Summary Protein translocation across the bacterial membrane, mediated by the SecYEG translocon and the SecA ATPase1–4, is enhanced by proton-motive force (PMF)5,6 and membrane-integrated SecDF7–9, which associates with SecYEG. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, which revealed a pseudo-symmetrical, 12-helix transmembrane (TM) domain belonging to the RND superfamily and major periplasmic domains (P1 and P4). Higher resolution analysis of the latter suggested that P1, which proved to bind an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and PMF. Electrophysiological analyses revealed that SecDF conducts protons in a pH- and unfolded protein-dependent fashion, in which conserved Asp and Arg residues at the TM SecD/SecF-interface play essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by PMF, to achieve ATP-independent protein translocation.

A membrane-integrated Sec component, SecDF, associates with the SecYEG protein-conducting channel and facilitates protein secretion and membraneprotein integration. SecDF contains 12 transmembrane helices and two periplasmic domains. The first periplasmic domain (P1) plays an important role in protein translocation. Here, the overexpression, purification and crystallization of the P1 domain of Thermus thermophilus SecDF are reported. The crystals diffracted X-rays to 2.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 161.1, b = 35.8, c = 181.6 Å , suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by the multiple-wavelength anomalous dispersion method using selenomethionine-labelled crystals.

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TheBiophysical Society of Japan General IncorporatedAssociation studieg that show a cLsruption of the alignrnent oi biidg under R) irradiauon[4] 2) Eschenciua Cole DNA Photolw se The DNA photo]yase Tg one of the most mterestmg model systems beL iuse it has veiy "milar strucLure with Cryptochrome which has been Tdentihed as strong candidate ot the photochemical cornpasb for dnimal navigationsL241 In the system we have suLLessfully ebserved the g]gn]ticant MFE thdt is attmbuted to RPM[IIWWiltgchko RWiltschko J Conrp Phisiot A 191 67S(200S) L2]TRitz etal Bioph)s J 78 707(2000) L3] K Maeda K Henbegt etai Ndttire 453 387(2008) [4]TRit7 etat Ndture 429 177(2e04) IS1KBHenbest KMaeda etai PIVAS105 14395<2008) 2SA2 Ol KoJi Yamano Sctence IVago)a Vn!verstty) Mitochondria aie two membrdne bounded organLl]es consistmg of 1 OOO 2 OOO dfferent pioteins >99% o[ which are synthesized m the Lytogol as precursor proteLnb ind subsLquent]y imported mto mitochondna MitoLhondnal protein import and sortmg to one of the four sub Lompartments thc outer membrane intermembrane space (IMS) innei membranL and matnx are medidted by protemaceous maLhmeries La]led trang]ocatorg in the outer and mner mitochondnal rnernbranes and solubie factors oi Lhaperones ]n the cytogol IMS and matTix The translocators cooperate edLh other and w]th soluble factois to achieve precise as vvell as efficient pietein dehvLry to their intramitochondiTal debtmauonb fo11owing in mogt eagcg the five megor import pathways thL TOM40 TIM23 TOM40 TIM22 TOM40 SAM TOM40 TIM40 andOXApathways ln this talk 1 mdke a bnef overview on the accumu]ated knovvledge on thL translocatois and import pathwayg most ot which have been identidiLd and chaTaLtented foT tungal and mammalian nlltoLhondna Then I present our reLent rcsults on cooperation of the translocdtorb and goiuble factors in mitochondmi] pTotem1rnporttsortmg 1ooo ptkos)./sovaet ±s)ft6:-F=)iFu7F7/xmo' -eoRpa Mitochondrial translocators that mediate sortmg of 1000 ditterent mitochondnalproteins (1} Endo Toshiya (1) {rl) Department ofChcmistn Graduat[ SLkoot of 2SA202 vaecWU*}HscoSS,] enpt eefiEitea6Lol-x7Ao asfiteefiE StruLture and funLtion or the Lol system cdtalyzing tl]e mcmbrane sortingofbactenalTipoprotems Hngime Tokuda {D ((IJ lnstitaie of ndoiecular ared Cellttlar Bios"[nLes Ln-ersitv of 7bk)o)The Lol system consisung of fJve LD] pTotcing LelA through LolE Latalyteg the sortmg of 1ipoprotanb to the outer rnembrane An ATP bmding Lagsette (ABC) trangporter the LolCDZ Lompkx releageg the outer menibranL gpecitic 1ipoprotems leadmg to the fermauon ot a so]uble complex with a periplagrnic Lhtperone LolA Asp at position 2 of 1ipoproteing functions as a LolCDE avoidance signal which causes the retention of hpoproteins in the mncr rnernbrane The LelA 1ipopiotein Lomplex reaches the outer rnembrane to which i hpoprotcin ieceptor LolB is anchored LolB accepts a 1ipoprotem from Lo]A and incorpofates it into the outer mLmbrane The overall stiuLtures of LD]A and LolB are very similar md compnse a novel fold comprising ll antiparall...

Japan General IncorporatedAssociation in terms of the water entrop} The order of the mcasure ls ln accord w]th thdt of the thermdl Htdbihty Ith c,sl < PH (ss2 < HT css2 < AA csss Ho"ever AA csss has LhL lowLst packing ethciency of heme and the hele IikL btructure ot apo polypepude whiLh ]s unfavorable for the water entropy The highest stabilit} of AA L"s ts cibuLbed

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