molecular architecture of the active mitochondrial protein gate...2015/03/09 · science...

foraging traits as indicated for alpine bumblebees (15). The model predicts changes in the en-ergetic advantage of generalization with floraldensity. Long-tongued bumble bees exhibit grea-ter specialization than that of short-tongued bees(16, 30). Across a range of flight speed and plantcommunity composition (15), the advantage ofgeneralizing increases as flower density declines(Fig. 4). Theoretical and empirical studies alikesuggest that with lower floral resources, fitnessadvantages of long-tongued specialist phenotypeshave diminished, potentially driving the rapidevolution of shorter-tongued bees. We have doc-umented decreases in bumble bee tongue lengthwithin species and communities on three peaksin the Rocky Mountains. Our analyses suggest thatreduced flower density at the landscape scale isdriving this shift in tongue length. Although pop-ulations of long-tongued bees are undergoingwidespread decline (1, 3), shifts foraging strategiesmay allow alpine bumble bees to cope with envi-ronmental change. We see broader bumble beeforaging niches, immigration by short-tonguedbumble bees, and shorter tongue length withinresident bee populations as floral resources havedwindled. In remote mountain habitats—largelyisolated from habitat destruction, toxins, andpathogens (31)—evolution is helping wild beeskeep pace with climate change.

REFERENCES AND NOTES

1. S. A. Cameron et al., Proc. Natl. Acad. Sci. U.S.A. 108, 662–667(2011).

2. E. F. Ploquin, J. M. Herrera, J. R. Obeso, Oecologia 173,1649–1660 (2013).

3. R. Bommarco, O. Lundin, H. G. Smith, M. Rundlöf, Proc. Biol.Sci. 279, 309–315 (2012).

4. J. C. Biesmeijer et al., Science 313, 351–354 (2006).5. J. C. Grixti, L. T. Wong, S. A. Cameron, C. Favret, Biol. Conserv.

142, 75–84 (2009).6. C. Matsumura, J. Yokoyama, I. Washitani, Glob. Environ. Res. 8,

51–66 (2004).7. M. Stang, P. G. L. Klinkhamer, N. M. Waser, I. Stang,

E. van der Meijden, Ann. Bot. (Lond.) 103, 1459–1469 (2009).8. D. P. Vázquez, N. Blüthgen, L. Cagnolo, N. P. Chacoff, Ann. Bot.

(Lond.) 103, 1445–1457 (2009).9. M. A. Rodríguez-Gironés, A. L. Llandres, PLOS ONE 3, e2992

(2008).10. L. D. Harder, Oecologia 57, 274–280 (1983).11. N. Muchhala, J. D. Thomson, Proc. R. Soc. Biol. Sci. 276,

2147–2152 (2009).12. V. Grant, E. J. Temeles, Proc. Natl. Acad. Sci. U.S.A. 89,

9400–9404 (1992).13. R. B. Miller, Evolution 35, 763–774 (1981).14. F. S. Gilbert, Ecol. Entomol. 6, 245–262 (1981).15. Materials and methods are available as supplementary

materials on Science Online.16. N. E. Miller-Struttmann, C. Galen, Oecologia 176, 1033–1045

(2014).17. P. A. Byron, thesis, University of Colorado, Boulder (1980).18. L. W. Macior, Melanderia 15, 1–59 (1974).19. R. MacArthur, R. Levins, Am. Nat. 101, 377–385 (1967).20. D. W. Inouye, Ecology 59, 672–678 (1978).21. C. W. Kopp, E. E. Cleland, J. Veg. Sci. 25, 135–146 (2014).22. A. J. Miller-Rushing, D. W. Inouye, Am. J. Bot. 96, 1821–1829

(2009).23. T. T. Høye, E. Post, N. M. Schmidt, K. Trojelsgaard,

M. C. Forchhammer, Nat. Clim. Change 3, 759–763 (2013).24. D. W. Inouye, Ecology 89, 353–362 (2008).25. C. Fontaine, C. L. Collin, I. Dajoz, J. Ecol. 96, 1002–1010 (2008).26. C. J. Essenberg, Am. Nat. 180, 153–166 (2012).27. C. R. McGuire, C. R. Nufio, M. D. Bowers, R. P. Guralnick,

PLOS ONE 7, e44370 (2012).28. J. C. Geib, J. P. Strange, C. Galen, Ecol. Appl. 25, 768–778 (2015).29. P. R. Elsen, M. W. Tingley, Nat. Clim. Change. 5, 772–776

(2015).

30. G. H. Pyke, D. W. Inouye, J. D. Thomson, Environ. Entomol. 41,1332–1349 (2012).

31. D. Goulson, E. Nicholls, C. Botías, E. L. Rotheray, Science 347,1255957 (2015).

ACKNOWLEDGMENTS

We acknowledge L. W. Macior and P. A. Byron for their meticulouswork on Rocky Mountain bumble bees; J. Myrick, J. Guinnup,A. Drew, L. Rimmer, J. Stoehr, M. Pallo, L. Hesh, and B. Lubinskifor laboratory and fieldwork; and the Mountain Research Station,University of Colorado and Mount Evans Field Station, DenverUniversity for research facilities. The Arapaho National Forest,Niwot Ridge Long-Term Ecological Research (NSF grant DEB-1027341) and Mountain Area Land Trust (Pennsylvania Mountain)provided access to research sites. The Canadian NationalCollection of Insects; Rocky Mountain Herbarium, University ofWyoming; Kathryn Kalmbach Herbarium, Denver Botanic Garden;

University of Colorado Herbarium; Colorado State UniversityHerbarium; and the Missouri Botanical Garden loaned specimens.Research was supported by NSF (grants DEB-79-10786 and1045322). Data and specific code are archived at DOI: 10.5061/dryad.10278 PRISM Climate Group data for Mount Evans andPennsylvania Mountain are from www.prism.oregonstate.edu.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/349/6255/1541/suppl/DC1Materials and MethodsFigs. S1 to S4Tables S1 to S9References (32–59)

9 March 2015; accepted 27 August 201510.1126/science.aab0868

MITOCHONDRIAL IMPORT

Molecular architecture of the activemitochondrial protein gateTakuya Shiota,1,2 Kenichiro Imai,3 Jian Qiu,4* Victoria L. Hewitt,1† Khershing Tan,1

Hsin-Hui Shen,1 Noriyuki Sakiyama,3‡ Yoshinori Fukasawa,3 Sikander Hayat,5§Megumi Kamiya,2 Arne Elofsson,5 Kentaro Tomii,3 Paul Horton,3 Nils Wiedemann,4,6

Nikolaus Pfanner,4,6 Trevor Lithgow,1|| Toshiya Endo2,7||

Mitochondria fulfill central functions in cellular energetics,metabolism, and signaling.The outermembrane translocator complex (the TOM complex) imports most mitochondrial proteins,but its architecture is unknown. Using a cross-linking approach, we mapped the activetranslocator down to single amino acid residues, revealing different transport paths forpreproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from thecytosol through the channel to recruit chaperones from the intermembrane space that guide thetransfer of hydrophobic preproteins.The translocator contains three Tom40 b-barrel channelssandwiched between a central a-helical Tom22 receptor cluster and external regulatory Tomproteins.The preprotein-translocating trimeric complex exchanges with a dimeric isoform toassemble new TOM complexes. Dynamic coupling of a-helical receptors, b-barrel channels, andchaperones generates a versatile machinery that transports about 1000 different proteins.

Mitochondria are pivotal for cellular aden-osine triphosphate (ATP) production,numerous metabolic pathways and reg-ulatory processes, and programmed celldeath. Most mitochondrial proteins are

synthesized as preproteins in the cytosol and areimported into mitochondria. Preproteins eithercontain N-terminal targeting sequences (pre-sequences) or internal targeting information in themature part (1–3). The protein translocator of theoutermembrane (the TOMcomplex) functions asthe main entry gate of mitochondria (1–3). Over90% of all mitochondrial proteins are importedby the TOM complex, followed by transfer todistinct translocators for individual classes ofpreproteins.Whereas all structurally knownmem-braneprotein complexes consist of eithera-helicalor b-barrel proteins, the TOM complex is com-posed of both a-helical and b-barrel integralmembrane proteins. The complex consists of thechannel-forming b-barrel protein Tom40 and sixother subunits, each containing single a-helicaltransmembrane (TM) segments: the receptor pro-teins Tom20, Tom22, and Tom70 and the reg-ulatory small Tom proteins (1–3). The molecular

architecture of the complex has not been elu-cidated. How a-helical and b-barrel membraneproteins can be combined into a functional

