Structural and mutational analysis of the ribosome-arresting human XBP1u

Abstract

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 ...

EDF1 coordinates cellular responses to ribosome collisions

“…In an orthogonal set of reporter experiments, loss of EDF1, but not ZNF598, led to an increase in the bulk translation output (as measured by a significant increase in Renilla luciferase (RLuc) activity) on a reporter with an unspliced Xbp1 mRNA (Xbp1u) peptide-pausing sequence ( Shanmuganathan et al, 2019 ; Yanagitani et al, 2011 ) previously reported to trigger ribosome collisions ( Han et al, 2020 ; Figure 6—figure supplement 2H for relative RLuc activity, and Figure 6—figure supplement 2I for Fluc:RLuc ratios). Taken together, these data establish that EDF1 recruits the translational repressors GIGYF2•EIF4E2 to multiple types of problematic mRNA sequences.…”

Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

“…However, the uL22Δloop ribosomes showed an increased IRD when 20 or more amino acids were present before the EPDP (Figure 3D, orange lines). Since previous studies suggested that the uL22 β-hairpin loop interacts with the nascent polypeptides at a 10-20 aa distance from the PTC (Bhushan et al, 2011; Seidelt et al, 2009; Shanmuganathan et al, 2019; Sohmen et al, 2015; Su et al, 2017), our data suggest that the interactions between the nascent polypeptide and the tunnel constriction site contribute to the elongation continuity beyond the IRD sequence, perhaps by changing the stability of the ribosome.…”

“…In contrast to the initial suggestion that the tunnel interior is inert in terms of interactions with the nascent polypeptide (Nissen et al, 2000), in reality, the tunnel interacts with nascent polypeptide chains, thus contributing to biological regulation (Chiba et al, 2009; Gong and Yanofsky, 2002; Ishii et al, 2015; Nakatogawa and Ito, 2002; Onouchi et al, 2005; Yanagitani et al, 2011, Ito and Chiba, 2013). Cryo-EM structures of ribosomes stalled by ribosome arresting peptides (RAPs) have revealed a range of interactions between the nascent chains and the ribosomal interior components at the PTC and/or the exit tunnel, such as the uL22/uL4 constriction site (Bhushan et al, 2011; Seidelt et al, 2009; Shanmuganathan et al, 2019; Sohmen et al, 2015; Su et al, 2017). Recent analyses using ribosome profiling (Dao Duc and Song, 2018; Han et al, 2020; Requião et al, 2016; Sabi and Tuller, 2015) and direct detection of accumulating peptidyl-tRNAs (Chadani et al, 2016) have suggested the widespread occurrence of translation pausing, to which nascent polypeptides may contribute.…”

Cotranslational folding of a periplasmic protein domain inEscherichia coli

“…chain length – a force profile (FP) – provides a map of cotranslational force-generating events with up to single-residue resolution. Further, by using APs of different stalling strengths, FPs can be fine-tuned to optimally reflect different kinds of force-generating events [35, 36].…”