University of Groningen Sec translocase in action Komarudin, Amalina Ghaisani

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(1)University of Groningen. Sec translocase in action Komarudin, Amalina Ghaisani. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2019 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Komarudin, A. G. (2019). Sec translocase in action: Translocation initiation and processivity. University of Groningen.. Copyright Other 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 the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 28-06-2021.

(2) Sec translocase in action Translocation initiation and processivity. Amalina Ghaisani Komarudin.

(3) Sec translocase in action Translocation initiation and processivity. Amalina Ghaisani Komarudin PhD thesis University of Groningen July 2019. ISBN: 978-94-034-1773-8 (Printed version) ISBN: 978-94-034-1772-1 (Electronic version). The research presented in this thesis was performed in the research group of Molecular Microbiology of the Groningen Biomolecular Science and Biotechnology, University of Groningen, UIF/FUIFSMBOETBOEXBTmOBODJBMMZTVQQPSUFECZUIFJODFOUJWFTDIFNFPGUIF;FSOJLF*OTUJUVUFGPS Advanced Materials and by the Foundation of Life Sciences of the Netherlands Organization for 4DJFOUJmD3FTFBSDI "-8/80 . $PWFSEFTJHO%JOB.BOJBS EJOBNBOJBS!HNBJMDPN BOE%JOBSB/VSBJTIB"EIBSJT -BZPVUEFTJHO"OOB#MFFLFS]XXXQFSTPPOMJKLQSPFGTDISJGUOM 1SJOUJOH*QTLBNQQSJOUJOH XXXQSPFGTDISJGUFOOFU. © 2019, Amalina Ghaisani Komarudin "MMSJHIUTSFTFSWFE/PQBSUPGUIJTUIFTJTNBZCFSFQSPEVDFE TUPSFE PSUSBOTNJUUFEJOBOZGPSN or by any means without the prior permission of the copyright holder, or when applicable, of the QVCMJTIFSTPGUIFTDJFOUJmDQBQFST.

(4) Sec translocase in action Translocation initiation and processivity. PhD thesis. to obtain the degree of PhD at the University of Groningen on the authority of the 5HFWRU0DJQL¿FXVSURI(6WHUNHQ and in accordance with WKHGHFLVLRQE\WKH&ROOHJHRI'HDQV This thesis will be defended in public on 7XHVGD\-XO\DWKRXUV. by. Amalina Ghaisani Komarudin born on 24 February 1990 in Bandung, Indonesia.

(5) Supervisor 3URI$-0'ULHVVHQ. Assessment Committee 3URI-0YDQ'LMO 3URI*0DJOLD 3URI$.HGURY.

(6) For my family.

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(8) TABLE OF CONTENTS. Chapter 1 SecA Mediated Protein Translocation Through SecYEG Channel. 10. Chapter 2 Engineering of the Two-helix Finger of SecA ATPase. 40. Chapter 3 4FD:&(.FEJBUFT5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJO.PEJmFEXJUI/PO1PMZQFQUJEF Segments. 54. Chapter 4 Dynamics of the Interaction between the Two-helix Finger of SecA ATPase and SecYEG. 74. Chapter 5 Summary. 94. Samenvatting. 101. 3JOHLBTBO. 107. Appendices "DLOPXMFEHFNFOUT. 116. About the Author. 121. List of Publications. 121.

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(10) CHAPTER 1 SecA Mediated Protein Translocation Through SecYEG Channel "NBMJOB(IBJTBOJ,PNBSVEJOBOE"SOPME+.%SJFTTFO submitted.

(11) Chapter 1. ABSTRACT *OCBDUFSJB QSPUFJOTBSFTZOUIFTJ[FEBUSJCPTPNFTMPDBMJ[FEJOUIFDZUPTPM1SPUFJOTUIBU reside outside the cytoplasmic membrane need to be targeted to the Sec translocase, UIFNBKPSTZTUFNGPSUIFUSBOTMPDBUJPOPGQSPUFJOTBDSPTTUIFDZUPQMBTNJDNFNCSBOF5IF Sec translocase consists of a hetero-trimeric protein conducting channel SecYEG, the peripheral ATP-dependent motor protein SecA, and the SecDF complex that accelerates USBOTMPDBUJPOBUUIFFYQFOTFPGUIFQSPUPONPUJWFGPSDF5PHFUIFSUIFTFQSPUFJOTGPSNUIF IPMPUSBOTMPDPO)FSF XFQSPWJEFBOPWFSWJFXPGUIFDVSSFOULOPXMFEHFPOUIFGVODUJPO and structure of the Sec translocase, with an emphasis on the minimal functional unit of 4FD"BOE4FD:&(. 10.

(12) SecA Mediated Protein Translocation Through SecYEG Channel. INTRODUCTION 1SPUFJOUSBOTQPSUJTBOPSDIFTUSBUFEQSPDFTTDPNNPOUPBMMEPNBJOTPGMJGF<>1SPUFJOTUIBU OFFEUPGVODUJPOPVUTJEFUIFDZUPTPMBSFUSBOTMPDBUFEBDSPTTUIFMJQJENFNCSBOFCBSSJFS This transport process is mediated by the translocon domain of the Sec translocase: 4FD:&(JOCBDUFSJB<> 4FD:&ЄJOBSDIBFB<>BOE4FDЃЄЅJOUIFNFNCSBOFPGUIF FOEPQMBTNJDSFUJDVMVNPGFVLBSZPUJDDFMMT<>5IFUSBOTMPDPOGPSNTBQSPUFJODPOEVDUJOH channel in the membrane, and conducts the membrane passage of unfolded signal peptide bearing preproteins and are subsequently released at the trans side of the membrane where they fold into their native structure once the signal peptide has been SFNPWFE<>*OBEEJUJPO UIF4FDUSBOTMPDBTFNFEJBUFTUIFDPUSBOTMBUJPOBMJOTFSUJPOPG OBTDFOUNFNCSBOFQSPUFJOTJOUPUIFDZUPQMBTNJDNFNCSBOF*OHFOFSBM UXPNPEFTPG activity can be distinguished: translocation of preproteins after they are fully synthesized (post translational pathway) and insertion of membrane proteins while they are still being USBOTMBUFECZUIFSJCPTPNFT DPUSBOTMBUJPOBMQBUIXBZ 'JH In the post-translational pathway, preproteins are synthesized with a cleavable aminoUFSNJOBMTJHOBMTFRVFODF5IFTFQSPUFJOTBSFCPVOECZUIFNPMFDVMBSDIBQFSPOF4FD# which maintains them in a translocation-competent conformation [6] corresponding to BTUBUFMBDLJOHTUBCMFUFSUJBSZTUSVDUVSF4FD#UBSHFUTQSFQSPUFJOTUPUIF4FD:&(CPVOE 4FD" < > 4FD" JT B NPUPS QSPUFJO BOE JUT "51BTF BDUJWJUZ QSPWJEFT UIF FOFSHZ GPS UIFUSBOTMPDBUJPOQSPDFTT<>"51JOEVDFEDPOGPSNBUJPOBMDIBOHFTPG4FD"ESJWFUIF TUFQXJTFUSBOTMPDBUJPOPGQSFQSPUFJOTUISPVHIUIFUSBOTMPDPO<>%VSJOHUSBOTMPDBUJPO the signal sequence is cleaved off by a membrane bound signal peptidase to yield the mature protein at the transTJEFPGUIFNFNCSBOF<>4FD:&(BMTPBTTPDJBUFTXJUI BDDFTTPSZ4FDDPNQPOFOUT NPTUOPUBCMZUIF4FD%'ZBK$DPNQMFY<>-BSHFTFHNFOUT of the preprotein may be translocated in the presence of the PMF once SecA has released UIFQSFQSPUFJO5IJTQSPDFTTJOWPMWFTUIF4FD%'NFNCSBOFQSPUFJODPNQMFY<> In the co-translational pathway, the nascent proteins are guided to the translocon by TJHOBMSFDPHOJUJPOQBSUJDMF 431 "TTPPOBTBOBTDFOUNFNCSBOFQSPUFJOFNFSHFT GSPN UIF SJCPTPNF JUT mSTU USBOTNFNCSBOF EPNBJO PS B IJHIMZ IZESPQIPCJD TJHOBM TFRVFODF JTSFDPHOJ[FECZ431UPGPSNBUFSOBSZOBTDFOUDIBJO431SJCPTPNF 3/$ DPNQMFY431UBSHFUTUIJTDPNQMFYUPUIF431SFDFQUPS 'UT:BUUIFNFNCSBOFXIFSF 431BOE'UT:GPSNBIFUFSPEJNFS4VCTFRVFOUMZ(51CJOEJOHSFBDUJPOTUPUIF431'UT: IFUFSPEJNFSSFTVMUJOUIFSFMFBTFPGUIFOBTDFOUDIBJOGSPN431BOEBUSBOTGFSUPUIF USBOTMPDPO&VLBSZPUFTNBZVTFUIJTQBUIXBZCPUIGPSQSPUFJOTFDSFUJPOBOENFNCSBOF. 11. 1.

(13) Chapter 1. protein insertion whereas in bacteria it is mostly used for membrane protein insertion <>5IJTSFWJFXGPDVTTFTPOQSPUFJOUSBOTMPDBUJPO. Fig. 1 The Sec pathway. (A) Post translational pathway: after complete synthesis of the preproteins at the ribosome, the unfolded preprotein is recognized by the molecular chaperone SecB (blue) and UBSHFUFEUP4FD" HSFFO 4FD"HVJEFTUIFQSFQSPUFJOUISPVHIUIF4FD:&(QPSF MJNF FNQMPZJOHUIF FOFSHZGSPN"51CJOEJOHBOEIZESPMZTJT5IFTJHOBMQFQUJEFJTDMFBWFECZUIFTJHOBMQFQUJEBTF 41 ZFMMPX 4FD%' QJOL QVMMUIFQSFQSPUFJOBUUIFFYQFOTFPGUIF1.' # $PUSBOTMBUJPOBMQBUIXBZ once a hydrophobic transmembrane domain of a nascent membrane protein emerges from the SJCPTPNFT TJHOBMSFDPHOJUJPOQBSUJDMF 431 CJOETUPUIFSJCPTPNFOBTDFOUDIBJOBOEHVJEFTUIF DPNQMFYUPUIF43SFDFQUPS'UT: CSPXO BUUIFDZUPQMBTNJDNFNCSBOFXIFSFUIFSJCPTPNFOBTDFOU DIBJOJTSFMFBTFEGSPN431BOEUSBOTGFSSFEUPUIF4FD:&(DIBOOFMVQPOUIFCJOEJOHPG(51UPUIF 431'UT:IFUFSPEJNFS/FYU NFNCSBOFQSPUFJOTZOUIFTJTBUUIFSJCPTPNFJTEJSFDUMZMJOLFEUPUIF 4FD:&(EFQFOEFOUJOTFSUTJOUPUIFNFNCSBOF. 12.

