title: Post-translational membrane insertion of an endogenous YidC substrate.
abstract: 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 F0c (subunit c of the F0F1 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 F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c 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.
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Robinson
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address: cheryl.woolhead@glasgow.ac.uk.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Woolhead
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D006180:Proton-Translocating ATPases; D012995:Solubility; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D004075:Diglycerides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C412443:YIDC protein, E coli; D006180:Proton-Translocating ATPases
keywords: 1-2-dioleoyl-sn-glycerol; DAG; DDM; F(0)c; IPTG; LB; Membrane insertion; PK; RNC; Reconstitution; SRP; TBS; Targeting; YidC; isopropyl β-d-1-thiogalactopyranoside; lysogeny broth; n-dodecyl-β-maltoside; proteinase K; ribosome nascent chain; signal recognition particle; subunit c of the F(0)F(1) ATP synthase; tris buffered saline