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Supporting data on prion protein translocation mechanism revealed by pulling force studies
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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334574/ https://www.ncbi.nlm.nih.gov/pubmed/32642528 http://dx.doi.org/10.1016/j.dib.2020.105931 |
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author | Kriegler, Theresa Lang, Sven Notari, Luigi Hessa, Tara |
author_facet | Kriegler, Theresa Lang, Sven Notari, Luigi Hessa, Tara |
author_sort | Kriegler, Theresa |
collection | PubMed |
description | 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 ((Sec)PrP) different pathognomonic, membrane-embedded variants ((Ntm)PrP and (Ctm)PrP) 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. |
format | Online Article Text |
id | pubmed-7334574 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-73345742020-07-07 Supporting data on prion protein translocation mechanism revealed by pulling force studies Kriegler, Theresa Lang, Sven Notari, Luigi Hessa, Tara Data Brief Biochemistry, Genetics and Molecular Biology 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 ((Sec)PrP) different pathognomonic, membrane-embedded variants ((Ntm)PrP and (Ctm)PrP) 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. Elsevier 2020-06-27 /pmc/articles/PMC7334574/ /pubmed/32642528 http://dx.doi.org/10.1016/j.dib.2020.105931 Text en © 2020 The Authors. Published by Elsevier Inc. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Biochemistry, Genetics and Molecular Biology Kriegler, Theresa Lang, Sven Notari, Luigi Hessa, Tara Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title | Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title_full | Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title_fullStr | Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title_full_unstemmed | Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title_short | Supporting data on prion protein translocation mechanism revealed by pulling force studies |
title_sort | supporting data on prion protein translocation mechanism revealed by pulling force studies |
topic | Biochemistry, Genetics and Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334574/ https://www.ncbi.nlm.nih.gov/pubmed/32642528 http://dx.doi.org/10.1016/j.dib.2020.105931 |
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