tailieunhanh - Báo cáo khoa học: Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated fromEscherichia coli(the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexa-meric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. | Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation Yuki Moritani1 2 Shin-ichiro M. Nomura1 Ikuo Morita3 4 and Kazunari Akiyoshi1 4 1 Institute of Biomaterials and Bioengineering Tokyo Medicaland DentalUniversity Japan 2 Research Laboratories NTT DOCOMO Inc. Kanagawa Japan 3 Department of Cellular PhysiologicalChemistry Tokyo Medicaland DentalUniversity Japan 4 GlobalCenter of Excellence GCOE Program InternationalResearch Center for Molecular Science in Tooth and Bone Diseases Tokyo Medicaland DentalUniversity Japan Keywords cell-free membrane protein synthesis chaperone-like function for hemichannels connexin liposome membrane protein orientation Correspondence K. Akiyoshi Institute of Biomaterials and Bioengineering Tokyo Medicaland Dental University 2-3-10 Kanda-Surugadai Chiyoda-ku Tokyo 101-0062 Japan Fax 81 3 5280 8027 Tel 81 3 5280 8020 E-mail Present address Institute for Integrated Cell-Material Sciences iCeMS Kyoto University Japan Received 18 March 2010 revised 31 May 2010 accepted 9 June 2010 doi 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 .