Evidence for recycling of cytochrome P450 sterol 14-demethylase from the cis-Golgi compartment to the endoplasmic reticulum (ER) upon saturation of the ER-retention mechanism.
Cytochrome P450 sterol 14-demethylase (P450-CYP51) is the enzyme that catalyzes 14alpha demethylation of lanosterol, a step in ergosterol biosynthesis, on the cytoplasmic side of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. To investigate its localization and the localization mechanism(s), we constructed a chimera by inserting a 30-residue segment, Leu(283)-Leu(312) of P450-CYP51 containing a potential N-glycosylation site in the cytoplasmic region, into the N-terminus of the same protein and tagging the C-terminus with three repeats of a hemagglutinin epitope. This chimera complements gene disruption on a single-copy vector and undergoes N-glycosylation, showing that it functions normally in vivo. Indirect immunofluorescence microscopy revealed that this chimera is localized exclusively to the ER when it is expressed on either a single-copy or multicopy vector. We carried out pulse-chase experiments and found that this chimera, when expressed on a multicopy plasmid, gradually undergoes alpha1-->6 glycosylation, a cis-Golgi-specific modification, but not alpha1-->;3 glycosylation, a medial Golgi-specific modification. In contrast, a single-copy expression of this chimera does not lead to the cis-Golgi-specific modification. These findings suggest that, when expressed on a multicopy plasmid, a fraction of this chimera is transported from the ER to the cis-Golgi compartment and subsequently recycled to the ER, but when expressed on a single-copy plasmid, no significant transport of this protein from the ER takes place. We thus suggest the possibility that cytochrome P450 is retained in the ER by a saturable static mechanism.
Homma
K
K
Division of Molecular, Cell Pharmacology, National Children's Medical Research Center, Taishido, Setagaya-ku, Tokyo 154-8509, Japan.
Yoshida
Y
Y
Nakano
A
A
D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002451:Cell Compartmentation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D058886:Sterol 14-Demethylase
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases