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.

The mammalian prion protein (PrP) engages with the ribosome-Sec61 translocation channel complex to generate different topological variants that are either physiological, or involved in neurodegenerative diseases. Here, we describe cotranslational folding and translocation mechanisms of PrP coupled to an Xbp1-based arrest peptide (AP) as folding sensor, to measure forces acting on PrP nascent chain. Our data reveal two main pulling events followed by a minor third one exerted on the nascent chains during their translocation. Using those force landscapes, we show that a specific sequence within an intrinsically disordered region, containing a polybasic and glycine-proline rich residues, modulates the second pulling event by interacting with TRAP complex. This work also delineates the sequence of events involved in generation of PrP toxic transmembrane topologies during its synthesis. Our results shed new insight into the folding of such a topological complex protein, where marginal pulling by the signal sequence, together with the flanking downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt alternative topologies.

The Prion protein (PrP) is a highly conserved cell surface glycoprotein. To enter the secretory pathway, the PrP precursor relies on the Sec61 complex and multiple accessory factors all gathering at the membrane of the Endoplasmic reticulum (ER). PrP topogenesis results in the formation of different PrP isoforms. Aside from the typical secretory variant (SecPrP) different pathognomonic, membrane-embedded variants (NtmPrP and CtmPrP) that are associated with neurodegenerative diseases can be found [1]. In this article, we provide supportive data related to “Prion Protein Translocation Mechanism Revealed by Pulling Force Studies” (Kriegler et al., May 2020)[2], where we utilize Xbp1 arrest peptide (AP)-mediated ribosomal stalling to study the co-translational folding experienced by PrP during its insertion into the ER. We measure translocation efficiency and characterize the force exerted on PrP nascent chain so called “pulling force profile”. Here, we describe the method of AP-mediated ribosomal stalling assay together with additional experimental data to the main article. Furthermore, we describe the combination of AP-mediated ribosomal stalling and semi-permeabilized Hela cells (SPCs) as ER membrane source. Using this experimental set-up one can directly determine the contribution of a specific membrane component, e.g. subunits of the ER protein translocase, as pulling factor exerting force on the PrP nascent chain.

The data presented here covers (a) the SDS-PAGE gel images visualized by autoradiography, (b) quantification of the different populations of PrP species observed in the AP-mediated ribosomal stalling method, and (c) calculation formulas of the pulling force profiles measured in SPCs in comparison to dog pancreas microsomes as ER membrane donor. Finally, Western Blot analysis and quantification of siRNA knockdown levels compared to control conditions of various translocation components are shown.

Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-boxbinding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAP beta subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.