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.
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.
Berrier
Catherine
C
Groupe Canaux ioniques, IBBMC, UMR CNRS 8619, Bât 430, Université Paris-Sud 11, 91405 Orsay Cedex, France.
Guilvout
Ingrid
I
Bayan
Nicolas
N
Park
Kyu-Ho
KH
Mesneau
Agnes
A
Chami
Mohamed
M
Pugsley
Anthony P
AP
Ghazi
Alexandre
A
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
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