Malfolded cytochrome P-450(M1) localized in unusual membrane structures of the endoplasmic reticulum in cultured animal cells.
A conserved region containing three to five proline residues is present just behind the signal-anchor sequence in the amino terminal portion of most microsomal cytochrome P-450s. We have shown that the proline residues are crucial for correct folding in Schizosaccharomyces pombe cells by using mutants of P-450(M1) in which one to three of the proline residues were changed to alanine. To examine the effects of the mutations on the intracellular localization of P-450s, they were expressed in COS-7 cells. They were found to be localized only in the perinuclear loci as patched structures like the Golgi apparatus, while the wild-type P-450(M1) is localized in the reticular structures which are typical for the ER membrane. However, treatment of the cells with Brefeldin A had no effect on the patched structures. Upon co-expression with another ER membrane protein, CD4D, which possesses a double lysine motif, the expressed CD4D was localized not only in the patched structures as the mutated P-450(M1)s, but also in the reticular structures of ER. When the cells were homogenized and then fractionated, the mutated P-450(M1) was recovered mainly in the low-speed precipitate and in the fractions of much higher density than the normal ER membrane. On electron microscopic observation, unusual membranous bodies were observed near the nucleus only when the mutated P-450(M1) was expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishihara
N
N
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Yamashina
S
S
Sakaguchi
M
M
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D017510:Protein Folding; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D009838:Oligodeoxyribonucleotides; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase