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4. Read, B.A., Kegel, J., Klute, M.J., Kuo, A., 11. Quince, C., Lanzen, A., Davenport, R.J., and 17. Mangot, J.F., Domaizon, I., Taib, N., Marouni, N., Lefebvre, S.C., Maumus, F., Mayer, C., Turnbaugh, P.J. (2011). Removing noise from Duffaud, E., Bronner, G., and Debroas, D. (2013). Miller, J., Monier, A., Salamov, A., et al. (2013). pyrosequenced amplicons. BMC Short-term dynamics of diversity patterns: Pan genome of the phytoplankton Emiliania Bioinformatics 12, 38. evidence of continual reassembly within underpins its global distribution. Nature 499, 12. Koeppel, A.F., and Wu, M. (2013). Surprisingly lacustrine small . Environ. Microbiol. 209–213. extensive mixed phylogenetic and ecological 15, 1745–1758. 5. Sogin, M.L., Morrison, H.G., Huber, J.A., Mark signals among bacterial Operational 18. Nolte, V., Pandey, R.V., Jost, S., Medinger, R., Welch, D., Huse, S.M., Neal, P.R., Arrieta, J.M., Taxonomic Units. Nucleic Acids Res. 41, Ottenwalder, B., Boenigk, J., and and Herndl, G.J. (2006). Microbial diversity 5175–5188. Schlotterer, C. (2010). Contrasting seasonal in the deep sea and the underexplored ‘‘rare 13. Stoeck, T., Bass, D., Nebel, M., Christen, R., niche separation between rare and abundant biosphere’’. Proc. Natl. Acad. Sci. USA 103, Jones, M.D., Breiner, H.W., and Richards, T.A. taxa conceals the extent of protist diversity. 12115–12120. (2010). Multiple marker parallel tag Mol. Ecol. 19, 2908–2915. 6. Pedros-Alio, C. (2012). The rare bacterial environmental DNA sequencing reveals a 19. Chow, C.E., Sachdeva, R., Cram, J.A., biosphere. Annu. Rev. Mar. Sci. 4, 449–466. highly complex eukaryotic community in Steele, J.A., Needham, D.M., Patel, A., 7. Vergin, K.L., Beszteri, B., Monier, A., Cameron marine anoxic water. Mol. Ecol. 19 (Suppl 1 ), Parada, A.E., and Fuhrman, J.A. (2013). Thrash, J., Temperton, B., Treusch, A.H., 21–31. Temporal variability and coherence of euphotic Kilpert, F., Worden, A.Z., and Giovannoni, S.J. 14. Stoeck, T., Behnke, A., Christen, R., zone bacterial communities over a decade in (2013). High-resolution SAR11 ecotype Amaral-Zettler, L., Rodriguez-Mora, M.J., the Southern California Bight. ISME J. 7, dynamics at the Bermuda Atlantic Chistoserdov, A., Orsi, W., and Edgcomb, V.P. 2259–2273. Time-series Study site by phylogenetic (2009). Massively parallel tag sequencing 20. Not, F., Gausling, R., Azam, F., Heidelberg, J.F., placement of pyrosequences. ISME J. 6, reveals the complexity of anaerobic and Worden, A.Z. (2007). Vertical distribution 481–492. marine protistan communities. BMC Biol. of picoeukaryotic diversity in the Sargasso Sea. 8. Caron, D.A., and Countway, P.D. (2009). 7, 72. Environ. Microbiol. 9, 1233–1252. Hypotheses on the role of the protistan rare 15. Lecroq, B., Lejzerowicz, F., Bachar, D., biosphere in a changing world. Aquat. Microb. Christen, R., Esling, P., Baerlocher, L., 1 Ecol. 57, 227–238. Osteras, M., Farinelli, L., and Pawlowski, J. Monterey Bay Aquarium Research Institute 9. Lennon, J.T., and Jones, S.E. (2011). Microbial (2011). Ultra-deep sequencing of foraminiferal (MBARI), Moss Landing, CA 95039, USA. seed banks: the ecological and evolutionary microbarcodes unveils hidden richness of early 2Integrated Microbial Biodiversity Program, implications of dormancy. Nat. Rev. Microbiol. monothalamous lineages in deep-sea Canadian Institute for Advanced Research, 9, 119–130. sediments. Proc. Natl. Acad. Sci. USA 108, 10. Huse, S.M., Welch, D.M., Morrison, H.G., and 13177–13182. Toronto, M5G 1Z8, Canada. Sogin, M.L. (2010). Ironing out the wrinkles 16. Jones, S.E., and Lennon, J.T. (2010). Dormancy *E-mail: [email protected] in the rare biosphere through improved contributes to the maintenance of microbial OTU clustering. Environ. Microbiol. 12, diversity. Proc. Natl. Acad. Sci. USA 107, 1889–1898. 5881–5886. http://dx.doi.org/10.1016/j.cub.2014.03.029

