURFHVVLQJ $
(15) EmisTJPOTQFDUSBSFDPSEJOHGPS*"/#% EPOPS DPOKVHBUFE4FD"NVUBOUTBOE5FYBT3FE$NBMFJNJEF 4FD:NVUBOUT5IFMFGUBOESJHIUTQFDUSBDPSSFTQPOEUP4FD"2$*"/#%BOE4FD",$ *"/#% SFTQFDUJWFMZ XJUI4FD:($5FYBT3FE*"/#%MBCFMFE4FD"JTFYDJUFEBUONBOE UIFFNJTTJPOTQFDUSBXBTHFOFSBUFEXJUIBTJOHMFQFBLNBYJNVNFNJTTJPOBU CMBDLMJOF "EEJUJPOPG4FD:5FYBT3FEEFDSFBTFTUIFFNJTTJPOPGEPOPSnVPSPQIPSF BOESFTVMUTJOFOIBODFE FNJTTJPOBUONJOEJDBUJOHFOFSHZUSBOTGFS '3&5 UPUIBUPGBDDFQUPSnVPSPQIPSF SFEMJOF "EEJUJPOPGOVDMFPUJEF ".11/1 CMVFMJOF EJEOPUTJHOJmDBOUMZDIBOHFUIF'3&55IFTFTQFDUSB XFSFDPSSFDUFECZCBDLHSPVOEFNJTTJPOPGCVGGFSBOE4FD:5FYBT3FEBMPOFFYDJUFEBUON (B)'3&5XBTNFBTVSFEBTSBUJPPGnVPSFTDFODFJOUFOTJUZBUON BDDFQUPS UPUIBUPGON EPOPS 5IJTCBSQMPUDPSSFTQPOETUP4FD"2$*"/#%TVQQMFNFOUFEXJUIWBSJPVT4FD:5FYBT 3FENVUBOUTBOEPUIFSUSBOTMPDBUJPODPNQPOFOUT(C)'3&5NFBTVSFNFOUPG4FD",$*"/#% TVQQMFNFOUFEXJUIWBSJPVT4FD:5FYBT3FENVUBOUTBOEPUIFSUSBOTMPDBUJPODPNQPOFOUT. 85.
(16) Chapter 4. 5PBTTVSFUIBUUIF'3&5TJHOBMSFTVMUFEGSPNUIF4FD"4FD:JOUFSBDUJPO BDPNQFUJUJPO FYQFSJNFOUXBTQFSGPSNFEXJUIUIF*"/#%MBCFMFE4FD"2$NVUBOU"EEJUJPOPGB GPMEFYDFTTPGVOMBCFMFE4FD"UPUIF4FD"4FD:NJYUVSFFMJNJOBUFEUIFnVPSFTDFODF BUONXJUIBDPSSFTQPOEJOHJODSFBTFJOUIFnVPSFTDFODFBUON 'JH -JLFXJTF UIF'3&5TJHOBMXBTSFEVDFEXIFO4FD:DPOUBJOJOH*.7TXFSFBEEFEUPUIFNJYUVSF PG*"/#%MBCFMFE4FD"2$BOEGPMEVOMBCFMFE4FD"2$5IJTTVHHFTUTUIBU 4FD"4FD:JOUFSBDUJPOJTEZOBNJD BOEUIBUUIFSFDPSEFE'3&5TJHOBMTFNFSHFEGSPNB TQFDJmDJOUFSBDUJPOCFUXFFO4FD"BOE4FD:. Fig. 6. Unlabeled SecA quenches the FRET signal between Q801C-IANBD and SecY G109C-Texas Red. 5IF'3&5TJHOBMXBTSFEVDFEBGUFSUIFBEEJUJPOPGGPMENPMBSFYDFTTPGVOMBCFMFE4FD"2$UPNJYUVSFPG4FD:($5FYBT3FEBOE4FD"2$*"/#%(A) or Sec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cisTJEFPGUIFUSBOTMPDPO<> "HBJO UIFTFDPOEJUJPOTEJEOPUMFBEUPBOBMUFSFE'3&5TJHOBM 'JH#BOE$ 5IFTF EBUBEFNPOTUSBUFBTUSPOH'3&5TJHOBMCFUXFFOUIF)'SFHJPOPG4FD"BOEnVPSPQIPSFT positioned at TM 2-4 at the cis side of SecY, but indicate that no further changes in the EJTUBODFCFUXFFOUIF)'SFHJPOPG4FD"BOEUIF5.PG4FD:PODFUIFJOUFSBDUJPO IBTCFFOFTUBCMJTIFE. 86.
