Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL

• Published in: Biochimica et biophysica acta. Biomembranes 2011-01, Vol.1808 (1), p.41-46
• Main Authors: Berrier, Catherine, Guilvout, Ingrid, Bayan, Nicolas, Park, Kyu-Ho, Mesneau, Agnes, Chami, Mohamed, Pugsley, Anthony P., Ghazi, Alexandre
• Format: Article
• Language: English
• Subjects: Cell-free synthesis; Insertase; Ion channel; Liposome; Membrane protein
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2010.09.018

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. ►The channel MscL can be produced in a cell-free system in the presence of liposomes. ►It inserts directly in the lipid bilayer in the absence of the insertase YidC. ►The inserted channels are highly active, as evidenced by patch-clamp experiments.