Post-translational membrane insertion of an endogenous YidC substrate

Robinson, P.J. and Woolhead, C.A. (2013) Post-translational membrane insertion of an endogenous YidC substrate. Biochimica et Biophysica Acta: Molecular Cell Research, 1833(12), pp. 2781-2788. (doi:10.1016/j.bbamcr.2013.07.003)

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Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F<sub>0</sub>c (subunit c of the F<sub>0</sub>F1 ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F<sub>0</sub>c and (iii) show that incubation of F<sub>0</sub>c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F<sub>0</sub>c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Woolhead, Professor Cheryl and Robinson, Dr Philip
Authors: Robinson, P.J., and Woolhead, C.A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Biochimica et Biophysica Acta: Molecular Cell Research

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
496961Folding and insertion of a bacterial inner membrane proteinCheryl WoolheadBiotechnology and Biological Sciences Research Council (BBSRC)BB/G011281/1RI MOLECULAR CELL & SYSTEMS BIOLOGY