(17) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. DISCUSSION 4FD"JTUIFNPUPSEPNBJOPGUIF4FDUSBOTMPDBTF*USFDPHOJ[FTBOEBDDFQUTQSFQSPUFJO by means of the signal peptide-binding domain that involves residues of the preprotein DSPTTMJOLJOHEPNBJO 119% BOEIFMJDBMTDBGGPMEEPNBJO )4% <>5PHFUIFSXJUIUIF IFMJDBMXJOHEPNBJO )8% UIFTFSFHJPOTGPSNBQFQUJEFCJOEJOHHSPPWF<>0ODFUIF QSFQSPUFJOJTCPVOE UIF119%TXJOHTBXBZGSPNUIF)8%UPXBSEOVDMFPUJEFCJOEJOH domain 2 (NBD2) in order to release signal sequence and forms a clamp, creating continuous polypeptide guiding channel that is further stabilized by interaction with 4FD:&(< >6QPOUIFSFMFBTF UIFTJHOBMTFRVFODFJOTFSUTJOUPUIFMBUFSBMHBUFPG SecY, causing the displacement of the plug domain and the widening of a central channel <>#ZDPNQBSJOHNPMFDVMBSTUSVDUVSFTPG4FD"BOEUIF4FD"4FD:&(DPDPNQMFY WBSJPVTDPOGPSNBUJPOBMDIBOHFTPG4FD"IBWFCFFOQSPQPTFE'VSUIFSJODPOKVODUJPO XJUIEJTVMmEFCSJEHFDSPTTMJOLJOHFYQFSJNFOUT JUXBTTVHHFTUFEUIBUUIFUJQPGUIF)' of SecA directly interact with the translocating polypeptide and it was proposed that this structure moves up-and-down during the ATPase cycle to pushes the polypeptide to go UISPVHI4FD:&(DIBOOFM< > 5PUFTUUIFBCPWFIZQPUIFTJT XFIBWFVTFE'3&5FYQFSJNFOUTUPEJSFDUMZBEESFTTUIF NPWFNFOUPGUIF)'SFMBUJWFUPmYFEQPTJUJPOPO4FD:5ISPVHIUIFVTFPGBQQSPQSJBUF nVPSFTDFOU EZFT NPWFNFOUT XJUIJO BO BUPNJD SBOHF DBO CF NFBTVSFE BMUIPVHI SFMBUJWFNPUJPOPGUIFQSPCFTBOEFYUFOUPGFOFSHZUSBOTGFSDPVMEMJNJUUIFBDDVSBDZ<> /FWFSUIFMFTT '3&5DBOCFVTFEBTBNPMFDVMBSSVMFSUPEFUFDUTVDIDPOGPSNBUJPOBM DIBOHFTUIBUDBOCFQSFEJDUFEPOUIFCBTJTPGUIFTUSVDUVSBMJOGPSNBUJPO.PWFNFOU PG$EPNBJOIBTBMSFBEZCFFOPCTFSWFECZ'3&5XIFSFNPEVMBUJPOPGUFNQFSBUVSF BOEOVDMFPUJEFCJOEJOHDIBOHFUIFQSPYJNJUZPGUIF119%BOEUIF$EPNBJO<>8F IBWFFOHJOFFSFEFJHIUTJOHMFDZTUFJOFNVUBOUTDPOTJTUJOHPGmWFNVUBOUTJOUIF)'PG SecA and three mutants on cis interface of SecY, using the structural and sequence homology of the E. coli proteins to that of T. maritima 4FD"BOE4FD:&( 1%#%*/ "T FMFDUSPTUBUJD JOUFSBDUJPO GPS CJOEJOH PG 4FD: XJUI UIF 119% BOE )4% EPNBJOT PG SecA mostly occurred through loop of TM 6-7 and 8-9 [37], the cysteine substitutions BOEnVPSFTDFOUMBCFMJOHBUcis ends of TM 2b, 3 and 4 of SecY will not inhibit binding or JOUFSGFSFXJUIUSBOTMPDBUJPOBDUJWJUZBTJOEFFEWFSJmFEXJUIUSBOTMPDBUJPOBTTBZTVTJOHUIF $ZNBMFJNJEFBOE5FYBT3FE$2NBMFNJEFMBCFMFE4FD:NVUBOUT 'JH 0OUIFPUIFSIBOE UIF)'BQQFBSTBTBDBUBMZUJDBMMZBDUJWFEPNBJOBTTPNFPGUIF cysteine mutations resulted in a decreased activity of SecA, in particular the Y794C NVUBUJPO 'JH " "MTP UIF JODPSQPSBUJPO PG UIF CVMLZ nVPSPQIPSF $ZNBMFJNJEF. 87. 4.
(18) Chapter 4. EJTSVQUFEUIF4FD"GVODUJPO CVUDPOKVHBUJPOXJUIUIFMFTTCVMLZnVPSPQIPSF*"/#% ZJFMEFEBTFUPGMBCFMFE4FD"DZTUFJOFNVUBOUTXJUIIJHIBDUJWJUZ3FNBSLBCMZ MBCFMJOH of the Y794C mutant with IANBD even restored some of the activity of this catalytically JOBDUJWFNVUBOU5IJTmOEJOHJTJOBHPPEBHSFFNFOUXJUIBQSFWJPVTTUVEZUIBUTIPXFE 5ZSDPVMECFSFQMBDFECZCVMLZBNJOPBDJET<> Using combinations of donor labeled SecA proteins and inner membrane vesicles DPOUBJOJOHUIFBDDFQUPSMBCFMFE4FD:QSPUFJO '3&5FYQFSJNFOUTXFSFDBSSJFEPVU*OUIF BTTBZ TUSPOH'3&5TJHOBMTXFSFPCTFSWFEXIFO4FD"XBTBEEFEUPUIF*.7TDPOUBJOJOH PWFSFYQSFTTFE4FD: 'JH" JOEJDBUJOHUIFCJOEJOHFWFOUDBOCFSFDPSEFECZ'3&5 4FD"2$*"/#%TIPXFEBIJHIFS'3&5SBUJPXJUIUIFWBSJPVTMBCFMFEQPTJUJPOTPO 4FD:UIBO4FD",$*"/#% .PSFPWFS UIJT '3&5 TJHOBM JT TQFDJmD GPS UIF 4FD" CJOEJOH SFBDUJPO BT JU DPVME CF RVFODIFECZVOMBCFMFE4FD"UIBUDPNQFUFTXJUIMBCFMFE4FD"GPSCJOEJOHUP4FD:&( 3FNBSLBCMZ BOZDPNCJOBUJPOPGOVDMFPUJEFBOETVCTUSBUFUFTUFE JODMVEJOHUIFGPSNBUJPO PGBOBSSFTUFEQSP0NQ"%)'3JOUFSNFEJBUF EJEOPUSFTVMUJOBOBMUFSFE'3&5TJHOBM which suggests that potential conformational changes involving the positioning of the )'BMSFBEZPDDVSBUUIFTUBHFPG4FD"CJOEJOHUP4FD:&( 5IFTFPCTFSWBUJPOTBSFOPUJOBDDPSEBODFXJUIUIFIZQPUIFTJTUIBUUIF)'BDUTBTBO ATP-dependent lever, but does not exclude a lever action in the initial stages of SecA4FD:&(CJOEJOH5IFMBDLPGBO"51FGGFDUPOUIFQPTJUJPOPGUIF)'SFMBUJWFUP4FD: positions at the cis interface of TM2b, TM3 and TM4, is more in line with a study where UIFUJQPG4FD")'XBTDZTUFJOFDSPTTMJOLFEUPUIFMPPQPG5.PG4FD: BOEUIBUXBT GPVOEUPSFNBJOGVMMZGVODUJPOBM<>"TXFPCTFSWFEMBDLPGDIBOHFPG'3&5TJHOBM JU JOEJDBUFEOPDIBOHFJOEJTUBODFCFUXFFO4FD")'BOEUIFcis interface of TM1-5 of 4FD:)FODF UIFQSPUFJODPOGPSNBUJPOSFNBJOFETJNJMBS5IFSFGPSF XFQSPQPTFUIFOFFE GPSGVSUIFSFYQMPSBUJPOPGEFMJDBUFGVODUJPOJOHPGUIF)'JOGBDJMJUBUJOHUIFDIBOOFMPQFOJOH. 88.
