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What’s in? What’s out? And how did it get there?: Studies on topologies and insertion of membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
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: urn:nbn:se:su:diva-42321ISBN: 978-91-7447-138-0 (print)OAI: oai:DiVA.org:su-42321DiVA: diva2:346255
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
List of papers
1. A global topology map of the Saccharomyces cerevisiae membrane proteome
Open this publication in new window or tab >>A global topology map of the Saccharomyces cerevisiae membrane proteome
2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 30, 11142-11147 p.Article in journal (Refereed) Published
Abstract [en]

The yeast Saccharomyces cerevisiae is, arguably, the best understood eukaryotic model organism, yet comparatively little is known about its membrane proteome. Here, we report the cloning and expression of 617 S. cerevisiae membrane proteins as fusions to a C-terminal topology reporter and present experimentally constrained topology models for 546 proteins. By homology, the experimental topology information can be extended to ≈15,000 membrane proteins from 38 fully sequenced eukaryotic genomes.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24329 (URN)10.1073/pnas.0604075103 (DOI)
Note
Part of urn:nbn:se:su:diva-6875Available from: 2007-05-24 Created: 2007-05-15 Last updated: 2017-12-13Bibliographically approved
2. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae.
Open this publication in new window or tab >>Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae.
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 30, 11148-11153 p.Article in journal (Refereed) Published
Abstract [en]

Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing ≈600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells’ response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies.

Keyword
Caffeine/pharmacology, Cluster Analysis, Fungal Proteins/chemistry, Gene Expression Regulation; Fungal, Genome; Fungal, Green Fluorescent Proteins/metabolism, Membrane Proteins/biosynthesis/*chemistry, Oxidative Stress, Phenotype, Plasmids/metabolism, Protein Structure; Tertiary, Saccharomyces cerevisiae/*metabolism, Saccharomyces cerevisiae Proteins/chemistry, Salts/pharmacology
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-18775 (URN)10.1073/pnas.0604078103 (DOI)16847257 (PubMedID)
Available from: 2007-12-27 Created: 2007-12-27 Last updated: 2017-12-13Bibliographically approved
3. Insertion of model helices into the mitochondrial inner membrane: the rules of the game
Open this publication in new window or tab >>Insertion of model helices into the mitochondrial inner membrane: the rules of the game
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:su:diva-42431 (URN)
Available from: 2010-08-31 Created: 2010-08-31 Last updated: 2010-08-31Bibliographically approved
4. Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
Open this publication in new window or tab >>Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
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.

Keyword
Mgm1p, Mitochondria, TIM23, Transmembrane helix, Yeast, Saccharomyces cerevisiae
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-68816 (URN)10.1016/j.febslet.2011.03.056 (DOI)000289505400021 ()
Note
authorCount :4Available from: 2012-01-09 Created: 2012-01-07 Last updated: 2017-12-08Bibliographically approved

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