Interaction of modified tail-anchored proteins with liposomes : Effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b_5(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b_5 (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH_2-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial liposomes.

著者

• Sakamoto Yoichi

  Department of Physics, Faculty of Science, Kochi University

• Miura Masahiro

  Department Of Chemistry Graduate School Of Science Kobe University

• Park Sam-yong

  Protein Design Lab. Yokohama City Univ.

• Takeuchi Fusako

  Department Of Pharmacy College Of Pharmaceutical Sciences Ritsumeikan University

• Tsubaki Motonari

  Department Of Chemistry Graduate School Of Science Kobe University

• Miura Masahiro

  Departmemt Of Chemistiy Graduate School Of Science Kobe University

• Park Sam-yong

  Protein Design Laboratory Department Of Supramolecular Biology Graduate School Of Nanobioscience Yok

• Takeuchi Fusako

  Institute for Promotion of Higher Education, Kobe University

• Sakamoto Yoichi

  Departmemt Of Chemistiy Graduate School Of Science Kobe University

• Takeuchi Fusako

  Institute For Promotion Of Higher Education Kobe University

• Tsubaki Motonari

  Departmemt of Chemistiy, Graduate School of Science, Kobe University

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