title: Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL MscL does not need the insertase YidC for insertion in vitro.
abstract: The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.
authors.affiliation: Groupe Canaux ioniques, IBBMC, UMR CNRS 8619, Bât 430, Université Paris-Sud 11, 91405 Orsay Cedex, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Berrier
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bayan
authors.forename: Kyu-Ho
authors.initials: KH
authors.lastname: Park
authors.forename: Agnes
authors.initials: A
authors.lastname: Mesneau
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ghazi
mesh_terms: D001671:Biochemistry / Q000379:methods; D002474:Cell-Free System; D002736:Chloroplasts / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005614:Freeze Fracturing; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009995:Osmosis; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D026901:Membrane Transport Proteins; C086596:MscL protein, E coli; D010455:Peptides; C412443:YIDC protein, E coli