In situ topology of cytochrome b5 in the endoplasmic reticulum membrane

Rieko Kuroda, Jun Ya Kinoshita, Masanori Honsho, Jun Ya Mitoma, Akio Ito

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Abstract

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. 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-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immune-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 carboxy-terminal 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.

Original languageEnglish
Pages (from-to)828-833
Number of pages6
JournalJournal of biochemistry
Volume120
Issue number4
DOIs
Publication statusPublished - Jan 1 1996

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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