Abstract

Cytochrome P450 sterol 14-demethylase (P450-CYP51) is the enzyme that catalyzes 14α 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-resi-due 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-tenni-nus with three repeats of a hemagglutinin epitope. This chimera complements gene disruption on a single-copy vector and undergoes JV-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 αl→6 glycosylation, a cis-Golgi-specific modification, but not αl→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 Key words cytochrome P450 vesicular transport yeast Saccharomyces cerevisiae © 2000 by the Japanese Biochemical Society « Previous | Next Article » Table of Contents This Article J Biochem (2000) 127 (5): 747-754. » Abstract Free Full Text (PDF) Free Classifications Regular Paper Services Article metrics Alert me when cited Alert me if corrected Find similar articles Similar articles in Web of Science Similar articles in PubMed Add to my archive Download citation Request Permissions Citing Articles Load citing article information Citing articles via CrossRef Citing articles via Scopus Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Homma, K. Articles by Nakano, A. Search for related content PubMed PubMed citation Articles by Homma, K. Articles by Yoshida, Y. Articles by Nakano, A. 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