(14) SecA Mediated Protein Translocation Through SecYEG Channel. SECYEG, THE PROTEIN CONDUCTING CHANNEL 4FD:&(GPSNTUIFDPSFPGUIF4FDUSBOTMPDBTF5IJTDPNQMFYIBTCFFODSZTUBMJ[FEJO different states, and structures have been obtained from both bacterial and archaeal USBOTMPDPOT3FDFOUTUVEJFTTVHHFTUUIBUUIFTFTUSVDUVSFTUIBUDBOCFDBUFHPSJ[FEJOUP BSFTUJOHPSDMPTFETUBUF 1%#FOUSZ3) <> BQSFPQFOFETUBUFQSJNFECZ SecA (PDB entry 3DIN) [15], and 3) an active state engaged or in complex with a signal QFQUJEFTVCTUSBUF<> 1%#FOUSZ&6- 'JH SecYEG structure #BTFEPOUIF9SBZTUSVDUVSFPG4FD:&ЄGSPNMethanocaldococcus jannaschii, and all other structures, SecY consists of two halves formed by transmembrane segments (TMS) BOE<>5IFUXPIBMWFTBSFDPOOFDUFECZBMPPQPG5.4SFTVMUJOHJOB DMBNTIFMMMJLFTUSVDUVSFPGUIFUSBOTMPDPO.PMFDVMBSEZOBNJDTTJNVMBUJPOTTVHHFTUUIBU UIFTIPSUMPPQBUUIFIJOHF )- BMMPXTDMBNTIFMMPQFOJOH<>4FD:JTTIBQFEMJLFBO IPVSHMBTTXJUIBGVOOFMMJLFFOUSBODFBOEBTVCDFOUSBMDPOTUSJDUJPO%VSJOHUSBOTMPDBUJPO clamshell opening results in a widening of the subcentral constriction allowing the QSFQSPUFJOUPJOTFSUJOUPUIFDIBOOFM5IFGSPOUPG4FD:CFUXFFO5.4BOEGPSNTB MBUFSBMHBUFXIJDIPQFOTUPXBSETUIFMJQJECJMBZFS<>"UUIFQFSJQMBTNJDGBDFPGUIF NFNCSBOF UIFFYJUTJUFPGUIFGVOOFMTIBQFEQPSFJTDMPTFECZBOЃIFMJDBMQMVH 5.4B UIBUGPMETCBDLJOUPUIFDIBOOFM< >5IFM. jannaschii4FD:&ЄTUSVDUVSFJTDPOTJEFS UPCFJOBSFTUJOHDMPTFETUBUFXJUIBTFBMFEQPSFXIFSFTJYIZESPQIPCJDSFTJEVFTDMPTFE UIFDFOUSBMDPOTUSJDUJPOSJOHBOEXJUIUIFQMVHDMPTJOHUIFFYJUGVOOFM<> 4FD&TVSSPVOET4FD:BUUIFCBDLPGUIFDPNQMFYBOEFNCSBDFTUIF4FD:DMBNTIFMM structure with a long transmembrane helix that via a hinge is connected to a surfaceFYQPTFE BNQIJQIBUJD IFMJY UIBU DPOUBDUT MPPQT PG 4FD: 4FD& PG UIF BSDIBFPO M. jannaschii consist of only one TMS while the bacterial Escherichia coli SecE has three TMS <>5IFGVODUJPOPGUIFUXPBEEJUJPOBM/UFSNJOBMIFMJDFTJTTUJMMVOLOPXOBTUSVODBUJPO of these two helices in the E. coli SecE does not interfere with functionality although this USVODBUJPOSFOEFSTUIFDPNQMFYMFTTTUBCMF<>*OBDSZPFMFDUSPONJDSPTDPQZTUSVDUVSFPG the ribosome bound SecYEG complex reconstituted in lipid nanodiscs, the two additional 5.4PG4FD&BSFQFSJQIFSBMMZMPDBMJ[FEGSPNUIFDPNQMFYDPOUBDUJOHUIFSJCPTPNF<> 5IFUIJSETVCVOJU4FD(JTQFSJQIFSBMMZCPVOEUP4FD:5IJTTVCVOJUJTOPUFTTFOUJBMGPS cell viability [23,24] but appears to stabilize the resting channel [25] whereby the loop SFHJPOUIBUDPOOFDUTUIFUXP5.4PG4FD(GPMETCBDLJOUPUIFUSBOTMPDBUJPODIBOOFMBU the cisTJEFPGUIFNFNCSBOF<>. 13. 1.

(15) Chapter 1. SecYEG channel opening 5IFMBUFSBMHBUFPQFOJOHNBZGVMmMNVMUJQMFGVODUJPOT*UDSFBUFTBQBUIXBZGPSUIFJOTFSUJPO of transmembrane domains of integral proteins into the phospholipid bilayer [27], and provides a binding site for the incoming signal sequence of a translocating preprotein < >4JHOBMTFRVFODFTDBOCFDSPTTMJOLFEUP5.4BOE5.4PGUIF4FDQ UIF ZFBTU4FD:IPNPMPHJOUIFFOEPQMBTNJDSFUJDVMVN<>5IFTJHOBMTFRVFODFJOUFSDBMBUFT into the lateral gate causes a conformational change involving TMS 7 and 10 and the QMVHEPNBJO5IFNPWFNFOUPG5.4UPXBSET5.4BDUJWBUFTUIFDIBOOFMBOESFTVMUT JOBOPQFOJOHPGUIFDFOUSBMDIBOOFM<>5ISFFPGUIFTJYQPSFSJOHSFTJEVFTBSFMPDBUFE on TMS2 and TMS7 and thus intercalation of the signal sequence between these TMSs DBOCFEJSFDUMZDPVQMFEUPDIBOOFMPQFOJOH<>4FD&QSFTVNBCMZTUBCJMJ[FTUIFUXP halves of SecY when the channel opens and the plug domain is displaced from its DFOUSBMQPTJUJPO< >$IBOOFMPQFOJOHJTBMTPJOnVFODFECZ4FD"BTFWJEFODFECZ the structure of the Thermotoga maritima4FD"4FD:&(DPNQMFY 'JH# <>*OUIJT structure, the C-terminal halve of SecY has moved outwards resulting in a destabilization of the pore ring and a movement of the plug towards TMS7 but in this structure the pore IBTOPUZFUGVMMZPQFOFE5IFMBUFSBMHBUFXJEFOTUPBQQSPYJNBUFMZ¯QSPWJEJOHBHBQ UIBUNBZBMMPXUIFTJHOBMTFRVFODFUPJOTFSUBOEUPTBNQMFUIFQIPTQIPMJQJECJMBZFS )PXFWFS GPSUSBOTMPDBUJPO UIFMBUFSBMHBUFOFFETUPPQFOBMMPXBHBQPGBCPVU¯ CFUXFFO5.4BOE5.4<><> The structure of (FPCBDJMMVTUIFSNPEFOJUSJmDBOTSecYE-SecA complex with a covalently MJOLFETJHOBMTFRVFODFJOUIFUSBOTMPDBUJPODIBOOFMTIPXTUIBUUIFMBUFSBMHBUFVOEFSHPFT MBSHFDPOGPSNBUJPOBMDIBOHFT<>$PNQBSFEUPUIFQPTJUJPOPG5.4JOUIFDMPTFE channel state of the M. jannaschii SecY, this helix in the (UIFSNPEFOJUSJmDBOTSecY has TJHOJmDBOUMZDIBOHFEQPTJUJPO5IFQMVHJTTIJGUFEUPUIFCBDLPGUIFDIBOOFMBOEOPXJTJO DMPTFQSPYJNJUZUPUIF5.4PG4FD&JOMJOFXJUIDSPTTMJOLJOHTUVEJFT< >$PNQBSFEUP the T. maritima SecA-SecYEG structure, TMS7 of the (UIFSNPEFOJUSJmDBOT SecY is tilted 10° relatively to the membrane and the periplasmic ends of TMS3 and TMS7 are now JODMPTFQSPYJNJUZ5IJTDIBOHFHFOFSBUFTBMBSHFPQFOJOHJOUIFMBUFSBMHBUFUIBUBMMPXT GPSTJHOBMTFRVFODFJOUFSDBMBUJPO<>*OUFSFTUJOHMZ UIFQMVHPG(UIFSNPEFOJUSJmDBOT 4FD:JTJOЄTUSBOETTUSVDUVSFXIJDIJTEJGGFSFOUGSPNUIFQSFWJPVTMZЃIFMJDBMTUSVDUVSFT < >)PXFWFS UIFBNJOPBDJETSFTJEVFTJOUIFQMVHEPNBJOBSFQPPSMZDPOTFSWFE [14], while plug domain deletion in yeast Sec61p and E. coli SecY does not to interfere XJUIDFMMWJBCJMJUZCVUJTPOMZJNQPSUBOUGPSFGmDJFOUUSBOTMPDBUJPO< >5IFQMVHEPNBJO is important for signal sequence recognition [35,37,38] as also suggested by molecular dynamics simulations which indicate that the plug domain samples the hydrophobicity PG UIF JODPNJOH QPMZQFQUJEF SFHJPO <> 8IFO UIF JODPNJOH QPMZQFQUJEF SFHJPO JT. 14.

(16) SecA Mediated Protein Translocation Through SecYEG Channel. highly hydrophobic, plug displacement does not occur and the polypeptide segment JTEJSFDUFEUPUIFMBUFSBMHBUFGPSNFNCSBOFJOTFSUJPO$SPTTMJOLJOHTUVEJFTTVHHFTUT BWFSZMBSHFNPWFNFOUPGUIFQMVHBSPVOEŶUPUIF$UFSNJOBMMPPQPG4FD&UP DSFBUFBOVOPCTUSVDUFEQBUIGPSUIFQPMZQFQUJEFUPDSPTTUIFDIBOOFM< >)PXFWFS in vitro translocation is not impaired when the plug is immobilized inside the channel <> JOEJDBUJOHUIBUUIJTMBSHFEJTQMBDFNFOUJTOPUDSJUJDBMGPSUSBOTMPDBUJPO5IFQMVH domain role might mostly serve to stabilize the closed state of SecY [35], and to act as a QFSJQMBTNJDTFBMUPQSFWFOUTVOEFTJSFEJPOMFBLBHFVOEFSUIPTFDPOEJUJPOT. Fig. 2 Structural stages of the translocation channel. (A-C)5IF4FD:&(ЄDSZTUBMTUSVDUVSFT viewed from the membrane. SecY TMS 1-5 (blue), TMS 6-10 (green), plug domain (red), SecE ZFMMPX BOE4FD(Є PSBOHF %' DBSUPPOJMMVTUSBUJPOPG4FD:&(Є5IFJMMVTUSBUJPOTEFQJDUUIF opening of the constriction and movement of the plug domain depending on the state of the USBOTMPDPO "BOE% Methanococcus jannaschii4FD:&Є 1%#FOUSZ3) LOPXOBTUIFDMPTFE PSSFTUJOHDPOGPSNBUJPO #BOE& Thermotoga maritima SecYEG co-crystalized with SecA (not TIPXO JOB.H"%1#F'YCPVOEUSBOTJUJPOTUBUF 1%#FOUSZ%*/ BTBQSFPQFODPOGPSNBUJPO (C and F) (FPCBDJMMVTUIFSNPEFOJUSJmDBOT SecYEG co-crystalized with SecA (not shown) and a signal sequence (magenta) latched into the lateral gate (PDB entry 5EUL), resembling an actively FOHBHFEUSBOTMPDBUJPODIBOOFM. SecYEG pore constriction and width "TFSJFTPGNVUBOUTIBWFCFFOJEFOUJmFEJOsec genes that allow the translocation of preproteins with a defective signal sequence or even when the signal sequence is DPNQMFUFMZ BCTFOU 5IF QSPUFJO MPDBMJ[BUJPO 1SM NVUBUJPOT JNQBJS UIF USBOTMPDBUJPO QSPPGSFBEJOHBDUJWJUZBOEEPOPUEJSFDUMZSFTUPSFTJHOBMTFRVFODFSFDPHOJUJPO<o>. 15. 1.

(17) Chapter 1. 1SMNVUBUJPOIBWFCFFOJEFOUJmFEJOsecY (prlA), secE (prlG), secA (prlD) and secG QSM) The most dominant prlWBSJBOUTBSFGPVOEJO4FD:<o>5IFTFBQQFBSUPEFTUBCJMJ[F UIFDMPTFETUBUFPGUIFUSBOTMPDBUJPODIBOOFM< >1SM"JTUIFNPTUTUVEJFEprl NVUBOUBOEJUTBOBMZTJTIBTZJFMEFENBKPSJOTJHIUTJOUIFNFDIBOJTNPGTVQQSFTTJPO TJHOBMTFRVFODF<>1SM"DBSSZJOHUXPNVUBUJPOT ':JO5.4BOE*/JO5.4 XJUI UIF MPDBMJ[BUJPO BU UIF GBDJOH TJEF PG 5.4 BOE UIF QMVH EPNBJO 5.4B 5IF I408N mutation is responsible for the suppressor activity [49], while the F286Y mutation SFEVDFTUIFTUSFOHUIPG*/NVUBUJPO5IFPQFODPOGPSNBUJPOPG4FD:&(JTTUBCJMJ[FE CZUIFTJHOBMTFRVFODF 4FD"PSUIFSJCPTPNFBOEJOWPMWFTNVMUJQMFJOUFSBDUJPOT<>*O DPOUSBTU UIF1SM"NVUBUJPOBMUFSTUIFDIBOOFMDPOGPSNBUJPO1SM"TIPXTBOJODSFBTFE CJOEJOHBGmOJUZGPS4FD" BOFMFWBUFE4FD""51BTFBDUJWJUZBOEUIF4FD:&DPNQMFYJT EFTUBCJMJ[FE< >*NQPSUBOUMZ 1SM"BMMPXTBNPSFFGmDJFOUUSBOTMPDBUJPOPGOBUJWF preproteins and to a lesser extent also supports translocation of preproteins with a EFGFDUJWF TJHOBM TFRVFODF < > *O UIJT NVUBOU USBOTMPDBUJPO JT MFTT EFQFOEFODF POUIF1.'<>5IJTDBOCFJOUFSQSFUFEBTBSFEVDFEQSPPGSFBEJOHBDUJWJUZ BTTJHOBM TFRVFODFSFDPHOJUJPOJTMFTTTUSJOHFOUJO1SM"NVUBOUTMJLFMZCFDBVTFUIFDIBOOFMJT BMSFBEZJOBNPSFPQFOTUBUF Many of the PrlA mutants cluster around the pore constriction, and indeed electrophysiological studies indicate that these mutations cause an increased ionMFBLBHFBMUIPVHIUIFDIBOOFMJTSBUIFSSFTJMJFOUBHBJOTUJOEJWJEVBMNVUBUJPOT<>5IF IZESPQIPCJDDPOTUSJDUJPOSJOHBQQFBSTUPGVODUJPOBTBHBTLFUUPTFBMUIFUSBOTMPDBUJPO channel when a polypeptide crosses through the pore [14,18] preventing further ion MFBLBHFEVSJOHUSBOTMPDBUJPO5IFQPSFFYIJCJUTBIJHIQMBTUJDJUZBTJUDBOCFXJEFOFE TVDIUIBUJUTVQQPSUTUIFUSBOTMPDBUJPOPGQPMZQFQUJEFTXJUIBOJOUFSOBMEJTVMmEFCSJEHF PSBGPMEJOEVDFECZBDIFNJDBMDSPTTMJOLFS< > BMUIPVHIUIJTUSBOTMPDBUJPOJTIJHIMZ 1.'EFQFOEFOU4FD:BMTPTVQQPSUTUIFUSBOTMPDBUJPOPGQSFQSPUFJOTEFSJWBUJ[FEXJUIB CVMLZnVPSPQIPSFTBUUIF$UFSNJOVT<>"NPMFDVMBSEZOBNJDTTJNVMBUJPOTVHHFTUT UIBUUIFQPSFDBOBDDPNNPEBUFTVCTUSBUFTXJUIBDSPTTTFDUJPOPGVQUP_¯ XJUIPVU SFRVJSJOHPQFOJOHPGUIFMBUFSBMHBUF< >"OFYQFSJNFOUBMTUVEZEFNPOTUSBUFTUIBU TVCTUSBUFVQUP_"<>DBOQBTTUIFQPSFJOBQSPDFTTUIBUMJLFMZJOWPMWFTUIFPQFOFE MBUFSBMHBUFBTBQPSFFYUFOTJPO Oligomeric state of SecYEG The oligomeric state of the SecYEG translocon has been a topic of long debate and DPOUSPWFSTZ 4FD:&( DBO CF QVSJmFE BT B NPOPNFS < > CVU EFQFOEJOH PO UIF EFUFSHFOUDPODFOUSBUJPOBMTPEJNFSTBOEIJHIFSPMJHPNFSTDBOCFPCTFSWFE<o> $SZTUBMMPHSBQIZBOEDSPTTMJOLJOHFYQFSJNFOUTIBWFTVHHFTUFEUIBU4FD:&(NBZGPSN. 16.

(18) SecA Mediated Protein Translocation Through SecYEG Channel. EJNFSTUIBUBSFPSHBOJ[FEJOFJUIFSBACBDLUPCBDL< >PSAGSPOUUPGSPOUBSSBOHFNFOU <>*OUIFACBDLUPCBDLBSSBOHFNFOU UIFEJNFSJOUFSGBDFJTGPSNFECZUIF4FD&QSPUFJOT PGCPUIQSPUPNFST<>"DSZP&.TUVEZPGUIFE. coli SecYEG bound to a ribosomeOBTDFOUQPMZQFQUJEFDPNQMFYTVHHFTUFEBAGSPOUUPGSPOUBSSBOHFNFOUCVUPOMZPOFPG UIFDIBOOFMTDBSSJFEBUSBOTMPDBUJOHQPMZQFQUJEF<>*UXBTTVHHFTUFEUIBUUIFMBUFSBM PQFOJOHNBZDPIFSFTUPHFOFSBUFBMBSHFSQPSF< >)PXFWFS TVDIBOBSSBOHFNFOU would hinder the interaction of SecY with accessories Sec proteins which interact with UIFMBUFSBMHBUF<>"MUIPVHI 4FD:&(NBZBHHSFHBUFJOUPBEJNFSJDTUSVDUVSF UIF GVODUJPOBMSPMFPGUIFEJNFSIBTSFNBJOFEPCTDVSF"DSPTTMJOLJOHTUVEZTIPXFEUIBU POMZPOFDIBOOFMJTVTFEUPUSBOTMPDBUFUIFQPMZQFQUJEFDIBJO< >#ZUIFVTFPG TJOHMF4FD:&(DPNQMFYFTSFDPOTUJUVUFEJOUPOBOPEJTDTBTXFMMBTTJOHMFNPMFDVMF'3&5 TUVEJFT BTJOHMFDPQZPG4FD:&(DPNQMFYXBTTIPXOUPCFTVGmDJFOUGPS4FD"CJOEJOH BOEUSBOTMPDBUJPO BTXFMMBTGPS3/$CJOEJOH<o>5IJTJTJOBHSFFNFOUXJUISFDFOU DSZPFMFDUSPONJDSPTDPQJDTUVEJFTPOUIFTUSVDUVSFPG4FD:&(CPVOEUPB3/$< > and thus ample evidence supports the monomeric SecYEG complex as the minimal GVODUJPOBMVOJU. SECA, AN ATP-DEPENDENT MOTOR PROTEIN SecA is a molecular motor that drives protein translocation by the conversion of chemical energy in the form of ATP into the movement of the polypeptide chain across UIFNFNCSBOF<>"TBTPMVCMFNFNCSBOFQFSJQIFSBMQSPUFJO 4FD"BTTPDJBUFTXJUI the membrane channel SecYEG but it also binds on its own to the phospholipid bilayer BOEUPSJCPTPNFT)PX4FD"FYBDUMZESJWFTQSPUFJOUSBOTMPDBUJPOJTTUJMMVOLOPXO5IF structure of a soluble form SecA has been resolved in various states and from different species, as well as SecA-SecYEG co-structures, providing a glimpse on how function SFMBUFTUPTUSVDUVSF SecA structure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

