An N-terminal glycosylation signal on cytochrome P450 is restricted to the endoplasmic reticulum in a luminal orientation.
The mechanism of retention of cytochrome P450 in the endoplasmic reticulum is unknown, and the membrane topology of the N-terminal region remains controversial. To address these problems, a sequence of 29 amino acids encoding an internal N-glycosylation site of rabbit cytochrome P450 2C2 was attached to the N terminus of cytochrome P450 2C1. This protein is glycosylated at a single site in a cell-free translation system containing microsomal membranes, as indicated by gel mobility and sensitivity to endoglycosidase H. When expressed in COS1 cells, an immunoreactive species with the same gel mobility as the in vitro synthesized glycosylated product was detected. Treatment with endoglycosidase H changed its mobility to that of unglycosylated hybrid cytochrome P450 2C1. These results indicate that in intact cells, as in the cell-free system, the N terminus of cytochrome P450 is luminally oriented which is not consistent with a hairpin loop conformation. Sensitivity of the glycosylated protein to endoglycosidase H suggests that the protein does not reach the Golgi compartments. When transfected cells were incubated at low temperatures to inhibit retrograde transport from the intermediate pre-Golgi compartment into the endoplasmic reticulum, localization of cytochrome P450 was not changed, as assayed by subcellular fractionation and immunofluorescent staining. These observations suggest that cytochrome P450 is restricted to the endoplasmic reticulum membrane by a mechanism different from recycling through the intermediate compartment, which is a pathway utilized by soluble endoplasmic reticulum proteins.
Szczesna-Skorupa
E
E
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
Kemper
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation; D002460:Cell Line; D002474:Cell-Free System; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D014162:Transfection
D010446:Peptide Fragments; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D006596:Hexosaminidases