In situ topology of cytochrome b5 in the endoplasmic reticulum membrane.
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-permeabilized 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 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.
Kuroda
R
R
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Kinoshita
J
J
Honsho
M
M
Mitoma
J
J
Ito
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins; D019556:COS Cells / Q000378:metabolism / Q000648:ultrastructure; D015786:Cytochromes b5 / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D017830:Octoxynol; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis; D051381:Rats; D013301:Streptolysins
D000906:Antibodies; D001426:Bacterial Proteins; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D013301:Streptolysins; C025964:streptolysin O; D017830:Octoxynol; D015786:Cytochromes b5