(19) Chapter 1. 4FD"SFDPHOJ[FTQSFQSPUFJOTVCTUSBUFTWJBJUTQSFQSPUFJODSPTTMJOLJOHEPNBJO 119% <>5IF119%JTUIPVHIUUPIBWFBEZOBNJDSPMFJOUIFBDUJWBUJPOPGUIF"51BTFBDUJWJUZ PG4FD"UIBUJTNPEVMBUFECZQSFQSPUFJOCJOEJOH<>5IFDBSCPYZEPNBJO $EPNBJO PG 4FD"DBOCFEJWJEFEJOUPGPVSTVCEPNBJOTUIFЃIFMJDBMTDBGGPMEEPNBJO )4% XIJDI JOUFSDPOOFDUTBMMPUIFSEPNBJOTPG4FD"<> UIFЃIFMJDBMXJOHEPNBJO )8% UIF JOUSBNPMFDVMBSSFHVMBUPSPG"51IZESPMZTJT *3" PSUXPIFMJYmOHFS )' XIJDIIBT been proposed to contact the preprotein during translocation [76], and the C-terminal MJOLFSEPNBJO $5- *OE. coli UIF$5-IBSCPSTB[JODmOHFSXIJDIQMBZTSPMFJOJOUFSBDUJPO XJUI4FD#< >BOEQIPTQIPMJQJET<> 4FD"FYIJCJUTBMPXCBTBM"51BTFBDUJWJUZ<>5IFBDUJWJUZJTTUJNVMBUFEJOBMMPTUFSJD way by the binding of SecA to the molecular chaperone SecB, SecYEG, preprotein and BOJPOJDQIPTQIPMJQJET< o>5IF"51BTFBDUJWJUZPGDZUPTPMJD4FD"JTJOIJCJUFECZ *3" PSUIF)'XIJDIGPSNTBIFMJYMPPQIFMJYTUSVDUVSFPGUIF)4%UIBUDPOUBDUTCPUI /#%BOE119%4FD"NVUBOUTXJUIBEFGFDUJOUIF*3"/#%JOUFSBDUJPOTIPXBO increased basal ATPase activity and are thus largely uncoupled from preprotein binding JOUIFJS"51BTFBDUJWJUZ<>. Fig. 3. Conformational states of SecA. Structures of (A) SecA from Bacillus subtilis (PDB entry 1M6N), (B) Mg-ADP-BeFx-bound SecA co-crystalized with SecYEG (not shown) from T. maritima (PDB 3DIN), and (C) Mg-ADP-BeFx bound SecA from B. subtilis engaged with the G. thermodeOJUSJmDBOT4FD:&(BOEBTJHOBMTFRVFODF OPUTIPXO 1%#FOUSZ&6- 5IFMPDBUJPOTPGUIF119% ZFMMPX /#% SFE /#% CMVF )8% HSFFO )4% QVSQMF BOE)' DZBO BSFJOEJDBUFE" MBSHFNPWFNFOUPGUIF119% ZFMMPX TVHHFTUTBDMPTFE " BOEPQFO # $ DPOGPSNBUJPOPG4FD". Oligomeric state of SecA 5IF GVODUJPOBM PMJHPNFSJD TUBUF PG 4FD" IBT CFFO B NBJO FMFNFOU PG DPOUSPWFSTZ *NQPSUBOUMZ XIFO 4FD" JT QVSJmFE GSPN DFMMT JU JT NBJOMZ EJNFSJD <> " NVMUJUVEF PGTUVEJFTEFNPOTUSBUFUIBU4FD"GVODUJPOTBTBEJNFS<o>*OTPMVUJPO 4FD"JT GPVOEBTBOBOUJQBSBMMFMIPNPEJNFS< >4FD"GSPNThermus thermophilus has been DSZTUBMMJ[FEBTBQBSBMMFMEJNFS<>5IF4FD"NPOPNFSEJNFSFRVJMJCSJVNJTBGGFDUFECZ UIFMJHBOETPG4FD"4FD"IBTCFFOSFQPSUFEUPCFEJNFSJDJOUIFQSFTFODFPGTZOUIFUJD. 18.

(20) SecA Mediated Protein Translocation Through SecYEG Channel. TJHOBMQFQUJEFT<>PSXIFOCPVOEUPQIPTQIPMJQJEJOUIFQSFTFODFPGOVDMFPUJEFT<> Convergingly, other studies suggests the opposite, that SecA monomerization occurs JOUIFQSFTFODFPGQIPTQIPMJQJET<>BOETZOUIFUJDTJHOBMQFQUJEFT<>*UIBTCFFO suggested that electrostatic and hydrophobic interactions maintain the SecA dimer, as the SecA monomer-dimer equilibrium is sensitive to high ionic strength and low UFNQFSBUVSF< >0OUIFPUIFSIBOE JUJTBMTPLOPXOUIBU4FD"JTIJHIMZUIFSNPMBCJMF JOUIFQSFTFODFPGQIPTQIPMJQJETBOESBQJEMZMPTFTJUTBDUJWJUZ)PXFWFS UIJTFGGFDUJT DPVOUFSBDUFECZQSFQSPUFJO BOEUIBUBDUJWJUZJTUFSNFE4FD"MJQJE"51BTF<>4JODF most of the studies on soluble SecA do not include the functional SecYEG bound state, UIFTJHOJmDBODFPGUIFPGUFODPOnJDUJOHPCTFSWBUJPOTPOUIFRVBUFSOBSZTUBUFPG4FD"JO TPMVUJPOJOSFMBUJPOUPUIFBDUVBMUSBOTMPDBUJPONFDIBOJTNSFNBJOTPCTDVSF 0UIFSTUVEJFTBEESFTTFEUIFPMJHPNFSJDTUBUFPG4FD"XIJMFCPVOEUP4FD:&(4JOHMF molecule analysis demonstrate that SecA remains dimeric during translocation [87,94] BOEJTBDUJWFBTEJNFS< >.VUBUJPOJOEVDFENPOPNFSJ[BUJPOTFWFSFMZSFEVDFT the SecA activity [96], but this defect can be overcome by non-physiologically high DPODFOUSBUJPOTPG4FD"UIBUSFTUPSFTUIFEJNFSJDTUBUF< >5IFNPMFDVMBSDIBQFSPOF SecB interacts with the SecYEG bound dimeric SecA and in this process the two positively DIBSHFE$UFSNJOJPGCPUI4FD"QSPUPNFSTCJOEUPUIFPQQPTJOHBOJPOJDnBUTVSGBDFT PGUIF4FD#IPNPUFUSBNFS<>"TJOHMFNPMFDVMFTUVEZTIPXFEUIBUUIFEJNFSJD4FD" CJOETUIF4FD:&(XJUIIJHIBGmOJUZ XIFSFPOFPGUIFQSPUPNFSTCJOETUJHIMZUP4FD:&( BOEUIFPUIFSQSPUPNFSJTCPVOEUPUIF4FD:&(CPVOE4FD"<>"EEJUJPOBMMZ BSFDFOU in vivo study demonstrated that SecA functions as a discrete anti-parallel dimer to drive QSPUFJOUSBOTMPDBUJPO<>"MUIPVHIBTUSVDUVSFPGUIF4FD"4FD:&(DPNQMFYXJUIB4FD" NPOPNFSXBTTPMWFE< > UIFIJHITBMUVTFEGPSUIFDSZTUBMMJ[BUJPOMJLFMZJOEVDFTUIF EJTTPDJBUJPOPGUIF4FD"EJNFS Binding partners of SecA 5IF/UFSNJOBMTJHOBMTFRVFODFPGQSFQSPUFJOTGVODUJPOTBTBUBSHFUJOHTJHOBM< > Signal sequences have a conserved tripartite structure that consists of a hydrophobic ЃIFMJDBMSFHJPO )EPNBJO UIBUJTnBOLFECZBO/BOE$EPNBJO<>5IF$EPNBJO DPOUBJOTIZESPQIJMJDSFTJEVFTBOEJTVTVBMMZEFWPJEPGQPTJUJWFMZDIBSHFESFTJEVFT5IJT region contains the signal peptidase cleavage site that meets the -1,-3 rule where these UXP QPTJUJPOT DPOUBJO BNJOP BDJET XJUI B TNBMM TJEF DIBJO < > 5IF /EPNBJO contains positively charged amino acid residues that help to orient the signal sequence in response to the transmembrane electrical potential, when the charged residues remain at the cisTJEFPGUIFNFNCSBOF5IFMFOHUIPGUIFTJHOBMTFRVFODFWBSJFT IPXFWFS the minimum length of E. coli signal sequence is 15-16 amino acids, with median of 22. 19. 1.

(21) Chapter 1. BNJOPBDJET<>0UIFSTUVEJFTTVHHFTUUIBUUIFUBSHFUJOHJOGPSNBUJPOJTDPOUBJOFEJO the mature region of a preprotein, and that the signal sequence only serves to initiate translocation [105,106] Prior the translocation, the preprotein is maintained in an unfolded conformation, a state that is preserved by the molecular chaperone SecB or an attribute of the mature domain JOUIFQIZTJDPDIFNJDBMTVSSPVOEJOHTPGUIFDZUPTPM5IF4FD:&(CPVOE4FD"EJSFDUMZ interacts with SecB through its C-terminal zinc binding domain, and this interaction SFTVMUTJOBUSBOTGFSPGUIFVOGPMEFEQSFQSPUFJOGSPN4FD#UP4FD"<>4FD#FYJTUTBTB IPNPUFUSBNFSXIJDIJTBSSBOHFEBTBEJNFSPGEJNFS<>5IFUFUSBNFSDPOUBJOTUXP HSPPWFTPGBQQSPYJNBUFMZ¯MPOH XIJDISVOBMPOHFJUIFSTJEFPGUIFUFUSBNFSBOEUIBU BSFUIPVHIUUPCFUIFQFQUJEFCJOEJOHEPNBJOT"QPMZQFQUJEFNBZCJOEBUCPUIHSPPWFT BOEUIFOMJLFMZJTXSBQQFEBSPVOEUIF4FD#DPNQMFY< >*OUIJTSFTQFDU 4FD# binds preproteins in their folding core thus preventing folding when the protein emerges GSPNUIFSJCPTPNF<>%VSJOHUIF"51EFQFOEFOUJOJUJBUJPOPGUSBOTMPDBUJPO 4FD#JT SFMFBTFEJOUPUIFDZUPTPMUPCJOEBOPUIFSQSFQSPUFJO The interaction of SecA with phospholipids plays an important role in translocation as 4FD"CJOETUP4FD:&(WJBBQIPTQIPMJQJECPVOEJOUFSNFEJBUFTUBUF< >4FD" binds to phospholipids through ionic interactions that involves the amphiphatic positively DIBSHFE/UFSNJOVT<>%FMFUJPOPGUIF/UFSNJOBMBNJOPBDJETPG4FD"SFTVMUT in a complete inactivation of SecA [93], but the activity can be restored by replacing UIJTSFHJPOCZBOBSUJmDJBMNFNCSBOFUFUIFS<>)PXFWFS NFNCSBOFUFUIFSJOHJTOPU TVGmDJFOU BTJOTFSUJPOPGBnFYJCMFMJOLFSCFUXFFOUIF/UFSNJOVTBOEUIFSFNBJOEFSPG UIF4FD"QSPUFJOEPFTOPUSFTUPSFUSBOTMPDBUJPO<>5IFBOJPOJDQIPTQIPMJQJEJOUFSBDUJPO of the N-terminus and its insertion into the membrane serves to enforce a conformational DIBOHFPOUP4FD"XIFSFVQPOJUJTQSJNFEGPSIJHIBGmOJUZ4FD:&(CJOEJOHBOE"51 CJOEJOH5IFQIPTQIPMJQJECPVOEDBUBMZUJDJOUFSNFEJBUFPG4FD"MJLFMZBMTPTFSWFTBTB membrane que of SecA-preprotein complexes before SecA delivers the preprotein to 4FD:&(GPSUSBOTMPDBUJPO Structural mechanisms of SecA function SecA undergoes a multitude of conformation changes during translocation [15], and various movements of the SecA motor domain have been suggested to occur upon "51CJOEJOHBOE"%1SFMFBTF< >*OUIFT. maritima SecA co-crystal structure with 4FD:&( XIFSF4FD"JTJOUSBOTJUJPOTUBUF JF BO"%1CFSZMMJVNnVPSJEFCPVOETUBUF UIF119%NPWFTUPXBSETUIF/#%BOEBXBZGSPNUIF)8%< >XIJMFUIF/#%T BSFJODMPTFQSPYJNJUZXJUIUIF119% 'JH# 5IVT UIJTTUSVDUVSFJTDPOTJEFSFEBTUIF. 20.

(22) SecA Mediated Protein Translocation Through SecYEG Channel. PQFODPOGPSNBUJPOPG4FD"$PNQBSFEUPUIFDMPTFEDPOGPSNBUJPOPG4FD"EFSJWFEGSPN the crystal structure of the Bacillus subtilis4FD" 1%#FOUSZ./ 'JH" UIF119% JTMPDBUFEOFBSUIF)8%<>5IFOVDMFPUJEFCJOEJOHQPDLFUCFUXFFO/#%BOE/#% JTGPSNFEBMMPXJOHUIFQSFQSPUFJOUPCJOEJOBHSPPWFCFUXFFOUIF/#%BOE119%5IJT opening stabilizes the preprotein-SecA interaction and increasing the rate of nucleotide FYDIBOHFSFTVMUJOHJOBOBDUJWBUJPOPGUIF"51BTFBDUJWJUZPG4FD"< >5IFTUSVDUVSF of the (UIFSNPEFOJUSJmDBOTSecYE engaged with B. subtilis SecA [16] shows that SecA does not undergo further dramatic conformational changes as compared to T. maritima 4FD"4FD:&(TUSVDUVSF 'JH$ *UIBTCFFOQSPQPTFEUIBU4FD"JOJUT"51CPVOE state prevents the two halves of SecY from further movements that would drive these UXPIBMWFTBQBSU 5IF)'PG4FD"JTJOTFSUFEJOUPUIFDZUPQMBTNJDPQFOJOHPGUIF4FD:DIBOOFM 'JH XIFSFUIFMPPQPGUIF)'JTJODMPTFQSPYJNJUZUPUIFUSBOTMPDBUJOHQSFQSPUFJOBUUIF FOUSBODFPGUIF4FD:&(QPSF<>5IF)'NBLFTDPOUBDUXJUIUIFDZUPTPMJDMPPQPG SecY, and the insertion results in the opening of lateral gate by a rigid body movement PG5.4oSFMBUJWFUP5.4o<>5IFUJQPGUIFMPPQPGUIF)'DPOUBJOTBIJHIMZ conserved tyrosine residue which is crucial for translocation and can be replaced only CZPUIFSCVMLZIZESPQIPCJDBNJOPBDJET<>5IJTIBTMFEUPUIFQSPQPTJUJPOUIBUUIF)' interacts with the unfolded polypeptide chain through hydrophobic interaction with side chains, although this model does not explain that SecA can mediate the translocation of preproteins with large stretches of glycine residues [117] which would only allow main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

(23) Chapter 1. Fig. 4 Structure of T.maritima SecA-SecYEG complex. SecA penetrates into the SecYEG chanOFM SFE WJBUIFTPDBMMFEAUXPIFMJYmOHFS )' MJHIUCMVF 5IF4FD"119% ZFMMPX BMTPCJOETUP 5.MPPQPG4FD:&(5IFDPOTFSWFEUZSPTJOFJTEFQJDUFEJOHSFFO. TRANSLOCATION MODELS *OUIFSFDFOUZFBST NBKPSQSPHSFTTIBTCFFONBEFJOUIFTUSVDUVSBMFMVDJEBUJPOPGUIF proteins involved in protein translocation, and the possible mechanism of channel PQFOJOH)PXFWFS UIFFYBDUNFDIBOJTNCZXIJDI4FD"ESJWFTUSBOTMPDBUJPOSFNBJOT UPCFFMVDJEBUFE*OUIJTTFDUJPO XFXJMMEJTDVTTWBSJPVTNPEFMTGPSUIF4FD"NFEJBUFE USBOTMPDBUJPONFDIBOJTN Power stroke model "MBSHFDMBTTPG"51BTFTDPOUBJOB3FD"MJLFTUSVDUVSBMEPNBJOBOEVTFUIFFOFSHZPG OVDMFPUJEFCJOEJOHBOEIZESPMZTJTUPNPWFQPMZQFQUJEFTPSOVDMFJDBDJET<>4FD"IBT a DEAD box typically found in helicase, and thus a DNA helicase molecular mechanism <> IBT CFFO QSPQPTFE UIBU DPVME QPUFOUJBMMZ BMTP CF BQQMJFE UP 4FD" *O UIJT TP DBMMFEQPXFSTUPLFNFDIBOJTN 4FD"JTTVHHFTUFEUPBDUBTNFDIBOJDBMQVNQUIBU QVTIFTQSFQSPUFJOJOUP4FD:&(QPSF<>)FSFJO UIFDPVQMFEDPOGPSNBUJPOBMDIBOHFT of SecA induced by ATP binding and hydrolysis generate a mechanical force that drives USBOTMPDBUJPO"TEJTDVTTFEJOUIFQSFWJPVTTFDUJPO UIF)'NBZGVODUJPOBTBO"51 EFQFOEFOUMFWFSUIBUXPVMETVQQPSUTVDIQPXFSTUSPLFNFDIBOJTNBOESFTVMUJOTUFQXJTF USBOTMPDBUJPO 'JH" 5PBQQMJFEUIF%/"IFMJDBTFQSJODJQMFUP4FD" 4FD"JTSFRVJSFE to multimerize in order to have multiple substrate binding site,s since monomeric SecA BQQFBSTUPIBWFPOMZPOFTVCTUSBUFCJOEJOH< >0OF4FD"QSPUPNFSDPVMEBDUBT the clamp and move the segment of the preprotein while the other SecA protomer traps UIFQPMZQFQUJEFDIBJOJOUIFDIBOOFMTJODF4FD:&(JTOPUBCMFUPNBLFBTUBCMFBODIPS GPSQSFQSPUFJOT<>*OUIJTNPEFM UIFQSFQSPUFJOOFFETUPCFQSFWFOUFEGSPNCBDLTMJEJOH JOUIFDIBOOFMPODF4FD"SFTFUTUPJUTQSFUSBOTMPDBUJPONPEF5IVT BIJHIDPPQFSBUJWJUZ. 22.

(24) SecA Mediated Protein Translocation Through SecYEG Channel. is needed between the two protomers of SecA so that the preprotein is bound to one of UIFQSPUPNFSTBUBOZHJWFOUJNF Brownian ratchet model *OUIF#SPXOJBOSBUDIFUNPEFM 'JH# 4FD"BDUTBTUIFSFHVMBUPSGPSDIBOOFMPQFOJOH of SecYEG [124], while translocation occurs by Brownian movement of the unfolded QSFQSPUFJOUISPVHIUIFDIBOOFM*OUIFDPTUSVDUVSF4FD"4FD:&( UIF)'JTJODPOUBDU XJUIUIF$MPPQPG4FD:UIBUDPOOFDUT5.4BOE<>5IVT NPWFNFOUPGUIF)' DPVMEQPUFOUJBMMZSFTVMUJOBOPQFOJOHPGUIFDIBOOFM#BDLTMJEJOHPGUIFQSFQSPUFJOXPVME be prevented by the SecA association and this may assure that the diffusion will only occur in only one direction, possibly further facilitated by folding of the polypeptide at the cisTJEFPGUIFNFNCSBOFBOEPSCZBTTPDJBUJPOTXJUIUIFQFSJQMBTNJDEPNBJOTPGUIF 4FD%'DPNQMFY<>5IJTNPEFMFYQMBJOTUIFQSPNJTDVJUZPGUIFTZTUFNGPSQPMZQFQUJEF DPNQPTJUJPO<>CVUEPFTOPUFYQMBJOTUFQTJ[FUSBOTMPDBUJPO< > Push and Slide model "OPUIFSNPEFMPG4FD"NFEJBUFEQSPUFJOUSBOTMPDBUJPOJTUIFTPDBMMFEAQVTIBOETMJEF NFDIBOJTN<> 'JH$ 5IJTNPEFMDPNCJOFTUIFQPXFSTUSPLFBOE#SPXOJBO3BUDIFU models, and explains earlier observations that SecA mediated translocation occurs stepwise, whereas in the absence of SecA association, the preprotein may slide within the USBOTMPDBUJPODIBOOFM<>.FDIBOJTUJDBMMZ UIF)'IBTCFFOQSPQPTFEUPJOEVDFB QPXFSTUSPLFCZJOUFSBDUJOHXJUIUIFQSFQSPUFJOBOEQVTIJOHJUUISPVHIUIF4FD:&(QPSF < >0ODF"51JTIZESPMZ[FE UIF)'XPVMESFUVSOUPJUTQSFUSBOTMPDBUJPOQPTJUJPO and dissociate from the preprotein to allows passive sliding of the polypeptide chain into UIFDIBOOFM5IJTNPEFM IPXFWFS EPFTOPUFYQMBJOUIBUDSPTTMJOLJOHPGUIF4FD")'UP UIFJOUFSBDUJOHMPPQTPO4FD:EPFTOPUJOUFSGFSFXJUIUSBOTMPDBUJPO<>*UJTBMTPOPUDMFBS IPXUIFDPOTFSWFEUZSPTJOFBUUIFUJQPGUIF)'XPVMESFMFBTFUIFQPMZQFQUJEFBGUFS"51 IZESPMZTJTBOEIPXJUXPVMEQSFWFOUTCBDLTMJEJOHJOUIF4FD:&(DIBOOFM"MUFSOBUJWFMZ step-wise translocation may arise from binding and release of SecA to and from the 4FD:&(DIBOOFMBTTVHHFTUFECZCJPDIFNJDBMTUVEJFT<> Reciprocating piston model As mentioned at the previous section, SecA exists a dimer during translocation < > CVU BMTP NPOPNFSJD TUBUFT IBWF CFFO SFQPSUFE < > 5IF SFDJQSPDBUJOHQJTUPONPEFMDPNCJOFTUIFQPXFSTUSPLFNPEFMXJUIUIF4FD"NPOPNFS EJNFSUSBOTJUJPO<> 'JH% 5SBOTMPDBUJPOJTJOJUJBUFECZUIFCJOEJOHPGUIFEJNFSJD 4FD"UP4FD:&(/FYU "51IZESPMZTJTJOEVDFT4FD"NPOPNFSJ[BUJPOXIFSFPOFPGUIF 4FD"NPOPNFSTSFNBJOTBODIPSFEUP4FD:&(UPQSFWFOUTCBDLTMJEJOHPGUIFQBSUJBMMZ. 23. 1.

(25) Chapter 1. translocated preprotein, whereas the other monomer dissociates into the cytosol or UP UIF NFNCSBOF /FYU SFCJOEJOH PG BOPUIFS 4FD" NPOPNFS UP 4FD:&(4FD" preprotein complex promotes ATP independent translocation of a preprotein segment, XIJMFTVCTFRVFOUCJOEJOHPG"51ESJWFTUIFUSBOTMPDBUJPOBDDPSEJOHUPBQPXFSTUSPLF NFDIBOJTN5IFTFTUFQTBSFSFQFBUFEVOUJMUIFQSFQSPUFJOJTGVMMZUSBOTMPDBUFEUIPVHI UIFDIBOOFM5IJTNPEFMFYQMBJOTUIFUXPUSBOTMPDBUJPOTUBHFTJOUIFQSPDFTTUIBUXFSF observed biochemically, i.e, translocation induced by SecA binding to the preprotein, BOEBO"51EFQFOEFOUUSBOTMPDBUJPOTUFQ< >'VSUIFSNPSF DPNQMFUFEJTTPDJBUJPO of SecA from SecYEG may allow translocation by Brownian diffusion and permit PMFESJWFOUSBOTMPDBUJPOBTXJMMCFEJTDVTTFEJOUIFGPMMPXJOHTFDUJPO. 24.

