title: Reprogramming chaperone pathways to improve membrane protein expression in Escherichia coli.
abstract: Because membrane proteins are difficult to express, our understanding of their structure and function is lagging. In Escherichia coli, α-helical membrane protein biogenesis usually involves binding of a nascent transmembrane segment (TMS) by the signal recognition particle (SRP), delivery of the SRP-ribosome nascent chain complexes (RNC) to FtsY, a protein that serves as SRP receptor and docks to the SecYEG translocon, cotranslational insertion of the growing chain into the translocon, and lateral transfer, packing and folding of TMS in the lipid bilayer in a process that may involve chaperone YidC. Here, we explored the feasibility of reprogramming this pathway to improve the production of recombinant membrane proteins in exponentially growing E. coli with a focus on: (i) eliminating competition between SRP and chaperone trigger factor (TF) at the ribosome through gene deletion; (ii) improving RNC delivery to the inner membrane via SRP overexpression; and (iii) promoting substrate insertion and folding in the lipid bilayer by increasing YidC levels. Using a bitopic histidine kinase and two heptahelical rhodopsins as model systems, we show that the use of TF-deficient cells improves the yields of membrane-integrated material threefold to sevenfold relative to the wild type, and that whereas YidC coexpression is beneficial to the production of polytopic proteins, higher levels of SRP have the opposite effect. The implications of our results on the interplay of TF, SRP, YidC, and SecYEG in membrane protein biogenesis are discussed.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington 98195-1750, USA.
authors.forename: Brent L
authors.initials: BL
authors.lastname: Nannenga
authors.forename: François
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D057005:Bioengineering; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D017510:Protein Folding; D012172:Retinaldehyde / Q000378:metabolism; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; C412443:YIDC protein, E coli; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase; D012172:Retinaldehyde