(19) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. REFERENCES %SJFTTFO"+./PVXFO/ 1SPUFJO5SBOTMPDBUJPO"DSPTTUIF#BDUFSJBM$ZUPQMBTNJD .FNCSBOFAnnu. Rev. Biochem. 77 o $BCFMMJ3+ $IFO- 5BJ1$0MJWFS%# 4FD"QSPUFJOJTSFRVJSFEGPSTFDSFUPSZQSPUFJO translocation into E. coliNFNCSBOFWFTJDMFTCell 55 o 'FLLFT 1 %SJFTTFO " 1SPUFJO UBSHFUJOH UP UIF CBDUFSJBM DZUPQMBTNJD NFNCSBOF Microbiol. Mol. Biol. Rev. 63 o 0MJWFS%##FDLXJUI+ 3FHVMBUJPOPGBNFNCSBOFDPNQPOFOUSFRVJSFEGPSQSPUFJO secretion in Escherichia coliCell 30 o -JMM3 %PXIBO88JDLOFS8 5IF"51BTFBDUJWJUZPG4FD"JTSFHVMBUFECZBDJEJD QIPTQIPMJQJET TFD: BOEUIFMFBEFSBOENBUVSFEPNBJOTPGQSFDVSTPSQSPUFJOTCell 60, o -JMM 3 $VOOJOHIBN , #SVOEBHF -" *UP , 0MJWFS % 8JDLOFS 8 4FD" QSPUFJO hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coliEMBO J. 8 o )BSUM'6 -FDLFS4 4DIJFCFM& )FOESJDL+18JDLOFS8 5IFCJOEJOHDBTDBEFPG 4FD#UP4FD"UP4FD:&NFEJBUFTQSFQSPUFJOUBSHFUJOHUPUIF&DPMJQMBTNBNFNCSBOFCell 63 o EF,FZ[FS+ WBOEFS4MVJT&0 4QFMCSJOL3&+ /JKTUBE/ EF,SVJKGG# /PVXFO/ WBOEFS%PFT $%SJFTTFO"+. $PWBMFOUMZEJNFSJ[FE4FD"JTGVODUJPOBMJOQSPUFJOUSBOTMPDBUJPOJ. Biol. Chem. 280 o 3BKBQBOEJ5 %PMBO,.0MJWFS%# 5IFmSTUHFOFJOUIFEscherichia coli secA operon, HFOF9 FODPEFTBOPOFTTFOUJBMTFDSFUPSZQSPUFJOJ. Bacteriol. 173 o &OHFMNBO %. 4UFJU[ 5" 5IF TQPOUBOFPVT JOTFSUJPO PG QSPUFJOT JOUP BOE BDSPTT NFNCSBOFT5IFIFMJDBMIBJSQJOIZQPUIFTJTCell 23 o /BUBMF1 4XBWJOH+ 7BO%FS%PFT$ %F,FZ[FS+%SJFTTFO" #JOEJOHPG4FD"UP UIF4FD:&($PNQMFY"DDFMFSBUFTUIF3BUFPG/VDMFPUJEF&YDIBOHFPO4FD"J. Biol. Chem. 279 o 'FLLFT1 WBOEFS%PFT$%SJFTTFO"+ 5IFNPMFDVMBSDIBQFSPOF4FD#JTSFMFBTFE GSPNUIFDBSCPYZUFSNJOVTPG4FD"EVSJOHJOJUJBUJPOPGQSFDVSTPSQSPUFJOUSBOTMPDBUJPOEMBO J. 16 o 4DIJFCFM& %SJFTTFO"+. )BSUM'68JDLOFS8 ϯ)
(20) BOE"51GVODUJPOBUEJGGFSFOU TUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o 7BO%FS8PML+18 %F8JU+(%SJFTTFO" 5IFDBUBMZUJDDZDMFPGUIFEscherichia coli 4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUTEMBO J. 16 o "SLPXJU[3" +PMZ+$8JDLOFS8 5SBOTMPDBUJPODBOESJWFUIFVOGPMEJOHPGBQSFQSPUFJO EPNBJOEMBO J. 12 o 1BQBOJLPV& ,BSBNBOPV4 #BVE$ 'SBOL. 4JBOJEJT( ,FSBNJTBOPV% ,BMPEJNPT$( ,VIO"&DPOPNPV" *EFOUJmDBUJPOPGUIFQSFQSPUFJOCJOEJOHEPNBJOPG4FD"J. Biol. Chem. 280 o. 89. 4.