(26) SecA Mediated Protein Translocation Through SecYEG Channel. 1. Fig. 5. Proposed models of SecA-mediated protein translocation. (A) Power stroke: ATP binding and hydrolysis induces conformational changes of SecA that result in a mechanical force POUIFQSFQSPUFJOQVTIJOHJUUISPVHIUIF4FD:&(DIBOOFM*OUIJTNPEFM PMJHPNFSJ[BUJPOPG4FD". 25.

(27) Chapter 1. JTSFRVJSFEUPQSFWFOUCBDLTMJEJOHPGUIFQSFQSPUFJO B) Brownian ratchet: SecA regulates the 4FD:&(DIBOOFMPQFOJOHWJBUIF)'PG4FD"BOEBMMPXJOHUIFQSPUFJOUSBOTMPDBUJPOWJBEJGGVTJPO Trapping and release of the translocating preprotein at the cis-side results in translocation, while 4FD"NBZGVMmMBOBEEJUJPOBMGVODUJPOCZPQFOJOHUIFUSBOTMPDBUJPODIBOOFM The oligomeric state PG4FD"JTOPUFYQMJDJUMZTIPXOJOUIJTNPEFM (C) Push and slide: This model uses both SecA deQFOEFOUQVTIJOHBOE#SPXOJBONPUJPO5IFPMJHPNFSJDTUBUFPG4FD"JTOPUFYQMJDJUMZTIPXOJOUIJT NPEFM(D) Reciprocating piston: 5IJTNPEFMJTBDPNCJOBUJPOPGBQPXFSTUSPLFNFDIBOJTNXJUI UIFDPOWFSTJPOPGEJNFSJDNPOPNFSJD4FD"3FQFBUFEDZDMFTPG4FD"NPOPNFSJ[BUJPOSFCJOEJOH BOE "51 CJOEJOHIZESPMZTJT ZJFMET JO B TUFQXJTF USBOTMPDBUJPO QSPDFTT *O OPOF PG UIF BCPWF NPEFMT UIFFYBDUSPMFPGUIF1.'BOE4FD%'JTJODMVEFEXIJDIDPOUSJCVUFUPFGmDJFOUUSBOTMPDBUJPO. ROLE OF THE SECDF COMPLEX *NQPSUBOUMZ UIFBGPSFNFOUJPOFENPEFMTEPOPUUBLFUIFSPMFPGUIF1.'JOUPBDDPVOU Although SecA can drive translocation in vitro on its own, protein translocation in vivo is TUSPOHMZEFQFOEFOUPOUIF1.'In vivo, SecA may mainly serve to initiate translocation by releasing the signal sequence domain and the early mature domain of a preprotein as a looped structure into the SecY pore then allow for a directed diffusional or power TUSPLFUSBOTMPDBUJPONFDIBOJTNTUIBUJTGVSUIFSBDDFMFSBUFECZUIF1.'BOEUIF4FD%' DPNQMFY<>*OEFFE in vitro preprotein translocation at low SecA concentrations is highly PMF dependent while in the presence of high concentrations of SecA, ATP-driven translocation dominates [129,130] SecDF is a subcomplex that associates with the SecYEG translocon to form the holoUSBOTMPDPODPNQMFY<>5IFDPNQMFYFYJTUTBTUXPJOEJWJEVBMQSPUFJOT4FD%BOE4FD' PS BT B MBSHF 4FD%' GVTJPO QSPUFJO *O UIF DSZTUBM TUSVDUVSF PG 4FD%' GSPN Thermus thermophilus and Deinococcus radiodurans, SecDF exists as a monomer with 12 TMS, 5.4FBDIJOCPUI4FD%BOE4FD'5IFQSPUFJO DPNQMFY BMTPDPOUBJOTQFSJQMBTNJD EPNBJO 11 XIFSF1BOE1GPSNBQFSJQMBTNJDQSPUSVEJOHTUSVDUVSF< >1 has been proposed to interact with the polypeptide substrate, and a conformational change or movement of P1 may results in a PMF dependent pulling action by SecDF BUUIFQFSJQMBTNJDTJEFPGUIFNFNCSBOF< >*OEFFE UIFMBUFTUBHFTPGQSPUFJO USBOTMPDBUJPOQSPDFTTDBOPDDVSXJUIPVU"51BOEBSF4FD%'BOE1.'EFQFOEFOU<> *OUIJTQSPDFTT MBSHFVOGPMEFESFHJPOTPGUIFQSFQSPUFJODBOCFUSBOTMPDBUFE. CONCLUDING REMARKS A multitude of biochemical and biophysical studies has been made to understand UIFNPMFDVMBSNFDIBOJTNPG4FD"JOQSPUFJOUSBOTMPDBUJPO5IF9SBZTUSVDUVSBMEBUB of different states of the SecA protein both in the absence and presence of SecYEG association has revealed various conformations providing further insight into structural. 26.

(28) SecA Mediated Protein Translocation Through SecYEG Channel. CBTJTPGQSPUFJOUSBOTMPDBUJPO)PXFWFS TUJMMNBOZPGUIFNFDIBOJTUJDRVFTUJPOTPO4FD" mediated protein translocation have remained unresolved, and although translocation FYIJCJUTGFBUVSFTUZQJDBMPCTFSWFEGPSBQPXFSTUSPLFBOE#SPXOJBOEJGGVTJPONFDIBOJTNT JUSFNBJOTVODMFBSIPXUIJTQSPDFTTJTEJSFDUFECZUIF4FD"QSPUFJO"MTP UIFFYBDUSPMF PGUIF4FD"EJNFSSFNBJOTUPCFSFTPMWFE5PVOJGZQPUFOUJBMMZDPOnJDUJOHSFTVMUT UIF process needs to be examined at the single molecule level to reveal the dynamic interplay CFUXFFOUIFDPNQPOFOUTBOEJEFOUJGZUIFJSSPMFBUUIFEJGGFSFOUTUBHFTPGUIFQSPDFTT Also, current mechanistic insights should be integrated and combined with in vivo studies on protein translocation, in order to understand how this process is coordinated XJUIJOUIFDSPXEFEJOUSBDFMMVMBSFOWJSPONFOUPGUIFDZUPTPMBOEDZUPQMBTNJDNFNCSBOF Considering the dual role of the translocon in protein translocation and membrane protein integration, a further unresolved questions is whether translocons exist in the membrane with a specialized function or whether the translocon composition is highly dynamics BOEBTTFNCMFEPOEFNBOE. SCOPE OF THIS THESIS This study aims to decipher the molecular mechanism of SecA-mediated protein USBOTMPDBUJPOVTJOHDPNCJOFECJPDIFNJDBMBOEFO[ZNBUJDBQQSPBDIFT Chapter 1 provides a review on the current understanding of the structure and function of the Sec translocon, with the focus on the protein conducting channel SecYEG and UIFNPUPSQSPUFJO4FD" Chapter 2JOWFTUJHBUFTUIFSPMFPGUIF)'PG4FD"JOQSPUFJOUSBOTMPDBUJPO)FSF XF BOBMZ[FEUIFJNQPSUBODFPGUIF)'JOQSPUFJOUSBOTMPDBUJPOCZFYUFOEJOHBOEUSVODBUJOH UIFMFOHUIPGUIF)' In vivo complementation assay were employed to analyze the BDUJWJUZPGUIF4FD")'NVUBOUTJODFMMWJBCJMJUZ'VSUIFSNPSF 4FD")'NVUBOUTXFSF tested in vitroXJUIUSBOTMPDBUJPOBOE4FD:&(CJOEJOHBTTBZT0VSEBUBEFNPOTUSBUFB DPNQMFUFGVODUJPOBMJUZPGB4FD"WBSJBOUXJUIBTZNNFUSJDBMMZTIPSUFE)'CVUTVHHFTUT UIBUUIFUSVODBUJPOSFTVMUTJOBOJODSFBTFEFOFSHFUJDSFRVJSFNFOUGPSUSBOTMPDBUJPO Chapter 3 analyzes the tolerance of SecYEG for the preproteins that were chemically NPEJmFEXJUIOPOQPMZQFQUJEFDPOTUJUVFOUT"nVPSPQIPSFBOEQPMZNFSJDIZESPQIJMJD PMJHPFUIZMFOFHMZDPMDIBJOTXFSFJOUSPEVDFEJOUPUIFNBUVSFSFHJPOPGQSP0NQ"0VSEBUB EFNPOTUSBUFTUIBUUIFUSBOTMPDPOJTSFNBSLBCMZQSPNJTDVPVTGPSJUTQPMZQFQUJEFTVCTUSBUFT BOE BDDFQUT TVDI DIFNJDBM NPEJmDBUJPOT FYDFQU QSFTFOU JO BU UIF FYUSFNF NBUVSF /UFSNJOVTPGUIFQSFQSPUFJOXIFSFJUQPTTJCMZJOUFSGFSFTXJUIJOJUJBUJPOPGUSBOTMPDBUJPO. 27. 1.

(29) Chapter 1. Chapter 4 QSFTFOUT B TUVEZ UP GPMMPX UIF JOUFSBDUJPO CFUXFFO UIF )' PG 4FD" XJUI 4FD:&( VTJOH B '3&5 CBTFE BQQSPBDI )FSFJO B TFU PG NPOPDZTUFJOF 4FD" BOE 4FD:NVUBOUTXFSFHFOFSBUFEBOEMBCFMFEXJUIBEPOPSBOEBOBDDFQUPSnVPSPQIPSF SFTQFDUJWFMZ'3&5XBTVTFEUPFYBNJOFQPTTJCMFEZOBNJDDIBOHFTJOUIFJOUFSBDUJPOCVU PODFUIFJOUFSBDUJPOXBTFTUBCMJTIFE OPUSBOTMPDBUJPOEFQFOEFOU'3&5DIBOHFTXFSF PCTFSWFETVHHFTUJOHUIF4FD")'JOUFSBDUTTUBCMZXJUI4FD:&( Finally, in Chapter 5 mOEJOHT SFQPSUFE JO UIJT UIFTJT BSF TVNNBSJ[FE XJUI B GVUVSF QFSTQFDUJWFPOUIFTUVEJFTPOUIFNFDIBOJTNPGUIF4FD"NFEJBUFEQSPUFJOUSBOTMPDBUJPO. 28.

