Abstract
The unspliced XBP1 mRNA encodes the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1u. Besides acting as a negative regulator for UPR, XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) facilitating IRE1-mediated splicing. Yet, its membrane association is controversial. Here, we use cell-free translocation assays and living cells to define a moderately hydrophobic stretch in XBP1u that is sufficient for insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that determine XBP1u for an ER-associated degradation route centered around the signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by recording residue-specific amide exchange kinetics, where data was evaluated by a novel semi-automated method. As compared to the previously employed procedure, this method reduces the time needed for data processing by approximately an order of magnitude. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by its conformational flexibility, but also by side chain interactions near the cleavage site with the enzyme’s active site.
