| 1. |
- Kriegler, Theresa, et al.
(author)
-
Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide
- 2018
-
In: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:7, s. 880-890
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 2. |
- Kriegler, Theresa, et al.
(author)
-
Prion Protein Translocation Mechanism Revealed by Pulling Force Studies
- 2020
-
In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:16, s. 4447-4465
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 3. |
- Kriegler, Theresa, 1990-
(author)
-
Pulling Force Studies of Secretory Protein Translocation into the Endoplasmic Reticulum
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- More than 30% of human genes encode secretory or membrane proteins. Most secretory proteins are targeted to the Endoplasmic reticulum (ER) membrane via cleavable N-terminal signal sequences either in a co- or post-translational manner. They enter or cross the membrane using a protein translocating channel (translocon). Although the core of the translocon, formed by the Sec61 complex, was identified some time ago, the details of how signal sequences can facilitate channel opening and initiate protein translocation still remain unclear. Interestingly, the signal sequences of different proteins do not share any sequence homology—only general motifs have been described—but the precise sequence has been found to substantially affect the efficiency of translocation initiation. Many proteins require auxiliary components in order to enter the ER lumen. During ER stress conditions, these weakly gating proteins are prevented from entering, reducing the load of unfolded protein within the ER and protecting the cell. Consequently, it is tempting to hypothesize that the “inefficiencies” of signal sequences may actually provide a different message that works as a protective mechanism during ER stress conditions.Here, we employed a translational arrest peptide, which pauses the ribosome until a force—such as the interaction of the signal sequence with the translocon—acts on the nascent chain. We analyzed the different forces that are experienced by efficient and inefficient signal sequences during their biosynthesis in vitro. Our data shows that the efficient signal sequence of prolactin (Prl) experiences a strong biphasic pulling force while less efficient sequences, such as the ones from the Prion protein (PrP) or insulin, are pulled to a much lesser extent, indicating different modes of engagement with the translocon. The Prl signal sequence interacts first with a hydrophobic patch within the channel (the first pulling event), next it is inverted and intercalates into the lateral gate of the translocon, facilitating channel opening both laterally and axially. In the case of PrP or insulin, the initiation of translocation is delayed, suggesting that the opening of the channel might require auxiliary components. In order to explore this, we made use of semi-permeabilized cells (SPCs) prepared after siRNA knockdown of components of the translocation machinery and studied the effect on the observed pulling events and translocation efficiency. We found that the translocon-associated protein (TRAP) complex enhanced translocation of client proteins bearing weakly gating signal sequences that contained more glycine and proline residues. Additionally, we showed that TRAP plays a role in the translocation of intrinsically disordered domains with a high content of proline and glycine residues, and other regions of the mature protein enriched in positively charged amino acids. Chemical crosslinking revealed that TRAP contacts the insulin nascent chain before it enters the translocon channel suggesting that TRAP scans along the translocating protein and provides sequence-dependent assistance in facilitating channel opening and as a ratchet for challenging regions of the mature protein. Taken together, all of this data expands our understanding of the interplay between the signal sequence and the mature protein during translocation and protein folding and how the cell may take advantage of this to regulate translocation during ER stress.
|
|
| 4. |
- Kriegler, Theresa, et al.
(author)
-
Supporting data on prion protein translocation mechanism revealed by pulling force studies
- 2020
-
In: Data in Brief. - : Elsevier BV. - 2352-3409. ; 31
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 5. |
- Kriegler, Theresa, et al.
(author)
-
Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin
- 2020
-
In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:24
-
Journal article (peer-reviewed)abstract
- 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.
|
|
