Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms

J Biochem. 1999 Dec;126(6):1080-9. doi: 10.1093/oxfordjournals.jbchem.a022553.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cytochrome P-450 Enzyme System / metabolism*
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / genetics
  • Gene Expression Regulation, Fungal*
  • HSP70 Heat-Shock Proteins / biosynthesis*
  • HSP70 Heat-Shock Proteins / genetics
  • Protein Folding*
  • Saccharomyces cerevisiae

Substances

  • Fungal Proteins
  • HSP70 Heat-Shock Proteins
  • KAR2 protein, yeast
  • Cytochrome P-450 Enzyme System