1544 25 SEPTEMBER 2015 • VOL 349 ISSUE 6255 sciencemag.org SCIENCE

1Biomedicine Discovery Institute and Department ofMicrobiology, Monash University, Melbourne, Victoria 3800,Australia. 2Department of Chemistry, Graduate School ofScience, Nagoya University, Chikusa-ku, Nagoya 464-8602,Japan. 3Biotechnology Research Institute for Drug Discovery,National Institute of Advanced Industrial Science andTechnology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.4Institut für Biochemie und Molekularbiologie, UniversitätFreiburg, 79104 Freiburg, Germany. 5Department ofBiochemistry and Biophysics and Science for LifeLaboratory, Stockholm University, Box 1031, 17121 Solna,Sweden. 6Centre for Biological Signalling Studies, UniversitätFreiburg, 79104 Freiburg, Germany. 7Faculty of Life Sciences,Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku,Kyoto 603-8555, Japan.*Present address: Swiss Federal Institute of Technology, 1015Lausanne, Switzerland. †Present address: Department of Bio-medical Science, The University of Sheffield, Sheffield S10 2TN,UK. ‡Present address: Biomedical Department Cloud ServicesDivision, IT Infrastructure Services Unit, Mitsui Knowledge IndustryCompany, 2-5-1 Atago, Minato-ku, Tokyo 105-6215, Japan.§Present address: Computational Biology Program, MemorialSloan-Kettering Cancer Center, New York, NY, USA.||Corresponding author. E-mail: [email protected](T.L.); [email protected] (T.E.)

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complex and how diverse classes of preproteinscan be transported by the same TM channel isunclear.Todefine the architecture of the functional TOM

complex, we mapped the interactions of Tom40with preproteins in transit and a-helical subunitsby invivoand inorganello site-specific cross-linking.Photoactivatable p-benzoylphenylalanine (BPA)was introduced at 108 different positions in the387-residue Tom40 in yeast cells (fig. S1) (4–6). Astructural model based on homology and cysteinescanning shows 19 antiparallel b strands with thefirst and last b strands annealing in parallel ar-rangement (7–9) (fig. S2A). In TM b strands, everysecond residue faces the pore lumen, and alter-nate residues face outward. BPA cross-linking toTom22 revealed outward orientationof side chains(Fig. 1A and fig. S1). Whether preproteins transitthrough the lumen of the Tom40 b barrel or viathe interstitial space between multiple b barrelsthat make up the TOM complex has been con-

troversial (10, 11). To resolve this, we accumulatedthe model preproteins presequence of subunit9-dihydrofolate reductase (pSu9-DHFR) andADP-ATP carrier (AAC)–DHFR without a pre-sequence, in the TOM complex (10, 12, 13) andirradiated it with ultraviolet (UV) light. Residuescross-linked to pSu9 and AACwere only found atpositions facing the pore interior (Fig. 1, B to D;red residues face the pore). Similar results wereobtained by sulfhydryl group (SH)–directed chem-ical cross-linking (fig. S2B). Thus, preproteins intransit are located inside the b-barrel pore ofTom40 and not in the interstitial space betweenTom40 molecules.Do presequence-containing and carrier-family

preproteins use the samepath through the Tom40channel? The cross-linking results revealed a non-identical pattern for pSu9-DHFR and AAC-DHFR(Fig. 1, D and E, and fig. S2C). Negatively chargedresidues are aligned in the pore from the cyto-solic side to the intermembrane space (IMS) side,

