caa a22 4500 397558112 CHVBK 20180308164809.0 cr unu---uuuuu 161202e199610 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021486 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021486 In Situ Topology of Cytochrome b5 in the Endoplasmic Reticulum Membrane [Elektronische Daten] [Rieko Kuroda, Jun-ya Kinoshita, Masanori Honsho, Jun-ya Mitoma, Akio Ito] Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-per-meabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxyterminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence cytochrome b5 nationallicence endoplasmic reticulum nationallicence immunofluorescence microscopy nationallicence membrane topology nationallicence tail-anchored protein nationallicence Kuroda Rieko Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut Kinoshita Jun-ya Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut Honsho Masanori Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut Mitoma Jun-ya Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut Ito Akio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut The Journal of Biochemistry Oxford University Press 120/4(1996-10), 828-833 0021-924X 120:4<828 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021486 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021486 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kuroda Rieko Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut NATIONALLICENCE 700 1- Kinoshita Jun-ya Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut NATIONALLICENCE 700 1- Honsho Masanori Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut NATIONALLICENCE 700 1- Mitoma Jun-ya Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut NATIONALLICENCE 700 1- Ito Akio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku Fukuoka 812-81 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/4(1996-10), 828-833 0021-924X 120:4<828 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford