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Structural and Functional Profiling of the Lateral Gate of the Sec61 Translocon
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 22, p. 15845-15855Article in journal (Refereed) Published
Abstract [en]

The evolutionarily conserved Sec61 translocon mediates the translocation and membrane insertion of proteins. For the integration of proteins into the membrane, the Sec61 translocon opens laterally to the lipid bilayer. Previous studies suggest that the lateral opening of the channel is mediated by the helices TM2b and TM7 of a pore-forming subunit of the Sec61 translocon. To map key residues in TM2b and TM7 in yeast Sec61 that modulate lateral gating activity, we performed alanine scanning and in vivo site-directed photocross-linking experiments. Alanine scanning identified two groups of critical residues in the lateral gate, one group that leads to defects in the translocation and membrane insertion of proteins and the other group that causes faster translocation and facilitates membrane insertion. Photocross-linking data show that the former group of residues is located at the interface of the lateral gate. Furthermore, different degrees of defects for the membrane insertion of single- and double-spanning membrane proteins were observed depending on whether the mutations were located in TM2b or TM7. These results demonstrate subtle differences in the molecular mechanism of the signal sequence binding/opening of the lateral gate and membrane insertion of a succeeding transmembrane segment in a polytopic membrane protein.

Place, publisher, year, edition, pages
2014. Vol. 289, no 22, p. 15845-15855
Keywords [en]
Endoplasmic Reticulum (ER), Membrane, Membrane Protein, Protein Translocation, Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-106102DOI: 10.1074/jbc.M113.533794ISI: 000337465400063OAI: oai:DiVA.org:su-106102DiVA, id: diva2:735315
Note

AuthorCount:5;

Available from: 2014-07-25 Created: 2014-07-21 Last updated: 2022-03-23Bibliographically approved
In thesis
1. Membrane Protein Biogenesis in Saccharomyces cerevisiae
Open this publication in new window or tab >>Membrane Protein Biogenesis in Saccharomyces cerevisiae
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membranes are hydrophobic barriers that define the outer boundaries and internal compartments of living cells. Membrane proteins are the gates in these barriers, and they perform vital functions in the highly regulated transport of matter and information across membranes. Membrane proteins destined for the endoplasmic reticulum are targeted either co- or post-translationally to the Sec61 translocon, the major translocation machinery in eukaryotic cells, which allows for lateral partitioning of hydrophobic segments into the lipid bilayer. This thesis aims to acquire insights into the mechanism of membrane protein insertion and the role of different translocon components in targeting, insertion and topogenesis, using the yeast Saccharomyces cerevisiae as a model organism.

By measuring the insertion efficiency of a set of model proteins, we studied the sequence requirements for Sec61-mediated insertion of an α-helical transmembrane segment and established a ‘biological hydrophobicity scale’ in yeast, which describes the individual contributions of the 20 amino acids to insertion. Systematic mutagenesis and photo-crosslinking of the Sec61 translocon revealed key residues in the lateral gate that modulate the threshold hydrophobicity for membrane insertion and transmembrane segment orientation. Further, my studies demonstrate that the translocon-associated Sec62 is important not only for post-translational targeting, but also for the insertion and topogenesis of moderately hydrophobic signal anchor proteins and the C-terminal translocation of multi-spanning membrane proteins. Finally, nuclearly encoded mitochondrial membrane proteins were found to evade mis-targeting to the endoplasmic reticulum by containing short C-terminal tails.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. p. 72
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-95376 (URN)978-91-7447-798-6 (ISBN)
Public defence
2013-12-13, Nordenskiöld Lecture Hall, Geo-Science Building, Svante Arrhenius väg 12, Stockholm, 13:00 (English)
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Supervisors
Note

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript

Available from: 2013-11-21 Created: 2013-10-26 Last updated: 2022-02-24Bibliographically approved

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