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The divergent outputs of Ire1 during the Unfolded Protein Response in fission yeast and budding yeast

  • Author(s): Crotty, Kelly
  • Advisor(s): Walter, Peter
  • et al.
Abstract

In metazoans, the Unfolded Protein Response (UPR) restores homeostasis in the endoplasmic reticulum (ER) by both increasing the protein-folding capacity of the ER and by reducing the protein folding burden. The UPR in S. cerevisiae exclusively relies on this first solution to survive ER stress. Remarkably, S. pombe utilizes the second, though the UPR in both species is mediated through site-specific RNA cleavage by the evolutionarily conserved kinase/RNase transmembrane protein Ire1. While most of the S. pombe Ire1 mRNA substrates are degraded post cleavage, the mRNA encoding the ER chaperone protein BiP1 evades decay and the 5’ fragment accumulates after truncation. Though the mechanism behind this stabilization remains unresolved, we have discovered that the relevance of bip1 mRNA processing to the cell’s survival may be greater than previously estimated. We have compared the Ire1 kinase/RNase domain from each species in vitro and discovered key structural differences we can modulate to make the S. cerevisiae Ire1 behave like S. pombe Ire1. First, we characterized differences in cofactor sensitivity in the kinase domains of the two enzymes. Additionally, we exploited the toxicity of S. pombe Ire1 in bacterial cells to identify mutations in the more stringent S. cerevisiae Ire1 protein that would cause toxicity and relax substrate stringency. We identified a pair of mutations at the RNase dimer interface that expanded the S. cerevisiae enzyme’s substrate repertoire and coincided with an increase in its propensity to dimerize. This interface could represent an under-utilized target for drug design to modulate the RNase activity of metazoan Ire1.

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