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Dissecting Stop Transfer versus Conservative Sorting Pathways for Mitochondrial Inner Membrane Proteins in Vivo
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 3, 1521-1532 p.Article in journal (Refereed) Published
Abstract [en]

Mitochondrial inner membrane proteins that carry an N-terminal presequence are sorted by one of two pathways: stop-transfer or conservative sorting. However, the sorting pathway is known for only a small number of proteins, in part due to lack of robust experimental tools with which to study. Here we present an approach that facilitates determination of inner membrane protein sorting pathways in vivo by fusing a mitochondrial inner membrane protein to the C-terminal part of Mgm1p containing the rhomboid cleavage region. We validated the Mgm1 fusion approach using a set of proteins for which the sorting pathway is known, and determined sorting pathways of inner membrane proteins for which the sorting mode is previously uncharacterized. For Sdh4p, a multi-spanning membrane protein, our results suggest that both conservative sorting and stop-transfer mechanisms are required for insertion. Furthermore, the sorting process of Mgm1 fusion proteins was analyzed under different growth conditions and yeast mutant strains that were defective in the import motor or the m-AAA protease function. Our results show that the sorting of mitochondrial proteins carrying moderately hydrophobic transmembrane segments is sensitive to cellular conditions, implying that mitochondrial import and membrane sorting in the physiological environment may be dynamically tuned.

Place, publisher, year, edition, pages
2012. Vol. 288, no 3, 1521-1532 p.
National Category
Biochemistry and Molecular Biology Biophysics
URN: urn:nbn:se:su:diva-83479DOI: 10.1074/jbc.M112.409748ISI: 000313751400010OAI: diva2:575956
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2013-03-19Bibliographically approved
In thesis
1. Translocation of proteins into and across the bacterial and mitochondrial inner membranes
Open this publication in new window or tab >>Translocation of proteins into and across the bacterial and mitochondrial inner membranes
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]


Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening.

This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively.

These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane.

A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease.

Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 86 p.
Escherichia coli, mitochondria, Saccharomyces cerevisiae, SecYEG, TIM23, transmembrane helix
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:su:diva-83234 (URN)978-91-7447-600-2 (ISBN)
Public defence
2013-01-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)

At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 1: Manuscript; Paper nr. 4: Manuscript

Available from: 2012-12-20 Created: 2012-12-06 Last updated: 2012-12-21Bibliographically approved

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Calado Botelho, Salomé
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