forming acidic patches near the cross-linkedsites for pSu9-DHFR (red in Fig. 1F and fig. S3),whereas hydrophobic patches (green) are nearthe cross-linked sites for AAC-DHFR and partlyfor pSu9-DHFR [presequences form positivelycharged amphiphilic helices (1–3)]. Homologymodels for animal and plant Tom40 indicatesimilar acidic and hydrophobic patches insidethe channel pore (fig. S4). We conclude that pos-itively charged presequences follow an acidic pathon the inner wall of the Tom40 pore, whereascarrier proteins interact withmostly hydrophobicresidues. Thus, Tom40 canhandle and chaperone(14) diverse classes of preproteins by providingdistinct translocation paths.Systematic analysis of the cross-linking partners

of BPA-bearing Tom40 revealed that a-helicalTom proteins interact with the outside of the bbarrel or loops of Tom40 (Fig. 2A and fig. S1).Unexpectedly, the IMS protein Tim10 was cross-linked to the N-terminal segment of Tom40 (Fig.

SCIENCE sciencemag.org 25 SEPTEMBER 2015 • VOL 349 ISSUE 6255 1545

Fig. 1. The pore interior of the b barrel of Tom40 is the pro-tein import channel for preproteins. (A) In vivo photo–cross-linking of BPA at the indicated positions of Tom40 with Tom22analyzed by SDS-PAGE followed by immunoblotting (top) andquantification (bottom). (B and C) [35S]pSu9-DHFR (Su9presequence fused to dihydrofolate reductase) (B) or [35S]AAC-DHFR (ADP-ATP carrier fused to DHFR) (C) was incubated with mitochondria[pretreated with valinomycin for (B)] with BPA-bearing Tom40 at 4°C (B) or25°C in the presence of 1 mM methotrexate and 1 mM NADPH (C), for 10 min,and UV-irradiated. Affinity-purified cross-linked products (asterisk) were sub-jected to SDS-PAGE and radioimaging. Variation in the apparent molecularweight of the cross-linked products may reflect different configurations.b-strand numbers and BPA positions (red and green, side chain facing thepore interior or membrane, respectively) are indicated. The cross-links of the

neighboring residues 356 (weak) and 357 (strong) with Tom22 may suggestimperfections in the last b strand 19 around this position. p, precursor form; i,processing intermediate form; m, processed mature form. (D) Quantificationof the cross-linked products in (B) and (C). (E) The side chains of the Tom40residues cross-linked to pSu9-DHFR or AAC-DHFR are shown with colorreflecting the amount of cross-linked products detected. (F) The acidic patches(red) and hydrophobic patches (green) are shown in the pore interior of Tom40,in relation to the position of Tom22TM (Fig. 4, inset).



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2, A and B), suggesting that this segment extendsfrom the cytosolic side through the b-barrel poreof Tom40 to the IMS (8). We generated yeastmutants with N-terminal truncations of Tom40and found that, whereas a 62-residue deletion(tom40D62) inhibited import of both presequence-

containing and presequence-less preproteins, a57-residue deletion selectively inhibited importof presequence-less preproteins (Fig. 2, C and D,and fig. S5). The tom40D57 yeast strain becamesensitive to overexpression of carrier proteins, notof a presequence-containing preprotein, being un-

able to cope with the increased load of hydropho-bic preproteins (Fig. 2E). Tim10 is a subunit of thehexameric Tim9-Tim10 (small TIM) chaperone inthe IMS, functioning to guide presequence-lesspreproteins through the aqueous IMS (1–3). Wethus conclude that the recruitment of these


Fig. 2. The N-terminal segment of Tom40 interacts with elementsof the carrier protein import pathway. (A) Topology model and BPAcross-linking results of Tom40. Circles, pentagons, and squares in-dicate residues in the loop, a helix, or b strand, respectively. BPA-incorporated residues are labeled in bold. Cross-linked partners areindicated by different colors. (B) UV-dependent in vivo photo–cross-linking of BPA at the indicated position of Tom40 detected by immu-noblotting (top) and quantification (bottom). (C and D) In vitro importof [35S]labeled presequence-containing (C) and presequence-less(D) preproteins into the tom40 mutant mitochondria at 25°C. Afterproteinase K treatment, imported proteins were analyzed by SDS-PAGE and radioimaging. The imported amount at the longest incu-bation time was set to 100% (control).Values are mean SEM (n = 3).