Protein Translocation: The / binds to cytosolic loops of the translocon, whereupon the signal SecYEG Translocon Caught in the Act sequence mediates pore opening and initiates transfer of the growing The Sec61/SecYEG complex mediates both the translocation of newly polypeptide from the ribosome through synthesized across the membrane and the integration of the channel. Hydrophobic segments transmembrane segments into the . New cryo-electron microscopy trigger lateral opening of the channel studies show ribosome–channel complexes in action and reveal their repertoire and integrate into the membrane as TM of conformational states. segments. Exactly how these steps work mechanistically is not known. The translocon is composed of Martin Spiess released into the lipid bilayer. From subunits SecY, E, and G in bacteria with extensive biochemical analyses and ten, one, and one or two TM domains, Biological membranes separate crystal structures of the closed, idle respectively, corresponding to Sec61a, cellular compartments, generating and translocon, a general picture of these g, and b in eukaryotes [1]. The first preserving concentration gradients dynamic processes has been pieced crystal structure of an idle translocon, and electrical potentials. How are entire together. Two new studies [3,4] now from Methanocaldococcus jannaschii polypeptides transported across or show cryo-electron microscopy (EM) 10 years ago [5], changed the view of inserted into membranes while structures of translocons in action, the translocation pore dramatically. maintaining the barrier? This task is arrested either at the point of signal Rather than an oligomer of several Sec accomplished by a conserved sequence insertion, polypeptide complexes forming a wide water-filled -conducting channel — the translocation, or transmembrane channel, it was found to be a compact SecYEG complex at the plasma segment integration, letting us watch helix bundle of a single heterotrimer membrane of , or the Sec61 the translocon at work more directly with the potential to open a narrow pore translocon at the endoplasmic than ever. This work confirms that the (Figure 1A). The ten TM segments of reticulum of eukaryotes [1,2]. picture that emerged from previous SecY form an hourglass shape with an translating secretory or biochemical data is encouragingly empty vestibule on the cytosolic side membrane proteins are targeted to accurate. and a lumenal cavity occupied by a the translocon by signal . As a hydrophobic signal sequence short hydrophobic helix — the Hydrophilic sequences are threaded emerges from the translating ribosome, so-called plug. The two cavities are through a polar channel, while apolar it is bound by the signal recognition separated by a central constriction of transmembrane (TM) segments stop particle (SRP) and targeted to SRP six apolar amino acid side chains. further translocation and are laterally receptors in the membrane. The SecY appears to be composed of two Current Biology Vol 24 No 8 R318

In the new studies, Park et al. [3] and Gogala et al. [4] used cryo-EM and single-particle analysis of defined translocation intermediates to explore and visualize the ribosome-bound translocon in action. They improved on previous cryo-EM structures [18,19] with a number of elegant tricks. Park et al. [3] produced early translocation intermediates in living Escherichia coli cells by inducing expression of a 100-amino acid with an amino-terminal signal sequence and a carboxy-terminal translational stalling sequence. Engineered cysteines were oxidized to form a stabilizing crosslink between the end of the signal sequence and the plug (with the risk of introducing a structural bias), before solubilization and sequential purification for affinity tags in the ribosome and the translocon. Similarly, Gogala et al. [4] translated stalled Figure 1. The translocon in successive functional states. nascent chains with two amino- Schematic representation of conformational states of a ribosome-bound idle translocon (A) or terminal TM segments followed by a translocons engaged with a signal sequence (B), with a translocating chain (C), or with an translocating chain with or without a inserting TM domain (D). The amino- and carboxy-terminal halves of the translocon are shown third TM domain into dog microsomal in blue and red, respectively; the main gate helices TM2 and TM7 as a blue and a red bar; the plug in yellow; and the nascent chains in purple with the signal and TM helices as purple bars. membranes. Upon solubilization, an Side views onto the lateral gate (with part of the ribosomes in gray) are shown in the upper row affinity tag allowed purification of and top views from the in the lower row. associated ribosome–translocon complexes, and glycosylation sites confirmed the expected membrane- rather symmetrical halves of five TMs Crosslinking the lateral gate shut spanning state of the substrates. connected on one side, where SecE/ abolished secretory protein Resolutions were sufficient to resolve Sec61g also clamps the structure, translocation, whereas crosslinking a-helices of the translocons and extra leaving a single potential lateral outlet across the gate with a spacer of R5A˚ densities of the nascent chains, on