Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kriegler, Theresa, Lang, Sven, Notari, Luigi, Hessa, Tara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
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
_version_ 1783553954264645632
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
work_keys_str_mv AT krieglertheresa supportingdataonprionproteintranslocationmechanismrevealedbypullingforcestudies
AT langsven supportingdataonprionproteintranslocationmechanismrevealedbypullingforcestudies
AT notariluigi supportingdataonprionproteintranslocationmechanismrevealedbypullingforcestudies
AT hessatara supportingdataonprionproteintranslocationmechanismrevealedbypullingforcestudies