(E) Serial dilutions of the Tom40 N-terminal truncation mutant cells with overexpression of the indicated proteins were spotted on SCD (-Ura, -Trp) (glucose),SCGal (-Ura, -Trp) (galactose), and SCGly + 0.05% D-glucose (-Ura, -Trp) (glycerol + galactose) media and grown at 30°C for 3 days (glucose and galactose) or4 days (glycerol + galactose). WT, corresponding TOM40-(His)10 strain; PiC, phosphate carrier; DiC, dicarboxylate carrier.



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chaperones to the TOM channel exit promotesan efficient transfer of hydrophobic preproteins.To define the subunit organization within the

TOM complex, we probed the interactions of theTom40 molecule with Tom40 itself and withthe core receptor Tom22. We asked whetherTom40 molecules are close enough to make di-rect contactwith each other, like bacterial b-barrelproteins (15), by chemical cross-linking with theSH-directed homobifunctional cross-linkers BMBor M2M. We introduced Cys at membrane-facing

positions as well as pore-facing positions of aCys-free Tom40 variant (8). Cross-linked productswere evident for the pairs of membrane-facingCys residues in endogenous Tom40 (Fig. 3A) orimported Tom40 (fig. S6A). The distances be-tween theSg atomsof cross-linkedCys are~6.9 and~12.0 Å for M2M and BMB cross-linking, respec-tively (16), indicating that two Tom40 moleculesare located within a distance of ~6.9 Å (Fig. 3B).Because the TM helix of Tom22 (Tom22TM)

interacts with two Tom40 molecules (17), we an-

alyzed the geometrical arrangement of Tom22and Tom40. We introduced BPA into Tom40 attwo of the positions 86, 309, 350, and 357 in thenarrow vertical Tom22-interacting regions alongthe b-barrel axis (Fig. 3C and fig. S6B) simulta-neously. UV irradiation generated cross-linkedTom40:[Tom22]2 oligomers for BPA positions86 and 309 (fig. S6B). Simultaneous introductionof BPA into Tom40 and Tom22 in their inter-acting regions (Fig. 3, C and D), generated cross-linked Tom40:[Tom22]2 and [Tom40]2:Tom22

SCIENCE sciencemag.org 25 SEPTEMBER 2015 • VOL 349 ISSUE 6255 1547

Fig. 3. The native TOM complex dynamically exchanges with the Tom40dimeric complex. (A) Mitochondria with the indicated Tom40 Cys residueswere treated with (+) or without (–) the cross-linker (1,4-bismaleimidobutane)[BMB (XL)]. Proteins were analyzed by SDS-PAGE and immunoblotting withantibodies to Tom40. Cross-linked products are indicated by the asterisk andarrowheads. (B) Schematic models for (A). (C) (Top) The Tom40 b barrelshowing the residues cross-linked with Tom22. (Botom) Tom22-interactingregions of Tom40 (blue and yellow) with the Tom22-cross-linked residues

(pink circles). (D) Tom40-interacting regions of Tom22TM (orange and green)(18). (E) In vivo cross-linking of BPA in Tom40 and Tom22 was detected byimmunoblotting. Cross-linked products are indicated. (F) Schematic modelsfor (E). (G) Mitochondria with the Tom40 Cys mutations were treated with (+)or without (–) the cross-linker BMOE [bis(maleimido)ethane]. Proteins wereanalyzed by SDS-PAGE (lanes 1 to 4), BN-PAGE (lanes 5 and 6), or 2D-PAGE(first- BN-PAGE and second-dimensional SDS-PAGE) and immunoblotting.Cross-linked products are indicated by the asterisk and arrowheads.