the opposite side between TMs 2/3 could still support translocation, allowing molecular dynamics flexible and 7/8. indicating that transport requires some fitting of structural models into the Clearly, the idle translocon expansion of the pore with slight experimental density maps. represents a closed state, and opening opening of the gate [9]. The plug could Both studies [3,4] revealed that the potential central channel requires be deleted without loss of translocon ribosomes were bound to single removal of the plug and widening of the functionality [10]: the plug was found to SecYEG/Sec61 complexes within central constriction. The hydrophobic be able to move out of its cavity and detergent–lipid micelles without signal sequence and TM sequences contact SecE, but, surprisingly, it did inducing major structural changes. The must further induce lateral gate not have to do so, since fixing it inside signal sequence was found as a helix opening to allow exit into the lipid by a disulfide crosslink was compatible inside the lateral gate exposed to lipids phase. Additional structures of with function [11]. Probing the [3] (Figure 1B). Translocon opening fortuitous crystal packing with a bound environment of the plug suggested that involved mostly rigid body movement antibody [6] or with the amino-terminal the plug actually prefers to stay inside of its two halves by a large rotation and sequence of SecY mutually inserted the cavity [12]. tilt. The plug moved very little, perhaps as helices into adjacent units [7] Signal sequences and TM domains because it was crosslinked, but the suggested how the gate might ‘crack of nascent chains arrested inside the splaying out of the translocon opened a open’. channel could be crosslinked to the gap sufficient for a translocating A wealth of biochemical translocon as well as to lipids [13], peptide. Interestingly, the extra length experiments, particularly site-specific suggesting positions in contact with of the nascent chain was not detected crosslinking between substrate both. Indeed, these domains could as a loop on the lumenal side of the peptides and the translocon or lipids, be crosslinked to the gate helices in channel, but appeared to loop out added detail to an emerging model how surprisingly defined positions [14,15], underneath the ribosome (which might the translocon works. For instance, it even when extended downstream illustrate that a flexible polypeptide was confirmed that the translocating sequences had already been cannot be pushed into the pore by the polypeptide moves through the center synthesized. In some cases, the translating ribosome, but may require a of a single SecY subunit [8], rather than interaction appeared to persist until the pulling force or ratchet). through a pore formed by exterior next TM pushed the previous one The Sec61 translocon containing a surfaces of multiple complexes. out [16,17]. hydrophilic chain in arrested transit Dispatch R319

(Figure 1C) was almost closed with the 2. Shao, S., and Hegde, R.S. (2011). Membrane 13. Martoglio, B., Hofmann, M.W., Brunner, J., and ˚ protein insertion at the . Dobberstein, B. (1995). The protein-conducting gate laterally open by less than 4A and Annu. Rev. Cell Dev. Biol. 27, 25–56. channel in the membrane of the endoplasmic the plug not detectably shifted [4]. 3. Park, E., Me´ ne´ tret, J.-F., Gumbart, J.C., reticulum is open laterally toward the lipid However, the changes appeared to be Ludtke, S.J., Li, W., Whynot, A., Rapoport, T.A., bilayer. Cell 81, 207–214. and Akey, C.W. (2014). Structure of the SecY 14. Plath, K., Mothes, W., Wilkinson, B.M., sufficient to produce a small gap in channel during initiation of protein Stirling, C.J., and Rapoport, T.A. (1998). Signal front of the plug. The nascent chain was translocation. Nature 506, 102–106. sequence recognition in posttranslational 4. Gogala, M., Becker, T., Beatrix, B., protein transport across the yeast ER not visible, suggesting a flexible and Armache, J.-P., Barrio-Garcia, C., membrane. Cell 94, 795–807. extended conformation, and neither Berninghausen, O., and Beckmann, R. (2014). 