(21) Chapter 4. .VTJBM4JXFL. 3VTDI4-,FOEBMM%" 1SPCJOHUIFBGmOJUZPG4FD"GPSTJHOBMQFQUJEF JOEJGGFSFOUFOWJSPONFOUTBiochemistry 44 o (FMJT* #POWJO".++ ,FSBNJTBOPV% ,PVLBLJ. (PVSJEJT( ,BSBNBOPV4 &DPOPNPV" ,BMPEJNPT$( 4USVDUVSBMCBTJTGPSTJHOBMTFRVFODFSFDPHOJUJPOCZUIFL%" USBOTMPDBTFNPUPS4FD"BTEFUFSNJOFECZ/.3Cell 131 o ,PPOJO&7(PSCBMFOZB"& "VUPHFOPVTUSBOTMBUJPOSFHVMBUJPOCZEscherichia coli "51BTF4FD"NBZCFNFEJBUFECZBOJOUSJOTJD3/"IFMJDBTFBDUJWJUZPGUIJTQSPUFJOFEBS Lett. 298 o 4JBOJEJT ( ,BSBNBOPV 4 7SPOUPV & #PVMJBT , 3FQBOBT , ,ZSQJEFT / 1PMJUPV "4 &DPOPNPV" $SPTTUBMLCFUXFFODBUBMZUJDBOESFHVMBUPSZFMFNFOUTJOB%&"%NPUPS EPNBJOJTFTTFOUJBMGPS4FD"GVODUJPOEMBO J. 20 o $PPQFS%# 4NJUI7' $SBOF+. 3PUI)$ -JMMZ""3BOEBMM-- 4FD" UIFNPUPSPGUIF TFDSFUJPONBDIJOF CJOETEJWFSTFQBSUOFSTPOPOFJOUFSBDUJWFTVSGBDFJ. Mol. Biol. 382 o &SMBOETPO,+ .JMMFS4#. /BN: 0TCPSOF"3 ;JNNFS+3BQPQPSU5" "SPMFGPSUIF UXPIFMJYmOHFSPGUIF4FD""51BTFJOQSPUFJOUSBOTMPDBUJPONature 455 o 6DIJEB, .PSJ).J[VTIJNB4 4UFQXJTFNPWFNFOUPGQSFQSPUFJOTJOUIFQSPDFTT of translocation across the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 270, o 4JNPO4. 1FTLJO$40TUFS(' 8IBUESJWFTUIFUSBOTMPDBUJPOPGQSPUFJOT Proc. Natl. Acad. Sci. U. S. A. 89 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XP way communication between SecY and SecA suggests a Brownian ratchet mechanism for QSPUFJOUSBOTMPDBUJPOElife 5 #BOFZY ' (FPSHJPV ( In vivo degradation of secreted fusion proteins by the Escherichia coliPVUFSNFNCSBOFQSPUFBTF0NQ5J. Bacteriol. 172 o )BOBIBO% 4UVEJFTPOUSBOTGPSNBUJPOPGEscherichia coliXJUIQMBTNJETJ. Mol. Biol. 166 o ;IBOH94UVEJFS'8 .FDIBOJTNPGJOIJCJUJPOPGCBDUFSJPQIBHF53/"QPMZNFSBTF CZ5MZTP[ZNFJ. Mol. Biol. 269 o ,VTUFST* WBOEFO#PHBBSU( EF8JU+ ,SBTOJLPW7 1PPMNBO#%SJFTTFO" 1VSJmDBUJPO BOEGVODUJPOBMSFDPOTUJUVUJPOPGUIFCBDUFSJBMQSPUFJOUSBOTMPDBUJPOQPSF UIF4FD:&(DPNQMFY Methods Mol. Biol. 619 o #PM3 8JU+(%F%SJFTTFO"+. 5IF"DUJWF1SPUFJODPOEVDUJOH$IBOOFMPGEscherichia coli$POUBJOTBO"QPMBS1BUDI Ƒ282 o WBOEFS4MVJT&0 /PVXFO/%SJFTTFO"+. 4FD:4FD:BOE4FD:4FD(DPOUBDUT SFWFBMFECZTJUFTQFDJmDDSPTTMJOLJOHFEBS Lett. 527 o ,FZ[FS+%F %PFT$7BO%FS%SJFTTFO"+. ,JOFUJD"OBMZTJTPGUIF5SBOTMPDBUJPOPG Fluorescent Precursor Proteins into Escherichia coli.FNCSBOF7FTJDMFT 277 o ,VTUFST* WBOEFO#PHBBSU( ,FESPW" ,SBTOJLPW7 'VMZBOJ' 1PPMNBO#%SJFTTFO"+. (2011) Quaternary structure of SecA in solution and bound to SecYEG probed at the single NPMFDVMFMFWFMStructure 19 o. 90.