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oligomers only for a specific combination of in-troduced BPA (Fig. 3E). These results pose a geo-metrical constraint that is consistent only with athreefold rotational symmetric arrangement ofthree molecules each of Tom40 and Tom22 (Fig.3F), in which the distance between the Tom40molecules bridged by Tom22must be larger than22 Å, which is incompatible with a distance of~6.9 Å between the Tom40 molecules (fig. S6A).How can the opposing cross-linking results be

explained? We hypothesized that both dimericand trimeric isoforms of TOM complexes, previ-ously observed by single-particle electronmicros-copy analyses (18–20), exist in organello. Tom40Cysmutantmitochondriawere subjected to cross-linking and analyzed by SDS–polyacrylamide gelelectrophoresis (PAGE) and blue native (BN)–PAGE (Fig. 3G). SDS-PAGE demonstrated thecross-linked dimers, and BN-PAGE revealed thatthe cross-linked Tom40 dimers arose from a~100-kD (100K) subcomplex, not from the mature(large) TOM complex. The two Tom40 moleculesare thus close together, bridged by short chem-ical cross-linkers only in the 100K complex (Fig.3B). The 100K complex contains Tom40 and smallTom proteins, but not Tom22; it functions as alate assembly intermediate of newly importedTom40 on the pathway to the mature TOM com-plex (17, 21). Thus, the 100K complex containingthe dimer exchanges with the mature trimericTOM complex. A dynamic exchange between di-meric and trimeric forms provides the means fortemplate-driven assembly of new subunits throughtheir exchange for old subunits.The Tom22TM has been conserved through evo-

lution, with an invariant Pro (Pro112), flanked bybasic residues on the cytosolic side and acidic re-sidues on the IMS side (fig. S7). We generatedyeast strains with mutant Tom22 and analyzeddestabilization of the TOM complex and in vitroimport of presequence-containing andpresequence-

less preproteins by amino acid replacements ofthose residues (fig. S8). These analyses suggest thatthe full-sized mature TOM complex tethered byTom22 (Fig. 4, inset) is required for efficient pre-protein import. We also determined the inter-actions of Tom40 with the receptor Tom20 andthe small subunits Tom5, Tom6, and Tom7 (sum-marized in Fig. 2A and figs. S1 and S9). A com-plete model of the subunit arrangement in theTOM core complex is shown in Fig. 4 (middlepanel).We conclude that the trimeric mature TOM

complex dynamically exchanges with a dimericTom22-free form that provides an assembly plat-form for the integration of new subunits (Fig. 4,right panel). The dynamic a/b organization ofthe TOM complex favors both assembly of thecomplex and cooperative preprotein transfer fromreceptors to the import channel and IMS chap-erones, ensuring the efficient translocation of dif-ferent classes of preproteins into mitochondria.

REFERENCES AND NOTES

1. A. Chacinska, C. M. Koehler, D. Milenkovic, T. Lithgow,N. Pfanner, Cell 138, 628–644 (2009).

2. W. Neupert, J. M. Herrmann, Annu. Rev. Biochem. 76, 723–749(2007).

3. T. Endo, K. Yamano, Biol. Chem. 390, 723–730 (2009).4. T. Shiota, S. Nishikawa, T. Endo, Methods Mol. Biol. 1033,

207–217 (2013).5. S. Chen, P. G. Schultz, A. Brock, J. Mol. Biol. 371, 112–122

(2007).6. J. W. Chin et al., Science 301, 964–967 (2003).7. R. Ujwal et al., Proc. Natl. Acad. Sci. U.S.A. 105, 17742–17747

(2008).8. J. Qiu et al., Cell 154, 596–608 (2013).9. S. W. Lackey et al., J. Biol. Chem. 289, 21640–21650

(2014).10. M. Harner, W. Neupert, M. Deponte, EMBO J. 30, 3232–3241

(2011).11. D. Rapaport, J. Cell Biol. 171, 419–423 (2005).12. T. Kanamori et al., Proc. Natl. Acad. Sci. U.S.A. 96, 3634–3639

(1999).13. N. Wiedemann, N. Pfanner, M. T. Ryan, EMBO J. 20, 951–960

(2001).

