Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation

Yuki Moritani, Shin Ichiro M. Nomura, Ikuo Morita, Kazunari Akiyoshi

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)

Abstract

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free-synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins.

Original languageEnglish
Pages (from-to)3343-3352
Number of pages10
JournalFEBS Journal
Volume277
Issue number16
DOIs
Publication statusPublished - 2010 Aug
Externally publishedYes

Keywords

  • cell-free membrane protein synthesis
  • chaperone-like function for hemichannels
  • connexin
  • liposome
  • membrane protein orientation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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