1 Scientific RepoRts | 6:38399 | DOI: 10.1038/srep38399 www.nature.com/scientificreports A central cavity within the holo- translocon suggests a mechanism for membrane protein insertion Mathieu Botte 1,* , Nathan R. Zaccai 2,* , Jelger Lycklama à. Nijeholt 1 , Remy Martin 2 , Kèvin Knoops 1 , Gabor papai 3 , Juan Zou 4 , Aurélien Deniaud 1 , Manikandan Karuppasamy 1 , Qiyang Jiang 1 , Abhishek singha Roy 5 , Klaus schulten 5 , patrick schultz 3 , Juri Rappsilber 4,6 , Giuseppe Zaccai 7,8 , Imre Berger 1,2 , Ian Collinson 2 & Christiane Schaftzel 1,2 the conserved secYeG protein-conducting channel and the accessory proteins secDF-YajC and YidC constitute the bacterial holo-translocon (HtL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (sANs), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. the results guided the placement of X-ray structures of individual HtL components and allowed the proposal of a model of the functional translocon. their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. the periplasmic domains of YidC and secD are poised at the protein- channel exit-site of secY, presumably to aid the emergence of translocating polypeptides. the secY lateral gate for membrane-insertion is adjacent to the membrane ‘insertase’ YidC. Absolute-scale sANs employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact confgurations around an adaptable central lipid-flled chamber, wherein polytopic membrane- proteins could fold, sheltered from aggregation and proteolysis. Te hetero-trimeric Sec protein-conducting channel translocates integral inner membrane proteins and secretory proteins into or across the membrane 1,2 . Doung and Wickner discovered that in bacteria additional factors associate with this complex to facilitate efcient protein translocation and named the supercomplex “preprotein translocase holoenzyme” 3 . Tey co-immunoprecipitated SecYEG, YajC and SecDF as well as a ~60 kDa protein which was subsequently identifed as YidC 4 from radiolabeled Escherichia coli membranes using an anti-SecG antibody. Further study, however, was impeded by the lack of means to produce holo-translocon in the quality and quantity required for its biochemical and structural characterization. More recently, using recombinant highly purifed SecYEG-SecDFYajC-YidC holo-translocon, it was shown that the complex is active in co- and post-translational translocation 5 . A recent proteomics study in E. coli based on absolute protein synthesis rates provided protein copy number estimates 6 (Figure S1). Tis data is consistent with a molar ratio of SecY, SecE, SecG, SecD, SecF, YajC and YidC of ~4:4:4:1:1:10:3 in the membrane, suggesting that as much as ~25% of all SecYEG could be complexed in HTL.Under optimal growth conditions, the protein synthesis of HTL could amount to 2,600 copies of HTL per generation 6 ; the real copy number is likely smaller, since this number does not take into account any protein turnover. Even accounting for a high level of turnover, this is in stark contrast to a previous copy number 1 European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38042 Grenoble, France. 2 School of Biochemistry, University of Bristol, BS8 1TD, United Kingdom. 3 Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), U964 INSERM, UMR7104 CNRS; University of Strasbourg, 1 Rue Laurent Fries, BP10142, 67404 Illkirch, France. 4 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, United Kingdom. 5 Department of Physics, University of Illinois Urbana Champaign, 3217 Beckman Institute, 405 N Mathews Ave., Urbana, IL 61801, USA. 6 Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany. 7 Institut Laue Langevin, 71 Avenue des Martyrs, F-38042 Grenoble, France. 8 CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to I.C. (email: ian.collinson@bristol.ac.uk) or C.S. (email: christiane.berger-schaftzel@bristol. ac.uk) Received: 28 July 2016 Accepted: 08 November 2016 Published: 07 December 2016 opeN