title: Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms.
abstract: Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae. Using various KAR2 promoter constructs in front of the Escherichia coli beta-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region. Additionally, a less pronounced induction was detected for the UPR element (UPRE). As expected, this response was absent in the ire1 disruptant strain. However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway. Western blotting confirmed that Kar2p levels were increased to the same extent in the ire1 disruptant and in the non-disruptant strain. Removal of the P450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained. The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P450 domain and to the membrane-integrated mutant P450, respectively.
authors.affiliation: The University of Tokyo, Department of Biotechnology, Cellular Genetics Laboratory, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Zimmer
authors.forename: A
authors.initials: A
authors.lastname: Ogura
authors.forename: A
authors.initials: A
authors.lastname: Ohta
authors.forename: M
authors.initials: M
authors.lastname: Takagi
mesh_terms: D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D017510:Protein Folding; D012441:Saccharomyces cerevisiae
chemical_list: D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D003577:Cytochrome P-450 Enzyme System