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bioRxiv · 10.1101/322107

Metastable XBP1u transmembrane domain mediates insertion into the ER membrane and intramembrane proteolysis by the signal peptide peptidase

Yucel, S. S.·Stelzer, W.·Lorenzoni, A.·Wozny, M.·Langosch, D.·Lemberg, M. K.
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Abstract

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 enzymes active site.

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Yucel, S. S., Stelzer, W., Lorenzoni, A., Wozny, M., Langosch, D., Lemberg, M. K.. 2018-05-14. Metastable XBP1u transmembrane domain mediates insertion into the ER membrane and intramembrane proteolysis by the signal peptide peptidase. https://doi.org/10.1101/322107

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