title: Interaction of modified tail-anchored proteins with liposomes: effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b₅.
abstract: 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.
authors.affiliation: Departmemt of Chemistry, Graduate School of Science, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Miura
authors.forename: Fusako
authors.initials: F
authors.lastname: Takeuchi
authors.forename: Sam-Yong
authors.initials: SY
authors.lastname: Park
authors.forename: Motonari
authors.initials: M
authors.lastname: Tsubaki
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D055355:Opsins / Q000235:genetics; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D055355:Opsins; D015786:Cytochromes b5; D014357:Trypsin