14. M. Esaki et al., Nat. Struct. Biol. 10, 988–994 (2003).15. G. Meng, R. Fronzes, V. Chandran, H. Remaut, G. Waksman,

Mol. Membr. Biol. 26, 136–145 (2009).16. N. S. Green, E. Reisler, K. N. Houk, Protein Sci. 10, 1293–1304

(2001).17. T. Shiota, H. Mabuchi, S. Tanaka-Yamano, K. Yamano, T. Endo,

Proc. Natl. Acad. Sci. U.S.A. 108, 15179–15183 (2011).18. K. P. Künkele et al., Cell 93, 1009–1019 (1998).19. U. Ahting et al., J. Cell Biol. 147, 959–968 (1999).20. K. Model, C. Meisinger, W. Kühlbrandt, J. Mol. Biol. 383,

1049–1057 (2008).21. K. Model et al., Nat. Struct. Biol. 8, 361–370 (2001).

ACKNOWLEDGMENTS

We thank C. Stubenrauch, M. Belousoff, A. Traven, and themembers of the Endo lab for discussions and critical commentson the manuscript. We are grateful to P. G. Schultz for thematerials for in vivo cross-linking. This work was supported byGrants-in-Aid for Scientific Research from the Japan Society forthe Promotion of Science (JSPS) and a CREST Grant from theJapan Science and Technology Agency (JST) (T.E.); the Platformfor Drug Discovery, Informatics, and Structural Life Sciencefrom the Ministry of Education, Culture, Sports, Science andTechnology and Japan Agency for Medical Research andDevelopment (K.T., K.I., and Y.F.); Grants-in-Aid for ScientificResearch on Innovative Areas (“Matryoshka-type evolution,”no. 3308) (K.I., K.T., and Y.F.); the Strategic Japanese-SwedishCooperative Program on “Multidisciplinary BIO” (JST-Verket FörInnovationssystem/Swedish Foundation for Strategic Research)(K.I., N.S., Y.F., S.H., A.E., K.T., P.H., and T.E.); the DeutscheForschungsgemeinschaft (PF 202/8-1), Sonderforschungsbereiche746 and 1140; and the Excellence Initiative of the Germanfederal and state governments (EXC 294 BIOSS, GSC-4 SpemannGraduate School) (N.W., N.P., and J.Q.). T.S. is a ResearchFellow of the JSPS and was supported by the Toyobo BioFoundation, H.S.S. is an Australian Research Council (ARC) SuperScience Fellow (FS110200015), and T.L. is an ARC AustralianLaureate Fellow (FL130100038). The data presented in this paperare tabulated in the main paper and the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/349/6255/1544/suppl/DC1Materials and MethodsFigs. S1 to S9Tables S1 to S5References (22–39)

24 May 2015; accepted 21 August 201510.1126/science.aac6428


Fig. 4. Subunit organization of the TOM complex. Subunit arrangement of the Tom40 b barrel and TM a helices of Tom5,Tom6,Tom7, and Tom22, based onthe BPA cross-linking results and the proposed model for the exchange of the Tom40-Tom22 trimeric complex with the Tom40 dimer.The inset shows possibleinteractions between Tom22TM and the Tom40 b barrel, with key residues suggested from fig. S8. Basic residues, acidic residues, and others are colored in blue,red, and pink, respectively.The Tom22TM a helix, possibly bent at Pro

112, tethers two Tom40 molecules through the interactions of its N-terminal and C-terminalparts with conserved residues of adjacent Tom40 molecules.



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Molecular architecture of the active mitochondrial protein gate

Trevor Lithgow and Toshiya EndoFukasawa, Sikander Hayat, Megumi Kamiya, Arne Elofsson, Kentaro Tomii, Paul Horton, Nils Wiedemann, Nikolaus Pfanner, Takuya Shiota, Kenichiro Imai, Jian Qiu, Victoria L. Hewitt, Khershing Tan, Hsin-Hui Shen, Noriyuki Sakiyama, Yoshinori

DOI: 10.1126/science.aac6428 (6255), 1544-1548.349Science

, this issue p. 1544Science have worked out the architecture and mechanism of the mitochondrial protein import channel.et al.Shiota

synthesized proteins need to be imported across the organelle's membrane through dedicated protein import machinery. Mitochondria, the powerhouses of the cell, are mainly composed of proteins made in the cytosol. These newly

Dissecting the mitochondrial entry portal

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