XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

26 XBP1u, a central component of the unfolded protein response (UPR), is a 27 mammalian protein containing a functionally critical translational arrest 28 peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human 29 XBP1u AP. It forms a unique turn in the upper part of the ribosomal exit tunnel 30 and causes a subtle distortion of the peptidyl transferase center, explaining 31 the temporary translational arrest induced by XBP1u. During ribosomal 32 pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but 33 fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan 34 of the XBP1u AP revealed that only 10 out of 21 mutagenized positions in the 35 XBP1u AP are optimal with respect to translational arrest activity. Thus, 36 XBP1u has evolved to induce an intermediate level of translational arrest, 37 allowing efficient targeting by SRP without activating the Sec61 channel and 38 thereby serving its central function in the UPR. 39 42 towards their respective destinations. The ER handles approximately one-third 43 of the proteome and the flux of proteins entering the ER lumen varies widely, 44primarily depending on the demands of the specific cell type. The ER is also 45 responsible for maintaining calcium homeostasis and is involved in lipid 46 biosynthesis (Fagone and Jackowski, 2009; Görlach et al., 2006). A number 47 of circumstances can alter the folding and modifying capacity of the ER, such 48 as glucose deprivation, calcium imbalance, hypoxia, or viral infection, and 49 thereby alter the demand on ER activity, as shown, e.g., in B cell 50 differentiation (Grootjans et al., 2016). The central cellular response 51 mechanism that alleviates ER stress and adjusts ER activity levels is the 52 unfolded protein response (UPR) (Walter and Ron, 2011). In mammalian 53 cells, this pathway is mainly mediated by three transmembrane sensors that 54 are located in the ER membrane: inositol requiring enzyme 1 alpha (IRE1α), 55 activating transcription factor 6 (ATF6), and pancreatic endoplasmic reticulum 56 kinase (PERK) (Walter and Ron, 2011). 57Of these three sensors, the evolutionarily most conserved is IRE1 (here, IRE1 58 denotes mammalian IRE1α and/or yeast Ire1); in lower eukaryotes such as 59 yeast, it is the only known sensor mediating the UPR (Mori, 2009). IRE1 is a 60 single-spanning membrane protein with three domains: a luminal unfolded 61 protein-sensing domain and cytosolic bifunctional serine/threonine kinase and 62 endo-ribonuclease domains. In unstressed cells, Hsp70 family chaperone BiP 63 binds the luminal region of IRE1 and keeps IRE1 in an inactive monomeric 64 4 state. Increasing levels of misfolded proteins during ER stress sequester BiP 65 away from, leading to active dimer (Bertolotti et al., 2000; Okamura et al., 66 2000) and further highly activated by cluster formation (Aragón et al., 2009; 67 Credle et al., 2005; Kimata et al., 2007; Korennykh et al., 2009; Li et al., 2010) 68 In yeast, direct binding of unfolded proteins to the luminal core ...

Secretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation machinery. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient versus inefficient signal sequencing, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phase event whereby the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products. Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.

SummarySecretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation channel complex. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER-targeting signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation initiation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient, versus inefficient (poorly hydrophobic) signal sequences, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phases event in which the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products.Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Mycobacterium tuberculosis (Mtb) bacilli are the causative agent of tuberculosis (TB), a major killer of mankind. Although it is widely accepted that local interactions between Mtb and the immune system in the tuberculous granuloma determine whether the outcome of infection is controlled or disseminated, these have been poorly studied due to methodological constraints. We have recently used a spatial transcriptomic technique, in situ sequencing (ISS), to define the spatial distribution of immune transcripts in TB mouse lungs. To further contribute to the understanding of the immune microenvironments of Mtb and their local diversity, we here present two complementary automated bacteria-guided analysis pipelines. These position 33 ISS-identified immune transcripts in relation to single bacteria and bacteria clusters. The analysis was applied on new ISS data from lung sections of Mtb-infected C57BL/6 and C3HeB/FeJ mice. In lungs from C57BL/6 mice early and late post infection, transcripts that define inflammatory macrophages were enriched at subcellular distances to bacteria, indicating the activation of infected macrophages. In contrast, expression patterns associated to antigen presentation were enriched in non-infected cells at 12 weeks post infection. T-cell transcripts were evenly distributed in the tissue. In Mtb-infected C3HeB/FeJ mice, transcripts characterizing activated macrophages localized in apposition to small bacteria clusters, but not in organized granulomas. Despite differences in the susceptibility to Mtb, the transcript patterns found around small bacteria clusters of C3HeB/FeJ and C57BL/6 mice were similar. Altogether, the presented tools allow us to characterize in depth the immune cell populations and their activation that interact with Mtb in the infected lung.