The Metastable XBP1u Transmembrane Domain Defines Determinants for Intramembrane Proteolysis by Signal Peptide Peptidase

Cell Rep. 2019 Mar 12;26(11):3087-3099.e11. doi: 10.1016/j.celrep.2019.02.057.


Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side-chain interactions near the scissile peptide bond with the enzyme's active site.

Keywords: ERAD; GxGD aspartic intramembrane protease; HO1; SPP; XBP1u; exosite; regulated intramembrane proteolysis; subsite; transmembrane helix dynamics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid Endopeptidases / metabolism*
  • Endoplasmic Reticulum / metabolism
  • HEK293 Cells
  • Heme Oxygenase-1 / metabolism
  • Humans
  • Intracellular Membranes / metabolism*
  • Mutation
  • Protein Domains
  • Proteolysis*
  • SEC Translocation Channels / metabolism
  • X-Box Binding Protein 1 / chemistry
  • X-Box Binding Protein 1 / genetics
  • X-Box Binding Protein 1 / metabolism*


  • SEC Translocation Channels
  • X-Box Binding Protein 1
  • XBP1 protein, human
  • Heme Oxygenase-1
  • Aspartic Acid Endopeptidases
  • signal peptide peptidase