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(34) SecA Mediated Protein Translocation Through SecYEG Channel. 5BVmL * ,FESPW " &YUFSLBUF . %SJFTTFO "+. .POJUPSJOH UIF BDUJWJUZ PG TJOHMF USBOTMPDPOTJ. Mol. Biol. 425 o 5PNLJFXJD[% /PVXFO/%SJFTTFO"+. 1VTIJOH QVMMJOHBOEUSBQQJOHo.PEFTPG NPUPSQSPUFJOTVQQPSUFEQSPUFJOUSBOTMPDBUJPOFEBS Lett. 581 o 4BUP, .PSJ) :PTIJEB..J[VTIJNB4 $IBSBDUFSJ[BUJPOPGBQPUFOUJBMDBUBMZUJD SFTJEVF "TQ JOUIFIJHIBGmOJUZ"51CJOEJOHTJUFPG Escherichia coli SecA, translocation "51BTFJ. Biol. Chem. 271 o )VOU +' 8FJOLBVG 4 )FOSZ - 'BL ++ .D/JDIPMBT 1 0MJWFS %# %FJTFOIPGFS + /VDMFPUJEFDPOUSPMPGJOUFSEPNBJOJOUFSBDUJPOTJOUIFDPOGPSNBUJPOBMSFBDUJPODZDMFPG4FD" Science 297 o :F+ 0TCPSOF"3 (SPMM.3BQPQPSU5" 3FD"MJLFNPUPS"51BTFTMFTTPOTGSPN TUSVDUVSFTBiochim. Biophys. Acta 1659 o #BVFS#83BQPQPSU5" .BQQJOHQPMZQFQUJEFJOUFSBDUJPOTPGUIF4FD""51BTFEVSJOH USBOTMPDBUJPOProc. Natl. Acad. Sci. U. S. A. 106 o %JOH) .VLFSKJ*0MJWFS% /VDMFPUJEFBOE1IPTQIPMJQJE%FQFOEFOU$POUSPMPG119% BOE$%PNBJO"TTPDJBUJPOGPS4FD""51BTFBiochemistry 42 o 1BQBOJLPMBV: 1BQBEPWBTJMBLJ. 3BWFMMJ3#( .D$BSUIZ"" $VTBDL4 &DPOPNPV" Petratos K (2007) Structure of Dimeric SecA, the Escherichia coli Preprotein Translocase .PUPSJ. Mol. Biol. 366 o &SMBOETPO,+ .JMMFS4#. /BN: 0TCPSOF"3 ;JNNFS+3BQPQPSU5" "SPMFGPSUIF UXPIFMJYmOHFSPGUIF4FD""51BTFJOQSPUFJOUSBOTMPDBUJPONature 455 o #SFVLJOL& /PVXFO/ WBO3BBMUF" .J[VTIJNB4 5PNNBTTFO+EF,SVJKGG# 5IF $UFSNJOVTPG4FD"JTJOWPMWFEJOCPUIMJQJECJOEJOHBOE4FD#CJOEJOHJ. Biol. Chem. 270, o (PME7". 3PCTPO" $MBSLF"3$PMMJOTPO* "MMPTUFSJD3FHVMBUJPOPG4FD"J. Biol. Chem. 282 o .JMMFS" 8BOH-,FOEBMM%" 4FD#NPEVMBUFTUIFOVDMFPUJEFCPVOETUBUFPG4FD" BOETUJNVMBUFT"51BTFBDUJWJUZBiochemistry 41 o (FMJT* #POWJO".++ ,FSBNJTBOPV% ,PVLBLJ. (PVSJEJT( ,BSBNBOPV4 &DPOPNPV" ,BMPEJNPT$( 4USVDUVSBM#BTJTGPS4JHOBM4FRVFODF3FDPHOJUJPOCZUIF5SBOTMPDBTF .PUPS4FD"BT%FUFSNJOFECZ/.3Cell 131 o 7BO%FS8PML+18 %F8JU+(%SJFTTFO" 5IFDBUBMZUJDDZDMFPGUIF&TDIFSJDIJBDPMJ 4FD""51BTFDPNQSJTFTUXPEJTUJODUQSFQSPUFJOUSBOTMPDBUJPOFWFOUTEMBO J. 16 o ,BSBNBOPV4 7SPOUPV& 4JBOJEJT( #BVE$ 3PPT5 ,VIO" 1PMJUPV"4&DPOPNPV" "NPMFDVMBSTXJUDIJO4FD"QSPUFJODPVQMFT"51IZESPMZTJTUPQSPUFJOUSBOTMPDBUJPO Mol. Microbiol. 34 o 8PPECVSZ3- $PNQMFYCFIBWJPSJOTPMVUJPOPGIPNPEJNFSJD4FD"Protein Sci. 11, o 8BOH) /B# :BOH)5BJ1$ "EEJUJPOBMin vitro and in vivo evidence for SecA functioning as dimers in the membrane: dissociation into monomers is not essential for protein USBOTMPDBUJPOJO&TDIFSJDIJBDPMJJ. Bacteriol. 190 o. 33. 1.

(35) Chapter 1. ,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 Driessen AJ (1993) SecA, the peripheral subunit of the Escherichia coli precursor protein USBOTMPDBTF JTGVODUJPOBMBTBEJNFSBiochemistry 32 o EF,FZ[FS+ WBOEFS4MVJT&0 4QFMCSJOL3&+ /JKTUBE/ EF,SVJKGG# /PVXFO/ WBOEFS%PFT $%SJFTTFO"+. $PWBMFOUMZEJNFSJ[FE4FD"JTGVODUJPOBMJOQSPUFJOUSBOTMPDBUJPOJ. Biol. Chem. 280 o 7BTTZMZFW%( .PSJ) 7BTTZMZFWB./ 5TVLB[BLJ5 ,JNVSB: 5BIJSPW5)*UP, Crystal Structure of the Translocation ATPase SecA from Thermus thermophilus3FWFBMTB 1BSBMMFM )FBEUP)FBE%JNFSJ. Mol. Biol. 364 o #FOBDI+ $IPV:5F 'BL++ *ULJO" /JDPMBF%% 4NJUI1$ 8JUUSPDL( 'MPZE%- (PMTB[$. (JFSBTDI-.)VOU+' 1IPTQIPMJQJEJOEVDFENPOPNFSJ[BUJPOBOETJHOBMQFQUJEF JOEVDFEPMJHPNFSJ[BUJPOPG4FD"J. Biol. Chem. 278 o #V; 8BOH-,FOEBMM%" /VDMFPUJEFCJOEJOHJOEVDFTDIBOHFTJOUIFPMJHPNFSJD state and conformation of Sec A in a lipid environment: A small-angle neutron-scattering TUVEZJ. Mol. Biol. 332 o .VTJBM4JXFL. 3VTDI4-,FOEBMM%" 1SPCJOHUIFBGmOJUZPG4FD"GPSTJHOBMQFQUJEF JOEJGGFSFOUFOWJSPONFOUTBiochemistry 44 o 8PXPS"+ :V% ,FOEBMM%"$PMF+- &OFSHFUJDTPG4FD"EJNFSJ[BUJPOJ. Mol. Biol. 408 o ,PDI4 EF8JU+( 7PT* #JSLOFS+1 (PSEJJDIVL1 )FSSNBOO" WBO0JKFO".%SJFTTFO "+. -JQJET"DUJWBUF4FD"GPS)JHI"GmOJUZ#JOEJOHUPUIF4FD:&($PNQMFYJ. Biol. Chem. 291 o +JMBWFBOV-# ;JUP$30MJWFS% %JNFSJD4FD"JTFTTFOUJBMGPSQSPUFJOUSBOTMPDBUJPO Proc. Natl. Acad. Sci. 102 o ,BSBNBOPV4 4JBOJEJT( (PVSJEJT( 1P[JEJT$ 1BQBOJLPMBV: 1BQBOJLPV&&DPOPNPV A (2005) Escherichia coli4FD"USVODBUFEBUJUTUFSNJOJJTGVODUJPOBMBOEEJNFSJDFEBS Lett. 579 o 0S& #PZE% (PO4 #FDLXJUI+3BQPQPSU5 5IFCBDUFSJBM"51BTF4FD"GVODUJPOT BTBNPOPNFSJOQSPUFJOUSBOTMPDBUJPOJ. Biol. Chem. 280 o (PVSJEJT ( ,BSBNBOPV 4 4BSEJT .' 4DIÊSFS ." $BQJUBOJ ( &DPOPNPV " 2VBUFSOBSZEZOBNJDTPGUIF4FD"NPUPSESJWFUSBOTMPDBTFDBUBMZTJTMol. Cell 52 o 'FLLFT1 EF8JU+( #PPSTNB" 'SJFTFO3)&%SJFTTFO"+. ;JOD4UBCJMJ[FTUIF 4FD##JOEJOH4JUFPG4FD"Biochemistry 38 o #BOFSKFF5 -JOEFOUIBM$0MJWFS% 4FD"GVODUJPOT in vivo as a discrete anti-parallel EJNFSUPQSPNPUFQSPUFJOUSBOTQPSUMol. Microbiol. 103 o )FHEF34#FSOTUFJO)% 5IFTVSQSJTJOHDPNQMFYJUZPGTJHOBMTFRVFODFTTrends Biochem. Sci. 31 o 0XKJ) /F[BGBU/ /FHBIEBSJQPVS. )BKJFCSBIJNJ"(IBTFNJ: "DPNQSFIFOTJWF SFWJFXPGTJHOBMQFQUJEFT4USVDUVSF SPMFT BOEBQQMJDBUJPOTEur. J. Cell Biol. 97 o. 34.

(36) SecA Mediated Protein Translocation Through SecYEG Channel. .BSUPHMJP#%PCCFSTUFJO# 4JHOBMTFRVFODFT.PSFUIBOKVTUHSFBTZQFQUJEFTTrends Cell Biol. 8 o WPO)FJKOF( 5IFTJHOBMQFQUJEFJ. Membr. Biol. 115 o $SBOGPSE4NJUI5)VCFS% 5IFXBZJTUIFHPBMIPX4FD"USBOTQPSUTQSPUFJOTBDSPTT UIFDZUPQMBTNJDNFNCSBOFJOCBDUFSJBFEMS Microbiol. Lett. 365 $IBU[J ,& 4BSEJT .' 5TJSJHPUBLJ " ,PVLBLJ . ÀPÝUBSJ˂ / ,POJKOFOCFSH " 4PCPUU ' ,BMPEJNPT $( ,BSBNBOPV 4 &DPOPNPV " 1SFQSPUFJO NBUVSF EPNBJOT DPOUBJO USBOTMPDBTFUBSHFUJOHTJHOBMTUIBUBSFFTTFOUJBMGPSTFDSFUJPOJ. Cell Biol. 216 o 'FTTM5 8BULJOT% 0BUMFZ1 "MMFO8+ $PSFZ3" )PSOF+ #BMEXJO4" 3BEGPSE4& $PMMJOTPO *5VNB3 %ZOBNJDBDUJPOPGUIF4FDNBDIJOFSZEVSJOHJOJUJBUJPO QSPUFJOUSBOTMPDBUJPO BOEUFSNJOBUJPOElife 7 9V; ,OBGFMT+%:PTIJOP, $SZTUBMTUSVDUVSFPGUIFCBDUFSJBMQSPUFJOFYQPSUDIBQFSPOF TFD#Nat. Struct. Biol. 7 o $SBOF +. 4VP : -JMMZ "" .BP $ )VCCFMM 8- 3BOEBMM -- 4JUFT PG *OUFSBDUJPO of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin -BCFMJOHJ. Mol. Biol. 363 o 7BO %FS 4MVJT &0 %SJFTTFO "+. 4UFQXJTF FWPMVUJPO PG UIF 4FD NBDIJOFSZ JO 1SPUFPCBDUFSJBTrends Microbiol. 14 o #FDIUMVGU1 WBO-FFVXFO3() 5ZSFNBO. 5PNLJFXJD[% /PVXFO/ 5FQQFS)- %SJFTTFO "+.5BOT4+ %JSFDUPCTFSWBUJPOPGDIBQFSPOFJOEVDFEDIBOHFTJOBQSPUFJOGPMEJOH QBUIXBZScience 318 o #BVFS#8 4IFNFTI5 $IFO:3BQPQPSU5" "Ai1VTIBOE4MJEFw.FDIBOJTN "MMPXT4FRVFODF*OTFOTJUJWF5SBOTMPDBUJPOPG4FDSFUPSZ1SPUFJOTCZUIF4FD""51BTFCell 157 o )FOESJDL+18JDLOFS8 4FD"QSPUFJOOFFETCPUIBDJEJDQIPTQIPMJQJETBOE4FD:& QSPUFJOGPSGVODUJPOBMIJHIBGmOJUZCJOEJOHUPUIFEscherichia coliQMBTNBNFNCSBOFJ. Biol. Chem. 266 o 4JBOJEJT ( ,BSBNBOPV 4 7SPOUPV & #PVMJBT , 3FQBOBT , ,ZSQJEFT / 1PMJUPV "4 &DPOPNPV" $SPTTUBMLCFUXFFODBUBMZUJDBOESFHVMBUPSZFMFNFOUTJOB%&"%NPUPS EPNBJOJTFTTFOUJBMGPS4FD"GVODUJPOEMBO J. 20 o $IFO: #BVFS#8 3BQPQPSU5"(VNCBSU+$ $POGPSNBUJPOBM$IBOHFTPGUIF$MBNQ PGUIF1SPUFJO5SBOTMPDBUJPO"51BTF4FD"J. Mol. Biol. (PME7". 8IJUFIPVTF4 3PCTPO"$PMMJOTPO* 5IFEZOBNJDBDUJPOPG4FD"EVSJOH UIFJOJUJBUJPOPGQSPUFJOUSBOTMPDBUJPOBiochem. J. 449 o 'BL++ *ULJO" $JPCBOV%% -JO&$ 4POH9+ $IPV:5 (JFSBTDI-.)VOU+' /VDMFPUJEFFYDIBOHFGSPNUIFIJHIBGmOJUZ"51CJOEJOHTJUFJO4FD"JTUIFSBUFMJNJUJOHTUFQ in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational TUBUFBiochemistry 43 o /PVXFO/ #FSSFMLBNQ(%SJFTTFO"+. $IBSHFEBNJOPBDJETJOBQSFQSPUFJOJOIJCJU 4FD"EFQFOEFOUQSPUFJOUSBOTMPDBUJPOJ. Mol. Biol. 386 o. 35. 1.

(37) Chapter 1. 8IJUFIPVTF4 (PME7B. 3PCTPO" "MMFO8+ 4FTTJPOT3#$PMMJOTPO* .PCJMJUZPG UIF4FD"IFMJYmOHFSJTOPUFTTFOUJBMGPSQPMZQFQUJEFUSBOTMPDBUJPOWJBUIF4FD:&(DPNQMFY J. Cell Biol. 199 o ;IBOH2 -BIJSJ4 #BOFSKFF5 4VO; 0MJWFS%.VLFSKJ* "MJHONFOUPGUIFQSPUFJO TVCTUSBUFIBJSQJOBMPOHUIF4FD"UXPIFMJYmOHFSQSJNFTQSPUFJOUSBOTQPSUJOEscherichia coli Proc. Natl. Acad. Sci. U. S. A. 114 o 0TCPSOF"3 $MFNPOT 8. 3BQPQPSU 5" " MBSHF DPOGPSNBUJPOBM DIBOHF PG UIF USBOTMPDBUJPO"51BTF4FD"Proc. Natl. Acad. Sci. U. S. A. 101 o (FMJT* #POWJO".++ ,FSBNJTBOPV% ,PVLBLJ. (PVSJEJT( ,BSBNBOPV4 &DPOPNPV" ,BMPEJNPT$( 4USVDUVSBMCBTJTGPSTJHOBMTFRVFODFSFDPHOJUJPOCZUIFL%B USBOTMPDBTFNPUPS4FD"BTEFUFSNJOFECZ/.3Cell 131 o 1BQBOJLPV& ,BSBNBOPV4 #BVE$ 'SBOL. 4JBOJEJT( ,FSBNJTBOPV% ,BMPEJNPT$( ,VIO"&DPOPNPV" *EFOUJmDBUJPOPGUIFQSFQSPUFJOCJOEJOHEPNBJOPG4FD"J. Biol. Chem. 280 o 4DIJFCFM& %SJFTTFO"+. )BSUM'68JDLOFS8 ϯ)

(38) BOE"51GVODUJPOBUEJGGFSFOU TUFQTPGUIFDBUBMZUJDDZDMFPGQSFQSPUFJOUSBOTMPDBTFCell 64 o "MMFO8+ $PSFZ3" 0BUMFZ1 4FTTJPOT3# 3BEGPSE4& 5VNB3$PMMJOTPO* 5XP way communication between SecY and SecA suggests a Brownian ratchet mechanism for QSPUFJOUSBOTMPDBUJPOElife 5 #PUUF . ;BDDBJ /3 /JKFIPMU +- Ë .BSUJO 3 ,OPPQT , 1BQBJ ( ;PV + %FOJBVE " ,BSVQQBTBNZ. +JBOH2 3PZ"4 4DIVMUFO, 4DIVMU[1 3BQQTJMCFS+ ;BDDBJ( #FSHFS * $PMMJOTPO*4DIBGmU[FM$ "DFOUSBMDBWJUZXJUIJOUIFIPMPUSBOTMPDPOTVHHFTUTB NFDIBOJTNGPSNFNCSBOFQSPUFJOJOTFSUJPOSci. Rep. 6 4JNPO4. 1FTLJO$40TUFS(' 8IBUESJWFTUIFUSBOTMPDBUJPOPGQSPUFJOT Proc. Natl. Acad. Sci. U. S. A. 89 o Duong F (2003) Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric 4FD:&(USBOTMPDBTFEMBO J. 22 o 0S& /BWPO"3BQPQPSU5 %JTTPDJBUJPOPGUIFEJNFSJD4FD""51BTFEVSJOHQSPUFJO USBOTMPDBUJPOBDSPTTUIFCBDUFSJBMNFNCSBOFEMBO J. 21 o .PSJ ) *UP , #JPDIFNJDBM DIBSBDUFSJ[BUJPO PG B NVUBUJPOBMMZ BMUFSFE QSPUFJO USBOTMPDBTFQSPUPONPUJWFGPSDFTUJNVMBUJPOPGUIFJOJUJBUJPOQIBTFPGUSBOTMPDBUJPOJ. Bacteriol. 185 o /JTIJZBNB ,* 'VLVEB " .PSJUB , 5PLVEB ) .FNCSBOF EFJOTFSUJPO PG 4FD" VOEFSMZJOHQSPUPONPUJWFGPSDFEFQFOEFOUTUJNVMBUJPOPGQSPUFJOUSBOTMPDBUJPOEMBO J. 18, o 'VSVLBXB" :PTIJLBJF, .PSJ5 .PSJ) .PSJNPUP:7 4VHBOP: *XBLJ4 .JOBNJOP5 4VHJUB : 5BOBLB:5TVLB[BLJ5 5VOOFM'PSNBUJPO*OGFSSFEGSPNUIF*'PSN4USVDUVSFTPGUIF 1SPUPO%SJWFO1SPUFJO4FDSFUJPO.PUPS4FD%'Cell Rep. 19 o 5TVLB[BLJ5 .PSJ) &DIJ[FO: *TIJUBOJ3 'VLBJ4 5BOBLB5 1FSFEFSJOB" 7BTTZMZFW%( ,PIOP 5 .BUVSBOB "% *UP , /VSFLJ 0 4USVDUVSF BOE GVODUJPO PG B NFNCSBOF DPNQPOFOU4FD%'UIBUFOIBODFTQSPUFJOFYQPSUNature 474 o 1BSL&3BQPQPSU5" .FDIBOJTNTPG4FD4FD:.FEJBUFE1SPUFJO5SBOTMPDBUJPO "DSPTT.FNCSBOFTAnnu. Rev. Biophys. 41 o. 36.

(39) SecA Mediated Protein Translocation Through SecYEG Channel. 5TVLB[BLJ5 4USVDUVSFCBTFEXPSLJOHNPEFMPG4FD%' BQSPUPOESJWFOCBDUFSJBMQSPUFJO USBOTMPDBUJPOGBDUPSFEMS Microbiol. Lett. 365. 1. 37.

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(41) CHAPTER 2 (QJLQHHULQJRIWKH7ZRKHOL[ÀQJHURI SecA ATPase "NBMJOB(IBJTBOJ,PNBSVEJO *MKB,VTUFST 4BCSJOB,PDI "SOPME+.%SJFTTFO.

(42) Chapter 2. ABSTRACT 5IF4FDQBUIXBZJTUIFNBKPSSPVUFGPSQSPUFJOUSBOTQPSUBDSPTTBOEJOUPUIFDZUPQMBTNJD NFNCSBOFPGCBDUFSJB*OJUTNJOJNBMGPSN 4FDUSBOTMPDBTFDPOTJTUTPGUIF4FD:&( complex that functions as a membrane embedded protein conducting channel that associates with the peripheral membrane protein SecA which acts as an ATP-driven NPUPS *U IBT CFFO TVHHFTUFE UIBU UIF UXPIFMJY mOHFS )' PG 4FD" NBZ QVTI UIF QSFQSPUFJOUISPVHIUIFQPSFJOBiQPXFSTUSPLFiNBOOFS"MUFSOBUJWFMZ NPWFNFOUPGUIF )' NBZ ESJWF UIF PQFOJOH PG UIF 4FD:&( QPSF BOE USBOTMPDBUJPO NBZ PDDVS CZ B iNPMFDVMBSSBUDIFUiNFDIBOJTN)FSF XFFOHJOFFSFEUIF)'PG4FD"UPFYBNJOFUIF JNQPSUBODFPGUIF)'JOQSPUFJOUSBOTMPDBUJPO0VSEBUBEFNPOTUSBUFTGVODUJPOBMJUZPGB 4FD"WBSJBOUXJUIBTZNNFUSJDBMMZTIPSUFE)'CVUXJUIJODSFBTFEFOFSHFUJDSFRVJSFNFOUT. 40.

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