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

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. [Display omitted] •XBP1u functionally interacts with Sec61, leading to membrane insertion•The membrane span of XBP1u and the tail-anchored protein HO1 are recognized by SPP•Introducing transmembrane leucine and glycine residues changes helix dynamics•SPP cleavage is governed by transmembrane helix dynamics and site-specific features Using XBP1u and determining its functional association with Sec61, Yücel et al. describe a model in which SPP requires both conformational flexibility and site-specific interactions to proteolyze its substrate.