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Mutational analysis of the human Xbp1 translational arrest peptide and construction of arrest-enhanced variants
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Xbp1, a protein involved in the unfolded protein response, is a rare example of a mammalian protein that contains a well-defined translational arrest peptide (AP). In order to define the critical residues in the Xbp1u AP, and to search for variants with stronger arrest potency than the wildtype Xbp1u AP, we have carried out a full mutagenesis scan where each residue in the AP was replaced by the other 19 natural amino acids. We find that 10 of the 21 mutagenized positions are optimal already in the wildtype Xbp1 AP, while certain mutations in the remaining residues lead to a strong increase in the arrest potency. Xbp1 has thus evolved to induce an intermediate level of translational arrest, and versions with much stronger arrest efficiency exist. We further show Xbp1- induced translational arrest is reduced in response to increased tension in the nascent chain, making it possible to carry out studies in mammalian systems of cotranslational processes such as membrane protein assembly and protein folding by using suitable Xbp1 AP variants as “force sensors”, as has been done previously in E. coli using bacterial APs.

Keyword [en]
Arrest peptide, translation, Xbp1
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-147391OAI: oai:DiVA.org:su-147391DiVA: diva2:1144445
Available from: 2017-09-26 Created: 2017-09-26 Last updated: 2017-09-26Bibliographically approved
In thesis
1. Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
Open this publication in new window or tab >>Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell.   To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize.  In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains.     

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry, Stockholm Universityand Biophysics, Stockholm University, 2017. 88 p.
Keyword
ribosome, membrane integration, translocation, arrest peptide, SecM, Xbp1
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-146869 (URN)978-91-7797-002-6 (ISBN)978-91-7797-003-3 (ISBN)
Public defence
2017-10-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2017-10-03 Created: 2017-09-14 Last updated: 2017-10-04Bibliographically approved

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