(22) %ZOBNJDTPGUIF*OUFSBDUJPOCFUXFFOUIF5XP)FMJY'JOHFSPG4FD""51BTFBOE4FD:&(. $VOOJOHIBN, -JMM3 $SPPLF& 3JDF. .PPSF, 8JDLOFS80MJWFS% 4FD"QSPUFJO a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional CJOEJOHBOEUSBOTMPDBUJPOPGQSP0NQ"EMBO J. 8 o ,FESPW " ,VTUFST * ,SBTOJLPW 7 7 %SJFTTFO "+. " TJOHMF DPQZ PG 4FD:&( JT TVGmDJFOUGPSQSFQSPUFJOUSBOTMPDBUJPOEMBO J. 30 o (PSZBTIDIFOLP"4 ,ISFOPWB.(4BWJUTLZ"1 %FUFDUJPOPGQSPUFBTFBDUJWJUZCZ nVPSFTDFOU QSPUFJO '3&5 TFOTPST GSPN DPNQVUFS TJNVMBUJPO UP MJWF DFMMT Methods Appl. Fluoresc. 6 ;JNNFS+ /BN:3BQPQPSU5" 4USVDUVSFPGBDPNQMFYPGUIF"51BTF4FD"BOEUIF QSPUFJOUSBOTMPDBUJPODIBOOFMNature 455 o )TJFI)7 4IFSNBO%# "OEBMV[4" "NJTT5+1JUOFS+# 'MVPSFTDFODFSFTPOBODF FOFSHZUSBOTGFSHMVDPTFTFOTPSGSPNTJUFTQFDJmDEVBMMBCFMJOHPGHMVDPTFHBMBDUPTFCJOEJOH QSPUFJOVTJOHMJHBOEQSPUFDUJPOJ. Diabetes Sci. Technol. 6 o .BMPWSI1 7JFSP( 4FSSB.% 1PEMFTFL; -BLFZ+) .BDFL1 .FOFTUSJOB("OEFSMVI G (2003) A novel mechanism of pore formation: membrane penetration by the N-terminal BNQIJQBUIJDSFHJPOPGFRVJOBUPYJOJ. Biol. Chem. 278 o 0TCPSOF "3 $MFNPOT 8. 3BQPQPSU 5" " MBSHF DPOGPSNBUJPOBM DIBOHF PG UIF USBOTMPDBUJPO"51BTF4FD"Proc. Natl. Acad. Sci. U. S. A. 101 o ,BSBNBOPV 4 (PVSJEJT ( 1BQBOJLPV & 4JBOJEJT ( (FMJT * ,FSBNJTBOPV % 7SPOUPV & ,BMPEJNPT$(&DPOPNPV" 1SFQSPUFJODPOUSPMMFEDBUBMZTJTJOUIFIFMJDBTFNPUPSPG 4FD"EMBO J. 26 o 5BN1$, .BJMMBSE"1 $IBO,,:%VPOH' *OWFTUJHBUJOHUIF4FD:QMVHNPWFNFOU BUUIF4FD:&(USBOTMPDBUJPODIBOOFMEMBO J. 24 o )FZEVL 5 .FBTVSJOH QSPUFJO DPOGPSNBUJPOBM DIBOHFT CZ '3&5-3&5 Curr. Opin. Biotechnol. 13 o %JOH) .VLFSKJ*0MJWFS% /VDMFPUJEFBOE1IPTQIPMJQJE%FQFOEFOU$POUSPMPG119% BOE$%PNBJO"TTPDJBUJPOGPS4FD""51BTFBiochemistry 42 o 8IJUFIPVTF4 (PME7B. 3PCTPO" "MMFO8+ 4FTTJPOT3#$PMMJOTPO* .PCJMJUZPG UIF4FD"IFMJYmOHFSJTOPUFTTFOUJBMGPSQPMZQFQUJEFUSBOTMPDBUJPOWJBUIF4FD:&(DPNQMFY J. Cell Biol. 199 o. 91. 4.
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