15. McCormick, P.J., Miao, Y., Shao, Y., Lin, J., and were the amino-terminal TM domains, Structures of the Sec61 complex engaged in Johnson, A.E. (2003). Cotranslational protein nascent peptide translocation or membrane integration into the ER membrane is mediated indicating that they had been released insertion. Nature 506, 107–110. by the binding of nascent chains to translocon into the hydrophobic phase. In 5. van den Berg, B., Clemons, W.M., Collinson, I., proteins. Mol. Cell 12, 329–341. Modis, Y., Hartmann, E., Harrison, S.C., and 16. Sadlish, H., Pitonzo, D., Johnson, A.E., and contrast, the TM domain that had just Rapoport, T.A. (2003). X-ray structure of Skach, W.R. (2005). Sequential triage of entered the translocon (Figure 1D) was a protein-conducting channel. Nature 427, transmembrane segments by Sec61alpha again detected as an extra density 36–44. during biogenesis of a native multispanning 6. Tsukazaki, T., Mori, H., Fukai, S., Ishitani, R., . Nat. Struct. Mol. Biol. 12, suggestive of a helix, which was Mori, T., Dohmae, N., Perederina, A., Sugita, Y., 870–878. intercalated into the lateral gate that Vassylyev, D.G., Ito, K., et al. (2008). 17. Hou, B., Lin, P.-J., and Johnson, A.E. (2012). w ˚ Conformational transition of Sec machinery Membrane protein TM segments are retained at was splayed open by 12 A. The plug inferred from bacterial SecYE structures. the translocon during integration until the had slightly moved, but remained Nature 455, 988–991. nascent chain cues FRET-detected release into inside the translocon, with gate 7. Egea, P.F., and Stroud, R.M. (2010). Lateral bulk lipid. Mol. Cell 48, 398–408. opening of a translocon upon entry of protein 18. Becker, T., Bhushan, S., Jarasch, A., opening providing most of the space suggests the mechanism of insertion into Armache, J.-P., Funes, S., Jossinet, F., for the passage of the chain. membranes. Proc. Natl. Acad. Sci. USA 107, Gumbart, J., Mielke, T., Berninghausen, O., 17182–17187. Schulten, K., et al. (2009). Structure of These new cryo-EM snapshots 8. Cannon, K.S., Or, E., Clemons, W.M., monomeric yeast and mammalian Sec61 reveal a spectrum of conformational Shibata, Y., and Rapoport, T.A. (2005). complexes interacting with the translating states of SecYEG/Sec61 translocons at Disulfide bridge formation between SecY and ribosome. Science 326, 1369–1373. a translocating polypeptide localizes the 19. Frauenfeld, J., Gumbart, J., Sluis, E.O.V.D., work. At the same time, they support a translocation pore to the center of SecY. J. Cell Funes, S., Gartmann, M., Beatrix, B., Mielke, T., number of conclusions derived from Biol. 169, 219–225. Berninghausen, O., Becker, T., Schulten, K., 9. du Plessis, D., Berrelkamp, G., Nouwen, N., and et al. (2011). Cryo-EM structure of the more indirect biochemical Driessen, A. (2009). The lateral gate of SecYEG ribosome-SecYE complex in the membrane experiments. What is still missing of opens during protein translocation. J. Biol. environment. Nat. Struct. Mol. Biol. 18, course is the dynamics. It has been Chem. 284, 15805–15814. 614–621. 10. Junne, T., Schwede, T., Goder, V., and 20. Hessa, T., Kim, H., Bihlmaier, K., Lundin, C., proposed that TM domain integration is Spiess, M. (2006). The plug domain of Boekel, J., Andersson, H., Nilsson, I., the result of dynamic equilibration yeast Sec61p is important for efficient White, S.H., and Heijne von, G. 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Animal Communication: Keep Your resolve rapid movements by prey, but Wings Off My Food! not so quickly that their returning echoes become masked by the next outgoing call [4]. They must also When foraging, male big brown bats produce ultrasonic social calls. The calls contend with the potentially masking repel rival bats from the caller and its prey, and increase the caller’s foraging effects of calls produced by other success during their high-speed aerial excursions. foraging bats [5]. Given these constraints, it may seem unlikely that David R. Wilson ‘echolocation’ [2,3]. During flight, bats flying bats would stress their vocal utter loud calls in rapid succession system further by producing and People have marveled for centuries at (often up to 200 calls per second), and perceiving acoustic signals that are not the ability of bats to hunt in complete then use the returning echoes to used directly in echolocation. Yet, darkness [1]. While navigating cluttered decipher the location and salient exciting new research in this issue of environments, they pursue prey with features of objects in their environment Current Biology by Genevieve Wright astounding agility and strike them with [3]. Although powerful, echolocation is and colleagues [6] shows that foraging lethal precision. These remarkable subject to a number of fundamental male big brown bats do supplement feats are possible because of a constraints. For example, bats must their echolocation calls during flight by complex biosonar system known as produce calls quickly enough to producing social calls that repel rivals