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Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
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.
2011 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 585, no 8, 1238-1242 p.Article in journal (Refereed) Published
Abstract [en]

Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique alternative topogenesis process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane.

Place, publisher, year, edition, pages
2011. Vol. 585, no 8, 1238-1242 p.
Keyword [en]
Mgm1p, Mitochondria, TIM23, Transmembrane helix, Yeast, Saccharomyces cerevisiae
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-68816DOI: 10.1016/j.febslet.2011.03.056ISI: 000289505400021OAI: oai:DiVA.org:su-68816DiVA: diva2:474265
Note
authorCount :4Available from: 2012-01-09 Created: 2012-01-07 Last updated: 2017-12-08Bibliographically approved
In thesis
1. What’s in? What’s out? And how did it get there?: Studies on topologies and insertion of membrane proteins
Open this publication in new window or tab >>What’s in? What’s out? And how did it get there?: Studies on topologies and insertion of membrane proteins
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Because of their hydrophobic and hydrophilic nature and the need for a lipid bilayer for retaining the native structure, membrane proteins are hard to study. Nevertheless, they are important, as many of our diseases are related to membrane proteins and around 60% of the different pharmaceutical drugs are directed against a membrane proteins [1]. There are many ways to study a protein, you can study function, structure, how the protein is targeted and inserted into its specific organelle, the interactions with other proteins or ligands etc. In the absence of a high-resolution structure, a topology model for a membrane protein is often useful. We have obtained reliable topologies for 546 of the membrane proteins going through the secretory pathways in S. cerevisiae by combining experimental data with topology prediction programs. In addition we have produced topology models for over 15,000 membrane proteins from 38 sequenced eukaryotic genomes using homology to the experimentally determined group.

We also examined the growth rates and tolerance to certain stress conditions for our large set of clones that over-express membrane proteins. This provides important information both for structural studies of membrane proteins where large amounts of protein is needed for further studies, and for getting some insight in the function of specific proteins. Finally we have studied the integration of membrane proteins by the Tim23 translocon in the inner membrane of mitochondria. We have investigated the hydrophobicity required for efficient integration of transmembrane (TM) helices by Tim23. From this data we have derived an in vivo hydrophobicity scale for the insertion of different amino acids into the inner membrane of the mitochondria, and have made a comparison with a previously determined hydrophobicity scale for the ER translocon Sec61. We concluded that charged residues flanking the TM segment are of major importance for insertion into the membrane.

We therefore further investigated the importance of charged residues flanking the first, weakly hydrophobic, TM segment in the mitochondrial inner membrane protein Mgm1p with regard to membrane insertion by the Tim23 complex.

Place, publisher, year, edition, pages
Stockholm: Department of biochemistry and biophysics, Stockholm University, 2010. 72 p.
Keyword
membrane protein, topology, yeast, mitochondria, TIM23
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42321 (URN)978-91-7447-138-0 (ISBN)
Public defence
2010-10-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: ManuscriptAvailable from: 2010-09-30 Created: 2010-08-24 Last updated: 2012-01-09Bibliographically approved
2. 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.
Keyword
Escherichia coli, mitochondria, Saccharomyces cerevisiae, SecYEG, TIM23, transmembrane helix
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
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)
Opponent
Supervisors
Note

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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